AbstractBackgroundWe have successfully used whole-genome sequencing to provide additional information for transmission pathways in infectious spread. We report and interpret genomic sequencing results in clinical context from a large outbreak of COVID-19 with 46 cases across staff and patients in a community hospital in the UK.MethodsFollowing multiple symptomatic cases within a two-week period, all staff and patients were screened by RT-PCR and staff subsequently had serology tests.ResultsThirty staff (25%) and 16 patients (62%) tested positive for COVID-19. Genomic sequencing data showed significant overlap of viral haplotypes in staff who had overlapping shift patterns. Patient haplotypes were more distinct from each other but had overlap with staff haplotypes.ConclusionsThis study includes clinical and genomic epidemiological detail that demonstrates the value of a combined approach. Viral genetic sequencing has identified that staff transmission of COVID-19 was important in this community hospital outbreak.Key pointsDetailed analysis of a large community hospital outbreak in older adults and staff with concurrent clinical and genomic data, including working patterns.Staff transmission was important in this community hospital outbreak.We found plausible associations between staff and patient cases.

BACKGROUND: Fetal hydrops is excessive extravasation of fluid into the third space in a fetus, which could be due to a wide differential of underlying pathology. IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome primarily affects males. It is a monogenic primary immunodeficiency syndrome of X-linked recessive inheritance due to FOXP3 gene variants. It is characterised by the development of multiple autoimmune disorders in affected individuals. CASE PRESENTATION: We present a rare cause of male fetal hydrops in the context of IPEX syndrome and discuss FOXP3 gene variants as a differential for 'unexplained' fetal hydrops that may present after the first trimester. DISCUSSION AND CONCLUSIONS: in all similar cases, the pathological process begins during intrauterine life. Furthermore, there are no survivors described. Consequently, this variant should be considered as a severe one, associated with intrauterine life onset and fatal course, i.e. the most severe IPEX phenotype.

EDEM3 encodes a protein that converts Man8GlcNAc2 isomer B to Man7-5GlcNAc2. It is involved in the endoplasmic reticulum-associated degradation pathway, responsible for the recognition of misfolded proteins that will be targeted and translocated to the cytosol and degraded by the proteasome. In this study, through a combination of exome sequencing and gene matching, we have identified seven independent families with 11 individuals with bi-allelic protein-truncating variants and one individual with a compound heterozygous missense variant in EDEM3. The affected individuals present with an inherited congenital disorder of glycosylation (CDG) consisting of neurodevelopmental delay and variable facial dysmorphisms. Experiments in human fibroblast cell lines, human plasma, and mouse plasma and brain tissue demonstrated decreased trimming of Man8GlcNAc2 isomer B to Man7GlcNAc2, consistent with loss of EDEM3 enzymatic activity. In human cells, Man5GlcNAc2 to Man4GlcNAc2 conversion is also diminished with an increase of Glc1Man5GlcNAc2. Furthermore, analysis of the unfolded protein response showed a reduced increase in EIF2AK3 (PERK) expression upon stimulation with tunicamycin as compared to controls, suggesting an impaired unfolded protein response. The aberrant plasma N-glycan profile provides a quick, clinically available test for validating variants of uncertain significance that may be identified by molecular genetic testing. We propose to call this deficiency EDEM3-CDG.

BACKGROUND: Coat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of proteins and lipids from the Golgi apparatus to the endoplasmic reticulum (ER). In recent years, coat proteins have been implicated in human diseases known collectively as "coatopathies". METHODS: Whole exome or genome sequencing of two families with a neuro-developmental syndrome, variable microcephaly and cataracts revealed biallelic variants in COPB1, which encodes the beta-subunit of COPI (β-COP). To investigate Family 1's splice donor site variant, we undertook patient blood RNA studies and CRISPR/Cas9 modelling of this variant in a homologous region of the Xenopus tropicalis genome. To investigate Family 2's missense variant, we studied cellular phenotypes of human retinal epithelium and embryonic kidney cell lines transfected with a COPB1 expression vector into which we had introduced Family 2's mutation. RESULTS: We present a new recessive coatopathy typified by severe developmental delay and cataracts and variable microcephaly. A homozygous splice donor site variant in Family 1 results in two aberrant transcripts, one of which causes skipping of exon 8 in COPB1 pre-mRNA, and a 36 amino acid in-frame deletion, resulting in the loss of a motif at a small interaction interface between β-COP and β'-COP. Xenopus tropicalis animals with a homologous mutation, introduced by CRISPR/Cas9 genome editing, recapitulate features of the human syndrome including microcephaly and cataracts. In vitro modelling of the COPB1 c.1651T>G p.Phe551Val variant in Family 2 identifies defective Golgi to ER recycling of this mutant β-COP, with the mutant protein being retarded in the Golgi. CONCLUSIONS: This adds to the growing body of evidence that COPI subunits are essential in brain development and human health and underlines the utility of exome and genome sequencing coupled with Xenopus tropicalis CRISPR/Cas modelling for the identification and characterisation of novel rare disease genes.

Kainate receptors (KARs) are glutamate-gated cation channels with diverse roles in the central nervous system. Bi-allelic loss of function of the KAR-encoding gene GRIK2 causes a nonsyndromic neurodevelopmental disorder (NDD) with intellectual disability and developmental delay as core features. The extent to which mono-allelic variants in GRIK2 also underlie NDDs is less understood because only a single individual has been reported previously. Here, we describe an additional eleven individuals with heterozygous de novo variants in GRIK2 causative for neurodevelopmental deficits that include intellectual disability. Five children harbored recurrent de novo variants (three encoding p.Thr660Lys and two p.Thr660Arg), and four children and one adult were homozygous for a previously reported variant (c.1969G>A [p.Ala657Thr]). Individuals with shared variants had some overlapping behavioral and neurological dysfunction, suggesting that the GRIK2 variants are likely pathogenic. Analogous mutations introduced into recombinant GluK2 KAR subunits at sites within the M3 transmembrane domain (encoding p.Ala657Thr, p.Thr660Lys, and p.Thr660Arg) and the M3-S2 linker domain (encoding p.Ile668Thr) had complex effects on functional properties and membrane localization of homomeric and heteromeric KARs. Both p.Thr660Lys and p.Thr660Arg mutant KARs exhibited markedly slowed gating kinetics, similar to p.Ala657Thr-containing receptors. Moreover, we observed emerging genotype-phenotype correlations, including the presence of severe epilepsy in individuals with the p.Thr660Lys variant and hypomyelination in individuals with either the p.Thr660Lys or p.Thr660Arg variant. Collectively, these results demonstrate that human GRIK2 variants predicted to alter channel function are causative for early childhood development disorders and further emphasize the importance of clarifying the role of KARs in early nervous system development.

Accurate classification of variants in cancer susceptibility genes (CSGs) is key for correct estimation of cancer risk and management of patients. Consistency in the weighting assigned to individual elements of evidence has been much improved by the American College of Medical Genetics (ACMG) 2015 framework for variant classification, UK Association for Clinical Genomic Science (UK-ACGS) Best Practice Guidelines and subsequent Cancer Variant Interpretation Group UK (CanVIG-UK) consensus specification for CSGs. However, considerable inconsistency persists regarding practice in the combination of evidence elements. CanVIG-UK is a national subspecialist multidisciplinary network for cancer susceptibility genomic variant interpretation, comprising clinical scientist and clinical geneticist representation from each of the 25 diagnostic laboratories/clinical genetic units across the UK and Republic of Ireland. Here, we summarise the aggregated evidence elements and combinations possible within different variant classification schemata currently employed for CSGs (ACMG, UK-ACGS, CanVIG-UK and ClinGen gene-specific guidance for PTEN, TP53 and CDH1). We present consensus recommendations from CanVIG-UK regarding (1) consistent scoring for combinations of evidence elements using a validated numerical 'exponent score' (2) new combinations of evidence elements constituting likely pathogenic' and 'pathogenic' classification categories, (3) which evidence elements can and cannot be used in combination for specific variant types and (4) classification of variants for which there are evidence elements for both pathogenicity and benignity.

BACKGROUND: Cilia are dynamic cellular extensions that generate and sense signals to orchestrate proper development and tissue homeostasis. They rely on the underlying polarisation of cells to participate in signalling. Cilia dysfunction is a well-known cause of several diseases that affect multiple organ systems including the kidneys, brain, heart, respiratory tract, skeleton and retina. METHODS: Among individuals from four unrelated families, we identified variants in discs large 5 (DLG5) that manifested in a variety of pathologies. In our proband, we also examined patient tissues. We depleted dlg5 in Xenopus tropicalis frog embryos to generate a loss-of-function model. Finally, we tested the pathogenicity of DLG5 patient variants through rescue experiments in the frog model. RESULTS: Patients with variants of DLG5 were found to have a variety of phenotypes including cystic kidneys, nephrotic syndrome, hydrocephalus, limb abnormalities, congenital heart disease and craniofacial malformations. We also observed a loss of cilia in cystic kidney tissue of our proband. Knockdown of dlg5 in Xenopus embryos recapitulated many of these phenotypes and resulted in a loss of cilia in multiple tissues. Unlike introduction of wildtype DLG5 in frog embryos depleted of dlg5, introduction of DLG5 patient variants was largely ineffective in restoring proper ciliation and tissue morphology in the kidney and brain suggesting that the variants were indeed detrimental to function. CONCLUSION: These findings in both patient tissues and Xenopus shed light on how mutations in DLG5 may lead to tissue-specific manifestations of disease. DLG5 is essential for cilia and many of the patient phenotypes are in the ciliopathy spectrum.

OBJECTIVE: Diagnostic germline RET analysis is offered to all patients with a diagnosis of medullary thyroid carcinoma (MTC), or other conditions associated with multiple endocrine neoplasia type 2 (MEN2) in the United Kingdom. Here, we report the experience of a single centre's germline RET analysis over a 21-year period. DESIGN: Retrospective case-note review. PATIENTS: all index patients referred to the Exeter Genomics Laboratory for diagnostic germline RET analysis between 1997 and 2018, and unaffected family members, undergoing predictive testing. MEASUREMENTS: the rate and nature of pathogenic variant detection were recorded, as well as the indication for testing. RESULTS: 1,058 index patients and 551 unaffected family members were tested. The overall rate of pathogenic variant detection was 10.2% amongst index patients and 29% amongst unaffected family members. The commonest indication was isolated MTC, and amongst the 690 patients with isolated MTC, 68 (9.9%) were found to harbour a RET pathogenic variant. of those with presumed sporadic MTC, 8.5% were found to harbour germline RET pathogenic variants, compared with 36.4% of those with a family history of MEN2-associated conditions. Pathogenic variants were identified in 3.6% and 0% of patients with isolated phaeochromocytoma and primary hyperparathyroidism, respectively. CONCLUSIONS: Although the detection rate of RET germline pathogenic variants in patients with presumed sporadic MTC was significant, the overall detection rate in those with MTC was lower than expected in this series. Advances in RET analysis in response to reports of new variants over the last two decades are likely to have improved the pick-up rate in recent years.

Background: Polycystic kidney disease with hyperinsulinaemic hypoglycaemia (HIPKD) is a recently described disease caused by a single nucleotide variant, c.-167G>T, in the promoter region of PMM2 (encoding phosphomannomutase 2), either in homozygosity or compound heterozygosity with a pathogenic coding variant in trans. All patients identified so far are of European descent, suggesting a possible founder effect. Methods: We generated high density genotyping data from 11 patients from seven unrelated families, and used this information to identify a common haplotype that included the promoter variant. We estimated the age of the promoter mutation with DMLE+ software, using demographic parameters corresponding to the European population. Results: all patients shared a 0.312 Mb haplotype which was absent in 503 European controls available in the 1000 Genomes Project. The age of this mutation was estimated as 105–110 generations, indicating its occurrence around 600 BC, a time of intense migration, which might explain the presence of the same mutations in Europeans around the globe. Conclusion: the shared unique haplotype among seemingly unrelated patients is consistent with a founder effect in Europeans.

BACKGROUND: ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane-anchored protein involved in diverse processes including mitochondrial dynamics, mitochondrial DNA organization, and cholesterol metabolism. Biallelic deletions (null), recessive missense variants (hypomorph), and heterozygous missense variants or duplications (antimorph) in ATAD3A lead to neurological syndromes in humans. METHODS: to expand the mutational spectrum of ATAD3A variants and to provide functional interpretation of missense alleles in trans to deletion alleles, we performed exome sequencing for identification of single nucleotide variants (SNVs) and copy number variants (CNVs) in ATAD3A in individuals with neurological and mitochondrial phenotypes. A Drosophila Atad3a Gal4 knockin-null allele was generated using CRISPR-Cas9 genome editing technology to aid the interpretation of variants. RESULTS: We report 13 individuals from 8 unrelated families with biallelic ATAD3A variants. The variants included four missense variants inherited in trans to loss-of-function alleles (p.(Leu77Val), p.(Phe50Leu), p.(Arg170Trp), p.(Gly236Val)), a homozygous missense variant p.(Arg327Pro), and a heterozygous non-frameshift indel p.(Lys568del). Affected individuals exhibited findings previously associated with ATAD3A pathogenic variation, including developmental delay, hypotonia, congenital cataracts, hypertrophic cardiomyopathy, and cerebellar atrophy. Drosophila studies indicated that Phe50Leu, Gly236Val, Arg327Pro, and Lys568del are severe loss-of-function alleles leading to early developmental lethality. Further, we showed that Phe50Leu, Gly236Val, and Arg327Pro cause neurogenesis defects. On the contrary, Leu77Val and Arg170Trp are partial loss-of-function alleles that cause progressive locomotion defects and whose expression leads to an increase in autophagy and mitophagy in adult muscles. CONCLUSION: Our findings expand the allelic spectrum of ATAD3A variants and exemplify the use of a functional assay in Drosophila to aid variant interpretation.

Objective: Neonatal diabetes mellitus (NDM) may be transient or permanent, and the majority is caused by genetic mutations. Early diagnosis is essential to select the patients who will respond to oral treatment. In this investigation, we aimed to present the phenotype and genotype of our patients with NDM and share our experience in a single tertiary center Methods: a total of 16 NDM patients from 12 unrelated families are included in the study. The clinical presentation, age at diagnosis, perinatal and family history, consanguinity, gender, hemoglobin A1c, C-peptide, insulin, insulin autoantibodies, genetic mutations, and response to treatment are retrospectively evaluated. Results: the median age at diagnosis of diabetes was five months (4 days-18 months) although six patients with a confirmed genetic diagnosis were diagnosed >6 months. Three patients had KCNJ11 mutations, six had ABCC8 mutations, three had EIF2AK3 mutations, and one had a de novo INS mutation. All the permanent NDM patients with KCNJ11 and ABCC8 mutations were started on sulfonylurea treatment resulting in a significant increase in C-peptide level, better glycemic control, and discontinuation of insulin. Conclusion: Although NDM is defined as diabetes diagnosed during the first six months of life, and a diagnosis of type 1 diabetes is more common between the ages of 6 and 24 months, in rare cases NDM may present as late as 12 or even 24 months of age. Molecular diagnosis in NDM is important for planning treatment and predicting prognosis. Therefore, genetic testing is essential in these patients.

OBJECTIVE: ABCC8 mutations cause neonatal diabetes mellitus that can be transient (TNDM) or, less commonly, permanent (PNDM); ∼90% of individuals can be treated with oral sulfonylureas instead of insulin. Previous studies suggested that people with ABCC8-PNDM require lower sulfonylurea doses and have milder neurological features than those with KCNJ11-PNDM. However, these studies were short-term and included combinations of ABCC8-PNDM and ABCC8-TNDM. We aimed to assess the long-term glycemic and neurological outcomes in sulfonylurea-treated ABCC8-PNDM. RESEARCH DESIGN AND METHODS: We studied all 24 individuals with ABCC8-PNDM diagnosed in the U.K. Italy, France, and U.S. known to transfer from insulin to sulfonylureas before May 2010. Data on glycemic control, sulfonylurea dose, adverse effects including hypoglycemia, and neurological features were analyzed using nonparametric statistical methods. RESULTS: Long-term data were obtained for 21 of 24 individuals (median follow-up 10.0 [range 4.1-13.2] years). Eighteen of 21 remained on sulfonylureas without insulin at the most recent follow-up. Glycemic control improved on sulfonylureas (presulfonylurea vs. 1-year posttransfer HbA1c 7.2% vs. 5.7%, P = 0.0004) and remained excellent long-term (1-year vs. 10-year HbA1c 5.7% vs. 6.5%, P = 0.04), n = 16. Relatively high doses were used (1-year vs. 10-year dose 0.37 vs. 0.25 mg/kg/day glyburide, P = 0.50) without any severe hypoglycemia. Neurological features were reported in 13 of 21 individuals; these improved following sulfonylurea transfer in 7 of 13. The most common features were learning difficulties (52%), developmental delay (48%), and attention deficit hyperactivity disorder (38%). CONCLUSIONS: Sulfonylurea treatment of ABCC8-PNDM results in excellent long-term glycemic control. Overt neurological features frequently occur and may improve with sulfonylureas, supporting early, rapid genetic testing to guide appropriate treatment and neurodevelopmental assessment.

Histone deacetylases play crucial roles in the regulation of chromatin structure and gene expression in the eukaryotic cell, and disruption of their activity causes a wide range of developmental disorders in humans. Loss-of-function alleles of HDAC4, a founding member of the class IIa deacetylases, have been reported in brachydactyly-mental retardation syndrome (BDMR). However, while disruption of HDAC4 activity and deregulation of its downstream targets may contribute to the BDMR phenotype, loss of HDAC4 function usually occurs as part of larger deletions of chromosome 2q37; BDMR is also known as chromosome 2q37 deletion syndrome, and the precise role of HDAC4 within the phenotype remains uncertain. Thus, identification of missense variants should shed new light on the role of HDAC4 in normal development. Here, we report seven unrelated individuals with a phenotype distinct from that of BDMR, all of whom have heterozygous de novo missense variants that affect a major regulatory site of HDAC4, required for signal-dependent 14-3-3 binding and nucleocytoplasmic shuttling. Two individuals possess variants altering Thr244 or Glu247, whereas the remaining five all carry variants altering Pro248, a key residue for 14-3-3 binding. We propose that the variants in all seven individuals impair 14-3-3 binding (as confirmed for the first two variants by immunoprecipitation assays), thereby identifying deregulation of HDAC4 as a pathological mechanism in a previously uncharacterized developmental disorder.

OBJECTIVE: Precursors of peptide hormones undergo posttranslational modifications within the trans-Golgi network (TGN). Dysfunction of proteins involved at different steps of this process cause several complex syndromes affecting the central nervous system (CNS). We aimed to clarify the genetic cause in a group of patients characterized by hypopituitarism in combination with brain atrophy, thin corpus callosum, severe developmental delay, visual impairment, and epilepsy. METHODS: Whole exome sequencing was performed in seven individuals of six unrelated families with these features. Postmortem histopathological and HID1 expression analysis of brain tissue and pituitary gland were conducted in one patient. Functional consequences of the homozygous HID1 variant p.R433W were investigated by Seahorse XF Assay in fibroblasts of two patients. RESULTS: Bi-allelic variants in the gene HID1 domain-containing protein 1 (HID1) were identified in all patients. Postmortem examination confirmed cerebral atrophy with enlarged lateral ventricles. Markedly reduced expression of pituitary hormones was found in pituitary gland tissue. Colocalization of HID1 protein with the TGN was not altered in fibroblasts of patients compared to controls, while the extracellular acidification rate upon stimulation with potassium chloride was significantly reduced in patient fibroblasts compared to controls. INTERPRETATION: Our findings indicate that mutations in HID1 cause an early infantile encephalopathy with hypopituitarism as the leading presentation, and expand the list of syndromic CNS diseases caused by interference of TGN function. ANN NEUROL 2021;90:149-164.

Despite the central role of chromosomal context in gene transcription, human noncoding DNA variants are generally studied outside of their genomic location. This limits our understanding of disease-causing regulatory variants. INS promoter mutations cause recessive neonatal diabetes. We show that all INS promoter point mutations in 60 patients disrupt a CC dinucleotide, whereas none affect other elements important for episomal promoter function. To model CC mutations, we humanized an ∼3.1-kb region of the mouse Ins2 gene. This recapitulated developmental chromatin states and cell-specific transcription. A CC mutant allele, however, abrogated active chromatin formation during pancreas development. A search for transcription factors acting through this element revealed that another neonatal diabetes gene product, GLIS3, has a pioneer-like ability to derepress INS chromatin, which is hampered by the CC mutation. Our in vivo analysis, therefore, connects two human genetic defects in an essential mechanism for developmental activation of the INS gene.

PURPOSE: Phosphatidylinositol Glycan Anchor Biosynthesis, class G (PIGG) is an ethanolamine phosphate transferase catalyzing the modification of glycosylphosphatidylinositol (GPI). GPI serves as an anchor on the cell membrane for surface proteins called GPI-anchored proteins (GPI-APs). Pathogenic variants in genes involved in the biosynthesis of GPI cause inherited GPI deficiency (IGD), which still needs to be further characterized. METHODS: We describe 22 individuals from 19 unrelated families with biallelic variants in PIGG. We analyzed GPI-AP surface levels on granulocytes and fibroblasts for three and two individuals, respectively. We demonstrated enzymatic activity defects for PIGG variants in vitro in a PIGG/PIGO double knockout system. RESULTS: Phenotypic analysis of reported individuals reveals shared PIGG deficiency-associated features. All tested GPI-APs were unchanged on granulocytes whereas CD73 level in fibroblasts was decreased. In addition to classic IGD symptoms such as hypotonia, intellectual disability/developmental delay (ID/DD), and seizures, individuals with PIGG variants of null or severely decreased activity showed cerebellar atrophy, various neurological manifestations, and mitochondrial dysfunction, a feature increasingly recognized in IGDs. Individuals with mildly decreased activity showed autism spectrum disorder. CONCLUSION: This in vitro system is a useful method to validate the pathogenicity of variants in PIGG and to study PIGG physiological functions.

Background:The KATPchannel plays a key role in glucose homeostasis by coupling metabolically generated changes in ATP to insulin secretion from pancreatic beta-cells.  Gain-of-function mutations in either the pore-forming (Kir6.2) or regulatory (SUR1) subunit of this channel are a common cause of transient neonatal diabetes mellitus (TNDM), in which diabetes presents shortly after birth but remits within the first few years of life, only to return in later life. The reasons behind this time dependence are unclear.Methods:In an attempt to understand the mechanism behind diabetes remission and relapse, we generated mice expressing the common TNDM mutation SUR1-R1183W. We employed Cre/LoxP technology for both inducible and constitutive expression of SUR1-R1183W specifically in mouse beta-cells, followed by investigation of their phenotype using glucose tolerance tests and insulin secretion from isolated islets. Results:We found that the R1183W mutation impaired inhibition of KATPchannels by ATP when heterologously expressed in human embryonic kidney cells. However, neither induced nor constitutive expression of SUR1-R1183W in mice resulted in changes in blood glucose homeostasis, compared to littermate controls. When challenged with a high fat diet, female mice expressing SUR1-R1183W showed increased weight gain, elevated blood glucose and impaired glycaemic control, but glucose-stimulated insulin secretion from pancreatic islets appeared unchanged.Conclusions:The mouse model of TNDM did not recapitulate the human phenotype. We discuss multiple potential reasons why this might be the case. Based on our findings, we recommend future TNDM mouse models employing a gain-of-function SUR1 mutation should be created using the minimally invasive CRISPR/Cas technology, which avoids many potential pitfalls associated with the Cre/LoxP system.

Deletion 1p36 (del1p36) syndrome is the most common human disorder resulting from a terminal autosomal deletion. This condition is molecularly and clinically heterogeneous. Deletions involving two non-overlapping regions, known as the distal (telomeric) and proximal (centromeric) critical regions, are sufficient to cause the majority of the recurrent clinical features, although with different facial features and dysmorphisms. SPEN encodes a transcriptional repressor commonly deleted in proximal del1p36 syndrome and is located centromeric to the proximal 1p36 critical region. Here, we used clinical data from 34 individuals with truncating variants in SPEN to define a neurodevelopmental disorder presenting with features that overlap considerably with those of proximal del1p36 syndrome. The clinical profile of this disease includes developmental delay/intellectual disability, autism spectrum disorder, anxiety, aggressive behavior, attention deficit disorder, hypotonia, brain and spine anomalies, congenital heart defects, high/narrow palate, facial dysmorphisms, and obesity/increased BMI, especially in females. SPEN also emerges as a relevant gene for del1p36 syndrome by co-expression analyses. Finally, we show that haploinsufficiency of SPEN is associated with a distinctive DNA methylation episignature of the X chromosome in affected females, providing further evidence of a specific contribution of the protein to the epigenetic control of this chromosome, and a paradigm of an X chromosome-specific episignature that classifies syndromic traits. We conclude that SPEN is required for multiple developmental processes and SPEN haploinsufficiency is a major contributor to a disorder associated with deletions centromeric to the previously established 1p36 critical regions.

OBJECTIVE: to use next generation sequencing (NGS) technology to identify undiagnosed, monogenic diseases in a cohort of children who suffered from acute liver failure (ALF) without an identifiable etiology. STUDY DESIGN: We identified 148 under 10 years of age admitted to King's College Hospital, London, with ALF of indeterminate etiology between 2000 and 2018. A custom NGS panel of 64 candidate genes known to cause ALF and/or metabolic liver disease was constructed. Targeted sequencing was carried out on 41 children in whom DNA samples were available. Trio exome sequencing was performed on 4 children admitted during 2019. A comparison of the clinical characteristics of those identified with biallelic variants against those without biallelic variants was then made. RESULTS: Homozygous and compound heterozygous variants were identified in 8 out of 41 children (20%) and 4 out of 4 children (100%) in whom targeted and exome sequencing were carried out, respectively. The genes involved were NBAS (3 children); DLD (2 children); and CPT1A, FAH, LARS1, MPV17, NPC1, POLG, SUCLG1, and TWINK (1 each). The 12 children who were identified with biallelic variants were younger at presentation and more likely to die in comparison with those who did not: median age at presentation of 3 months and 30 months and survival rate 75% and 97%, respectively. CONCLUSIONS: NGS was successful in identifying several specific etiologies of ALF. Variants in NBAS and mitochondrial DNA maintenance genes were the most common findings. In the future, a rapid sequencing NGS workflow could help in reaching a timely diagnosis and facilitate clinical decision making in children with ALF.

PURPOSE: to investigate if specific exon 38 or 39 KMT2D missense variants (MVs) cause a condition distinct from Kabuki syndrome type 1 (KS1). METHODS: Multiple individuals, with MVs in exons 38 or 39 of KMT2D that encode a highly conserved region of 54 amino acids flanked by Val3527 and Lys3583, were identified and phenotyped. Functional tests were performed to study their pathogenicity and understand the disease mechanism. RESULTS: the consistent clinical features of the affected individuals, from seven unrelated families, included choanal atresia, athelia or hypoplastic nipples, branchial sinus abnormalities, neck pits, lacrimal duct anomalies, hearing loss, external ear malformations, and thyroid abnormalities. None of the individuals had intellectual disability. The frequency of clinical features, objective software-based facial analysis metrics, and genome-wide peripheral blood DNA methylation patterns in these patients were significantly different from that of KS1. Circular dichroism spectroscopy indicated that these MVs perturb KMT2D secondary structure through an increased disordered to ɑ-helical transition. CONCLUSION: KMT2D MVs located in a specific region spanning exons 38 and 39 and affecting highly conserved residues cause a novel multiple malformations syndrome distinct from KS1. Unlike KMT2D haploinsufficiency in KS1, these MVs likely result in disease through a dominant negative mechanism.

OBJECTIVE: Identifying maturity-onset diabetes of the young (MODY) in pediatric populations close to diabetes diagnosis is difficult. Misdiagnosis and unnecessary insulin treatment are common. We aimed to identify the discriminatory clinical features at diabetes diagnosis of patients with glucokinase (GCK), hepatocyte nuclear factor-1A (HNF1A), and HNF4A MODY in the pediatric population. RESEARCH DESIGN AND METHODS: Swedish patients (n = 3,933) aged 1-18 years, diagnosed with diabetes May 2005 to December 2010, were recruited from the national consecutive prospective cohort Better Diabetes Diagnosis. Clinical data, islet autoantibodies (GAD insulinoma antigen-2, zinc transporter 8, and insulin autoantibodies), HLA type, and C-peptide were collected at diagnosis. MODY was identified by sequencing GCK, HNF1A, and HNF4A, through either routine clinical or research testing. RESULTS: the minimal prevalence of MODY was 1.2%. Discriminatory factors for MODY at diagnosis included four islet autoantibody negativity (100% vs. 11% not-known MODY; P = 2 × 10-44), HbA1c (7.0% vs. 10.7% [53 vs. 93 mmol/mol]; P = 1 × 10-20), plasma glucose (11.7 vs. 26.7 mmol/L; P = 3 × 10-19), parental diabetes (63% vs. 12%; P = 1 × 10-15), and diabetic ketoacidosis (0% vs. 15%; P = 0.001). Testing 303 autoantibody-negative patients identified 46 patients with MODY (detection rate 15%). Limiting testing to the 73 islet autoantibody-negative patients with HbA1c

BackgroundPathogenicity predictors are integral to genomic variant interpretation but, despite their widespread usage, an independent validation of performance using a clinically relevant dataset has not been undertaken.MethodsWe derive two validation datasets: an ‘open’ dataset containing variants extracted from publicly available databases, similar to those commonly applied in previous benchmarking exercises, and a ‘clinically representative’ dataset containing variants identified through research/diagnostic exome and panel sequencing. Using these datasets, we evaluate the performance of three recent meta-predictors, REVEL, GAVIN and ClinPred, and compare their performance against two commonly used in silico tools, SIFT and PolyPhen-2.ResultsAlthough the newer meta-predictors outperform the older tools, the performance of all pathogenicity predictors is substantially lower in the clinically representative dataset. Using our clinically relevant dataset, REVEL performed best with an area under the receiver operating characteristic curve of 0.82. Using a concordance-based approach based on a consensus of multiple tools reduces the performance due to both discordance between tools and false concordance where tools make common misclassification. Analysis of tool feature usage may give an insight into the tool performance and misclassification.ConclusionOur results support the adoption of meta-predictors over traditional in silico tools, but do not support a consensus-based approach as in current practice.

SWI/SNF-related intellectual disability disorders (SSRIDDs) are rare neurodevelopmental disorders characterized by developmental disability, coarse facial features, and fifth digit/nail hypoplasia that are caused by pathogenic variants in genes that encode for members of the SWI/SNF (or BAF) family of chromatin remodeling complexes. We have identified 12 individuals with rare variants (10 loss-of-function, 2 missense) in the BICRA (BRD4 interacting chromatin remodeling complex-associated protein) gene, also known as GLTSCR1, which encodes a subunit of the non-canonical BAF (ncBAF) complex. These individuals exhibited neurodevelopmental phenotypes that include developmental delay, intellectual disability, autism spectrum disorder, and behavioral abnormalities as well as dysmorphic features. Notably, the majority of individuals lack the fifth digit/nail hypoplasia phenotype, a hallmark of most SSRIDDs. To confirm the role of BICRA in the development of these phenotypes, we performed functional characterization of the zebrafish and Drosophila orthologs of BICRA. In zebrafish, a mutation of bicra that mimics one of the loss-of-function variants leads to craniofacial defects possibly akin to the dysmorphic facial features seen in individuals harboring putatively pathogenic BICRA variants. We further show that Bicra physically binds to other non-canonical ncBAF complex members, including the BRD9/7 ortholog, CG7154, and is the defining member of the ncBAF complex in flies. Like other SWI/SNF complex members, loss of Bicra function in flies acts as a dominant enhancer of position effect variegation but in a more context-specific manner. We conclude that haploinsufficiency of BICRA leads to a unique SSRIDD in humans whose phenotypes overlap with those previously reported.

Childhood dilated cardiomyopathy (DCM) is a leading cause of heart failure requiring cardiac transplantation and approximately 5% of cases result in sudden death. Knowledge of the underlying genetic cause can aid prognostication and clinical management and enables accurate recurrence risk counselling for the family. Here we used genomic sequencing to identify the causative genetic variant(s) in families with children affected by severe DCM. In an international collaborative effort facilitated by GeneMatcher, biallelic variants in PPP1R13L were identified in seven children with severe DCM from five unrelated families following exome or genome sequencing and inheritance-based variant filtering. PPP1R13L encodes inhibitor of apoptosis-stimulating protein of p53 protein (iASPP). In addition to roles in apoptosis, iASPP acts as a regulator of desmosomes and has been implicated in inflammatory pathways. DCM presented early (mean: 2 years 10 months; range: 3 months-9 years) and was progressive, resulting in death (n = 3) or transplant (n = 3), with one child currently awaiting transplant. Genomic sequencing technologies are valuable for the identification of novel and emerging candidate genes. Biallelic variants in PPP1R13L were previously reported in a single consanguineous family with paediatric DCM. The identification here of a further five families now provides sufficient evidence to support a robust gene-disease association between PPP1R13L and severe paediatric DCM. The PPP1R13L gene should be included in panel-based genetic testing for paediatric DCM.

PURPOSE: Diagnosis of genetic disorders is hampered by large numbers of variants of uncertain significance (VUSs) identified through next-generation sequencing. Many such variants may disrupt normal RNA splicing. We examined effects on splicing of a large cohort of clinically identified variants and compared performance of bioinformatic splicing prediction tools commonly used in diagnostic laboratories. METHODS: Two hundred fifty-seven variants (coding and noncoding) were referred for analysis across three laboratories. Blood RNA samples underwent targeted reverse transcription polymerase chain reaction (RT-PCR) analysis with Sanger sequencing of PCR products and agarose gel electrophoresis. Seventeen samples also underwent transcriptome-wide RNA sequencing with targeted splicing analysis based on Sashimi plot visualization. Bioinformatic splicing predictions were obtained using Alamut, HSF 3.1, and SpliceAI software. RESULTS: Eighty-five variants (33%) were associated with abnormal splicing. The most frequent abnormality was upstream exon skipping (39/85 variants), which was most often associated with splice donor region variants. SpliceAI had greatest accuracy in predicting splicing abnormalities (0.91) and outperformed other tools in sensitivity and specificity. CONCLUSION: Splicing analysis of blood RNA identifies diagnostically important splicing abnormalities and clarifies functional effects of a significant proportion of VUSs. Bioinformatic predictions are improving but still make significant errors. RNA analysis should therefore be routinely considered in genetic disease diagnostics.

Advances in technology have led to a massive expansion in the capacity for genomic analysis, with a commensurate fall in costs. The clinical indications for genomic testing have evolved markedly; the volume of clinical sequencing has increased dramatically; and the range of clinical professionals involved in the process has broadened. There is general acceptance that our early dichotomous paradigms of variants being pathogenic-high risk and benign-no risk are overly simplistic. There is increasing recognition that the clinical interpretation of genomic data requires significant expertise in disease-gene-variant associations specific to each disease area. Inaccurate interpretation can lead to clinical mismanagement, inconsistent information within families and misdirection of resources. It is for this reason that 'national subspecialist multidisciplinary meetings' (MDMs) for genomic interpretation have been articulated as key for the new NHS Genomic Medicine Service, of which Cancer Variant Interpretation Group UK (CanVIG-UK) is an early exemplar. CanVIG-UK was established in 2017 and now has >100 UK members, including at least one clinical diagnostic scientist and one clinical cancer geneticist from each of the 25 regional molecular genetics laboratories of the UK and Ireland. Through CanVIG-UK, we have established national consensus around variant interpretation for cancer susceptibility genes via monthly national teleconferenced MDMs and collaborative data sharing using a secure online portal. We describe here the activities of CanVIG-UK, including exemplar outputs and feedback from the membership.

Heterotaxy and congenital heart defects associated with pathogenic variants in the PKD1L1 gene (autosomal visceral heterotaxy type 8, MIM 617205) has been reported in only four individuals from three unrelated families. We describe a further family with two affected fetuses and novel compound heterozygous pathogenic variants in PKD1L1. PKD1L1 has been shown to function in the ciliary sensation of nodal flow at the embryo primitive node and in the restriction of NODAL signalling to the left lateral. plate mesoderm, mechanisms involved in the development of laterality in vertebrates. Individuals affected with this autosomal recessive condition have variable thoracic and abdominal situs. Features of CHD and other anomalies vary between and within families.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Short-rib polydactyly syndromes are a heterogeneous group of disorders characterized by narrow thorax with short ribs, polydactyly and often other visceral and skeletal malformations. To date there have only been six reported patients with homozygous and compound heterozygous variants in IFT81, causing a short-rib thoracic dysplasia, with, or without, polydactyly (SRTD19: OMIM 617895). IFT81 is a protein integral to the core of the intraflagellar transport complex B (IFT-B), which is involved in anterograde transport in the cilium. We describe the case of a male infant with compound heterozygous variants in IFT81, who presented with short long bones, a narrow thorax, polydactyly, and multiple malformations. Three novel clinical features are reported including complete situs inversus, micropenis, and rectal atresia, which have not previously been associated with variants in IFT81. We reviewed the literature and identified the most consistent clinical features associated with this rare ciliopathy syndrome. We postulate that dolichocephaly and sagittal craniosynostosis may be associated with this condition, and provide a clue to considering IFT81 as the causative gene when deciphering complex ciliopathies.

Pathogenic variants in HNRNPH1 were first reported in 2018. The reported individual, a 13 year old boy with a c.616C>T (p.R206W) variant in the HNRNPH1 gene, was noted to have overlapping symptoms with those observed in HNRNPH2-related X-linked intellectual disability, Bain type (MRXSB), specifically intellectual disability and dysmorphic features. While HNRNPH1 variants were initially proposed to represent an autosomal cause of MRXSB, we report an additional seven cases which identify phenotypic differences from MRXSB. Patients with HNRNPH1 pathogenic variants diagnosed via WES were identified using clinical networks and GeneMatcher. Features unique to individuals with HNRNPH1 variants include distinctive dysmorphic facial features; an increased incidence of congenital anomalies including cranial and brain abnormalities, genitourinary malformations, and palate abnormalities; increased incidence of ophthalmologic abnormalities; and a decreased incidence of epilepsy and cardiac defects compared to those with MRXSB. This suggests that pathogenic variants in HNRNPH1 result in a related, but distinct syndromic cause of intellectual disability from MRXSB, which we refer to as HNRNPH1-related syndromic intellectual disability.

OBJECTIVE: Heterozygous loss-of-function mutations in HNF1A cause maturity-onset diabetes of the young (MODY). Affected individuals can be treated with low-dose sulfonylureas. Individuals with homozygous HNF1A mutations causing MODY have not been reported. RESEARCH DESIGN AND METHODS: We phenotyped a kindred with young-onset diabetes and performed molecular genetic testing, a mixed meal tolerance test, a sulfonylurea challenge, and in vitro assays to assess variant protein function. RESULTS: a homozygous HNF1A variant (p.A251T) was identified in three insulin-treated family members diagnosed with diabetes before 20 years of age. Those with the homozygous variant had low hs-CRP levels (0.2-0.8 mg/L), and those tested demonstrated sensitivity to sulfonylurea given at a low dose, completely transitioning off insulin. In silico modeling predicted a variant of unknown significance; however, in vitro studies supported a modest reduction in transactivation potential (79% of that for the wild type; P < 0.05) in the absence of endogenous HNF1A. CONCLUSIONS: Homozygous hypomorphic HNF1A variants are a cause of HNF1A-MODY. We thus expand the allelic spectrum of variants in dominant genes causing diabetes.

Multiple Nucleotide Variants (MNVs) are miscalled by the most widely utilised next generation sequencing analysis (NGS) pipelines, presenting the potential for missing diagnoses. These variants, which should be treated as a single insertion-deletion mutation event, are commonly called as separate single nucleotide variants. This can result in misannotation, incorrect amino acid predictions and potentially false positive and false negative diagnostic results. Using simulated data and re-analysis of sequencing data from a diagnostic targeted gene panel, we demonstrate that the widely adopted pipeline, GATK best practices, results in miscalling of MNVs and that alternative tools can call these variants correctly. The adoption of calling methods that annotate MNVs correctly would present a solution for individual laboratories, however GATK best practices are the basis for important public resources such as the gnomAD database. We suggest integrating a solution into these guidelines would be the optimal approach.

Recent studies have identified both recessive and dominant forms of mitochondrial disease that result from ATAD3A variants. The recessive form includes subjects with biallelic deletions mediated by non-allelic homologous recombination. We report five unrelated neonates with a lethal metabolic disorder characterized by cardiomyopathy, corneal opacities, encephalopathy, hypotonia, and seizures in whom a monoallelic reciprocal duplication at the ATAD3 locus was identified. Analysis of the breakpoint junction fragment indicated that these 67 kb heterozygous duplications were likely mediated by non-allelic homologous recombination at regions of high sequence identity in ATAD3A exon 11 and ATAD3C exon 7. At the recombinant junction, the duplication allele produces a fusion gene derived from ATAD3A and ATAD3C, the protein product of which lacks key functional residues. Analysis of fibroblasts derived from two affected individuals shows that the fusion gene product is expressed and stable. These cells display perturbed cholesterol and mitochondrial DNA organization similar to that observed for individuals with severe ATAD3A deficiency. We hypothesize that the fusion protein acts through a dominant-negative mechanism to cause this fatal mitochondrial disorder. Our data delineate a molecular diagnosis for this disorder, extend the clinical spectrum associated with structural variation at the ATAD3 locus, and identify a third mutational mechanism for ATAD3 gene cluster variants. These results further affirm structural variant mutagenesis mechanisms in sporadic disease traits, emphasize the importance of copy number analysis in molecular genomic diagnosis, and highlight some of the challenges of detecting and interpreting clinically relevant rare gene rearrangements from next-generation sequencing data.

We present eight families with arthrogryposis multiplex congenita and myopathy bearing a TTN intron 213 extended splice-site variant (NM_001267550.1:c.39974-11T>G), inherited in trans with a second pathogenic TTN variant. Muscle-derived RNA studies of three individuals confirmed mis-splicing induced by the c.39974-11T>G variant; in-frame exon 214 skipping or use of a cryptic 3' splice-site effecting a frameshift. Confounding interpretation of pathogenicity is the absence of exons 213-217 within the described skeletal muscle TTN N2A isoform. However, RNA-sequencing from 365 adult human gastrocnemius samples revealed that 56% specimens predominantly include exons 213-217 in TTN transcripts (inclusion rate ≥66%). Further, RNA-sequencing of five fetal muscle samples confirmed that 4/5 specimens predominantly include exons 213-217 (fifth sample inclusion rate 57%). Contractures improved significantly with age for four individuals, which may be linked to decreased expression of pathogenic fetal transcripts. Our study extends emerging evidence supporting a vital developmental role for TTN isoforms containing metatranscript-only exons.

Background: Large contiguous gene deletions at the distal end of the short arm of chromosome 9 result in the complex multi-organ condition chromosome 9p deletion syndrome.  a range of clinical features can result from these deletions with the most common being facial dysmorphisms and neurological impairment. Congenital hyperinsulinism is a rarely reported feature of the syndrome with the genetic mechanism for the dysregulated insulin secretion being unknown.  Methods: We studied the clinical and genetic characteristics of 12 individuals with chromosome 9p deletions who had a history of neonatal hypoglycaemia. Using off-target reads generated from targeted next-generation sequencing of the genes known to cause hyperinsulinaemic hypoglycaemia (n=9), or microarray analysis (n=3), we mapped the minimal shared deleted region on chromosome 9 in this cohort. Targeted sequencing was performed in three patients to search for a recessive mutation unmasked by the deletion. Results: in 10/12 patients with hypoglycaemia, hyperinsulinism was confirmed biochemically. A range of extra-pancreatic features were also reported in these patients consistent with the diagnosis of the Chromosome 9p deletion syndrome. The minimal deleted region was mapped to 7.2 Mb, encompassing 38 protein-coding genes. In silico analysis of these genes highlighted SMARCA2 and RFX3 as potential candidates for the hypoglycaemia. Targeted sequencing performed on three of the patients did not identify a second disease-causing variant within the minimal deleted region. Conclusions: This study identifies 9p deletions as an important cause of hyperinsulinaemic hypoglycaemia and increases the number of cases reported with 9p deletions and hypoglycaemia to 15 making this a more common feature of the syndrome than previously appreciated.  Whilst the precise genetic mechanism of the dysregulated insulin secretion could not be determined in these patients, mapping the deletion breakpoints highlighted potential candidate genes for hypoglycaemia within the deleted region.

CONTEXT: Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene are responsible for a subset of familial isolated pituitary adenoma (FIPA) cases and sporadic pituitary neuroendocrine tumors (PitNETs). OBJECTIVE: to compare prospectively diagnosed AIP mutation-positive (AIPmut) PitNET patients with clinically presenting patients and to compare the clinical characteristics of AIPmut and AIPneg PitNET patients. DESIGN: 12-year prospective, observational study. PARTICIPANTS & SETTING: We studied probands and family members of FIPA kindreds and sporadic patients with disease onset ≤18 years or macroadenomas with onset ≤30 years (n = 1477). This was a collaborative study conducted at referral centers for pituitary diseases. INTERVENTIONS & OUTCOME: AIP testing and clinical screening for pituitary disease. Comparison of characteristics of prospectively diagnosed (n = 22) vs clinically presenting AIPmut PitNET patients (n = 145), and AIPmut (n = 167) vs AIPneg PitNET patients (n = 1310). RESULTS: Prospectively diagnosed AIPmut PitNET patients had smaller lesions with less suprasellar extension or cavernous sinus invasion and required fewer treatments with fewer operations and no radiotherapy compared with clinically presenting cases; there were fewer cases with active disease and hypopituitarism at last follow-up. When comparing AIPmut and AIPneg cases, AIPmut patients were more often males, younger, more often had GH excess, pituitary apoplexy, suprasellar extension, and more patients required multimodal therapy, including radiotherapy. AIPmut patients (n = 136) with GH excess were taller than AIPneg counterparts (n = 650). CONCLUSIONS: Prospectively diagnosed AIPmut patients show better outcomes than clinically presenting cases, demonstrating the benefits of genetic and clinical screening. AIP-related pituitary disease has a wide spectrum ranging from aggressively growing lesions to stable or indolent disease course.

Exome sequencing in diabetes presents a diagnostic challenge because depending on frequency, functional impact, and genomic and environmental contexts, HNF1A variants can cause maturity-onset diabetes of the young (MODY), increase type 2 diabetes risk, or be benign. A correct diagnosis matters as it informs on treatment, progression, and family risk. We describe a multi-dimensional functional dataset of 73 HNF1A missense variants identified in exomes of 12,940 individuals. Our aim was to develop an analytical framework for stratifying variants along the HNF1A phenotypic continuum to facilitate diagnostic interpretation. HNF1A variant function was determined by four different molecular assays. Structure of the multi-dimensional dataset was explored using principal component analysis, k-means, and hierarchical clustering. Weights for tissue-specific isoform expression and functional domain were integrated. Functionally annotated variant subgroups were used to re-evaluate genetic diagnoses in national MODY diagnostic registries. HNF1A variants demonstrated a range of behaviors across the assays. The structure of the multi-parametric data was shaped primarily by transactivation. Using unsupervised learning methods, we obtained high-resolution functional clusters of the variants that separated known causal MODY variants from benign and type 2 diabetes risk variants and led to reclassification of 4% and 9% of HNF1A variants identified in the UK and Norway MODY diagnostic registries, respectively. Our proof-of-principle analyses facilitated informative stratification of HNF1A variants along the continuum, allowing improved evaluation of clinical significance, management, and precision medicine in diabetes clinics. Transcriptional activity appears a superior readout supporting pursuit of transactivation-centric experimental designs for high-throughput functional screens.

We report the case of a consanguineous couple who lost four pregnancies associated with skeletal dysplasia. Radiological examination of one fetus was inconclusive. Parental exome sequencing showed that both parents were heterozygous for a novel missense variant, p.(Pro133Leu), in the SLC35D1 gene encoding a nucleotide sugar transporter. The affected fetus was homozygous for the variant. The radiological features were reviewed, and being similar, but atypical, the phenotype was classified as a 'Schneckenbecken-like dysplasia.' the effect of the missense change was assessed using protein modelling techniques and indicated alterations in the mouth of the solute channel. A detailed biochemical investigation of SLC35D1 transport function and that of the missense variant p.(Pro133Leu) revealed that SLC35D1 acts as a general UDP-sugar transporter and that the p.(Pro133Leu) mutation resulted in a significant decrease in transport activity. The reduced transport activity observed for p.(Pro133Leu) was contrasted with in vitro activity for SLC35D1 p.(Thr65Pro), the loss-of-function mutation was associated with Schneckenbecken dysplasia. The functional classification of SLC35D1 as a general nucleotide sugar transporter of the endoplasmic reticulum suggests an expanded role for this transporter beyond chondroitin sulfate biosynthesis to a variety of important glycosylation reactions occurring in the endoplasmic reticulum.

The centrosomal protein 55 kDa (CEP55 (OMIM 610000)) plays a fundamental role in cell cycle regulation and cytokinesis. However, the precise role of CEP55 in human embryonic growth and development is yet to be fully defined. Here we identified a novel homozygous founder frameshift variant in CEP55, present at low frequency in the Amish community, in two siblings presenting with a lethal foetal disorder. The features of the condition are reminiscent of a Meckel-like syndrome comprising of Potter sequence, hydranencephaly, and cystic dysplastic kidneys. These findings, considered alongside two recent studies of single families reporting loss of function candidate variants in CEP55, confirm disruption of CEP55 function as a cause of this clinical spectrum and enable us to delineate the cardinal clinical features of this disorder, providing important new insights into early human development.

The original version of this Article contained an error in the spelling of the author Laurence Faivre, which was incorrectly given as Laurence Faive. This has now been corrected in both the PDF and HTML versions of the Article.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life, and in utero obstruction to urine flow is a frequent cause of secondary upper urinary tract malformations. Here, using whole-exome sequencing, we identified three different biallelic mutations in CHRNA3, which encodes the α3 subunit of the nicotinic acetylcholine receptor, in five affected individuals from three unrelated families with functional lower urinary tract obstruction and secondary CAKUT. Four individuals from two families have additional dysautonomic features, including impaired pupillary light reflexes. Functional studies in vitro demonstrated that the mutant nicotinic acetylcholine receptors were unable to generate current following stimulation with acetylcholine. Moreover, the truncating mutations p.Thr337Asnfs∗81 and p.Ser340∗ led to impaired plasma membrane localization of CHRNA3. Although the importance of acetylcholine signaling in normal bladder function has been recognized, we demonstrate for the first time that mutations in CHRNA3 can cause bladder dysfunction, urinary tract malformations, and dysautonomia. These data point to a pathophysiologic sequence by which monogenic mutations in genes that regulate bladder innervation may secondarily cause CAKUT.

Congenital hyperinsulinism (CHI) is the most common cause of persistent hypoglycemia in infants and children. Recessive inactivating mutations in the ABCC8 and KCNJ11 genes account for approximately 50% of all CHI cases. Hyperinsulinaemic hypoglycaemia in infancy and diabetes in later life have been reported in patients with HNF1A, HNF4A and ABCC8 mutations. Herein, we present a child who was diagnosed with CHI at birth, then developed diabetes mellitus at the age of nine years due to a novel homozygous missense, p.L171F (c.511C>T) mutation in exon 4 of ABCC8. The parents and one sibling were heterozygous carriers, whilst a younger sibling who had transient neonatal hypoglycemia was homozygous for the mutation. The mother and (maternal) uncle, who was also heterozygous for the mutation, developed diabetes within their third decade of life. The preliminary results of sulphonylurea (SU) treatment was suggestive of SU responsiveness. Patients with homozygous ABCC8 mutations can present with CHI in the newborn period, the hyperinsulinism can show variability in terms of clinical severity and age at presentation and can cause diabetes later in life. Patients with homozygous ABCC8 mutations who are managed medically should be followed long-term as they may be at increased risk of developing diabetes after many years.

PURPOSE: Describe the clinical and molecular findings of patients with Kabuki syndrome (KS) who present with hypoglycemia due to congenital hyperinsulinism (HI), and assess the incidence of KS in patients with HI. METHODS: We documented the clinical features and molecular diagnoses of 9 infants with persistent HI and KS via a combination of sequencing and copy-number profiling methodologies. Subsequently, we retrospectively evaluated 100 infants with HI lacking a genetic diagnosis, for causative variants in KS genes. RESULTS: Molecular diagnoses of KS were established by identification of pathogenic variants in KMT2D (n = 5) and KDM6A (n = 4). Among the 100 infants with HI of unknown genetic etiology, a KS diagnosis was uncovered in one patient. CONCLUSIONS: the incidence of HI among patients with KS may be higher than previously reported, and KS may account for as much as 1% of patients diagnosed with HI. As the recognition of dysmorphic features associated with KS is challenging in the neonatal period, we propose KS should be considered in the differential diagnosis of HI. Since HI in patients with KS is well managed medically, a timely recognition of hyperinsulinemic episodes will improve outcomes, and prevent aggravation of the preexisting mild to moderate intellectual disability in KS.

The author Diva D. De Leon was incorrectly listed as instead of Diva D. De Leó-Critchlow in the original version of this paper.

The original version of this Article contained an error in the top left of Figure 2: the number 1 on the y-axis had been changed to 0 during the typesetting process. This has now been corrected in both the PDF and HTML versions of the Article.

Congenital hyperinsulinism (CHI) is the commonest cause of persistent and severe hypoglycemia in infancy due to unregulated insulin secretion from pancreatic β-cells. Prompt early diagnosis is important, as insulin reduces glucose supply to the brain, resulting in significant brain injury and risk of death. Histologically, CHI has focal and diffuse forms; in focal CHI, an inappropriate level of insulin is secreted from localized β-cell hyperplasia. We report a 4-month-old male infant, who presented with sudden illness and collapse without a recognized cause and died. Postmortem examination revealed pancreatic histopathology compatible with focal CHI. Immunofluoresence staining showed limited expression of p57kip2 β-cells reinforcing the diagnosis. Mutation testing for genes associated with CHI from DNA from the focal lesion was negative. This case highlights the recognition of focal CHI as a possible cause for sudden infant death. In children dying suddenly and unexpectedly, postmortem pancreatic sections should be carefully examined for focal CHI.

Sharing de-identified genetic variant data via custom-built online repositories is essential for the practice of genomic medicine and is demonstrably beneficial to patients. Robust genetic diagnoses that inform medical management cannot be made accurately without reference to genetic test results from other patients, population controls and correlation with clinical context and family history. Errors in this process can result in delayed, missed or erroneous diagnoses, leading to inappropriate or missed medical interventions for the patient and their family. The benefits of sharing individual genetic variants, and the harms of not sharing them, are numerous and well-established. Databases and mechanisms already exist to facilitate deposition and sharing of de-identified genetic variants, but clarity and transparency around best practice is needed to encourage widespread use, prevent inconsistencies between different communities, maximise individual privacy and ensure public trust. We therefore recommend that widespread sharing of a small number of genetic variants per individual, associated with limited clinical information, should become standard practice in genomic medicine. Information confirming or refuting the role of genetic variants in specific conditions is fundamental scientific knowledge from which everyone has a right to benefit, and therefore should not require consent to share. For additional case-level detail about individual patients or more extensive genomic information, which is often essential for individual clinical interpretation, it may be more appropriate to use a controlled-access model for such data sharing, with the ultimate aim of making as much information available as possible with appropriate governance.

Sharing de-identified genetic variant data is essential for the practice of genomic medicine and is demonstrably beneficial to patients. Robust genetic diagnoses that inform medical management cannot be made accurately without reference to genetic test results from other patients, as well as population controls. Errors in this process can result in delayed, missed or erroneous diagnoses, leading to inappropriate or missed medical interventions for the patient and their family. The benefits of sharing individual genetic variants, and the harms of not sharing them, are numerous and well-established. Databases and mechanisms already exist to facilitate deposition and sharing of pseudonomised genetic variants, but clarity and transparency around best practice is needed to encourage widespread use, prevent inconsistencies between different communities, maximise individual privacy and ensure public trust. We therefore recommend that widespread sharing of a small number of individual genetic variants associated with limited clinical information should become standard practice in genomic medicine. Information robustly linking genetic variants with specific conditions is fundamental biological knowledge, not personal information, and therefore should not require consent to share. For additional case-level detail about individual patients or more extensive genomic information, which is often essential for clinical interpretation, it may be more appropriate to use a controlled-access model for data sharing, with the ultimate aim of making as much information as open and de-identified as possible with appropriate consent.

Background: Mutations in the transcription factor hepatocyte nuclear factor 1B (HNF1B) are the most common inherited cause of renal malformations, yet also associated with renal tubular dysfunction, most prominently magnesium wasting with hypomagnesemia. The presence of hypomagnesemia has been proposed to help select appropriate patients for genetic testing. Yet, in a large cohort, hypomagnesemia was discriminatory only in adult, but not in pediatric patients. We therefore investigated whether hypomagnesemia and other biochemical changes develop with age. Methods: We performed a retrospective analysis of clinical, biochemical, and genetic results of pediatric patients with renal malformations tested for HNF1B mutations, separated into 4 age groups. Values were excluded if concurrent estimated glomerular filtration rate (eGFR) was

We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities.

PURPOSE: One of the greatest challenges currently facing those studying Mendelian disease is identifying the pathogenic variant from the long list produced by a next-generation sequencing test. We investigate the predictive ability of homozygosity mapping for identifying the regions likely to contain the causative variant. METHODS: We use 179 homozygous pathogenic variants from three independent cohorts to investigate the predictive power of homozygosity mapping. RESULTS: We demonstrate that homozygous pathogenic variants in our cohorts are disproportionately likely to be found within one of the largest regions of homozygosity: 80% of pathogenic variants are found in a homozygous region that is in the ten largest regions in a sample. The maximal predictive power is achieved in patients with 3 Mb from a telomere; this gives an area under the curve (AUC) of 0.735 and results in 92% of the causative variants being in one of the ten largest homozygous regions. CONCLUSION: This predictive power can be used to prioritize the list of candidate variants in gene discovery studies. When classifying a homozygous variant the size and rank of the region of homozygosity in which the candidate variant is located can also be considered as supporting evidence for pathogenicity.

Multiple Nucleotide Variants (MNVs) are miscalled by the most widely utilised next generation sequencing analysis (NGS) pipelines, presenting the potential for missing diagnoses that would previously have been made by standard Sanger (dideoxy) sequencing. These variants, which should be treated as a single insertion-deletion mutation event, are commonly called as separate single nucleotide variants. This can result in misannotation, incorrect amino acid predictions and potentially false positive and false negative diagnostic results. This risk will be increased as confirmatory Sanger sequencing of Single Nucleotide variants (SNVs) ceases to be standard practice. Using simulated data and re-analysis of sequencing data from a diagnostic targeted gene panel, we demonstrate that the widely adopted pipeline, GATK best practices, results in miscalling of MNVs and that alternative tools can call these variants correctly. The adoption of calling methods that annotate MNVs correctly would present a solution for individual laboratories, however GATK best practices are the basis for important public resources such as the gnomAD database. We suggest integrating a solution into these guidelines would be the optimal approach.

Proteins anchored to the cell surface via glycosylphosphatidylinositol (GPI) play various key roles in the human body, particularly in development and neurogenesis. As such, many developmental disorders are caused by mutations in genes involved in the GPI biosynthesis and remodeling pathway. We describe ten unrelated families with bi-allelic mutations in PIGB, a gene that encodes phosphatidylinositol glycan class B, which transfers the third mannose to the GPI. Ten different PIGB variants were found in these individuals. Flow cytometric analysis of blood cells and fibroblasts from the affected individuals showed decreased cell surface presence of GPI-anchored proteins. Most of the affected individuals have global developmental and/or intellectual delay, all had seizures, two had polymicrogyria, and four had a peripheral neuropathy. Eight children passed away before four years old. Two of them had a clinical diagnosis of DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, and seizures), a condition that includes sensorineural deafness, shortened terminal phalanges with small finger and toenails, intellectual disability, and seizures; this condition overlaps with the severe phenotypes associated with inherited GPI deficiency. Most individuals tested showed elevated alkaline phosphatase, which is a characteristic of the inherited GPI deficiency but not DOORS syndrome. It is notable that two severely affected individuals showed 2-oxoglutaric aciduria, which can be seen in DOORS syndrome, suggesting that severe cases of inherited GPI deficiency and DOORS syndrome might share some molecular pathway disruptions.

Physical stress, including high temperatures, may damage the central metabolic nicotinamide nucleotide cofactors [NAD(P)H], generating toxic derivatives [NAD(P)HX]. The highly conserved enzyme NAD(P)HX dehydratase (NAXD) is essential for intracellular repair of NAD(P)HX. Here we present a series of infants and children who suffered episodes of febrile illness-induced neurodegeneration or cardiac failure and early death. Whole-exome or whole-genome sequencing identified recessive NAXD variants in each case. Variants were predicted to be potentially deleterious through in silico analysis. Reverse-transcription PCR confirmed altered splicing in one case. Subject fibroblasts showed highly elevated concentrations of the damaged cofactors S-NADHX, R-NADHX and cyclic NADHX. NADHX accumulation was abrogated by lentiviral transduction of subject cells with wild-type NAXD. Subject fibroblasts and muscle biopsies showed impaired mitochondrial function, higher sensitivity to metabolic stress in media containing galactose and azide, but not glucose, and decreased mitochondrial reactive oxygen species production. Recombinant NAXD protein harbouring two missense variants leading to the amino acid changes p.(Gly63Ser) and p.(Arg608Cys) were thermolabile and showed a decrease in Vmax and increase in KM for the ATP-dependent NADHX dehydratase activity. This is the first study to identify pathogenic variants in NAXD and to link deficient NADHX repair with mitochondrial dysfunction. The results show that NAXD deficiency can be classified as a metabolite repair disorder in which accumulation of damaged metabolites likely triggers devastating effects in tissues such as the brain and the heart, eventually leading to early childhood death.

Background: the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) clinical variant interpretation guidelines established criteria for different types of evidence. This includes the strong evidence codes PS3 and BS3 for "well-established" functional assays demonstrating a variant has abnormal or normal gene/protein function, respectively. However, they did not provide detailed guidance on how functional evidence should be evaluated, and differences in the application of the PS3/BS3 codes are a contributor to variant interpretation discordance between laboratories. This recommendation seeks to provide a more structured approach to the assessment of functional assays for variant interpretation and guidance on the use of various levels of strength based on assay validation. Methods: the Clinical Genome Resource (ClinGen) Sequence Variant Interpretation (SVI) Working Group used curated functional evidence from ClinGen Variant Curation Expert Panel-developed rule specifications and expert opinions to refine the PS3/BS3 criteria over multiple in-person and virtual meetings. We estimated the odds of pathogenicity for assays using various numbers of variant controls to determine the minimum controls required to reach moderate level evidence. Feedback from the ClinGen Steering Committee and outside experts were incorporated into the recommendations at multiple stages of development. Results: the SVI Working Group developed recommendations for evaluators regarding the assessment of the clinical validity of functional data and a four-step provisional framework to determine the appropriate strength of evidence that can be applied in clinical variant interpretation. These steps are as follows: (1) define the disease mechanism, (2) evaluate the applicability of general classes of assays used in the field, (3) evaluate the validity of specific instances of assays, and (4) apply evidence to individual variant interpretation. We found that a minimum of 11 total pathogenic and benign variant controls are required to reach moderate-level evidence in the absence of rigorous statistical analysis. Conclusions: the recommendations and approach to functional evidence evaluation described here should help clarify the clinical variant interpretation process for functional assays. Further, we hope that these recommendations will help develop productive partnerships with basic scientists who have developed functional assays that are useful for interrogating the function of a variety of genes.

Background: Large contiguous gene deletions at the distal end of the short arm of chromosome 9 result in the complex multi-organ condition chromosome 9p deletion syndrome.  a range of clinical features can result from these deletions with the most common being facial dysmorphisms and neurological impairment. Congenital hyperinsulinism is a rarely reported feature of the syndrome with the genetic mechanism for the dysregulated insulin secretion being unknown.  Methods: We studied the clinical and genetic characteristics of 12 individuals with chromosome 9p deletions who had a history of neonatal hypoglycaemia. Using off-target reads generated from targeted next-generation sequencing of the genes known to cause hyperinsulinaemic hypoglycaemia (n=9), or microarray analysis (n=3), we mapped the minimal shared deleted region on chromosome 9 in this cohort. Targeted sequencing was performed in three patients to search for a recessive mutation unmasked by the deletion. Results: in 10/12 patients with hypoglycaemia, hyperinsulinism was confirmed biochemically. A range of extra-pancreatic features were also reported in these patients consistent with the diagnosis of the Chromosome 9p deletion syndrome. The minimal deleted region was mapped to 7.2 Mb, encompassing 38 protein-coding genes. In silico analysis of these genes highlighted SMARCA2 and RFX3 as potential candidates for the hypoglycaemia. Targeted sequencing performed on three of the patients did not identify a second disease-causing variant within the minimal deleted region. Conclusions: This study identifies 9p deletions as an important cause of hyperinsulinaemic hypoglycaemia and increases the number of cases reported with 9p deletions and hypoglycaemia to 15 making this a more common feature of the syndrome than previously appreciated.  Whilst the precise genetic mechanism of the dysregulated insulin secretion could not be determined in these patients, mapping the deletion breakpoints highlighted potential candidate genes for hypoglycaemia within the deleted region.

BACKGROUND: the significant variation in the clinical behaviour of sporadic medullary thyroid carcinoma (sMTC) causes uncertainty when planning the management of these patients. Several tumour genetic and epigenetic markers have been described, but their clinical usefulness remains unclear. The aim of this review was to evaluate the evidence for the use of molecular genetic and epigenetic profiles in the risk stratification and management of sMTC. METHODS: MEDLINE and Embase databases were searched using the MeSH terms "medullary carcinoma", "epigenetics", "molecular genetics", "microRNAs"; and free text terms "medullary carcinoma", "sporadic medullary thyroid cancer", "sMTC", "RET", "RAS" and "miR". Articles containing less than ten subjects, not focussing on sMTC, or not reporting clinical outcomes were excluded. Risk of bias was assessed using a modified version of the Newcastle-Ottawa Scale. RESULTS: Twenty-three studies met the inclusion criteria, and key findings were summarized in themes according to the genetic and epigenetic markers studied. There is good evidence that somatic RET mutations predict higher rates of lymph node metastasis and persistent disease, and worse survival. There are also several good quality studies demonstrating associations between certain epigenetic markers such as tumour miR-183 and miR-375 expression and higher rates of lymph node and distant metastasis, and worse survival. CONCLUSIONS: There is a growing body of evidence that tumour genetic and epigenetic profiles can be used to risk stratify patients with sMTC. Further research should focus on the clinical applicability of these findings by investigating the possibility of tailoring management to an individual's tumour mutation profile.

AIM: to examine the extent to which discriminatory testing using antibodies and Type 1 diabetes genetic risk score, validated in European populations, is applicable in a non-European population. METHODS: We recruited 127 unrelated children with diabetes diagnosed between 9 months and 5 years from two centres in Iran. All children underwent targeted next-generation sequencing of 35 monogenic diabetes genes. We measured three islet autoantibodies (islet antigen 2, glutamic acid decarboxylase and zinc transporter 8) and generated a Type 1 diabetes genetic risk score in all children. RESULTS: We identified six children with monogenic diabetes, including four novel mutations: homozygous mutations in WFS1 (n=3), SLC19A2 and SLC29A3, and a heterozygous mutation in GCK. All clinical features were similar in children with monogenic diabetes (n=6) and in the rest of the cohort (n=121). The Type 1 diabetes genetic risk score discriminated children with monogenic from Type 1 diabetes [area under the receiver-operating characteristic curve 0.90 (95% CI 0.83-0.97)]. All children with monogenic diabetes were autoantibody-negative. In children with no mutation, 59 were positive to glutamic acid decarboxylase, 39 to islet antigen 2 and 31 to zinc transporter 8. Measuring zinc transporter 8 increased the number of autoantibody-positive individuals by eight. CONCLUSIONS: the present study provides the first evidence that Type 1 diabetes genetic risk score can be used to distinguish monogenic from Type 1 diabetes in an Iranian population with a large number of consanguineous unions. This test can be used to identify children with a higher probability of having monogenic diabetes who could then undergo genetic testing. Identification of these individuals would reduce the cost of treatment and improve the management of their clinical course.

Abstract
. Despite the rapid expansion in recent years of databases reporting either benign or pathogenic genetic variations, the interpretation of novel missense variants remains challenging, particularly for clinical or genetic testing laboratories where functional analysis is often unfeasible. Previous studies have shown that thermodynamic analysis of protein structure in silico can discriminate between groups of benign and pathogenic missense variants. However, although structures exist for many human disease‒associated proteins, such analysis remains largely unexploited in clinical laboratories. Here, we analyzed the predicted effect of 338 known missense variants on the structure of menin, the MEN1 gene product. Results provided strong discrimination between pathogenic and benign variants, with a threshold of &gt;4 kcal/mol for the predicted change in stability, providing a strong indicator of pathogenicity. Subsequent analysis of seven novel missense variants identified during clinical testing of patients with MEN1 showed that all seven were predicted to destabilize menin by &gt;4 kcal/mol. We conclude that structural analysis provides a useful tool in understanding the effect of missense variants in MEN1 and that integration of proteomic with genomic data could potentially contribute to the classification of novel variants in this disease.

Neonatal diabetes mellitus (NDM) is a rare type of monogenic diabetes that presents in the first 6 months of life. Activating mutations in the KCNJ11 gene encoding for the Kir6.2 subunit of the ATP-sensitive potassium (KATP ) channel can lead to transient NDM (TNDM) or to permanent NDM (PNDM). A female infant presented on the 22nd day of life with severe hyperglycemia and ketoacidosis (glucose: 907mg/dL, blood gas pH: 6.84, HCO3: 6 mmol/L). She was initially managed with intravenous (IV) fluids and IV insulin. Ketoacidosis resolved within 48 hours and she was started on subcutaneous insulin injections with intermediate acting insulin NPH twice daily requiring initially 0.75-1.35 IU/kg/d. Pre-prandial C-peptide levels were 0.51 ng/mL (normal: 1.77-4.68). Insulin requirements were gradually reduced and insulin administration was discontinued at the age of 10 months with subsequent normal glucose and HbA1c levels. C-peptide levels normalized (pre-prandial: 1.6 ng/mL, postprandial: 2 ng/mL). Genetic analysis identified a novel missense mutation (p.Pro254Gln) in the KCNJ11 gene. We report a novel KCNJ11 mutation in a patient who presented in the first month of life with a phenotype of NDM that subsided at the age of 10 months. It is likely that the novel p.P254Q mutation results in mild impairment of the KATP channel function leading to TNDM.

AIMS/HYPOTHESIS: Treatment change following a genetic diagnosis of MODY is frequently indicated, but little is known about the factors predicting future treatment success. We therefore conducted the first prospective study to determine the impact of a genetic diagnosis on individuals with GCK-, HNF1A- or HNF4A-MODY in the UK, and to identify clinical characteristics predicting treatment success (i.e. HbA1c ≤58 mmol/mol [≤7.5%]) with the recommended treatment at 2 years. METHODS: This was an observational, prospective, non-selective study of individuals referred to the Exeter Molecular Genetic Laboratory for genetic testing from December 2010 to December 2012. Individuals from the UK with GCK- or HNF1A/HNF4A-MODY who were not on recommended treatment at the time of genetic diagnosis, and who were diagnosed below the age of 30 years and were currently aged less than 50 years, were eligible to participate. RESULTS: a total of 44 of 58 individuals (75.9%) changed treatment following their genetic diagnosis. Eight individuals diagnosed with GCK-MODY stopped all diabetes medication without experiencing any change in HbA1c (49.5 mmol/mol [6.6%] both before the genetic diagnosis and at a median of 1.25 years' follow-up without treatment, p = 0.88). A total of 36 of 49 individuals (73.5%) diagnosed with HNF1A/HNF4A-MODY changed treatment; however, of the 21 of these individuals who were being managed with diet or sulfonylurea alone at 2 years, only 13 (36.1% of the population that changed treatment) had an HbA1c ≤58 mmol/mol (≤7.5%). These individuals had a shorter diabetes duration (median 4.6 vs 18.1 years), lower HbA1c (58 vs 73 mmol/mol [7.5% vs 8.8%]) and lower BMI (median 24.2 vs 26.0 kg/m2) at the time of genetic diagnosis, compared with individuals (n = 23/36) with an HbA1c >58 mmol/mol (>7.5%) (or 69 mmol/mol (>8.5%) at the time of genetic diagnosis. CONCLUSIONS/INTERPRETATION: in participants with GCK-MODY, treatment cessation was universally successful, with no change in HbA1c at follow-up. In those with HNF1A/HNF4A-MODY, a shorter diabetes duration, lower HbA1c and lower BMI at genetic diagnosis predicted successful treatment with sulfonylurea/diet alone, supporting the need for early genetic diagnosis and treatment change. Our study suggests that, in individuals with HNF1A/HNF4A-MODY with a longer duration of diabetes (>11 years) at time of genetic test, rather than ceasing current treatment, a sulfonylurea should be added to existing therapy, particularly in those who are overweight or obese and have a high HbA1c.

OBJECTIVE: Comprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder. METHODS: Using massively parallel sequencing we identified 30 patients from 27 families with 2 pathogenic nonsense, frameshift and/or splice site TTN mutations in trans. We then undertook a detailed analysis of the clinical, histopathological and imaging features of these patients. RESULTS: all patients had prenatal or early onset hypotonia and/or congenital contractures. None had ophthalmoplegia. Scoliosis and respiratory insufficiency typically developed early and progressed rapidly, whereas limb weakness was often slowly progressive, and usually did not prevent independent walking. Cardiac involvement was present in 46% of patients. Relatives of 2 patients had dilated cardiomyopathy. Creatine kinase levels were normal to moderately elevated. Increased fiber size variation, internalized nuclei and cores were common histopathological abnormalities. Cap-like regions, whorled or ring fibers, and mitochondrial accumulations were also observed. Muscle magnetic resonance imaging showed gluteal, hamstring and calf muscle involvement. Western blot analysis showed a near-normal sized titin protein in all samples. The presence of 2 mutations predicted to impact both N2BA and N2B cardiac isoforms appeared to be associated with greatest risk of cardiac involvement. One-third of patients had 1 mutation predicted to impact exons present in fetal skeletal muscle, but not included within the mature skeletal muscle isoform transcript. This strongly suggests developmental isoforms are involved in the pathogenesis of this congenital/early onset disorder. INTERPRETATION: This detailed clinical reference dataset will greatly facilitate diagnostic confirmation and management of patients, and has provided important insights into disease pathogenesis. Ann Neurol 2018;83:1105-1124.

The article entitled "The Clinical Course of Patients with Preschool Manifestation of Type 1 Diabetes is Independent of the HLA DR-DQ Genotype" contained a calculation error in Table 2 and the statistical methods used were not completely described.[. ].

OBJECTIVE: Rare genetic disorders resulting in prenatal or neonatal death are genetically heterogeneous, but testing is often limited by the availability of fetal DNA, leaving couples without a potential prenatal test for future pregnancies. We describe our novel strategy of exome sequencing parental DNA samples to diagnose recessive monogenic disorders in an audit of the first 50 couples referred. METHOD: Exome sequencing was carried out in a consecutive series of 50 couples who had 1 or more pregnancies affected with a lethal or prenatal-onset disorder. In all cases, there was insufficient DNA for exome sequencing of the affected fetus. Heterozygous rare variants (MAF 

BACKGROUND: Persistent hyperinsulinemic hypoglycemia of infancy (PHHI), also known as congenital hyperinsulinism, has been known to go into spontaneous remission, with patients developing diabetes in later life. A temporary phase of hyperglycemia is, however, rarely reported. CASE PRESENTATION: We describe a 16-month-old child, a known case of diazoxide responsive PHHI, presenting with mixed hyperglycemic hyperosmolar coma and ketoacidosis with rhabdomyolysis while on diazoxide treatment. The patient required temporary cessation of diazoxide and initiation of insulin infusion, followed by a relapse of hypoglycemia again necessitating diazoxide therapy. CONCLUSIONS: Hyperosmolar coma with ketoacidosis is a rare side-effect of diazoxide therapy, documented even in patients with persistent hyperinsulinemic hypoglycemia of infancy.

BACKGROUND: KCNJ11 mutations cause permanent neonatal diabetes through pancreatic ATP-sensitive potassium channel activation. 90% of patients successfully transfer from insulin to oral sulfonylureas with excellent initial glycaemic control; however, whether this control is maintained in the long term is unclear. Sulfonylurea failure is seen in about 44% of people with type 2 diabetes after 5 years of treatment. Therefore, we did a 10-year multicentre follow-up study of a large international cohort of patients with KCNJ11 permanent neonatal diabetes to address the key questions relating to long-term efficacy and safety of sulfonylureas in these patients. METHODS: in this multicentre, international cohort study, all patients diagnosed with KCNJ11 permanent neonatal diabetes at five laboratories in Exeter (UK), Rome (Italy), Bergen (Norway), Paris (France), and Krakow (Poland), who transferred from insulin to oral sulfonylureas before Nov 30, 2006, were eligible for inclusion. Clinicians collected clinical characteristics and annual data relating to glycaemic control, sulfonylurea dose, severe hypoglycaemia, side-effects, diabetes complications, and growth. The main outcomes of interest were sulfonylurea failure, defined as permanent reintroduction of daily insulin, and metabolic control, specifically HbA1c and sulfonylurea dose. Neurological features associated with KCNJ11 permanent neonatal diabetes were also assessed. This study is registered with ClinicalTrials.gov, number NCT02624817. FINDINGS: 90 patients were identified as being eligible for inclusion and 81 were enrolled in the study and provided long-term (>5·5 years cut-off) outcome data. Median follow-up duration for the whole cohort was 10·2 years (IQR 9·3-10·8). At most recent follow-up (between Dec 1, 2012, and Oct 4, 2016), 75 (93%) of 81 participants remained on sulfonylurea therapy alone. Excellent glycaemic control was maintained for patients for whom we had paired data on HbA1c and sulfonylurea at all time points (ie, pre-transfer [for HbA1c], year 1, and most recent follow-up; n=64)-median HbA1c was 8·1% (IQR 7·2-9·2; 65·0 mmol/mol [55·2-77·1]) before transfer to sulfonylureas, 5·9% (5·4-6·5; 41·0 mmol/mol [35·5-47·5]; p

BACKGROUND: As KATP channel mutations are the most common cause of neonatal diabetes mellitus (NDM) and patients with these mutations can be treated with oral sulfonylureas, empiric therapy is a common practice for NDM patients. CASE PRESENTATION: a non-syndromic, small for gestational age baby born to first-degree consanguineous parents was diagnosed with NDM. Because of hypo- and hyperglycemic episodes and variability in insulin requirement, we initiated a trial of glibenclamide, with a presumptive diagnosis of NDM caused by a KATP channel mutation. However, this empiric sulfonylurea trial did not improve the patient's glycemic control and resulted in resistance to exogenous insulin. Genetic testing identified a previously reported homozygous INS promoter mutation (c.-331C>G), which was not responsive to sulfonylurea therapy. CONCLUSIONS: in light of our results, we recommend to confirm the genetic diagnosis as soon as possible and decide on sulfonylurea treatment after a genetic diagnosis is confirmed.

Background: Heterozygous mutations in the HNF1B gene are the most common monogenic cause of developmental kidney disease. Extrarenal phenotypes frequently occur, including diabetes mellitus and pancreatic hypoplasia; the latter is associated with subclinical exocrine dysfunction. We measured faecal elastase-1 in patients with HNF1B-associated disease regardless of diabetes status and assessed the degree of symptoms associated with pancreatic exocrine deficiency. Methods: Faecal elastase-1 was measured in 29 patients with a known HNF1B mutation. We defined a low faecal elastase-1 concentration based on the 2.5 percentile of 99 healthy control individuals (410 μg/g stool). Symptoms related to pancreatic exocrine dysfunction were assessed and a subset of the HNF1B cohort (n  =  6) underwent pancreatic imaging. Results: Faecal elastase-1 was below the 2.5 percentile of the control cohort in 18/29 (62%) patients with HNF1B-associated renal disease. A total of 8/29 (28%) had a measurement suggestive of exocrine pancreatic insufficiency at  

BACKGROUND: Neonatal diabetes mellitus (NDM) is a rare disorder worldwide where diabetes is diagnosed in the first 6 months of life. However, Oman has a relatively high incidence of NDM. METHODS: in this study, we investigated the genetic etiologies underlying NDM and their prevalence in Oman. We collected a cohort of 24 NDM patients, with and without genetic diagnosis, referred to our center from 2007 to 2015. All patients without a genetic diagnosis were tested for mutations in 23 NDM-associated genes using a custom-targeted next-generation sequencing (NGS) panel and methylation analysis of the 6q24 locus. RESULTS: a genetic abnormality was detected in 15/24 (62.5%) of our Omani NDM patients. We report the detection of 6q24 methylation abnormalities and KCNJ11 mutations for the first time in Omani NDM patients. Unlike Western populations where NDM is predominantly due to mutations in the KCNJ11, ABCC8 and INS genes, NDM due to homozygous GCK gene mutations were most prevalent in Oman, having been observed in seven out of 15 NDM patients in whom we established the genetic etiology. This reflects the high degree of consanguinity which makes recessive conditions more likely. CONCLUSIONS: the results of this study are likely to impact any future strategy to introduce genetic testing for NDM disorders within the national healthcare system in Oman.

Evaluating, optimising and benchmarking of next generation sequencing (NGS) variant calling performance are essential requirements for clinical, commercial and academic NGS pipelines. Such assessments should be performed in a consistent, transparent and reproducible fashion, using independently, orthogonally generated data. Here we present ICR142 Benchmarker, a tool to generate outputs for assessing germline base substitution and indel calling performance using the ICR142 NGS validation series, a dataset of Illumina platform-based exome sequence data from 142 samples together with Sanger sequence data at 704 sites. ICR142 Benchmarker provides summary and detailed information on the sensitivity, specificity and false detection rates of variant callers. ICR142 Benchmarker also automatically generates a single page report highlighting key performance metrics and how performance compares to widely-used open-source tools. We used ICR142 Benchmarker with VCF files outputted by GATK, OpEx and DeepVariant to create a benchmark for variant calling performance. This evaluation revealed pipeline-specific differences and shared challenges in variant calling, for example in detecting indels in short repeating sequence motifs. We next used ICR142 Benchmarker to perform regression testing with DeepVariant versions 0.5.2 and 0.6.1. This showed that v0.6.1 improves variant calling performance, but there was evidence of minor changes in indel calling behaviour that may benefit from attention. The data also allowed us to evaluate filters to optimise DeepVariant calling, and we recommend using 30 as the QUAL threshold for base substitution calls when using DeepVariant v0.6.1. Finally, we used ICR142 Benchmarker with VCF files from two commercial variant calling providers to facilitate optimisation of their in-house pipelines and to provide transparent benchmarking of their performance. ICR142 Benchmarker consistently and transparently analyses variant calling performance based on the ICR142 NGS validation series, using the standard VCF input and outputting informative metrics to enable user understanding of pipeline performance. ICR142 Benchmarker is freely available at https://github.com/RahmanTeamDevelopment/ICR142_Benchmarker/releases.

Evaluating, optimising and benchmarking of next generation sequencing (NGS) variant calling performance are essential requirements for clinical, commercial and academic NGS pipelines. Such assessments should be performed in a consistent, transparent and reproducible fashion, using independently, orthogonally generated data. Here we present ICR142 Benchmarker, a tool to generate outputs for assessing variant calling performance using the ICR142 NGS validation series, a dataset of exome sequence data from 142 samples together with Sanger sequence data at 704 sites. ICR142 Benchmarker provides summary and detailed information on the sensitivity, specificity and false detection rates of variant callers. ICR142 Benchmarker also automatically generates a single page report highlighting key performance metrics and how performance compares to widely-used open-source tools. We used ICR142 Benchmarker with VCF files outputted by GATK, OpEx and DeepVariant to create a benchmark for variant calling performance. This evaluation revealed pipeline-specific differences and shared challenges in variant calling, for example in detecting indels in short repeating sequence motifs. We next used ICR142 Benchmarker to perform regression testing with versions 0.5.2 and 0.6.1 of DeepVariant. This showed that v0.6.1 improves variant calling performance, but there was evidence of some minor changes in indel calling behaviour that may benefit from attention in future updates. The data also allowed us to evaluate filters to optimise DeepVariant calling, and we recommend using 30 as the QUAL threshold for base substitution calls when using DeepVariant v0.6.1. Finally, we used ICR142 Benchmarker with VCF files from two commercial variant calling providers to facilitate optimisation of their in-house pipelines and to provide transparent benchmarking of their performance. ICR142 Benchmarker consistently and transparently analyses variant calling performance based on the ICR142 NGS validation series, using the standard VCF input and outputting informative metrics to enable user understanding of pipeline performance. ICR142 Benchmarker is freely available at https://github.com/RahmanTeamDevelopment/ICR142_Benchmarker/releases.

The objective of this study was to describe the clinical characteristics of syndromic neonatal diabetes in a family with a GATA6 mutation. A girl, currently aged 12 years 3 months, was born with intrauterine growth retardation: weight 1600 g (–4.3 SDS) at term. After birth, foramen ovale and patent ductus arteriosus (PDA) were diagnosed by echocardiography. Diabetes was diagnosed on the 9th day after birth. Exocrine pancreatic insufficiency was clinically diagnosed at about 2 years of age and pancreatic agenesis was revealed later by magnetic resonance imaging. Her father had undergone surgery during infancy for PDA and had developed insulin dependent diabetes at 12 years of age. Ultrasound revealed a thin pancreas with normal length and anatomical structure. He has subclinical exocrine pancreatic insufficiency, low insulin needs and no late complications of diabetes up to the age of 40 years. Sequencing of GATA6 identified a heterozygous splicing mutation, 1136-2A>G, in the girl and her father. Testing of the paternal grandparents showed that the mutation was likely to have arisen de novo in the father. Identification of a GATA6 mutation explains the cardiac anomalies and diabetes in this family. This case highlights the marked intra-familial variability of both exocrine and endocrine pancreatic phenotypes in patients with GATA6 mutations.

Monogenic diabetes represents a heterogeneous group of disorders resulting from a single gene defect leading to disruption of insulin secretion or a reduction in the number of beta cells. Despite the classification of monogenic diabetes into neonatal diabetes or maturity onset diabetes of the young (MODY) according to age of onset, not every case can be classified into those 2 groups. We evaluated patients with monogenic diabetes diagnosed during the last 10 year period. Type 1 DM, MODY, and patients with negative autoantibodies and no mutation in a known gene were excluded from the study. Thirteen patients were diagnosed with monogenic diabetes in Department of Pediatric Endocrinology, Ankara University School of Medicine, Ankara, Turkey. Five of them were diagnosed after 6 months of age. Five had a KATP channel defect. Mutations in genes resulting in destruction of beta cells were detected in 7 patients, with 4 cases having a WFS, 2 an LRBA, and one a IL2RA mutation. Additional systemic findings were seen in 6/13 patients, with 5/6 having severe immune system dysfunction. Treatment with sulphonylurea was successful in two patients. The other patients were given insulin in differing doses. Four patients died during follow-up, three of which had immune system dysfunction. Monogenic diabetes can be diagnosed after 6 months of age, even with positive autoantibodies. Immune dysfunction was a common feature in our cohort and should be investigated in all patients with early-onset monogenic diabetes. Mortality of patients with monogenic diabetes and additional autoimmunity was high in our cohort and is likely to reflect the multisystem nature of these diseases.

Neonatal diabetes mellitus is a rare form of monogenic diabetes which is diagnosed in the first six months of life. Here we report three patients with neonatal diabetes; two with isolated pancreas agenesis due to mutations in the pancreas-specific transcription factor 1A (PTF1A) enhancer and one with developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome, due to a KCNJ11 mutation. The two cases with mutations in the distal enhancer of PTF1A had a homozygous g.23508363A>G and a homozygous g.23508437A>G mutation respectively. Previous functional analyses showed that these mutations can decrease expression of PTF1A which is involved in pancreas development. Both patients were born small for gestational age to consanguineous parents. Both were treated with insulin and pancreatic enzymes. One of these patients’ fathers was also homozygous for the PTF1A mutation, whilst his partner and the parents of the other patient were heterozygous carriers. In the case with DEND sydrome, a previosly reported heterozygous KCNJ11 mutation, p.Cys166Tyr (c.497G>A), was identified. This patient was born to nonconsanguineous parents with normal birth weight. The majority of neonatal diabetes patients with KCNJ11 mutations will respond to sulphonylurea treatment. Therefore Glibenclamide, an oral antidiabetic of the sulphonylurea group, was started. This treatment regimen relatively improved blood glucose levels and neurological symptoms in the short term. Because we could not follow the patient in the long term, we are not able to draw conclusions about the efficacy of the treatment. Although neonatal diabetes mellitus can be diagnosed clinically, genetic analysis is important since it is a guide for the treatment and for prognosis.

Context: Monogenic partial lipodystrophy is a genetically heterogeneous disease where only variants with specific genetic mechanisms are causative. Three heterozygous protein extending frameshift variants in PLIN1 have been reported to cause a phenotype of partial lipodystrophy and insulin resistance. Objective: We investigated if null variants in PLIN1 cause lipodystrophy. Methods: As part of a targeted sequencing panel test, we sequenced PLIN1 in 2208 individuals. We also investigated the frequency of PLIN1 variants in the gnomAD database, and the type 2 diabetes knowledge portal. Results: We identified 6/2208 (1 in 368) individuals with a PLIN1 null variant. None of these individuals had clinical or biochemical evidence of overt lipodystrophy. Additionally, 14/17,000 (1 in 1214) individuals with PLIN1 null variants in the type 2 diabetes knowledge portal showed no association with biomarkers of lipodystrophy. PLIN1 null variants occur too frequently in gnomAD (126/138,632; 1 in 1100) to be a cause of rare overt monogenic partial lipodystrophy. Conclusions: Our study suggests that heterozygous variants that are predicted to result in PLIN1 haploinsufficiency are not a cause of familial partial lipodystrophy and should not be reported as disease-causing variants by diagnostic genetic testing laboratories. This finding is in keeping with other known monogenic causes of lipodystrophy, such as PPARG and LMNA, where only variants with specific genetic mechanisms cause lipodystrophy.

The basic helix-loop-helix (bHLH) transcription factor, neuronal differentiation 1 (NEUROD1) (also known as BETA2) is involved in the development of neural elements and endocrine pancreas. Less than 10 reports of adult-onset non-insulin-dependent diabetes mellitus (NIDDM) due to heterozygous NEUROD1 mutations and 2 cases with permanent neonatal diabetes mellitus (PNDM) and neurological abnormalities due to homozygous NEUROD1 mutations have been published. A 13 year-old female was referred to endocrine department due to hyperglycemia. She was on insulin therapy following a diagnosis of neonatal diabetes mellitus (NDM) at the age of 9-weeks but missed regular follow-up. Parents are second cousin. There was a significant family history of adult onset NIDDM including patient's father. Auxological measurements were within normal ranges. On laboratory examination blood glucose was 33.2 mmol/L with undetectable c-peptide and glycosylated hemoglobin level of 8.9% (73.8 mmol/mol). She had developed difficulty in walking at the age of 4 years which had worsened over time. On further evaluation, a diagnosis of visual impairment, mental retardation, ataxic gait, retinitis pigmentosa and sensory-neural deafness were considered. Cranial magnetic resonance imaging revealed cerebellar hypoplasia. Molecular genetic analysis using targeted next generation sequencing detected a novel homozygous missense mutation, p.Ile150Asn(c.449T>A), in NEUROD1. Both parents and 2 unaffected siblings were heterozygous for the mutation. We report the third case of PNDM with neurological abnormalities caused by homozygous NEUROD1 mutation, the first caused by a missense mutation. Heterozygous carriers of the p.Ile150Asn mutation were either unaffected or diagnosed with diabetes in adulthood. It is currently unclear whether the NEUROD1 heterozygous mutation has contributed to diabetes development in these individuals.

AIMS/HYPOTHESIS: Diabetes is one of the cardinal features of thiamine-responsive megaloblastic anaemia (TRMA) syndrome. Current knowledge of this rare monogenic diabetes subtype is limited. We investigated the genotype, phenotype and response to thiamine (vitamin B1) in a cohort of individuals with TRMA-related diabetes. METHODS: We studied 32 individuals with biallelic SLC19A2 mutations identified by Sanger or next generation sequencing. Clinical details were collected through a follow-up questionnaire. RESULTS: We identified 24 different mutations, of which nine are novel. The onset of the first TRMA symptom ranged from birth to 4 years (median 6 months [interquartile range, IQR 3-24]) and median age at diabetes onset was 10 months (IQR 5-27). At presentation, three individuals had isolated diabetes and 12 had asymptomatic hyperglycaemia. Follow-up data was available for 15 individuals treated with thiamine for a median 4.7 years (IQR 3-10). Four patients were able to stop insulin and seven achieved better glycaemic control on lower insulin doses. These 11 patients were significantly younger at diabetes diagnosis (p = 0.042), at genetic testing (p = 0.01) and when starting thiamine (p = 0.007) compared with the rest of the cohort. All patients treated with thiamine became transfusion-independent and adolescents achieved normal puberty. There were no additional benefits of thiamine doses >150 mg/day and no reported side effects up to 300 mg/day. CONCLUSIONS/INTERPRETATION: in TRMA syndrome, diabetes can be asymptomatic and present before the appearance of other features. Prompt recognition is essential as early treatment with thiamine can result in improved glycaemic control, with some individuals becoming insulin-independent. DATA AVAILABILITY: SLC19A2 mutation details have been deposited in the Decipher database ( https://decipher.sanger.ac.uk/ ).

CHN is genetically heterogeneous and its genetic basis is difficult to determine on features alone. CNTNAP1 encodes CASPR, integral in the paranodal junction high molecular mass complex. Nineteen individuals with biallelic variants have been described in association with severe congenital hypomyelinating neuropathy, respiratory compromise, profound intellectual disability and death within the first year. We report 7 additional patients ascertained through exome sequencing. We identified 9 novel CNTNAP1 variants in 6 families: three missense variants, four nonsense variants, one frameshift variant and one splice site variant. Significant polyhydramnios occurred in 6/7 pregnancies. Severe respiratory compromise was seen in 6/7 (tracheostomy in 5). A complex neurological phenotype was seen in all patients who had marked brain hypomyelination/demyelination and profound developmental delay. Additional neurological findings included cranial nerve compromise: orobulbar dysfunction in 5/7, facial nerve weakness in 4/7 and vocal cord paresis in 5/7. Dystonia occurred in 2/7 patients and limb contractures in 5/7. All had severe gastroesophageal reflux, and a gastrostomy was required in 5/7. In contrast to most previous reports, only one patient died in the first year of life. Protein modelling was performed for all detected CNTNAP1 variants. We propose a genotype-phenotype correlation, whereby hypomorphic missense variants partially ameliorate the phenotype, prolonging survival. This study suggests that biallelic variants in CNTNAP1 cause a distinct recognisable syndrome, which is not caused by other genes associated with CHN. Neonates presenting with this phenotype will benefit from early genetic definition to inform clinical management and enable essential genetic counselling for their families.

BACKGROUND: Accurate diabetes prevalence estimates are important for health service planning and prioritisation. Available data have limitations, suggesting that the contemporary burden of diabetes in Australia is best assessed from multiple sources. AIMS: to use systematic active detection of diabetes cases in a postcode-defined urban area through the Fremantle Diabetes Study Phase II (FDS2) to complement other epidemiological and survey data in estimating the national prevalence of diabetes and its types. METHODS: People with known diabetes in a population of 157 000 were identified (n = 4639) from a variety of sources and those providing informed consent (n = 1668 or 36%) were recruited to the FDS2 between 2008 and 2011. All FDS2 participants were assigned a type of diabetes based on clinical and laboratory (including serological and genetic) features. Data from people identified through the FDS2 were used to complement Australian Health Survey and National Diabetes Services Scheme prevalence estimates (the proportions of people well controlled on no pharmacotherapy and registering with the National Diabetes Services Scheme respectively) in combination with Australian Bureau of Statistics data to generate the prevalence of diabetes in Australia. RESULTS: Based on data from multiple sources, 4.8% or 1.1 million Australians had diabetes in 2011-2012, of whom 85.8% had type 2 diabetes, 7.9% type 1 diabetes and 6.3% other types (latent autoimmune diabetes of adults, monogenic diabetes and secondary diabetes). CONCLUSIONS: Approximately 1 in 20 Australians has diabetes. Although most have type 2 diabetes, one in seven has other types that may require more specialised diagnosis and/or management.

BACKGROUND: Predictive tools to identify patients at risk for gene mutations related to pituitary adenomas are very helpful in clinical practice. We therefore aimed to develop and validate a reliable risk category system for aryl hydrocarbon receptor-interacting protein (AIP) mutations in patients with pituitary adenomas. METHODS: an international cohort of 2227 subjects were consecutively recruited between 2007 and 2016, including patients with pituitary adenomas (familial and sporadic) and their relatives. All probands (n=1429) were screened for AIP mutations, and those diagnosed with a pituitary adenoma prospectively, as part of their clinical screening (n=24), were excluded from the analysis. Univariate analysis was performed comparing patients with and without AIP mutations. Based on a multivariate logistic regression model, six potential factors were identified for the development of a risk category system, classifying the individual risk into low-risk, moderate-risk and high-risk categories. An internal cross-validation test was used to validate the system. RESULTS: 1405 patients had a pituitary tumour, of which 43% had a positive family history, 55.5% had somatotrophinomas and 81.5% presented with macroadenoma. Overall, 134 patients had an AIP mutation (9.5%). We identified four independent predictors for the presence of an AIP mutation: age of onset providing an odds ratio (OR) of 14.34 for age 0-18 years, family history (OR 10.85), growth hormone excess (OR 9.74) and large tumour size (OR 4.49). In our cohort, 71% of patients were identified as low risk (

Sirolimus has been reported to be effective in the treatment of the diffuse form of congenital hyperinsulinism (CHI), unresponsive to diazoxide and octreotide, without causing severe side effects. Two newborns with CHI due to homozygous ABCC8 gene mutations were started on sirolimus aged 21 and 17 days, due to lack of response to medical treatment. A good response to sirolimus was observed. At follow-up after ten and two months of treatment, liver enzymes were found to be increased [serum sirolimus level 1.4 ng/mL (normal range: 5-15), aspartate aminotransferase (AST): 298U/L, alanine aminotransferase (ALT): 302U/L and serum sirolimus level: 9.9 ng/mL, AST: 261U/L, ALT: 275U/L, respectively]. In Case 1, discontinuation of the drug resulted in normalization of liver enzymes within three days. Two days after normalization, sirolimus was restarted at a lower dose, which resulted in a repeated increase in transferases. In Case 2, a reduction of sirolimus dose caused normalization of liver enzymes within ten days. When the dose was increased, enzymes increased within three days. Sirolimus was discontinued in both cases.
The rapid normalization of liver enzyme levels after sirolimus withdrawal or dose reduction; elevation of transaminases after restart or dose increase and rapid normalization after sirolimus withdrawal were findings strongly suggestive of sirolimus-induced hepatitis.
To the best of our knowledge, this is the first report of sirolimus-induced hepatitis in CHI. Sirolimus is a promising drug for CHI patients who are unresponsive to medical treatment, but physicians should be vigilant for adverse effects on liver function.

Transient receptor potential vanilloid 6 (TRPV6) functions in tetramer form for calcium transport. Until now, TRPV6 has not been linked with skeletal development disorders. An infant with antenatal onset thoracic insufficiency required significant ventilatory support. Skeletal survey showed generalized marked undermineralization, hypoplastic fractured ribs, metaphyseal fractures, and extensive periosteal reaction along femoral, tibial, and humeral diaphyses. Parathyroid hormone (PTH) elevation (53.4-101 pmol/L) initially suggested PTH signaling disorders. Progressively, biochemical normalization with radiological mineralization suggested recovery from in utero pathophysiology. Genomic testing was undertaken and in silico protein modeling of variants. No abnormalities in antenatal CGH array or UPD14 testing. Postnatal molecular genetic analysis found no causative variants in CASR, GNA11, APS21, or a 336 gene skeletal dysplasia panel investigated by whole exome sequencing. Trio exome analysis identified compound heterozygous TRPV6 likely pathogenic variants: novel maternally inherited missense variant, c.1978G > C p.(Gly660Arg), and paternally inherited nonsense variant, c.1528C > T p.(Arg510Ter), confirming recessive inheritance. p.(Gly660Arg) generates a large side chain protruding from the C-terminal hook into the interface with the adjacent TRPV6 subunit. In silico protein modeling suggests steric clashes between interface residues, decreased C-terminal hook, and TRPV6 tetramer stability. The p.(Gly660Arg) variant is predicted to result in profound loss of TRPV6 activity. This first case of a novel dysplasia features severe but improving perinatal abnormalities. The TRPV6 compound heterozygous variants appear likely to interfere with fetoplacental calcium transfer crucial for in utero skeletal development. Astute clinical interpretation of evolving perinatal abnormalities remains valuable in complex calcium and bone pathophysiology and informs exome sequencing interpretation.

There is wide variation in the age at diagnosis of diabetes in individuals with maturity-onset diabetes of the young (MODY) due to a mutation in the HNF1A gene. We hypothesized that common variants at the HNF1A locus (rs1169288 [I27L], rs1800574 [A98V]), which are associated with type 2 diabetes susceptibility, may modify age at diabetes diagnosis in individuals with HNF1A-MODY. Meta-analysis of two independent cohorts, comprising 781 individuals with HNF1A-MODY, found no significant associations between genotype and age at diagnosis. However after stratifying according to type of mutation (protein-truncating variant [PTV] or missense), we found each 27L allele to be associated with a 1.6-year decrease (95% CI -2.6, -0.7) in age at diagnosis, specifically in the subset (n = 444) of individuals with a PTV. The effect size was similar and significant across the two independent cohorts of individuals with HNF1A-MODY. We report a robust genetic modifier of HNF1A-MODY age at diagnosis that further illustrates the strong effect of genetic variation within HNF1A upon diabetes phenotype.

Locus heterogeneity characterizes a variety of skeletal dysplasias often due to interacting or overlapping signaling pathways. Robinow syndrome is a skeletal disorder historically refractory to molecular diagnosis, potentially stemming from substantial genetic heterogeneity. All current known pathogenic variants reside in genes within the noncanonical Wnt signaling pathway including ROR2, WNT5A, and more recently, DVL1 and DVL3. However, ∼70% of autosomal-dominant Robinow syndrome cases remain molecularly unsolved. To investigate this missing heritability, we recruited 21 families with at least one family member clinically diagnosed with Robinow or Robinow-like phenotypes and performed genetic and genomic studies. In total, four families with variants in FZD2 were identified as well as three individuals from two families with biallelic variants in NXN that co-segregate with the phenotype. Importantly, both FZD2 and NXN are relevant protein partners in the WNT5A interactome, supporting their role in skeletal development. In addition to confirming that clustered -1 frameshifting variants in DVL1 and DVL3 are the main contributors to dominant Robinow syndrome, we also found likely pathogenic variants in candidate genes GPC4 and RAC3, both linked to the Wnt signaling pathway. These data support an initial hypothesis that Robinow syndrome results from perturbation of the Wnt/PCP pathway, suggest specific relevant domains of the proteins involved, and reveal key contributors in this signaling cascade during human embryonic development. Contrary to the view that non-allelic genetic heterogeneity hampers gene discovery, this study demonstrates the utility of rare disease genomic studies to parse gene function in human developmental pathways.

Context: to date, penetrance figures for medullary thyroid cancer (MTC) for variants in rearranged during transfection (RET) have been estimated from families ascertained because of the presence of MTC. Objective: to gain estimates of penetrance, unbiased by ascertainment, we analyzed 61 RET mutations assigned as disease causing by the American Thyroid Association (ATA) in population whole-exome sequencing data. Design: for the 61 RET mutations, we used analyses of the observed allele frequencies in ∼51,000 individuals from the Exome Aggregation Consortium (ExAC) database that were not contributed via the Cancer Genome Atlas (TCGA; non-TCGA ExAC), assuming lifetime penetrance for MTC of 90%, 50%, and unbounded. Setting: Population-based. Results: Ten of 61 ATA disease-causing RET mutations were present in the non-TCGA ExAC population with observed frequency consistent with penetrance for MTC of >90%. For p.Val804Met, the lifetime penetrance for MTC, estimated from the allele frequency observed, was 4% [95% confidence interval (CI), 0.9% to 8%]. Conclusions: Based on penetrance analysis in carrier relatives of p.Val804Met-positive cases of MTC, p.Val804Met is currently understood to have high-lifetime penetrance for MTC (87% by age 70), albeit of later onset of MTC than other RET mutations. Given our unbiased estimate of penetrance for RET p.Val804Met of 4% (95% CI, 0.9% to 8%), the current recommendation by the ATA of prophylactic thyroidectomy as standard for all RET mutation carriers is likely inappropriate.

Congenital hyperinsulinaemic hypoglycaemia (HH) can occur in isolation or it may present as part of a wider syndrome. For approximately 40%-50% of individuals with this condition, sequence analysis of the known HH genes identifies a causative mutation. Identifying the underlying genetic aetiology in the remaining cases is important as a genetic diagnosis will inform on recurrence risk, may guide medical management and will provide valuable insights into β-cell physiology. We sequenced the exome of a child with persistent diazoxide-responsive HH, mild aortic insufficiency, severe hypotonia, and developmental delay as well as the unaffected parents. This analysis identified a de novo mutation, p.G403D, in the proband's CACNA1D gene. CACNA1D encodes the main L-type voltage-gated calcium channel in the pancreatic β-cell, a key component of the insulin secretion pathway. The p.G403D mutation had been reported previously as an activating mutation in an individual with primary hyper-aldosteronism, neuromuscular abnormalities, and transient hypoglycaemia. Sequence analysis of the CACNA1D gene in 60 further cases with HH did not identify a pathogenic mutation. Identification of an activating CACNA1D mutation in a second patient with congenital HH confirms the aetiological role of CACNA1D mutations in this disorder. A genetic diagnosis is important as treatment with a calcium channel blocker may be an option for the medical management of this patient.

Neonatal diabetes mellitus is a rare monogenic disease with incidence of 1/90,000 newborns. A case of two months aged male infant with life threatening diabetic ketoacidosis is presented with novel ABCC8 gene mutation (p.F577L), successful transition from insulin to sulfonylurea and follow-up of three years.

The pancreatic ATP-sensitive K+ (K-ATP) channel is a key regulator of insulin secretion. Gain-of-function mutations in the genes encoding the Kir6.2 (KCNJ11) and SUR1 (ABCC8) subunits of the channel cause neonatal diabetes, whilst loss-of-function mutations in these genes result in congenital hyperinsulinism. We report two patients with neonatal diabetes in whom we unexpectedly identified recessively inherited loss-of-function mutations. The aim of this study was to investigate how a homozygous nonsense mutation in ABCC8 could result in neonatal diabetes. The ABCC8 p.Glu747. was identified in two unrelated Vietnamese patients. This mutation is located within the in-frame exon 17 and RNA studies confirmed (a) the absence of full length SUR1 mRNA and (b) the presence of the alternatively spliced transcript lacking exon 17. Successful transfer of both patients to sulphonylurea treatment suggests that the altered transcript expression enhances the sensitivity of the K-ATP channel to Mg-ADP/ATP. This is the first report of an ABCC8 nonsense mutation causing a gain-of-channel function and these findings extend the spectrum of K-ATP channel mutations observed in patients with neonatal diabetes.

Copy number variants (CNV) are a major cause of disease, with over 30,000 reported in the DECIPHER database. To use read depth data from targeted Next Generation Sequencing (NGS) panels to identify CNVs with the highest degree of sensitivity, it is necessary to account for biases inherent in the data. GC content and ambiguous mapping due to repetitive sequence elements and pseudogenes are the principal components of technical variability. In addition, the algorithms used favour the detection of multi-exon CNVs, and rely on suitably matched normal dosage samples for comparison. We developed a calling strategy that subdivides target intervals, and uses pools of historical control samples to overcome these limitations in a clinical diagnostic laboratory. We compared our enhanced strategy with an unmodified pipeline using the R software package ExomeDepth, using a cohort of 109 heterozygous CNVs (91 deletions, 18 duplications in 26 genes), including 25 single exon CNVs. The unmodified pipeline detected 104/109 CNVs, giving a sensitivity of 89.62% to 98.49% at the 95% confidence interval. The detection of all 109 CNVs by our enhanced method demonstrates 95% confidence the sensitivity is ≥96.67%, allowing NGS read depth analysis to be used for CNV detection in a clinical diagnostic setting.

AIMS: an early genetic diagnosis of neonatal diabetes guides clinical management and results in improved treatment in ~ 40% of patients. In the offspring of individuals with neonatal diabetes, a prenatal diagnosis allows accurate estimation of the risk of developing diabetes and, eventually, the most appropriate treatment for the baby. In this study, we performed non-invasive prenatal genetic testing for a fetus at risk of inheriting a paternal KCNJ11 p.R201C mutation causing permanent neonatal diabetes. METHODS: a droplet digital polymerase chain reaction assay was used to detect the presence of the mutation in cell-free circulating DNA (cfDNA) extracted from maternal plasma at 12 and 16 weeks' gestation. RESULTS: the mutation was not detected in the cfDNA samples, suggesting that the fetus had not inherited the KCNJ11 mutation. The fetal DNA fraction was estimated at 6.2% and 10.7%, which is above the detection limit of the assay. The result was confirmed by Sanger sequencing after the baby's birth, confirming that the baby's risk of developing neonatal diabetes was reduced to that of the general population. CONCLUSIONS: We report the first case of non-invasive prenatal testing in a family with neonatal diabetes. A prenatal diagnosis in families at high risk of monogenic diabetes informs both prenatal and postnatal management. Although the clinical impact of this novel technology still needs to be assessed, its implementation in clinical practice (including cases at risk of inheriting mutations from the mother) will likely have a positive impact upon the clinical management of families affected by monogenic diabetes.

BACKGROUND: Haploinsufficiency of the GATA6 transcription factor gene was recently found to be the most common cause of pancreatic agenesis, a rare cause of neonatal diabetes mellitus. Although most cases are de novo, we describe three siblings with inherited GATA6 haploinsufficiency and the rare finding of parental mosaicism. CASE PRESENTATION: the proband was born at term with severe intrauterine growth restriction, the first child of non-consanguineous parents. Diabetes occurred on day of life 1 with pancreatic exocrine insufficiency noted at several months of age. Pancreatic agenesis with absent gallbladder was confirmed when he underwent congenital diaphragmatic hernia and intestinal malrotation repair. A patent ductus arteriosus and pulmonary stenosis were repaired in infancy. Neurocognitive development has been normal. A second pregnancy was terminated due to tetralogy of Fallot and pulmonary hypoplasia secondary to congenital diaphragmatic hernia. The fetus also demonstrated severe pancreatic hypoplasia, gallbladder agenesis and intestinal rotation abnormalities. Despite severe hypoplasia, the pancreas demonstrated normal islet histology. Another sibling was found to have multiple cardiac abnormalities, requiring procedural intervention. Given the proband's spectrum of congenital anomalies, Sanger sequencing of the GATA6 gene was performed, revealing a novel heterozygous c.635_660del frameshift mutation (p.Pro212fs). The mutation is predicted to be pathogenic, resulting in inclusion of a premature stop codon and likely degradation of the gene transcript by nonsense-mediated decay. The abortus and the sibling with the cardiac defect were both found to have the mutation, while the father and remaining sibling were negative. The mother, who is healthy with no evidence of diabetes or cardiac disease, is mosaic for the mutation at a level of 11% in her peripheral leukocytes by next-generation sequencing. CONCLUSION: We highlight a rare mechanism of pancreatic agenesis, this being only the second report of parental mosaicism for a GATA6 mutation and one of a handful of inherited cases. We also further define the phenotypic variability of GATA6 haploinsufficiency, even in individuals carrying the same mutation. Mutations in GATA6 should be strongly considered in cases of diabetes due to pancreatic hypoplasia or agenesis, and potentially affected family members should be tested regardless of phenotype.

OBJECTIVE: to study the genetic mutations and clinical profile in children with neonatal diabetes mellitus. METHODS: Genetic evaluation, clinical management and follow-up of infants with neonatal diabetes. RESULTS: Eleven infants were studied of which eight had permanent neonatal diabetes. Median age at presentation was 8 weeks and mean (SD) birth weight was 2.4 (0.5) kg. Pathogenic genetic mutations were identified in 7 (63.6%) children; 3 infants with mutations in KCNJ11 gene and 1 in ABCC8 were switched to oral sulfonylureas; 2 infants had mutations in INS and 1 in ZFP57. CONCLUSION: Neonatal diabetes mellitus is a heterogeneous disorder. Identification of genetic cause guides clinical management.

BACKGROUND: Congenital hyperinsulinism (CHI) can present with considerable clinical heterogeneity which may be due to differences in the underlying genetic etiology. We present two siblings with hyperinsulinaemic hypoglycaemia (HH) and marked clinical heterogeneity caused by compound heterozygosity for the same two novel ABCC8 mutations. CASE PRESENTATION: the index patient is a 3-year-old boy with hypoglycaemic episodes presenting on the first day of life. HH was diagnosed and treatment with intravenous glucose and diazoxide was initiated. Currently he has normal physical and neurological development, with occasional hypoglycaemic episodes detected following continuous fasting on treatment with diazoxide. The first-born 8-year-old sibling experienced severe postnatal hypoglycaemia, generalised seizures and severe brain damage despite diazoxide treatment. The latter was stopped at 6-months of age with no further registered hypoglycaemia. Genetic testing showed that both children were compound heterozygotes for two novel ABCC8 missense mutations p.I60N (c.179T>A) and p.G1555V (c.4664G>T). CONCLUSIONS: These ABCC8 missense mutations warrant further studies mainly because of the variable clinical presentation and treatment response.

Neonatal diabetes is frequently part of a complex syndrome with extrapancreatic features: 18 genes causing syndromic neonatal diabetes have been identified to date. There are still patients with neonatal diabetes who have novel genetic syndromes. We performed exome sequencing in a patient and his unrelated, unaffected parents to identify the genetic etiology of a syndrome characterized by neonatal diabetes, sensorineural deafness, and congenital cataracts. Further testing was performed in 311 patients with diabetes diagnosed before 1 year of age in whom all known genetic causes had been excluded. We identified 5 patients, including the initial case, with three heterozygous missense mutations in WFS1 (4/5 confirmed de novo). They had diabetes diagnosed before 12 months (2 before 6 months) (5/5), sensorineural deafness diagnosed soon after birth (5/5), congenital cataracts (4/5), and hypotonia (4/5). In vitro studies showed that these WFS1 mutations are functionally different from the known recessive Wolfram syndrome-causing mutations, as they tend to aggregate and induce robust endoplasmic reticulum stress. Our results establish specific dominant WFS1 mutations as a cause of a novel syndrome including neonatal/infancy-onset diabetes, congenital cataracts, and sensorineural deafness. This syndrome has a discrete pathophysiology and differs genetically and clinically from recessive Wolfram syndrome.

© 2017 Elsevier Inc.Background Mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL) is an autosomal dominant systemic disorder characterized by prominent loss of subcutaneous fat, a characteristic facial appearance and metabolic abnormalities. This syndrome is caused by heterozygous de novo mutations in the POLD1 gene. To date, 19 patients with MDPL have been reported in the literature and among them 14 patients have been characterized at the molecular level. Twelve unrelated patients carried a recurrent in-frame deletion of a single codon (p.Ser605del) and two other patients carried a novel heterozygous mutation in exon 13 (p.Arg507Cys). Additionally and interestingly, germline mutations of the same gene have been involved in familial polyposis and colorectal cancer (CRC) predisposition. Patients and Methods We describe a male and a female patient with MDPL respectively affected with mild and severe phenotypes. Both of them showed mandibular hypoplasia, a beaked nose with bird-like facies, prominent eyes, a small mouth, growth retardation, muscle and skin atrophy, but the female patient showed such a severe and early phenotype that a first working diagnosis of Hutchinson-Gilford Progeria was made. The exploration was performed by direct sequencing of POLD1 gene exon 15 in the male patient with a classical MDPL phenotype and by whole exome sequencing in the female patient and her unaffected parents. Results Exome sequencing identified in the latter patient a de novo heterozygous undescribed mutation in the POLD1 gene (NM_002691.3: c.3209T > A), predicted to cause the missense change p.Ile1070Asn in the ZnF2 (Zinc Finger 2) domain of the protein. This mutation was not reported in the 1000 Genome Project, dbSNP and Exome sequencing databases. Furthermore, the Isoleucine1070 residue of POLD1 is highly conserved among various species, suggesting that this substitution may cause a major impairment of POLD1 activity. For the second patient, affected with a typical MDPL phenotype, direct sequencing of POLD1 exon 15 revealed the recurrent in-frame deletion (c.1812_1814del, p.S605del). Conclusion Our work highlights that mutations in different POLD1 domains can lead to phenotypic variability, ranging from dominantly inherited cancer predisposition syndromes, to mild MDPL phenotypes without lifespan reduction, to very severe MDPL syndromes with major premature aging features. These results also suggest that POLD1 gene testing should be considered in patients presenting with severe progeroid features.

BACKGROUND: Rare diseases may elude diagnosis due to unfamiliarity of the treating physicians with the specific disorder. Yet, advances in genetics have tremendously enhanced our ability to establish specific and sometimes surprising diagnoses. CASE PRESENTATION: We report a case of renal Fanconi syndrome associated with intermittent hypoglycemic episodes, the specific cause for which remained elusive for over 30 years, despite numerous investigations, including three kidney and one liver biopsy. The most recent kidney biopsy showed dysmorphic mitochondria, suggesting a mitochondrial disorder. When her son presented with hypoglycemia in the neonatal period, he underwent routine genetic testing for hyperinsulinemic hypoglycemia, which revealed a specific mutation in HNF4A. Subsequent testing of the mother confirmed the diagnosis also in her. CONCLUSION: Modern sequencing technologies that test multiple genes simultaneously enable specific diagnoses, even if the underlying disorder was not clinically suspected. The finding of mitochondrial dysmorphology provides a potential clue for the mechanism, by which the identified mutation causes renal Fanconi syndrome.

Finding new causes of monogenic diabetes helps understand glycaemic regulation in humans. To find novel genetic causes of maturity-onset diabetes of the young (MODY), we sequenced MODY cases with unknown aetiology and compared variant frequencies to large public databases. From 36 European patients, we identify two probands with novel RFX6 heterozygous nonsense variants. RFX6 protein truncating variants are enriched in the MODY discovery cohort compared to the European control population within ExAC (odds ratio = 131, P = 1 × 10-4). We find similar results in non-Finnish European (n = 348, odds ratio = 43, P = 5 × 10-5) and Finnish (n = 80, odds ratio = 22, P = 1 × 10-6) replication cohorts. RFX6 heterozygotes have reduced penetrance of diabetes compared to common HNF1A and HNF4A-MODY mutations (27, 70 and 55% at 25 years of age, respectively). The hyperglycaemia results from beta-cell dysfunction and is associated with lower fasting and stimulated gastric inhibitory polypeptide (GIP) levels. Our study demonstrates that heterozygous RFX6 protein truncating variants are associated with MODY with reduced penetrance.Maturity-onset diabetes of the young (MODY) is the most common subtype of familial diabetes. Here, Patel et al. use targeted DNA sequencing of MODY patients and large-scale publically available data to show that RFX6 heterozygous protein truncating variants cause reduced penetrance MODY.

OBJECTIVE: Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene are associated with pituitary adenoma, acromegaly and gigantism. Identical alleles in unrelated pedigrees could be inherited from a common ancestor or result from recurrent mutation events. DESIGN AND METHODS: Observational, inferential and experimental study, including: AIP mutation testing; reconstruction of 14 AIP-region (8.3 Mbp) haplotypes; coalescent-based approximate Bayesian estimation of the time to most recent common ancestor (tMRCA) of the derived allele; forward population simulations to estimate current number of allele carriers; proposal of mutation mechanism; protein structure predictions; co-immunoprecipitation and cycloheximide chase experiments. RESULTS: Nine European-origin, unrelated c.805_825dup-positive pedigrees (four familial, five sporadic from the UK, USA and France) included 16 affected (nine gigantism/four acromegaly/two non-functioning pituitary adenoma patients and one prospectively diagnosed acromegaly patient) and nine unaffected carriers. All pedigrees shared a 2.79 Mbp haploblock around AIP with additional haploblocks privately shared between subsets of the pedigrees, indicating the existence of an evolutionarily recent common ancestor, the 'English founder', with an estimated median tMRCA of 47 generations (corresponding to 1175 years) with a confidence interval (9-113 generations, equivalent to 225-2825 years). The mutation occurred in a small tandem repeat region predisposed to slipped strand mispairing. The resulting seven amino-acid duplication disrupts interaction with HSP90 and leads to a marked reduction in protein stability. CONCLUSIONS: the c.805_825dup allele, originating from a common ancestor, associates with a severe clinical phenotype and a high frequency of gigantism. The mutation is likely to be the result of slipped strand mispairing and affects protein-protein interactions and AIP protein stability.

The aryl hydrocarbon receptor interacting protein (AIP) founder mutation R304. (or p.R304. ; NM_003977.3:c.910C>T, p.Arg304Ter) identified in Northern Ireland (NI) predisposes to acromegaly/gigantism; its population health impact remains unexplored. We measured R304. carrier frequency in 936 Mid Ulster, 1,000 Greater Belfast (both in NI) and 2,094 Republic of Ireland (ROI) volunteers and in 116 NI or ROI acromegaly/gigantism patients. Carrier frequencies were 0.0064 in Mid Ulster (95%CI = 0.0027-0.013; P = 0.0005 vs. ROI), 0.001 in Greater Belfast (0.00011-0.0047) and zero in ROI (0-0.0014). R304. prevalence was elevated in acromegaly/gigantism patients in NI (11/87, 12.6%, P < 0.05), but not in ROI (2/29, 6.8%) versus non-Irish patients (0-2.41%). Haploblock conservation supported a common ancestor for all the 18 identified Irish pedigrees (81 carriers, 30 affected). Time to most recent common ancestor (tMRCA) was 2550 (1,275-5,000) years. tMRCA-based simulations predicted 432 (90-5,175) current carriers, including 86 affected (18-1,035) for 20% penetrance. In conclusion, R304. is frequent in Mid Ulster, resulting in numerous acromegaly/gigantism cases. tMRCA is consistent with historical/folklore accounts of Irish giants. Forward simulations predict many undetected carriers; geographically targeted population screening improves asymptomatic carrier identification, complementing clinical testing of patients/relatives. We generated disease awareness locally, necessary for early diagnosis and improved outcomes of AIP-related disease.

Insulin resistance is a key mediator of obesity-related cardiometabolic disease, yet the mechanisms underlying this link remain obscure. Using an integrative genomic approach, we identify 53 genomic regions associated with insulin resistance phenotypes (higher fasting insulin levels adjusted for BMI, lower HDL cholesterol levels and higher triglyceride levels) and provide evidence that their link with higher cardiometabolic risk is underpinned by an association with lower adipose mass in peripheral compartments. Using these 53 loci, we show a polygenic contribution to familial partial lipodystrophy type 1, a severe form of insulin resistance, and highlight shared molecular mechanisms in common/mild and rare/severe insulin resistance. Population-level genetic analyses combined with experiments in cellular models implicate CCDC92, DNAH10 and L3MBTL3 as previously unrecognized molecules influencing adipocyte differentiation. Our findings support the notion that limited storage capacity of peripheral adipose tissue is an important etiological component in insulin-resistant cardiometabolic disease and highlight genes and mechanisms underpinning this link.

Neonatal diabetes, defined as the onset of diabetes within the first six months of life, is very rarely caused by pancreatic agenesis. Homozygous truncating mutations in the PTF1A gene, which encodes a transcriptional factor, have been reported in patients with pancreatic and cerebellar agenesis, whilst mutations located in a distal pancreatic-specific enhancer cause isolated pancreatic agenesis. We report an infant, born to healthy non-consanguineous parents, with neonatal diabetes due to pancreatic agenesis. Initial genetic investigation included sequencing of KCNJ11, ABCC8 and INS genes, but no mutations were found. Following this, 22 neonatal diabetes associated genes were analyzed by a next generation sequencing assay. We found compound heterozygous mutations in the PTF1A gene: a frameshift mutation in exon 1 (c.437_462 del, p.Ala146Glyfs*116) and a mutation affecting a highly conserved nucleotide within the distal pancreatic enhancer (g.23508442A>G). Both mutations were confirmed by Sanger sequencing. Isolated pancreatic agenesis resulting from compound heterozygosity for truncating and enhancer mutations in the PTF1A gene has not been previously reported. This report broadens the spectrum of mutations causing pancreatic agenesis.

Hyperinsulinemic hypoglycemia (HI) and congenital polycystic kidney disease (PKD) are rare, genetically heterogeneous disorders. The co-occurrence of these disorders (HIPKD) in 17 children from 11 unrelated families suggested an unrecognized genetic disorder. Whole-genome linkage analysis in five informative families identified a single significant locus on chromosome 16p13.2 (logarithm of odds score 6.5). Sequencing of the coding regions of all linked genes failed to identify biallelic mutations. Instead, we found in all patients a promoter mutation (c.-167G>T) in the phosphomannomutase 2 gene (PMM2), either homozygous or in trans with PMM2 coding mutations. PMM2 encodes a key enzyme in N-glycosylation. Abnormal glycosylation has been associated with PKD, and we found that deglycosylation in cultured pancreatic β cells altered insulin secretion. Recessive coding mutations in PMM2 cause congenital disorder of glycosylation type 1a (CDG1A), a devastating multisystem disorder with prominent neurologic involvement. Yet our patients did not exhibit the typical clinical or diagnostic features of CDG1A. In vitro, the PMM2 promoter mutation associated with decreased transcriptional activity in patient kidney cells and impaired binding of the transcription factor ZNF143. In silico analysis suggested an important role of ZNF143 for the formation of a chromatin loop including PMM2 We propose that the PMM2 promoter mutation alters tissue-specific chromatin loop formation, with consequent organ-specific deficiency of PMM2 leading to the restricted phenotype of HIPKD. Our findings extend the spectrum of genetic causes for both HI and PKD and provide insights into gene regulation and PMM2 pleiotropy.

Aims/hypothesis: the majority of infants with neonatal diabetes mellitus present with severe ketoacidosis at a median of 6 weeks. The treatment is very challenging and can result in severe neurological sequelae or death. The genetic defects that cause neonatal diabetes are present from birth. We aimed to assess if neonatal diabetes could be diagnosed earlier by measuring glucose in a dried blood spot collected on day 5 of life. Methods: in this retrospective case–control study we retrieved blood spot cards from 11 infants with genetically confirmed neonatal diabetes (median age of diagnosis 6 [range 2–112] days). For each case we also obtained one (n = 5) or two (n = 6) control blood spot cards collected on the same day. Glucose was measured on case and control blood spot cards. We established a normal range for random glucose at day 5 of life in 687 non-diabetic neonates. Results: all 11 neonates with diabetes had hyperglycaemia present on day 5 of life, with blood glucose levels ranging from 10.2 mmol/l to >30 mmol/l (normal range 3.2–6.0 mmol/l). In six of these neonates the diagnosis of diabetes was made after screening at day 5, with the latest diagnosis made at 16 weeks. Conclusions/interpretation: Neonatal diabetes can be detected on day 5 of life, preceding conventional diagnosis in most cases. Earlier diagnosis by systematic screening could lead to prompt genetic diagnosis and targeted treatment, thereby avoiding the most severe sequelae of hyperglycaemia in neonates.

INTRODUCTION: Major histocompatibility complex class II genes are considered major genetic risk factors for autoimmune diabetes. We analysed Human Leukocyte Antigen (HLA) DR and DQ haplotypes in a cohort with early-onset (age < 5 years), long term type 1 diabetes (T1D) and explored their influence on clinical and laboratory parameters. METHODS: Intermediate resolution HLA-DRB1, DQA1 and DQB1 typing was performed in 233 samples from the German Paediatric Diabetes Biobank and compared with a local control cohort of 19,544 cases. Clinical follow-up data of 195 patients (diabetes duration 14.2 ± 2.9 years) and residual C-peptide levels were compared between three HLA risk groups using multiple linear regression analysis. RESULTS: Genetic variability was low, 44.6% (104/233) of early-onset T1D patients carried the highest-risk genotype HLA-DRB1*03:01-DQA1*05:01-DQB1*02:01/DRB1*04-DQA1*03:01-DQB1*03:02 (HLA-DRB1*04 denoting 04:01/02/04/05), and 231 of 233 individuals carried at least one of six risk haplotypes. Comparing clinical data between the highest (n = 83), moderate (n = 106) and low risk (n = 6) genotypes, we found no difference in age at diagnosis (mean age 2.8 ± 1.1 vs. 2.8 ± 1.2 vs. 3.2 ± 1.5 years), metabolic control, or frequency of associated autoimmune diseases between HLA risk groups (each p > 0.05). Residual C-peptide was detectable in 23.5% and C-peptide levels in the highest-risk group were comparable to levels in moderate to high risk genotypes. CONCLUSION: in this study, we saw no evidence for a different clinical course of early-onset T1D based on the HLA genotype within the first ten years after manifestation.

OBJECTIVE: to determine the prevalence of monogenic diabetes in an Australian community. DESIGN: Longitudinal observational study of a cohort recruited between 2008 and 2011. SETTING: Urban population of 157 000 people (Fremantle, Western Australia). PARTICIPANTS: 1668 (of 4639 people with diabetes) who consented to participation (36.0% participation). MAIN OUTCOME MEASURES: Prevalence of maturity-onset diabetes of the young (MODY) and permanent neonatal diabetes in patients under 35 years of age, from European and non-European ethnic backgrounds, who were at risk of MODY according to United Kingdom risk prediction models, and who were then genotyped for relevant mutations. RESULTS: Twelve of 148 young participants with European ethnic backgrounds (8%) were identified by the risk prediction model as likely to have MODY; four had a glucokinase gene mutation. Thirteen of 45 with non-European ethnic backgrounds (28%) were identified as likely to have MODY, but none had a relevant mutation (DNA unavailable for one patient). Two patients with European ethnic backgrounds (one likely to have MODY) had neonatal diabetes. The estimated MODY prevalence among participants with diagnosed diabetes was 0.24% (95% confidence interval [CI], 0.08-0.66%), an overall population prevalence of 89 cases per million; the prevalence of permanent neonatal diabetes was 0.12% (95% CI, 0.02-0.48%) and the population prevalence 45 cases per million. CONCLUSIONS: One in 280 Australians diagnosed with diabetes have a monogenic form; most are of European ethnicity. Diagnosing MODY and neonatal diabetes is important because their management (including family screening) and prognosis can differ significantly from those for types 1 and 2 diabetes.

A 2.4 kg baby boy born via Caesarian section at 35 weeks had the first onset of hypoglycemia at 2 hours of life. The infant required a glucose load of 30 mg/kg/min. Insulin level was 19.6 pmol/L (normal value 17.8-173.0) in the absence of ketosis. He was resistant to oral diazoxide but responded to octreotide infusion. The boy was found to be heterozygous for an ABCC8 nonsense mutation, p.R934. We present our experience on the use of subcutaneous octreotide for 2 years for the treatment of diazoxide resistant congenital hyperinsulinism (CHI).

Congenital hyperinsulinism (CHI) is the most common cause of neonatal persistent hypoglycemia caused by mutations in nine known genes. Early diagnosis and treatment are important to prevent brain injury. The clinical presentation and response to pharmacological therapy may vary depending on the underlying pathology. Genetic analysis is important in the diagnosis, treatment, patient follow-up, and prediction of recurrence risk within families. Our patient had severe hypoglycemia and seizure following birth. His diagnostic evaluations including genetic testing confirmed CHI. He was treated with a high-glucose infusion, high-dose diazoxide, nifedipine, and glucagon infusion. A novel homozygous mutation (p.F315I) in the KCNJ11 gene, leading to diazoxide-unresponsive CHI, was identified. Both parents were heterozygous for this mutation. Our patient’s clinical course was complicated by severe refractory hypoglycemia; he was successfully managed with sirolimus and surgical intervention was not required. Diazoxide, nifedipine, and glucagon were discontinued gradually following sirolimus therapy. The patient was discharged at 2 months of age on low-dose octreotide and sirolimus. His outpatient clinical follow-up continues with no episodes of hypoglycemia. We present a novel homozygous p.F315I mutation in the KCNJ11 gene leading to diazoxide-unresponsive CHI in a neonate. This case illustrates the challenges associated with the diagnosis and management of CHI, as well as the successful therapy with sirolimus.

Although aryl hydrocarbon receptor-interacting protein (AIP) mutations are rare in sporadic acromegaly, their prevalence among young patients is nonnegligible. The objectives of this study were to evaluate the frequency of AIP mutations in a cohort of Mexican patients with acromegaly with disease onset before the age of 30 and to search for molecular abnormalities in the AIP gene in teeth obtained from the "Tampico Giant". Peripheral blood DNA from 71 patients with acromegaly (51 females) with disease onset T (p.Arg304Ter), well-known truncating mutation was identified; in one of these two cases and her identical twin sister, the mutation proved to be a de novo event, since neither of their parents were found to be carriers. In the remaining three patients, new mutations were identified: a frameshift mutation (c.976_977insC, p.Gly326AfsTer), an in-frame deletion (c.872_877del, p.Val291_Leu292del) and a nonsense mutation (c.868A > T, p.Lys290Ter), which are predicted to be pathogenic based on in silico analysis. Patients with AIP mutations tended to have an earlier onset of acromegaly and harboured larger and more invasive tumours. A previously described genetic variant of unknown significance (c.869C > T, p.Ala299Val) was identified in DNA from the Tampico Giant. The prevalence of AIP mutations in young Mexican patients with acromegaly is similar to that of European cohorts. Our results support the need for genetic evaluation of patients with early onset acromegaly.

Maturity onset diabetes of the young (MODY) is a monogenic form of diabetes caused by a mutation in a single gene, often not requiring insulin. The aim of this study was to estimate the frequency and clinical characteristics of MODY at the Barbara Davis Center. A total of 97 subjects with diabetes onset before age 25, a random C-peptide ≥0.1 ng/mL, and negative for all diabetes autoantibodies (GADA, IA-2, ZnT8, and IAA) were enrolled, after excluding 21 subjects with secondary diabetes or refusal to participate. Genetic testing for MODY 1-5 was performed through Athena Diagnostics, and all variants of unknown significance were further analyzed at Exeter, UK. A total of 22 subjects [20 (21%) when excluding two siblings] were found to have a mutation in hepatocyte nuclear factor 4A (n = 4), glucokinase (n = 8), or hepatocyte nuclear factor 1A (n = 10). of these 22 subjects, 13 had mutations known to be pathogenic and 9 (41%) had novel mutations, predicted to be pathogenic. Only 1 of the 22 subjects had been given the appropriate MODY diagnosis prior to testing. Compared with MODY-negative subjects, the MODY-positive subjects had lower hemoglobin A1c level and no diabetic ketoacidosis at onset; however, these characteristics are not specific for MODY. In summary, this study found a high frequency of MODY mutations with the majority of subjects clinically misdiagnosed. Clinicians should have a high index of suspicion for MODY in youth with antibody-negative diabetes.

Heterozygous mutations of the HNF1B gene are the commonest known monogenic cause of developmental kidney disease. Half of patients have a deletion (approximately 1.3 Mb) of chromosome 17q12, encompassing HNF1B plus 14 additional genes. This 17q12 deletion has been linked with an increased risk of neurodevelopmental disorders, such as autism. Here we compared the neurodevelopmental phenotype of 38 patients with HNF1B-associated renal disease due to an intragenic mutation in 18 patients or due to 17q12 deletion in 20 patients to determine whether haploinsufficiency of HNF1B is responsible for the neurodevelopmental phenotype. Significantly, brief behavioral screening in children with the deletion showed high levels of psychopathology and its impact. Eight individuals (40%) with a deletion had a clinical diagnosis of a neurodevelopmental disorder compared to none with an intragenic mutation. The 17q12 deletions were also associated with more autistic traits. Two independent clinical geneticists were able to predict the presence of a deletion with a sensitivity of 83% and specificity of 79% when assessing facial dysmorphic features as a whole. Thus, the 17q12 deletions but not HNF1B intragenic mutations are associated with neurodevelopmental disorders. Hence, the HNF1B gene is not involved in the neurodevelopmental phenotype of these patients. Nephrologists need to be aware of this association to ensure appropriate referral to psychiatric services.

Objective: Mutations in the KATP channel genes is the most common cause of congenital hyperinsulinism (CHI) of infancy. Our aim was to report the clinical and genetic characteristics, treatment modalities, and long-term prognosis of patients with CHI. Methods: Clinical and biochemical findings, operation procedures, and results of genetic analysis were retrospectively evaluated in 22 CHI patients from two pediatric endocrine centers in Turkey. Results: Seven of the patients were born large for gestational age. Hypoglycemia was diagnosed within the first 24 hours of life in 9 patients and treatment with diazoxide (n=21) and/or somatostatin (n=8) had been attempted. Seven patients (31.8%) were unresponsive to medical treatment and underwent pancreatectomy. Histological examination of the pancreas confirmed diffuse disease in 6 patients. Diabetes developed in 3 patients following pancreatectomy (10 years, 2.5 years, and immediately after operation). The remaining four patients had neither recurrence of CHI nor of diabetes during the 3.67±0.7 years of follow-up. Sequence analysis identified mutations in 12 out of 19 patients (63%). Mutations in the ABCC8 gene were the most common finding and were found in 6 out of 7 patients who underwent pancreatectomy. Other mutations included a paternally inherited KCNJ11 mutation, a homozygous HADH mutation, and a heterozygous GLUD1 mutation. Conclusion: Mutations in the ABCC8 gene were the most common cause of CHI in our cohort. These mutations were identified in 85% of patients who underwent pancreatectomy. The development of diabetes mellitus after pancreatectomy may occur at any age and these patients should be screened regularly.

Aims: Neonatal diabetes mellitus (NDM) is a rare monogenic disorder, reported to affect less than 2 cases per 100,000 infants. There are two types, permanent (PNDM) and transient (TNDM). We describe our clinical experience in determining and comparing the genetic basis of diabetes in children with onset before 6 months versus those diagnosed between 6 and 12 months of age. Methods: We reviewed medical records of children with diabetes diagnosed before 12 months of age. Genetic testing was performed in all cases. Results: 12 patients were diagnosed with diabetes before 6 months of age (PNDM = 6; TNDM = 6), and 11 patients between 6 and 12 months (all with permanent diabetes). Among children with PNDM, we identified three different KCNJ11 mutations in 5 patients, and one novel ABCC8 mutation in a single patient. Among children with TNDM, we detected a KCNJ11 and ABCC8 mutation each in a single patient and methylation abnormalities at chromosome 6q24 in 4 patients.Among children with diabetes diagnosed between 6 and 12 months, 1 patient had an INS mutation and one patient was homozygous for an SLC19A2 mutation which confirmed a diagnosis of thiamine-responsive megaloblastic anaemia syndrome. Five of the patients with an ABCC8 or KCNJ11 mutation have successfully transferred from insulin to glibenclamide whist 1 child demonstrated a partial response to sulfonylurea treatment. Conclusions: Investigating the underlying genetic basis of diabetes in children with onset before 1 year is useful for choosing the most efficient treatment, the basis of Personalized Medicine.

Background: Isolated hyperinsulinaemic hypoglycaemia (HH) commonly results from recessively inherited mutations in the ABCC8 and KCNJ11 genes that are located on chromosome 11p15.1. More rarely, HH can feature in patients with Beckwith-Wiedemann syndrome (BWS), a congenital overgrowth disorder, resulting from defects at a differentially methylated region telomeric to the K-ATP channel genes at chromosome 11p15.5. Subject: We undertook genetic testing in a patient with diazoxide-unresponsive HH diagnosed at birth. Physical examination later revealed hemihypertrophy of the right arm, a feature of BWS. Results: We identified a novel mosaic, paternally-inherited KCNJ11 mutation(s) in the patient. Further analysis confirmed uniparental disomy (UPD) of chromosome 11, which extended across the KCNJ11 gene at 11p15.1 and the BWS locus at 11p15.5. Conclusion: These results highlight the importance of considering UPD as a mechanism of disease in patients with HH and a paternally inherited K-ATP channel mutation, especially when additional syndromic features are present.

BACKGROUND: Patients with Congenital Hyperinsulinism (CHI) due to mutations in K-ATP channel genes (K-ATP CHI) are increasingly treated by conservative medical therapy without pancreatic surgery. However, the natural history of medically treated K-ATP CHI has not been described; it is unclear if the severity of recessively and dominantly inherited K-ATP CHI reduces over time. We aimed to review variation in severity and outcomes in patients with K-ATP CHI treated by medical therapy. METHODS: Twenty-one consecutively presenting patients with K-ATP CHI with dominantly and recessively inherited mutations in ABCC8/KCNJ11 were selected in a specialised CHI treatment centre to review treatment outcomes. Medical treatment included diazoxide and somatostatin receptor agonists (SSRA), octreotide and somatuline autogel. CHI severity was assessed by glucose infusion rate (GIR), medication dosage and tendency to resolution. CHI outcome was assessed by glycaemic profile, fasting tolerance and neurodevelopment. RESULTS: CHI presenting at median (range) age 1 (1, 240) days resolved in 15 (71%) patients at age 3.1(0.2, 13.0) years. Resolution was achieved both in patients responsive to diazoxide (n = 8, 57%) and patients responsive to SSRA (n = 7, 100%) with earlier resolution in the former [1.6 (0.2, 13.0) v 5.9 (1.6, 9.0) years, p = 0.08]. In 6 patients remaining on treatment, diazoxide dose was reduced in follow up [10.0 (8.5, 15.0) to 5.4 (0.5, 10.8) mg/kg/day, p = 0.003]. GIR at presentation did not correlate with resolved or persistent CHI [14.9 (10.0, 18.5) v 16.5 (13.0, 20.0) mg/kg/min, p = 0.6]. The type of gene mutation did not predict persistence; resolution could be achieved in recessively-inherited CHI with homozygous (n = 3), compound heterozygous (n = 2) and paternal mutations causing focal CHI (n = 2). Mild developmental delay was present in 8 (38%) patients; adaptive functioning assessed by Vineland Adaptive Behavior Scales questionnaire showed a trend towards higher standard deviation scores (SDS) in resolved than persistent CHI [-0.1 (-1.2, 1.6) v -1.2 (-1.7, 0.03), p = 0.1]. CONCLUSIONS: in K-ATP CHI patients managed by medical treatment only, severity is reduced over time in the majority, including those with compound heterozygous and homozygous mutations in ABCC8/KCNJ11. Severity and treatment requirement should be assessed periodically in all children with K-ATP CHI on medical therapy.

Aims/Introduction: Monogenic diabetes accounts for approximately 1-2% of all diabetes, and is difficult to distinguish from type 1 and type 2 diabetes. Molecular diagnosis is important, as the molecular subtype directs appropriate treatment. Patients are selected for testing according to clinical criteria, but up to 80% of monogenic diabetes in the UK has not been correctly diagnosed. We investigated outcomes of genetic testing in our center to compare methods of selecting patients, and consider avenues to increase diagnostic efficiency. Materials and Methods: We reviewed 36 probands tested for monogenic diabetes in the last 10 years in a large adult diabetes outpatient clinic, serving an ethnically diverse urban population. We compared published clinical criteria and an online maturity onset diabetes of the young calculator applied to these 36 patients, and presented the predictions together with the molecular results. Results: the overall mutation detection rate was 42%, reflecting the strict clinical selection process applied before genetic testing. Both methods had high sensitivity for identifying patients with mutations: 88 and 89% for the clinical criteria and online calculator, respectively. Cascade testing in a total of 16 relatives led to diagnosis of a further 13 cases. Conclusions: Existing patient selection criteria were effective in identifying patients with monogenic forms of diabetes, but the number of patients missed using these strict criteria is unknown. Because of the potential savings resulting from correct molecular diagnosis, it is possible that testing a larger pool of patients using less stringent selection criteria would be cost-effective. Further evidence is required to inform this assessment.

AIMS/HYPOTHESIS: We aimed to identify microRNAs (miRNAs) under transcriptional control of the HNF1β transcription factor, and investigate whether its effect manifests in serum. METHODS: the Polish cohort (N = 60) consisted of 11 patients with HNF1B-MODY, 17 with HNF1A-MODY, 13 with GCK-MODY, an HbA1c-matched type 1 diabetic group (n = 9) and ten healthy controls. Replication was performed in 61 clinically-matched British patients mirroring the groups in the Polish cohort. The Polish cohort underwent miRNA serum level profiling with quantitative real-time PCR (qPCR) arrays to identify differentially expressed miRNAs. Validation was performed using qPCR. To determine whether serum content reflects alterations at a cellular level, we quantified miRNA levels in a human hepatocyte cell line (HepG2) with small interfering RNA knockdowns of HNF1α or HNF1β. RESULTS: Significant differences (adjusted p 

OBJECTIVE: Inactivating heterozygous mutations in the GCK gene are a common cause of MODY and result in mild fasting hyperglycaemia, which does not require treatment. We aimed to identify the frequency, clinical and molecular features of GCK mutations in a Turkish paediatric cohort. DESIGN AND PATIENTS: Fifty-four unrelated probands were selected based on the following criteria: age of diagnosis ≤17 years, family history of diabetes in at least two generations, anti-GAD/ICA negative, BMIT) were identified in 13/54 probands (24%). Twelve of these patients had a MODY probability score ≥75%. FBG level and 2-h glucose level in OGTT were 123 ± 14 mg/dl (6·8 ± 0·7 mmol/l) (107-157 mg/dl) and 181 ± 30 mg/dl (10·1 ± 1·6 mmol/l) (136-247 mg/dl), respectively. Average of glucose increment in OGTT was 58 ± 27 mg/dl (3·2 ± 1·5 mmol/l) (19-120 mg/dl), and mean HbA1c level was 6·5 ± 0·5% (47·5 ± 5·5 mmol/mol) (5·9-7·6%). Five novel missense mutations were identified (p.F123S, p.L58P, p.G246A, p.F419C, and p.S151C). Two patients treated with low-dose insulin before the molecular analysis were able to stop treatment. CONCLUSIONS: Approximately 1 in 4 MODY cases in this Turkish paediatric cohort have a GCK mutation. Selection of patients for GCK gene analysis using the MODY probability score was an effective way of identifying most (11/12) patients with a GCK mutation.

Neonatal diabetes mellitus (NDM) can be transient (TNDM) or permanent (PNDM). Data on NDM from the Gulf region are limited to few studies on PNDM.The objective of this study was to describe the genetic and clinical spectrum of NDM and estimate its incidence in AbuDhabi, capital of the United Arab Emirate (UAE). Patients were identified from the pediatric diabetes clinics and sequencing of known NDM genes was conducted in all families. Twenty-five patients were identified. Incidence during 1985-2013 was 1:29,241 Live births. Twenty-three out of twenty-five had PNDM (incidence 1:31,900) and 2/25 had TNDM (incidence 1:350,903). Eleven out of twenty-five had extra-pancreatic features and three had pancreatic aplasia. The genetic cause was detected in 21/25 (84%). of the PNDM patients, nine had recessive EIF2AK3 mutations, six had homozygous INS mutations, two with deletion of the PTF1A enhancer, one was heterozygous for KCNJ11 mutation, one harboured a novel ABCC8 variant, and 4/21 without mutations in all known PNDM genes. One TNDM patient had a 6q24 methylation defect and another was homozygous for the INS c-331C>G mutation. This mutation also caused permanent diabetes with variable age of onset from birth to 18 years. The parents of a child with Wolcott-Rallison syndrome had a healthy girl following pre-implantation genetic diagnosis. The child with KCNJ11 mutation was successfully switched from insulin to oral sulphonylurea. The incidence of PNDM in Abu Dhabi is among the highest in the world and its spectrum is different from Europe and USA. In our cohort, genetic testing has significant implications for the clinical management.

Non-syndromic pituitary gigantism can result from AIP mutations or the recently identified Xq26.3 microduplication causing X-linked acrogigantism (XLAG). Within Xq26.3, GPR101 is believed to be the causative gene, and the c.924G > C (p.E308D) variant in this orphan G protein-coupled receptor has been suggested to play a role in the pathogenesis of acromegaly.We studied 153 patients (58 females and 95 males) with pituitary gigantism. AIP mutation-negative cases were screened for GPR101 duplication through copy number variation droplet digital PCR and high-density aCGH. The genetic, clinical and histopathological features of XLAG patients were studied in detail. 395 peripheral blood and 193 pituitary tumor DNA samples from acromegaly patients were tested for GPR101 variants.We identified 12 patients (10 females and 2 males; 7.8 %) with XLAG. In one subject, the duplicated region only contained GPR101, but not the other three genes in found to be duplicated in the previously reported patients, defining a new smallest region of overlap of duplications. While females presented with germline mutations, the two male patients harbored the mutation in a mosaic state. Nine patients had pituitary adenomas, while three had hyperplasia. The comparison of the features of XLAG, AIP-positive and GPR101&AIP-negative patients revealed significant differences in sex distribution, age at onset, height, prolactin co-secretion and histological features. The pathological features of XLAG-related adenomas were remarkably similar. These tumors had a sinusoidal and lobular architecture. Sparsely and densely granulated somatotrophs were admixed with lactotrophs; follicle-like structures and calcifications were commonly observed. Patients with sporadic of familial acromegaly did not have an increased prevalence of the c.924G > C (p.E308D) GPR101 variant compared to public databases.In conclusion, XLAG can result from germline or somatic duplication of GPR101. Duplication of GPR101 alone is sufficient for the development of XLAG, implicating it as the causative gene within the Xq26.3 region. The pathological features of XLAG-associated pituitary adenomas are typical and, together with the clinical phenotype, should prompt genetic testing.

Background: Hyperinsulinemia is the commonest cause of persistent hypoglycemia in infancy. Inactivating mutations in the genes ABCC8 and KCNJ11 are the commonest cause. Mutation in the HADH gene, which encodes the short-chain-L-3-hydroxyacyl-CoA dehydrogenase, is a rare cause. Case characteristics: Two Indian sisters who presented with hyperinsulinemic hypoglycemia of infancy. Observation/Intervention: a novel homozygous missense mutation in the HADH gene was identified in both the sisters, while the parents were found to be heterozygous carriers. Outcome: Establishment of molecular diagnosis, optimization of therapy and counseling of parents regarding risk of recurrence in future pregnancy. Messages: HADH mutations are rare causes of hypoglycemia and can be mitigated with diazoxide and appropriate dietary therapy if identified early.

Homozygous truncating mutations in the helix-loop-helix transcription factor PTF1A are a rare cause of pancreatic and cerebellar agenesis. The correlation of Ptf1a dosage with pancreatic phenotype in a mouse model suggested the possibility of finding hypomorphic PTF1A mutations in patients with pancreatic agenesis or neonatal diabetes but no cerebellar phenotype. Genome-wide single nucleotide polymorphism typing in two siblings with neonatal diabetes from a consanguineous pedigree revealed a large shared homozygous region (31 Mb) spanning PTF1A Sanger sequencing of PTF1A identified a novel missense mutation, p.P191T. Testing of 259 additional patients using a targeted next-generation sequencing assay for 23 neonatal diabetes genes detected one additional proband and an affected sibling with the same homozygous mutation. All four patients were diagnosed with diabetes at birth and were treated with insulin. Two of the four patients had exocrine pancreatic insufficiency requiring replacement therapy but none of the affected individuals had neurodevelopmental delay. Transient transfection assays of the mutant protein demonstrated a 75% reduction in transactivation activity. This study shows that the functional severity of a homozygous mutation impacts the severity of clinical features found in patients.

AIMS/HYPOTHESIS: the pancreatic ATP-sensitive potassium (KATP) channel plays a pivotal role in linking beta cell metabolism to insulin secretion. Mutations in KATP channel genes can result in hypo- or hypersecretion of insulin, as in neonatal diabetes mellitus and congenital hyperinsulinism, respectively. To date, all patients affected by neonatal diabetes due to a mutation in the pore-forming subunit of the channel (Kir6.2, KCNJ11) are heterozygous for the mutation. Here, we report the first clinical case of neonatal diabetes caused by a homozygous KCNJ11 mutation. METHODS: a male patient was diagnosed with diabetes shortly after birth. At 5 months of age, genetic testing revealed he carried a homozygous KCNJ11 mutation, G324R, (Kir6.2-G324R) and he was successfully transferred to sulfonylurea therapy (0.2 mg kg(-1) day(-1)). Neither heterozygous parent was affected. Functional properties of wild-type, heterozygous and homozygous mutant KATP channels were examined after heterologous expression in Xenopus oocytes. RESULTS: Functional studies indicated that the Kir6.2-G324R mutation reduces the channel ATP sensitivity but that the difference in ATP inhibition between homozygous and heterozygous channels is remarkably small. Nevertheless, the homozygous patient developed neonatal diabetes, whereas the heterozygous parents were, and remain, unaffected. Kir6.2-G324R channels were fully shut by the sulfonylurea tolbutamide, which explains why the patient's diabetes was well controlled by sulfonylurea therapy. CONCLUSIONS/INTERPRETATION: the data demonstrate that tiny changes in KATP channel activity can alter beta cell electrical activity and insulin secretion sufficiently to cause diabetes. They also aid our understanding of how the Kir6.2-E23K variant predisposes to type 2 diabetes.

The long-read sequencers from Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT) offer the opportunity to phase mutations multiple kilobases apart directly from sequencing reads. In this study, we used long-range PCR with ONT and PacBio sequencing to phase two variants 9 kb apart in the RET gene. We also re-analysed data from a recent paper which had apparently successfully used ONT to phase clinically important haplotypes at the CYP2D6 and HLA loci. From these analyses, we demonstrate PCR-chimera formation during PCR amplification and reference alignment bias are pitfalls that need to be considered when attempting to phase variants using amplicon-based long-read sequencing technologies. These methodological pitfalls need to be avoided if the opportunities provided by long-read sequencers are to be fully exploited.

BACKGROUND: Hyperglycemia in premature infants is usually thought to reflect inadequate pancreatic development rather than monogenic neonatal diabetes. No studies, to our knowledge, have investigated the prevalence of monogenic forms of diabetes in preterm infants. METHODS: We studied 750 patients with diabetes diagnosed before 6 months of age. We compared the genetic etiology and clinical characteristics of 146 preterm patients born 32 weeks). Prematurity should not prevent referral for genetic testing as 37% have a potassium channel mutation and as a result can get improved control by replacing insulin with sulphonylurea therapy.

Clinical exome sequencing routinely identifies missense variants in disease-related genes, but functional characterization is rarely undertaken, leading to diagnostic uncertainty. For example, mutations in PPARG cause Mendelian lipodystrophy and increase risk of type 2 diabetes (T2D). Although approximately 1 in 500 people harbor missense variants in PPARG, most are of unknown consequence. To prospectively characterize PPARγ variants, we used highly parallel oligonucleotide synthesis to construct a library encoding all 9,595 possible single-amino acid substitutions. We developed a pooled functional assay in human macrophages, experimentally evaluated all protein variants, and used the experimental data to train a variant classifier by supervised machine learning. When applied to 55 new missense variants identified in population-based and clinical sequencing, the classifier annotated 6 variants as pathogenic; these were subsequently validated by single-variant assays. Saturation mutagenesis and prospective experimental characterization can support immediate diagnostic interpretation of newly discovered missense variants in disease-related genes.

BACKGROUND: Mucosal neuromas, thickened corneal nerves and marfanoid body habitus are characteristic phenotypic features of multiple endocrine neoplasia type 2B (MEN2B) and often provide an early clue to the diagnosis of the syndrome. Rarely, patients present with typical physical features of MEN2B but without associated endocrinopathies (medullary thyroid carcinoma or pheochromocytoma) or a RET gene mutation; this clinical presentation is thought to represent a distinct condition termed 'pure mucosal neuroma syndrome'. METHODS: Exome sequencing was performed in two unrelated probands with mucosal neuromas, thickened corneal nerves and marfanoid body habitus, but no MEN2B-associated endocrinopathy or RET gene mutation. Sanger sequencing was performed to confirm mutations detected by exome sequencing and to test in family members and 3 additional unrelated index patients with mucosal neuromas or thickened corneal nerves. RESULTS: a heterozygous SOS1 gene frameshift mutation (c.3266dup or c.3248dup) was identified in each proband. Sanger sequencing showed that proband 1 inherited the c.3266dup mutation from his affected mother, while the c.3248dup mutation had arisen de novo in proband 2. Sanger sequencing also identified one further novel SOS1 mutation (c.3254dup) in one of the 3 additional index patients. CONCLUSION: Our results demonstrate the existence of pure mucosal neuroma syndrome as a clinical entity distinct from MEN2B that can now be diagnosed by genetic testing.

CONTEXT: Recent reports have proposed that sporadic or familial germline Xq26.3 microduplications involving the GPR101 gene are associated with early-onset X-linked acrogigantism (XLAG) with a female preponderance. CASE DESCRIPTION: a 4-year-old boy presented with rapid growth over the previous 2 years. He complained of sporadic headaches and had coarse facial features. His height Z-score was +4.89, and weight Z-score was +5.57. Laboratory testing revealed elevated serum prolactin (185 μg/L; normal. 35.0 μg/L. Magnetic resonance imaging demonstrated a homogenous bulky pituitary gland (18 × 15 × 13 mm) without obvious adenoma. A pituitary biopsy showed hyperplastic pituitary tissue with enlarged cords of GH and prolactin cells. Germline PRKAR1A, MEN1, AIP, DICER1, CDKN1B, and somatic GNAS mutations were negative. Medical management was challenging until institution of continuous sc infusion of short-acting octreotide combined with sc pegvisomant and oral cabergoline. The patient remains well controlled with minimal side effects 7 years after presentation. His phenotype suggested XLAG, but his peripheral leukocyte-, saliva-, and buccal cell-derived DNA tested negative for microduplication in Xq26.3 or GPR101. However, DNA isolated from the pituitary tissue and forearm skin showed duplicated dosage of GPR101, suggesting that he is mosaic for this genetic abnormality. CONCLUSIONS: Our patient is the first to be described with somatic microduplication leading to typical XLAG phenotype. This patient demonstrates that a negative test for Xq26.3 microduplication or GPR101 duplication on peripheral blood DNA does not exclude the diagnosis of XLAG because it can result from a mosaic mutation affecting the pituitary.

AIMS: HNF1A gene mutations are the most common cause of maturity-onset diabetes of the young (MODY) in the UK. Persons with HNF1A-MODY display sensitivity to sulphonylurea therapy; however, the long-term efficacy is not established. There is limited literature as to the prevalence of micro- and macrovascular complications in this unique cohort. The aim of this study was to determine the natural progression and clinical management of HNF1A-MODY diabetes in a dedicated MODY clinic. METHODS: Sixty patients with HNF1A-MODY and a cohort of 60 BMI-, age-, ethnicity- and diabetes duration-matched patients with Type 1 diabetes mellitus participated in the study. All patients were phenotyped in detail. Clinical follow-up of the HNF1A-MODY cohort occurred on a bi-annual basis. RESULTS: Following a genetic diagnosis of MODY, the majority of the cohort treated with sulphonylurea therapy remained insulin independent at 84-month follow-up (80%). The HbA1c in the HNF1A-MODY group treated with sulphonylurea therapy alone improved significantly over the study period [from 49 (44-63) mmol/mol, 6.6 (6.2-7.9)% to 41 (31-50) mmol/mol, 5.9 (5-6.7)%; P = 0.003]. The rate of retinopathy was significantly lower than that noted in the Type 1 diabetes mellitus group (13.6 vs. 50%; P = 0.0001).There was also a lower rate of microalbuminuria and cardiovascular disease in the HNF1A-MODY group compared with the Type 1 diabetes mellitus group. CONCLUSIONS: This study demonstrates that the majority of patients with HNF1A-MODY can be maintained successfully on sulphonylurea therapy with good glycaemic control. We note a significantly lower rate of micro- and macrovascular complications than reported previously. The use of appropriate therapy at early stages of the disorder may decrease the incidence of complications.

AIMS/HYPOTHESIS: the finding that patients with diabetes due to potassium channel mutations can transfer from insulin to sulfonylureas has revolutionised the management of patients with permanent neonatal diabetes. The extent to which the in vitro characteristics of the mutation can predict a successful transfer is not known. Our aim was to identify factors associated with successful transfer from insulin to sulfonylureas in patients with permanent neonatal diabetes due to mutations in KCNJ11 (which encodes the inwardly rectifying potassium channel Kir6.2). METHODS: We retrospectively analysed clinical data on 127 patients with neonatal diabetes due to KCNJ11 mutations who attempted to transfer to sulfonylureas. We considered transfer successful when patients completely discontinued insulin whilst on sulfonylureas. All unsuccessful transfers received ≥0.8 mg kg(-1) day(-1) glibenclamide (or the equivalent) for >4 weeks. The in vitro response of mutant Kir6.2/SUR1 channels to tolbutamide was assessed in Xenopus oocytes. For some specific mutations, not all individuals carrying the mutation were able to transfer successfully; we therefore investigated which clinical features could predict a successful transfer. RESULTS: in all, 112 out of 127 (88%) patients successfully transferred to sulfonylureas from insulin with an improvement in HbA1c from 8.2% (66 mmol/mol) on insulin, to 5.9% (41 mmol/mol) on sulphonylureas (p = 0.001). The in vitro response of the mutation to tolbutamide determined the likelihood of transfer: the extent of tolbutamide block was 73% did transfer successfully. The few patients with these mutations who could not transfer had a longer duration of diabetes than those who transferred successfully (18.2 vs 3.4 years, p = 0.032). There was no difference in pre-transfer HbA1c (p = 0.87), weight-for-age z scores (SD score; p = 0.12) or sex (p = 0.17). CONCLUSIONS/INTERPRETATION: Transfer from insulin is successful for most KCNJ11 patients and is best predicted by the in vitro response of the specific mutation and the duration of diabetes. Knowledge of the specific mutation and of diabetes duration can help predict whether successful transfer to sulfonylureas is likely. This result supports the early genetic testing and early treatment of patients with neonatal diabetes aged under 6 months.

OBJECTIVE: Monogenic diabetes is rare but is an important diagnosis in pediatric diabetes clinics. These patients are often not identified as this relies on the recognition of key clinical features by an alert clinician. Biomarkers (islet autoantibodies and C-peptide) can assist in the exclusion of patients with type 1 diabetes and allow systematic testing that does not rely on clinical recognition. Our study aimed to establish the prevalence of monogenic diabetes in U.K. pediatric clinics using a systematic approach of biomarker screening and targeted genetic testing. RESEARCH DESIGN AND METHODS: We studied 808 patients (79.5% of the eligible population)

HNF4A mutations cause increased birth weight, transient neonatal hypoglycemia, and maturity onset diabetes of the young (MODY). The most frequently reported HNF4A mutation is p.R114W (previously p.R127W), but functional studies have shown inconsistent results; there is a lack of cosegregation in some pedigrees and an unexpectedly high frequency in public variant databases. We confirm that p.R114W is a pathogenic mutation with an odds ratio of 30.4 (95% CI 9.79-125, P = 2 × 10(-21)) for diabetes in our MODY cohort compared with control subjects. p.R114W heterozygotes did not have the increased birth weight of patients with other HNF4A mutations (3,476 g vs. 4,147 g, P = 0.0004), and fewer patients responded to sulfonylurea treatment (48% vs. 73%, P = 0.038). p.R114W has reduced penetrance; only 54% of heterozygotes developed diabetes by age 30 years compared with 71% for other HNF4A mutations. We redefine p.R114W as a pathogenic mutation that causes a distinct clinical subtype of HNF4A MODY with reduced penetrance, reduced sensitivity to sulfonylurea treatment, and no effect on birth weight. This has implications for diabetes treatment, management of pregnancy, and predictive testing of at-risk relatives. The increasing availability of large-scale sequence data is likely to reveal similar examples of rare, low-penetrance MODY mutations.

BACKGROUND: a syndrome of young-onset diabetes mellitus associated with microcephaly, epilepsy and intellectual disability caused by mutations in the tRNA methyltransferase 10 homologue a (TRMT10A) gene has recently been described. CASE REPORT: We report two siblings from the fourth family reported to have diabetes mellitus as a result of a TRMT10A mutation. A homozygous nonsense mutation p.Glu27Ter in TRMT10A was identified using targeted next-generation sequencing and confirmed by PCR/Sanger sequencing. Diabetes was diagnosed while the subjects were in their 20s and was characterized by insulin resistance. Epilepsy and intellectual disability were features in common. Mild microcephaly was present at birth but their final head circumferences were normal. CONCLUSION: Our report provides independent confirmation of the role of TRMT10A mutations in this syndrome and expands its phenotypic description. TRMT10A sequencing should be considered in children or adults with young-onset diabetes who have a history of intellectual disability, microcephaly and epilepsy. This report also shows the advantages of using a targeted panel to identify previously unsuspected monogenic diabetes among young-onset non-insulin-dependent diabetes in the absence of obesity and autoimmunity.

Unlike other congenital fatty acid oxidation defects, short-chain L-3-hydroxyacyl-CoA (SCHAD, HADH) deficiency is characterised by hypoglycemia with hyperinsulinism in the neonatal or infancy periods. The long-term and detailed clinical progression of the disease is largely unknown with almost 40 patients reported and only a few patients described clinically. We present clinical and laboratory findings together with the long-term clinical course of a case with a deep intronic HADH splicing mutation (c.636+471G>T) causing neonatal-onset hyperinsulinemic hypoglycemia with mild progression.

Background: Inheritance of two pathogenic ABCC8 alleles typically causes severe congenital hyperinsulinism. We describe a girl and her father, both homozygous for the same ABCC8 mutation, who presented with unusual phenotypes. Methods: Single nucleotide polymorphism microarray and Sanger sequencing were performed. Western blot, rubidium efflux, and patch clamp recordings interrogated the expression and activity of the mutant protein. Results: a 16-month-old girl of consanguineous descent manifested hypoglycemia. She had dysregulation of insulin secretion, with postprandial hyperglycemia followed by hypoglycemia. Microarray revealed homozygosity for the regions encompassing KCNJ11 and ABCC8. Her father had developed diabetes at 28 years of age. Sequencing of ABCC8 identified a homozygous missense mutation, p.R1419H, in both individuals. Functional studies showed absence of working KATP channels. Conclusion: This is the first description of a homozygous p.R1419H mutation. Our findings highlight that homozygous loss-of-function mutations of ABCC8 do not necessarily translate into early-onset severe hyperinsulinemia.

Rare disorders resulting in prenatal or neonatal death are genetically heterogeneous. For some conditions, affected fetuses can be diagnosed by ultrasound scan, but this is not usually possible until mid-gestation. There is often limited fetal DNA available for investigation. We investigated a strategy for diagnosing autosomal recessive lethal disorders in non-consanguineous pedigrees with multiple affected fetuses. Exome sequencing was performed to identify genes where each parent is heterozygous for a rare non-synonymous-coding or splicing variant. Putative pathogenic variants were tested for cosegregation in affected fetuses and unaffected siblings. In eight couples of European ancestry, we found on average 1.75 genes (range 0-4) where both parents were heterozygous for rare potentially deleterious variants. A proof-of-principle study detected heterozygous DYNC2H1 variants in a couple whose five fetuses had short-rib polydactyly. Prospective analysis of two couples with multiple pregnancy terminations for fetal akinesia syndrome was performed and a diagnosis was obtained in both the families. The first couple were each heterozygous for a previously reported GLE1 variant, p.Arg569His or p.Val617Met; both were inherited by their two affected fetuses. The second couple were each heterozygous for a novel RYR1 variant, c.14130-2A>G or p.Ser3074Phe; both were inherited by their three affected fetuses but not by their unaffected child. Biallelic GLE1 and RYR1 disease-causing variants have been described in other cases with fetal akinesia syndrome. We conclude that exome sequencing of parental samples can be an effective tool for diagnosing lethal recessive disorders in outbred couples. This permits early prenatal diagnosis in future pregnancies.

Rare disorders resulting in prenatal or neonatal death are genetically heterogeneous. For some conditions, affected fetuses can be diagnosed by ultrasound scan, but this is not usually possible until mid-gestation. There is often limited fetal DNA available for investigation. We investigated a strategy for diagnosing autosomal recessive lethal disorders in non-consanguineous pedigrees with multiple affected fetuses. Exome sequencing was performed to identify genes where each parent is heterozygous for a rare non-synonymous-coding or splicing variant. Putative pathogenic variants were tested for cosegregation in affected fetuses and unaffected siblings. In eight couples of European ancestry, we found on average 1.75 genes (range 0-4) where both parents were heterozygous for rare potentially deleterious variants. A proof-of-principle study detected heterozygous DYNC2H1 variants in a couple whose five fetuses had short-rib polydactyly. Prospective analysis of two couples with multiple pregnancy terminations for fetal akinesia syndrome was performed and a diagnosis was obtained in both the families. The first couple were each heterozygous for a previously reported GLE1 variant, p.Arg569His or p.Val617Met; both were inherited by their two affected fetuses. The second couple were each heterozygous for a novel RYR1 variant, c.14130-2A>G or p.Ser3074Phe; both were inherited by their three affected fetuses but not by their unaffected child. Biallelic GLE1 and RYR1 disease-causing variants have been described in other cases with fetal akinesia syndrome. We conclude that exome sequencing of parental samples can be an effective tool for diagnosing lethal recessive disorders in outbred couples. This permits early prenatal diagnosis in future pregnancies.

BACKGROUND/AIMS: Diagnosing hepatocyte nuclear factor 1β (HNF1B)-related disease is a challenging task due to the phenotypic variability and frequent absence of a family history. An HNF1B score has recently been developed to help select appropriate patients for genetic testing with a negative predictive value (NPV) of 99%. We aimed at testing the clinical utility of this score in a large number of referrals for HNF1B genetic testing to the UK diagnostic testing service for the HNF1B gene. METHODS: an HNF1B score was assigned for 686 UK referrals for HNF1B genetic testing using clinical information available at referral. The performance of the score was evaluated by receiver-operating characteristic curve analysis. The relative discriminatory ability of different clinical features for making a genetic diagnosis of HNF1B-related disease were estimated in the UK dataset alone and pooled with French data. RESULTS: the HNF1B score discriminated between patients with and without a mutation reasonably well with an area under the curve of 0.72. Applying the suggested cut-off score of ≥8 gave a NPV of 85%. In a pooled analysis, antenatal renal abnormalities, renal hyperechogenicity and cysts were discriminatory in children, whereas renal hypoplasia and cysts were discriminatory in adults. Pancreatic abnormalities were discriminatory in both, whereas other extra-renal characteristics had a large effect size only in adults. CONCLUSION: the HNF1B score was discriminatory for HNF1B mutations in a large cohort of individuals tested in a single UK centre. The lower NPV (85 vs. 99%) reduces its clinical utility in selecting patients for HNF1B genetic testing, although validation in a prospective cohort is required.

Neonatal diabetes is a highly genetically heterogeneous disorder. There are over 20 distinct syndromic and non-syndromic forms, including dominant, recessive and X-linked subtypes. Biallelic truncating or mis-sense mutations in the DNA-binding domain of the RFX6 transcription factor cause an autosomal recessive, syndromic form of neonatal diabetes previously described as Mitchell-Riley syndrome. In all, eight cases have been reported, with the age at onset of diabetes in the first 2 weeks of life. Here we report two individuals born to double first cousins in whom intestinal atresias consistent with a diagnosis of Mitchell-Riley syndrome were diagnosed at birth, but in whom diabetes did not present until the ages of 3 and 6 years. Novel compound heterozygous RFX6 nonsense mutations (p.Arg726X/p.Arg866X) were identified at the 3′ end of the gene. The later onset of diabetes in these patients may be due to incomplete inactivation of RFX6. Genetic testing for RFX6 mutations should be considered in patients presenting with intestinal atresias in the absence of neonatal diabetes.

Neonatal diabetes is a highly genetically heterogeneous disorder. There are over 20 distinct syndromic and non-syndromic forms, including dominant, recessive and X-linked subtypes. Biallelic truncating or mis-sense mutations in the DNA-binding domain of the RFX6 transcription factor cause an autosomal recessive, syndromic form of neonatal diabetes previously described as Mitchell-Riley syndrome. In all, eight cases have been reported, with the age at onset of diabetes in the first 2 weeks of life. Here we report two individuals born to double first cousins in whom intestinal atresias consistent with a diagnosis of Mitchell-Riley syndrome were diagnosed at birth, but in whom diabetes did not present until the ages of 3 and 6 years. Novel compound heterozygous RFX6 nonsense mutations (p.Arg726X/p.Arg866X) were identified at the 3' end of the gene. The later onset of diabetes in these patients may be due to incomplete inactivation of RFX6. Genetic testing for RFX6 mutations should be considered in patients presenting with intestinal atresias in the absence of neonatal diabetes.

BACKGROUND: Neonatal diabetes mellitus (NDM) is a rare form of monogenic diabetes and usually presents in the first 6 months of life. We aimed to describe the clinical characteristics and molecular genetics of a large Turkish cohort of NDM patients from a single centre and estimate an annual incidence rate of NDM in South-Eastern Anatolian region of Turkey. DESIGN AND METHODS: NDM patients presenting to Diyarbakir Children State Hospital between 2010 and 2013, and patients under follow-up with presumed type 1 diabetes mellitus, with onset before 6 months of age were recruited. Molecular genetic analysis was performed. RESULTS: Twenty-two patients (59% males) were diagnosed with NDM (TNDM-5; PNDM-17). Molecular genetic analysis identified a mutation in 20 (95%) patients who had undergone a mutation analysis. In transient neonatal diabetes (TNDM) patients, the genetic cause included chromosome 6q24 abnormalities (n=3), ABCC8 (n=1) and homozygous INS (n=1). In permanent neonatal diabetes (PNDM) patients, homozygous GCK (n=6), EIF2AK3 (n=3), PTF1A (n=3), and INS (n=1) and heterozygous KCNJ11 (n=2) mutations were identified. Pancreatic exocrine dysfunction was observed in patients with mutations in the distal PTF1A enhancer. Both patients with a KCNJ11 mutation responded to oral sulphonylurea. A variable phenotype was associated with the homozygous c.-331C>A INS mutation, which was identified in both a PNDM and TNDM patient. The annual incidence of PNDM in South-East Anatolian region of Turkey was one in 48 000 live births. CONCLUSIONS: Homozygous mutations in GCK, EIF2AK3 and the distal enhancer region of PTF1A were the commonest causes of NDM in our cohort. The high rate of detection of a mutation likely reflects the contribution of new genetic techniques (targeted next-generation sequencing) and increased consanguinity within our cohort.

Hyperinsulinaemic hypoglycaemia (HH) is characterised by inappropriate insulin secretion and is the most common cause for persistent neonatal hypoglycaemia. The only treatment available for medically unresponsive hypoglycaemia is a near-total pancreatectomy. A neonate with severe HH, due to a homozygous ABCC8 mutation, was not responsive to treatment with maximal doses of diazoxide and subcutaneous daily octreotide, and underwent a near-total pancreatectomy; however, hypoglycaemia persisted. Introduction of sirolimus, an mTOR (mammalian target of rapamycin) inhibitor, obviated the requirement for glucose infusion. Euglycaemia was achieved with no significant adverse events from the drug. Sirolimus therapy was ceased at 13 months of age. No episodes of persistent hypoglycaemia were observed after cessation of sirolimus. This report demonstrates the successful use of sirolimus for persistent hypoglycaemia in the critically ill infant post pancreatectomy. Sirolimus could be considered in patients with severe HH not responsive to conventional medical and surgical therapy. However, the long-term efficacy and safety with this immunosuppressive drug in very young patients are not assured.

CONTEXT: GLIS3 (GLI-similar 3) is a member of the GLI-similar zinc finger protein family encoding for a nuclear protein with 5 C2H2-type zinc finger domains. The protein is expressed early in embryogenesis and plays a critical role as both a repressor and activator of transcription. Human GLIS3 mutations are extremely rare. OBJECTIVE: the purpose of this article was determine the phenotypic presentation of 12 patients with a variety of GLIS3 mutations. METHODS: GLIS3 gene mutations were sought by PCR amplification and sequence analysis of exons 1 to 11. Clinical information was provided by the referring clinicians and subsequently using a questionnaire circulated to gain further information. RESULTS: We report the first case of a patient with a compound heterozygous mutation in GLIS3 who did not present with congenital hypothyroidism. All patients presented with neonatal diabetes with a range of insulin sensitivities. Thyroid disease varied among patients. Hepatic and renal disease was common with liver dysfunction ranging from hepatitis to cirrhosis; cystic dysplasia was the most common renal manifestation. We describe new presenting features in patients with GLIS3 mutations, including craniosynostosis, hiatus hernia, atrial septal defect, splenic cyst, and choanal atresia and confirm further cases with sensorineural deafness and exocrine pancreatic insufficiency. CONCLUSION: We report new findings within the GLIS3 phenotype, further extending the spectrum of abnormalities associated with GLIS3 mutations and providing novel insights into the role of GLIS3 in human physiological development. All but 2 of the patients within our cohort are still alive, and we describe the first patient to live to adulthood with a GLIS3 mutation, suggesting that even patients with a severe GLIS3 phenotype may have a longer life expectancy than originally described.

The use of targeted gene panels now allows the analysis of all the genes known to cause a disease in a single test. For neonatal diabetes, this has resulted in a paradigm shift with patients receiving a genetic diagnosis early and the genetic results guiding their clinical management. Exome and genome sequencing are powerful tools to identify novel genetic causes of known diseases. For neonatal diabetes, the use of these technologies has resulted in the identification of 2 novel disease genes (GATA6 and STAT3) and a novel regulatory element of PTF1A, in which mutations cause pancreatic agenesis.

The use of targeted gene panels now allows the analysis of all the genes known to cause a disease in a single test. For neonatal diabetes, this has resulted in a paradigm shift with patients receiving a genetic diagnosis early and the genetic results guiding their clinical management. Exome and genome sequencing are powerful tools to identify novel genetic causes of known diseases. For neonatal diabetes, the use of these technologies has resulted in the identification of 2 novel disease genes (. GATA6 and STAT3) and a novel regulatory element of PTF1A, in which mutations cause pancreatic agenesis.

BACKGROUND: Hyperinsulinaemic hypoglycaemia (HH) is a group of clinically and genetically heterogeneous disorders characterized by unregulated insulin secretion. Abnormalities in nine different genes (ABCC8, KCNJ11, GLUD1, GCK, HADH, SLC16A1, HNF4A, UCP2 and HNF1A) have been reported in HH, the most common being ABCC8 and KCNJ11. We describe the genetic aetiology and phenotype of Iranian patients with HH. METHODS: Retrospective clinical, biochemical and genetic information was collected on 23 patients with biochemically confirmed HH. Mutation analysis was carried out for the ATP-sensitive potassium (K(ATP)) channel genes (ABCC8 and KCNJ11), GLUD1, GCK, HADH and HNF4A. RESULTS: 78% of the patients were identified to have a genetic cause for HH. 48% of patients had mutation in HADH, whilst ABCC8/KCNJ11 mutations were identified in 30% of patients. Among the diazoxide-responsive patients (18/23), mutations were identified in 72%. These include two novel homozygous ABCC8 mutations. of the five patients with diazoxide-unresponsive HH, three had homozygous ABCC8 mutation, one had heterozygous ABCC8 mutation inherited from an unaffected father and one had homozygous KCNJ11 mutation. 52% of children in our cohort were born to consanguineous parents. Patients with ABCC8/KCNJ11 mutations were noted to be significantly heavier than those with HADH mutation (p = 0.002). Our results revealed neurodevelopmental deficits in 30% and epilepsy in 52% of all patients. CONCLUSIONS: to the best of our knowledge, this is the first study of its kind in Iran. We found disease-causing mutations in 78% of HH patients. The predominance of HADH mutation might be due to a high incidence of consanguineous marriage in this population. Further research involving a larger cohort of HH patients is required in Iranian population.

Heterozygous mutations in the gene that encodes the transcription factor hepatocyte nuclear factor 1β (HNF1B) represent the most common known monogenic cause of developmental kidney disease. Renal cysts are the most frequently detected feature of HNF1B-associated kidney disease; however, other structural abnormalities, including single kidneys and renal hypoplasia, and electrolyte abnormalities can also occur. Extra-renal phenotypes might also be observed; consequently, HNF1B-associated disease is considered a multi-system disorder. Other clinical features include early-onset diabetes mellitus, pancreatic hypoplasia, genital tract malformations, abnormal liver function and early-onset gout. Heterozygous mutations in the coding region or splice sites of HNF1B, and complete gene deletion, each account for ∼50% of all cases of HNF1B-associated disease, respectively, and often arise spontaneously. There is no clear genotype-phenotype correlation, consistent with haploinsufficiency as the disease mechanism. Data from animal models suggest that HNF1B has an important function during several stages of nephrogenesis; however, the precise signalling pathways remain to be elucidated. This Review discusses the genetics and molecular pathways that lead to disease development, summarizes the reported renal and extra-renal phenotypes, and identifies areas for future research in HNF1B-associated disease.

Heterozygous mutations in the gene that encodes the transcription factor hepatocyte nuclear factor 1β (HNF1B) represent the most common known monogenic cause of developmental kidney disease. Renal cysts are the most frequently detected feature of HNF1B-associated kidney disease; however, other structural abnormalities, including single kidneys and renal hypoplasia, and electrolyte abnormalities can also occur. Extra-renal phenotypes might also be observed; consequently, HNF1B-associated disease is considered a multi-system disorder. Other clinical features include early-onset diabetes mellitus, pancreatic hypoplasia, genital tract malformations, abnormal liver function and early-onset gout. Heterozygous mutations in the coding region or splice sites of HNF1B, and complete gene deletion, each account for ∼50% of all cases of HNF1B-associated disease, respectively, and often arise spontaneously. There is no clear genotype-phenotype correlation, consistent with haploinsufficiency as the disease mechanism. Data from animal models suggest that HNF1B has an important function during several stages of nephrogenesis; however, the precise signalling pathways remain to be elucidated. This Review discusses the genetics and molecular pathways that lead to disease development, summarizes the reported renal and extra-renal phenotypes, and identifies areas for future research in HNF1B-associated disease.

CONTEXT: Pituitary adenomas and pheochromocytomas/paragangliomas (pheo/PGL) can occur in the same patient or in the same family. Coexistence of the two diseases could be due to either a common pathogenic mechanism or a coincidence. OBJECTIVE: the objective of the investigation was to study the possible coexistence of pituitary adenoma and pheo/PGL. DESIGN: Thirty-nine cases of sporadic or familial pheo/PGL and pituitary adenomas were investigated. Known pheo/PGL genes (SDHA-D, SDHAF2, RET, VHL, TMEM127, MAX, FH) and pituitary adenoma genes (MEN1, AIP, CDKN1B) were sequenced using next generation or Sanger sequencing. Loss of heterozygosity study and pathological studies were performed on the available tumor samples. SETTING: the study was conducted at university hospitals. PATIENTS: Thirty-nine patients with sporadic of familial pituitary adenoma and pheo/PGL participated in the study. OUTCOME: Outcomes included genetic screening and clinical characteristics. RESULTS: Eleven germline mutations (five SDHB, one SDHC, one SDHD, two VHL, and two MEN1) and four variants of unknown significance (two SDHA, one SDHB, and one SDHAF2) were identified in the studied genes in our patient cohort. Tumor tissue analysis identified LOH at the SDHB locus in three pituitary adenomas and loss of heterozygosity at the MEN1 locus in two pheochromocytomas. All the pituitary adenomas of patients affected by SDHX alterations have a unique histological feature not previously described in this context. CONCLUSIONS: Mutations in the genes known to cause pheo/PGL can rarely be associated with pituitary adenomas, whereas mutation in a gene predisposing to pituitary adenomas (MEN1) can be associated with pheo/PGL. Our findings suggest that genetic testing should be considered in all patients or families with the constellation of pheo/PGL and a pituitary adenoma.

Congenital hyperinsulinism causes profound hypoglycemia, which may persist or resolve spontaneously. Among 13 children with congenital hyperinsulinism, elevated incretin hormone concentrations were detected in 2 with atypical, persistent disease. We suggest that incretin biomarkers may identify these patients, and that elevated hormone levels may contribute to their pathophysiology.

CONTEXT: Familial isolated pituitary adenoma (FIPA) due to aryl hydrocarbon receptor interacting protein (AIP) gene mutations is an autosomal dominant disease with incomplete penetrance. Clinical screening of apparently unaffected AIP mutation (AIPmut) carriers could identify previously unrecognized disease. OBJECTIVE: to determine the AIP mutational status of FIPA and young pituitary adenoma patients, analyzing their clinical characteristics, and to perform clinical screening of apparently unaffected AIPmut carrier family members. DESIGN: This was an observational, longitudinal study conducted over 7 years. SETTING: International collaborative study conducted at referral centers for pituitary diseases. PARTICIPANTS: FIPA families (n 216) and sporadic young-onset (30 y) pituitary adenoma patients (n 404) participated in the study. INTERVENTIONS: We performed genetic screening of patients for AIPmuts, clinical assessment of their family members, and genetic screening for somatic GNAS1 mutations and the germline FGFR4 p.G388R variant. MAIN OUTCOME MEASURE(S): We assessed clinical disease in mutation carriers, comparison of characteristics of AIPmut positive and negative patients, results of GNAS1, and FGFR4 analysis. RESULTS: Thirty-seven FIPA families and 34 sporadic patients had AIPmuts. Patients with truncating AIPmuts had a younger age at disease onset and diagnosis, compared with patients with nontruncating AIPmuts. Somatic GNAS1 mutations were absent in tumors from AIPmut-positive patients, and the studied FGFR4 variant did not modify the disease behavior or penetrance in AIPmut-positive individuals. A total of 164 AIPmut-positive unaffected family members were identified; pituitary disease was detected in 18 of those who underwent clinical screening. CONCLUSIONS: a quarter of the AIPmut carriers screened were diagnosed with pituitary disease, justifying this screening and suggesting a variable clinical course for AIPmut-positive pituitary adenomas.

Despite three decades of successful, predominantly phenotype-driven discovery of the genetic causes of monogenic disorders, up to half of children with severe developmental disorders of probable genetic origin remain without a genetic diagnosis. Particularly challenging are those disorders rare enough to have eluded recognition as a discrete clinical entity, those with highly variable clinical manifestations, and those that are difficult to distinguish from other, very similar, disorders. Here we demonstrate the power of using an unbiased genotype-driven approach to identify subsets of patients with similar disorders. By studying 1,133 children with severe, undiagnosed developmental disorders, and their parents, using a combination of exome sequencing and array-based detection of chromosomal rearrangements, we discovered 12 novel genes associated with developmental disorders. These newly implicated genes increase by 10% (from 28% to 31%) the proportion of children that could be diagnosed. Clustering of missense mutations in six of these newly implicated genes suggests that normal development is being perturbed by an activating or dominant-negative mechanism. Our findings demonstrate the value of adopting a comprehensive strategy, both genome-wide and nationwide, to elucidate the underlying causes of rare genetic disorders.

Background: Wolcott-Rallison syndrome (WRS) is caused by recessive EIF2AK3 mutations and characterized by early-onset diabetes and skeletal dysplasia. Hepatic dysfunction has been reported in 60% of patients. Aims: to describe a cohort of WRS patients and discuss the pattern and management of their liver disease. Methods: Detailed phenotyping and direct sequencing of EIF2AK3 gene were conducted in all patients. Results: Twenty-eight genetically confirmed patients (67% male; mean age 4.6 years) were identified. 17 different EIF2AK3 mutations were detected, of which 2 were novel. The p.S991N mutation was associated with prolonged survival and p.I650T with delayed onset. All patients presented before 25 months with diabetes with variation in the frequency and severity of 10 other features. Liver disease, first manifested as non-autoimmune hepatitis, was the commonest extra-pancreatic feature identified in 85.7% (24/28). 22/24 had at least one episode of acute hepatic failure which was the cause of death in all deceased patients (13/28). One child was treated by liver transplantation and had no liver disease and better diabetes control for the following 6 years. Conclusions: Liver disease in WRS is more frequent than previously described and carries high mortality. The first experience with liver transplantation in WRS is encouraging.

OBJECTIVE: Small studies using ultrasensitive C-peptide assays suggest endogenous insulin secretion is frequently detectable in patients with long-standing type 1 diabetes (T1D), but these studies do not use representative samples. We aimed to use the stimulated urine C-peptide-to-creatinine ratio (UCPCR) to assess C-peptide levels in a large cross-sectional, population-based study of patients with T1D. RESEARCH DESIGN AND METHODS: We recruited 924 patients from primary and secondary care in two U.K. centers who had a clinical diagnosis of T1D, were under 30 years of age when they received a diagnosis, and had a diabetes duration of >5 years. The median age at diagnosis was 11 years (interquartile range 6-17 years), and the duration of diabetes was 19 years (11-27 years). All provided a home postmeal UCPCR, which was measured using a Roche electrochemiluminescence assay. RESULTS: Eighty percent of patients (740 of 924 patients) had detectable endogenous C-peptide levels (UCPCR >0.001 nmol/mmol). Most patients (52%, 483 of 924 patients) had historically very low undetectable levels (UCPCR 0.0013-0.03 nmol/mmol); 8% of patients (70 of 924 patients) had a UCPCR ≥0.2 nmol/mmol, equivalent to serum levels associated with reduced complications and hypoglycemia. Absolute UCPCR levels fell with duration of disease. Age at diagnosis and duration of disease were independent predictors of C-peptide level in multivariate modeling. CONCLUSIONS: This population-based study shows that the majority of long-duration T1D patients have detectable urine C-peptide levels. While the majority of patients are insulin microsecretors, some maintain clinically relevant endogenous insulin secretion for many years after the diagnosis of diabetes. Understanding this may lead to a better understanding of pathogenesis in T1D and open new possibilities for treatment.

Hyperinsulinaemic hypoglycaemia (HH) is caused by mutations in the key genes involved in regulation of insulin secretion from the pancreatic β-cells and mutations in ABCC8 and KCNJ11 are the most common causes of HH. Mutations in HADH (which encodes the enzyme 3-hydroxyacyl-CoA dehydrogenase) are a rare cause of HH. We report three siblings (21, 9, and 7 years old) from a consanguineous Saudi family with HH due to a homozygous mutation in HADH. All three siblings presented with HH in the 1st year of life. HH responded well to medical therapy (diazoxide/octreotide) although the 1st sibling suffered neurological damage. The protein load test revealed protein sensitivity in the 21-year-old proband, the oldest reported patient with HH secondary to a HADH mutation. Genetic analysis revealed a homozygous HADH splicing mutation (c.133-1G>A) in all three siblings. HADH mutations can present in later infancy or childhood with severe HH that is usually diazoxide responsive. Severe neurological complications such as epilepsy and developmental delay can be associated with HADH mutations. This is the 1st report of HH due to HADH mutation in an adult suggesting that HH could persist into adulthood possibly becoming milder over the years.

Glucokinase-maturity-onset diabetes of the young (GCK-MODY), also known as MODY2, is caused by heterozygous inactivating mutations in the GCK gene. GCK gene mutations are present in ∼1 in 1,000 of the population, but most are not diagnosed. They are common causes of MODY (10-60%): persistent incidental childhood hyperglycemia (10-60%) and gestational diabetes mellitus (1-2%). GCK-MODY has a unique pathophysiology and clinical characteristics, so it is best considered as a discrete genetic subgroup. People with GCK-MODY have a defect in glucose sensing; hence, glucose homeostasis is maintained at a higher set point resulting in mild, asymptomatic fasting hyperglycemia (5.4-8.3 mmol/L, HbA1c range 5.8-7.6% [40-60 mmol/mol]), which is present from birth and shows slight deterioration with age. Even after 50 years of mild hyperglycemia, people with GCK-MODY do not develop significant microvascular complications, and the prevalence of macrovascular complications is probably similar to that in the general population. Treatment is not recommended outside pregnancy because glucose-lowering therapy is ineffective in people with GCK-MODY and there is a lack of long-term complications. In pregnancy, fetal growth is primarily determined by whether the fetus inherits the GCK gene mutation from their mother. Insulin treatment of the mother is only appropriate when increased fetal abdominal growth on scanning suggests the fetus is unaffected. The impact on outcome of maternal insulin treatment is limited owing to the difficulty in altering maternal glycemia in these patients. Making the diagnosis of GCK-MODY through genetic testing is essential to avoid unnecessary treatment and investigations, especially when patients are misdiagnosed with type 1 or type 2 diabetes.

Introduction: Hyperinsulinaemic hypoglycaemia (HH) is the most common cause of severe and persistent hypoglycaemia in neonates. The treatment of severe diazoxide unresponsive HH involves near total pancreatectomy. Mammalian target of rapamycin (mTOR) is a protein kinase that regulates cellular proliferation. mTOR inhibitors are used in cancer patients and recently found to be effective in the treatment of insulinoma and HH patients. Case: a 36 weeks large for gestational age neonate presented with severe hypoglycaemia on day 1 of life. The hypoglycaemia screen confirmed HH and genetic testing revealed compound heterozygous ABCC8 mutation, confirming diffuse disease. He was unresponsive to the maximal dose of diazoxide (15 mg/kg/day), hence needed treatment with higher concentration of intravenous glucose (25 mg/kg/min), intravenous glucagon and subcutaneous octreotide (30 μg/kg/day) infusions to maintain normoglycaemia. Sirolimus, a mTOR inhibitor, was commenced at 9 weeks of age following which he showed a marked improvement in his glycaemic control. After 4 weeks of sirolimus therapy, he was discharged home on subcutaneous octreotide injection (20 μg/kg/day) and oral sirolimus, thereby avoiding the need for a near total pancreatectomy. Conclusion: We report the first case of compound heterozygous ABCC8 mutation causing severe diffuse HH that responded to therapy with a mTOR inhibitor.

BACKGROUND: Traditional genetic testing focusses on analysis of one or a few genes according to clinical features; this approach is changing as improved sequencing methods enable simultaneous analysis of several genes. Neonatal diabetes is the presenting feature of many discrete clinical phenotypes defined by different genetic causes. Genetic subtype defines treatment, with improved glycaemic control on sulfonylurea treatment for most patients with potassium channel mutations. We investigated the effect of early, comprehensive testing of all known genetic causes of neonatal diabetes. METHODS: in this large, international, cohort study, we studied patients with neonatal diabetes diagnosed with diabetes before 6 months of age who were referred from 79 countries. We identified mutations by comprehensive genetic testing including Sanger sequencing, 6q24 methylation analysis, and targeted next-generation sequencing of all known neonatal diabetes genes. FINDINGS: Between January, 2000, and August, 2013, genetic testing was done in 1020 patients (571 boys, 449 girls). Mutations in the potassium channel genes were the most common cause (n=390) of neonatal diabetes, but were identified less frequently in consanguineous families (12% in consanguineous families vs 46% in non-consanguineous families; p4 years; p

In infants, especially with novel previously undescribed mutations of the KATP channel causing neonatal diabetes, in vitro studies can be used to both predict the response to sulphonylurea treatment and support a second trial of glibenclamide at higher than standard doses if the expected response is not observed.

Background: Wolfram syndrome is an autosomal recessive disorder caused by mutations in the WFS1 gene. Clinical heterogeneity has been reported both within and between families with WFS1 mutations. Subjects: the first case was diagnosed with insulin-dependent diabetes mellitus with positive for pancreatic autoantibodies and had a ketoacidotic attack in the follow-up period. The second case presented initially with optic atrophy and was diagnosed with behavioral and psychiatric problems at an early age. The third case had early onset insulin-dependent diabetes with multiple anomalies and congenital hypothyroidism. Many of these features have not been reported previously in patients with Wolfram syndrome. In all three patients homozygous mutations in WFS1 were identified. Conclusion: Wolfram syndrome is a disease where the characteristic features may present at different times. A diagnosis of Wolfram syndrome should therefore be considered even in the absence of the full spectrum of clinical features.

In the absence of specific metabolic disorders, predictors of response to ketogenic dietary therapies (KDT) are unknown. We aimed to determine whether variants in established candidate genes KCNJ11 and BAD influence response to KDT. We sequenced KCNJ11 and BAD in individuals without previously-known glucose transporter type 1 deficiency syndrome or other metabolic disorders, who received KDT for epilepsy. Hospital records were used to obtain demographic and clinical data. Two response phenotypes were used: ≥ 50% seizure reduction and seizure-freedom at 3-month follow-up. Case/control association tests were conducted with KCNJ11 and BAD variants with minor allele frequency (MAF)>0.01, using PLINK. Response to KDT in individuals with variants with MAF0.01. Eight variants in KCNJ11 and seven in BAD (of which three were previously-unreported) had MAF0.05 and effect size >3. A larger sample size is needed to detect associations from rare variants or those with smaller effect sizes.

Hyperinsulinemic hypoglycemia (HH) is the commonest cause of persistent hypoglycemia in the neonatal and infancy periods. Mutations in the ABCC8 and KCNJ11 genes, which encode subunits of the ATP- sensitive potassium channel in the pancreatic beta cell, are identified in approximately 50% of these patients. The first-line drug in the treatment of HH is diazoxide. Octreotide and glucagon can be used in patients who show no response to diazoxide. Nifedipine, a calcium-channel blocker, has been shown to be an effective treatment in a small number of patients with diazoxide-unresponsive HH. We report a HH patient with a homozygous ABCC8 mutation (p.W1339X) who underwent a near- total pancreatectomy at 2 months of age due to a lack of response to diazoxide and octreotide treatment. Severe hypoglycemic attacks continued following surgery, while the patient was being treated with octreotide. These attacks resolved when nifedipine was introduced. Whilst our patient responded well to nifedipine, the dosage could not be increased to 0.75 mg/kg/day due to development of hypotension, a reported side effect of this drug. Currently, our patient, now aged 4 years, is receiving a combination of nifedipine and octreotide treatment. He is under good control and shows no side effects. In conclusion, nifedipine treatment can be started in patients with HH who show a poor response to diazoxide and octreotide treatment. © Journal of Clinical Research in Pediatric Endocrinology, Published by Galenos Publishing.

BACKGROUND: Hyperinsulinemic hypoglycemia (HH), characterized by unregulated insulin secretion, is an important cause of persistent and severe hypoglycemia. The biochemical picture of HH is hypoketotic hypo-fatty-acidemic hypoglycemia along with elevated serum insulin. Not infrequently, serum insulin might be undetectable in HH despite the presence of evidence of insulin action (suppressed ketogenesis and lipolysis). However, autonomous activity of the downstream insulin signaling pathway without the presence of the ligand (insulin) will give rise to the same clinical and biochemical picture, apart from undetectable serum insulin/C-peptide. AKT2, a serine/threonine protein kinase, is involved downstream to the insulin receptor in mediating the physiological effects of insulin. AIM: We describe the second report of an activating AKT2 mutation leading to hypoinsulinemic hypoketotic hypoglycemia. PATIENTS AND METHODS: the proband presented with hemihypertrophy and symptomatic hypoglycemia. Investigations confirmed evidence of insulin action, despite absence of detectable serum insulin on multiple occasions. Molecular genetic testing for common causes of HH (ABCC8, KCNJ11, and GLUD1) was negative. Sequencing of AKT2 identified a de novo mosaic c.49G→A (p.E17K) mutation, consistent with the clinical and biochemical phenotype. CONCLUSIONS: This is the second report of an activating AKT2 mutation leading to hypoinsulinemic hypoketotic hypo-fatty-acidemic hypoglycemia. In patients presenting a clinical and biochemical picture of HH with undetectable serum insulin, consideration of autonomous activation of the downstream insulin signaling pathway should be made.

Monogenic causes of autoimmunity provide key insights into the complex regulation of the immune system. We report a new monogenic cause of autoimmunity resulting from de novo germline activating STAT3 mutations in five individuals with a spectrum of early-onset autoimmune disease, including type 1 diabetes. These findings emphasize the critical role of STAT3 in autoimmune disease and contrast with the germline inactivating STAT3 mutations that result in hyper IgE syndrome.

CONTEXT: Congenital hyperinsulinism (CHI) has two main histological types: diffuse and focal. Heterozygous paternally inherited ABCC8/KCNJ11 mutations (depending upon whether recessive or dominant acting and occurrence of somatic maternal allele loss) can give rise to either phenotype. However, the relative proportion of these two phenotypes in a large cohort of CHI patients due to paternally inherited heterozygous ABCC8/KCNJ11 mutations has not been reported. OBJECTIVE: the purpose of this study is to highlight the variable clinical phenotype and to characterise the distribution of diffuse and focal disease in a large cohort of CHI patients due to paternally inherited heterozygous ABCC8/KCNJ11 mutations. DESIGN: a retrospective chart review of the CHI patients due to heterozygous paternally inherited ABCC8/KCNJ11 mutations from 2000 to 2013 was conducted. RESULTS: Paternally inherited heterozygous ABCC8/KCNJ11 mutations were identified in 53 CHI patients. of these, 18 (34%) either responded to diazoxide or resolved spontaneously. Fluorine-18 l-3, 4-dihydroxyphenylalanine positron emission tomography computerised tomography 18F DOPA-PET CT) scanning in 3/18 children showed diffuse disease. The remaining 35 (66%) diazoxide-unresponsive children either had pancreatic venous sampling (n=8) or 18F DOPA-PET CT (n=27). Diffuse, indeterminate and focal disease was identified in 13, 1 and 21 patients respectively. Two patients with suspected diffuse disease were identified to have focal disease on histology. CONCLUSIONS: Paternally inherited heterozygous ABCC8/KCNJ11 mutations can manifest as a wide spectrum of CHI with variable 18F DOPA-PET CT/histological findings and clinical outcomes. Focal disease was histologically confirmed in 24/53 (45%) of CHI patients with paternally inherited heterozygous ABCC8/KCNJ11 mutations.

OBJECTIVE: Congenital hyperinsulinism (CHI) is the commonest cause of hyperinsulinaemic hypoglycaemia in the neonatal, infancy and childhood periods. Its clinical presentation, histology and underlying molecular biology are extremely heterogeneous. The aim of this study was to describe the clinical characteristics, analyse the genotype-phenotype correlations and describe the treatment outcome of Turkish CHI patients. DESIGN AND METHODS: a total of 35 patients with CHI were retrospectively recruited from four large paediatric endocrine centres in Turkey. Detailed clinical, biochemical and genotype information was collected. RESULTS: Diazoxide unresponsiveness was observed in nearly half of the patients (n=17; 48.5%). Among diazoxide-unresponsive patients, mutations in ABCC8/KCNJ11 were identified in 16 (94%) patients. Among diazoxide-responsive patients (n=18), mutations were identified in two patients (11%). Genotype-phenotype correlation revealed that mutations in ABCC8/KCNJ11 were associated with an increased birth weight and early age of presentation. Five patients had p.L1171fs (c.3512del) ABCC8 mutations, suggestive of a founder effect. The rate of detection of a pathogenic mutation was higher in consanguineous families compared with non-consanguineous families (87.5 vs 21%; P

AIMS/HYPOTHESIS: Heterozygous glucokinase (GCK) mutations cause mild, fasting hyperglycaemia from birth. Although patients are usually asymptomatic and have glycaemia within target ranges, some are put on pharmacological treatment. We aimed to investigate how many patients are on pharmacological treatment and the impact of treatment on glycaemic control. METHODS: Treatment details were ascertained for 799 patients with heterozygous GCK mutations. In a separate, longitudinal study, HbA1c was obtained for 16 consecutive patients receiving insulin (n = 10) or oral hypoglycaemic agents (OHAs) (n = 6) whilst on treatment, and again having discontinued treatment following a genetic diagnosis of GCK-MODY. For comparison, HbA1c before and after genetic testing was studied in a control group (n = 18) not receiving pharmacological therapy. RESULTS: at referral for genetic testing, 168/799 (21%) of patients were on pharmacological treatment (13.5% OHAs, 7.5% insulin). There was no difference in the HbA1c of these patients compared with those receiving no treatment(median [IQR]: 48 [43, 51] vs 46 [43, 50] mmol/mol, respectively; 6.5% [6.1%, 6.8%] vs 6.4% [6.1%, 6.7%]; p = 0.11). Following discontinuation of pharmacological treatment in 16 patients, HbA1c did not change. The mean change in HbA1c was -0.68 mmol/mol (95% CI: -2.97, 1.61) (-0.06% [95% CI: -0.27, 0.15]). CONCLUSIONS/INTERPRETATION: Prior to a genetic diagnosis, 21% of patients were on pharmacological treatment. HbA1c was no higher than in untreated patients and did not change when therapy was discontinued, suggesting no impact on glycaemia. The lack of response to pharmacological therapy is likely to reflect the regulated hyperglycaemia seen in these patients owing to their glucose sensing defect and is an example of pharmacogenetics.

Fanconi-Bickel Syndrome (FBS) is a rare autosomal recessive disorder of carbohydrate metabolism. The defect in the GLUT 2 receptors in the hepatocytes, pancreas and renal tubules leads to symptoms secondary to glycogen storage, glucose metabolism and renal tubular dysfunction. Derangement in glucose metabolism is classical with fasting hypoglycemia and post-prandial hyperglycemia. The authors report a 4-year-old boy who presented with failure to thrive, motor delay, protuberant abdomen and was noted to have huge hepatomegaly with glycogen deposition in liver, and renal tubular acidosis. Gene sequencing revealed homozygous mutation, c.1330T > C in SLC2A2 gene, thus confirming the diagnosis of FBS. Only three mutations have been reported from India so far. The primary reason for referral to authors' hospital was for liver transplantation, but an accurate diagnosis led to avoidance of the major surgery and streamlining of treatment with clinical benefit to the child and family. © 2014 Dr. K C Chaudhuri Foundation.

Fanconi-Bickel Syndrome (FBS) is a rare autosomal recessive disorder of carbohydrate metabolism. The defect in the GLUT 2 receptors in the hepatocytes, pancreas and renal tubules leads to symptoms secondary to glycogen storage, glucose metabolism and renal tubular dysfunction. Derangement in glucose metabolism is classical with fasting hypoglycemia and post-prandial hyperglycemia. The authors report a 4-year-old boy who presented with failure to thrive, motor delay, protuberant abdomen and was noted to have huge hepatomegaly with glycogen deposition in liver, and renal tubular acidosis. Gene sequencing revealed homozygous mutation, c.1330T > C in SLC2A2 gene, thus confirming the diagnosis of FBS. Only three mutations have been reported from India so far. The primary reason for referral to authors’ hospital was for liver transplantation, but an accurate diagnosis led to avoidance of the major surgery and streamlining of treatment with clinical benefit to the child and family.

The GATA family zinc finger transcription factors GATA4 and GATA6 are known to play important roles in the development of the pancreas. In mice, both Gata4 and Gata6 are required for pancreatic development. In humans, GATA6 haploinsufficiency can cause pancreatic agenesis and heart defects. Congenital heart defects also are common in patients with GATA4 mutations and deletions, but the role of GATA4 in the developing human pancreas is unproven. We report five patients with deletions (n = 4) or mutations of the GATA4 gene who have diabetes and a variable exocrine phenotype. In four cases, diabetes presented in the neonatal period (age at diagnosis 1-7 days). A de novo GATA4 missense mutation (p.N273K) was identified in a patient with complete absence of the pancreas confirmed at postmortem. This mutation affects a highly conserved residue located in the second zinc finger domain of the GATA4 protein. In vitro studies showed reduced DNA binding and transactivational activity of the mutant protein. We show that GATA4 mutations/deletions are a cause of neonatal or childhood-onset diabetes with or without exocrine insufficiency. These results confirm a role for GATA4 in normal development of the human pancreas.

BACKGROUND: Hepatocyte nuclear factor 4α (HNF4A) is a member of the nuclear receptor family of ligand-activated transcription factors. HNF4A mutations cause hyperinsulinaemic hypoglycaemia in early life and maturity-onset diabetes of the young. Regular screening of HNF4A mutation carriers using the oral glucose tolerance test has been recommended to diagnose diabetes mellitus at an early stage. Glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide are incretin hormones, responsible for up to 70% of the secreted insulin after a meal in healthy individuals. We describe, for the first time, gradual alteration of glucose homeostasis in a patient with HNF4A mutation after resolution of hyperinsulinaemic hypoglycaemia, on serial oral glucose tolerance testing. We also measured the incretin response to a mixed meal in our patient. CASE REPORT: Our patient was born with macrosomia and developed hyperinsulinaemic hypoglycaemia in the neonatal period. Molecular genetic analysis confirmed HNF4A mutation (p.M116I, c.317G>A) as an underlying cause of hyperinsulinaemic hypoglycaemia. Serial oral glucose tolerance testing, after the resolution of hyperinsulinaemic hypoglycaemia, confirmed the diagnosis of maturity-onset diabetes of the young at the age of 10 years. Interestingly, the intravenous glucose tolerance test revealed normal glucose disappearance rate and first-phase insulin secretion. Incretin hormones showed a suboptimal rise in response to the mixed meal, potentially explaining the discrepancy between the oral glucose tolerance test and the intravenous glucose tolerance test. CONCLUSIONS: Maturity-onset diabetes of the young can develop as early as the first decade of life in persons with an HNF4A mutation. Impaired incretin response might be contributory in the early stages of HNF4A maturity-onset diabetes of the young.

CONTEXT: Octreotide, a somatostatin analog, is commonly used in diazoxide unresponsive congenital hyperinsulinism (CHI) patients as a second-line therapy. OBJECTIVE: the aims of this study were to evaluate the dose range, side effects, and long-term follow-up of octreotide therapy in a large cohort of CHI patients. SETTING: the study was conducted at an international referral center for the management of CHI. PATIENTS: Twenty-eight (17 males) diazoxide unresponsive CHI patients (15 biallelic and 10 monoallelic ATP sensitive potassium channel mutation) managed with daily multidose octreotide therapy between 2001 and 2013 participated in the study. MAIN OUTCOME MEASURES: Regular follow-up of auxology, growth factors (serum IGF-1 and IGF binding protein 3 levels), thyroid functions, liver function tests, and hepatobiliary ultrasonography were measured. RESULTS: the median age of CHI diagnosis was 1 week (range 1-80 wk). The mean (±SD) dose of octreotide required was 17.8 (±7.5) μg/kg · d (range 7.5-30 μg/kg · d). The mean (±SD) duration of follow-up on octreotide therapy was 52.4 (±33.8) months (range 6 mo to 9.5 y). Elevation of liver enzymes was the most prevalent side effect (n = 13; 46.4%), which resolved spontaneously. Gallbladder pathology was detected in nine patients (32%). Mean (±SD) duration of octreotide therapy before the development of gallbladder pathology was 4.3 (±4.6 mo), whereas 19 patients were free of gallstones after a follow-up of 53.6 ± 32.9 months on octreotide therapy. There was no relationship between the dose or the duration of octreotide therapy and development of gallbladder pathology or liver dysfunction. CONCLUSIONS: Transient elevation of liver enzymes and asymptomatic gallbladder pathology were the most prevalent long-term side effects of octreotide therapy. There was no correlation between the dose or the duration of octreotide therapy and development of liver dysfunction and gallbladder pathology.

Objective Split-hand/foot malformation type 1 is an autosomal dominant condition with reduced penetrance and variable expression. We report three individuals from two families with split-hand/split-foot malformation (SHFM) in whom next generation sequencing was performed to investigate the cause of their phenotype. Methods and results the first proband has a de novo balanced translocation t(2;7)(p25.1;q22) identified by karyotyping. Whole genome sequencing showed that the chromosome 7 breakpoint is situated within the SHFM1 locus on chromosome 7q21.3. This separates the DYNC1I1 exons recently identified as limb enhancers in mouse studies from their target genes, DLX5 and DLX6. In the second family, X-linked recessive inheritance was suspected and exome sequencing was performed to search for a mutation in the affected proband and his uncle. No coding mutation was found within the SHFM2 locus at Xq26 or elsewhere in the exome, but a 106 kb deletion within the SHFM1 locus was detected through copy number analysis. Genome sequencing of the deletion breakpoints showed that the DLX5 and DLX6 genes are disomic but the putative DYNC1I1 exon 15 and 17 enhancers are deleted. Conclusions Exome sequencing identified a 106 kb deletion that narrows the SHFM1 critical region from 0.9 to 0.1 Mb and confirms a key role of DYNC1I1 exonic enhancers in normal limb formation in humans.

OBJECTIVE: Split-hand/foot malformation type 1 is an autosomal dominant condition with reduced penetrance and variable expression. We report three individuals from two families with split-hand/split-foot malformation (SHFM) in whom next generation sequencing was performed to investigate the cause of their phenotype. METHODS AND RESULTS: the first proband has a de novo balanced translocation t(2;7)(p25.1;q22) identified by karyotyping. Whole genome sequencing showed that the chromosome 7 breakpoint is situated within the SHFM1 locus on chromosome 7q21.3. This separates the DYNC1I1 exons recently identified as limb enhancers in mouse studies from their target genes, DLX5 and DLX6. In the second family, X-linked recessive inheritance was suspected and exome sequencing was performed to search for a mutation in the affected proband and his uncle. No coding mutation was found within the SHFM2 locus at Xq26 or elsewhere in the exome, but a 106 kb deletion within the SHFM1 locus was detected through copy number analysis. Genome sequencing of the deletion breakpoints showed that the DLX5 and DLX6 genes are disomic but the putative DYNC1I1 exon 15 and 17 enhancers are deleted. CONCLUSIONS: Exome sequencing identified a 106 kb deletion that narrows the SHFM1 critical region from 0.9 to 0.1 Mb and confirms a key role of DYNC1I1 exonic enhancers in normal limb formation in humans.

Monogenic diabetes describes a genetically heterogeneous set of disorders which are caused by a mutation in a single gene. For both major subtypes, maturity-onset diabetes of the young and neonatal diabetes, making a genetic diagnosis is important for clinical management because the genetic subtype defines the treatment. Mutations in at least 26 genes have been identified through studies using genetic linkage, candidate gene sequencing and most recently, exome sequencing. With the advent of next-generation sequencing technology, it is now possible to do a single test to identify mutations in any of the known genes, either for the purposes of a clinical diagnostic test or as a pre-screen in the search for novel disease genes. Exome sequencing focuses on the proteincoding regions of the genome and has been applied in a small number of monogenic diabetes studies. It requires a strategy for selecting those patients likely to have a monogenic aetiology, defining the likely mode of inheritance and filtering variants to identify possible deleterious variants for further investigation. The identification through exome sequencing of GATA6 mutations as the most common cause of pancreatic agenesis is likely to be the first of many new discoveries enabled by exome or genome sequencing.

CONTEXT: Congenital hyperinsulinism (CHI), the commonest cause of persistent hypoglycaemia, has two main histological subtypes: diffuse and focal. Diffuse CHI, if medically unresponsive, is managed with near-total pancreatectomy. Post-pancreatectomy, in addition to persistent hypoglycaemia, there is a very high risk of diabetes mellitus and pancreatic exocrine insufficiency. SETTING: International referral centre for the management of CHI. PATIENTS: Medically unresponsive diffuse CHI patients managed with near-total pancreatectomy between 1994 and 2012. INTERVENTION: Near-total pancreatectomy. MAIN OUTCOME MEASURES: Persistent hypoglycaemia post near-total pancreatectomy, insulin-dependent diabetes mellitus, clinical and biochemical (faecal elastase 1) pancreatic exocrine insufficiency. RESULTS: of more than 300 patients with CHI managed during this time period, 45 children had medically unresponsive diffuse disease and were managed with near-total pancreatectomy. After near-total pancreatectomy, 60% of children had persistent hypoglycaemia requiring medical interventions. The incidence of insulin dependent diabetes mellitus was 96% at 11 years after surgery. Thirty-two patients (72%) had biochemical evidence of severe pancreatic exocrine insufficiency (Faecal elastase 1

Mutations involving the insulin (INS) gene are a common cause of permanent neonatal diabetes (PND). Although INS mutations typically occur de novo and germline INS mutations transmitted to offspring by unaffected parents has been described, somatic mosaicism in a parent with an INS mutation has not been previously reported. We describe two siblings (one brother and one sister) with PND (26- and 19-yr old diagnosed at 3 and 7months old, respectively), whose parents were unaffected. We performed genetic analysis of leukocyte DNA for this family. Both patients were found to carry the novel heterozygous c.326G>A substitution in exon 3 of INS, resulting in a p.C109Y change of the insulin protein. Analyses of leukocyte DNA from the parents revealed low level mutation in the sequencing trace of the father, raising the possibility of somatic mosaicism. Real-time polymerase chain reaction (PCR) analysis showed he had approximately 73% of the mutant allele relative to his affected son. This first report of somatic mosaicism in an unaffected parent with an INS mutation suggests that parental mosaicism may be responsible for the transmission of PND in patients with de novo INS mutations. As such, appropriate counseling for recurrent risks should be considered and we recommend that molecular genetic testing for future siblings at birth should be offered to the parents of children with INS mutation. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Context Neonatal diabetes is a rare disorder with an incidence of about 1 in 100,000 live births. It is defined as diabetes diagnosed in the first 6 months of life and it is vital to differentiate this entity from type 1 diabetes to enable accurate diagnosis, prognosis, genetic counseling and treatment. Case report We describe a case of permanent neonatal diabetes mellitus due to a novel mutation affecting the ABCC8 gene that encodes the SUR1 subunit of potassium ATP channel (KATP).Conclusion This genetic diagnosis has therapeutic implications as patients can switch from insulin therapy to sulphonylurea, as described in this case report.

CONTEXT: Neonatal diabetes is a rare disorder with an incidence of about 1 in 100,000 live births. It is defined as diabetes diagnosed in the first 6 months of life and it is vital to differentiate this entity from type 1 diabetes to enable accurate diagnosis, prognosis, genetic counseling and treatment. CASE REPORT: We describe a case of permanent neonatal diabetes mellitus due to a novel mutation affecting the ABCC8 gene that encodes the SUR1 subunit of potassium ATP channel (KATP). CONCLUSION: This genetic diagnosis has therapeutic implications as patients can switch from insulin therapy to sulphonylurea, as described in this case report.

Many patients with monogenic diabetes are missed or misclassified. Herein, we report a 28-year-old Indian female who developed diabetes at the age of 3 months. An audit of our type 1 diabetes database led to her genetic testing. A KCNJ11 mutation was identified and she was successfully switched to sulphonylurea.

Many patients with monogenic diabetes are missed or misclassified. Herein, we report a 28-year-old Indian female who developed diabetes at the age of 3 months. An audit of our type 1 diabetes database led to her genetic testing. A KCNJ11 mutation was identified and she was successfully switched to sulphonylurea.

Mutations in glucokinase (GCK) cause a spectrum of glycemic disorders. Heterozygous loss-of-function mutations cause mild fasting hyperglycemia irrespective of mutation severity due to compensation from the unaffected allele. Conversely, homozygous loss-of-function mutations cause permanent neonatal diabetes requiring lifelong insulin treatment. This study aimed to determine the relationship between in vitro mutation severity and clinical phenotype in a large international case series of patients with homozygous GCK mutations. Clinical characteristics for 30 patients with diabetes due to homozygous GCK mutations (19 unique mutations, including 16 missense) were compiled and assigned a clinical severity grade (CSG) based on birth weight and age at diagnosis. The majority (28 of 30) of subjects were diagnosed before 9 months, with the remaining two at 9 and 15 years. These are the first two cases of a homozygous GCK mutation diagnosed outside infancy. Recombinant mutant GCK proteins were analyzed for kinetic and thermostability characteristics and assigned a relative activity index (RAI) or relative stability index (RSI) value. Six of 16 missense mutations exhibited severe kinetic defects (RAI ≤ 0.01). There was no correlation between CSG and RAI (r(2) = 0.05, P = 0.39), indicating that kinetics alone did not explain the phenotype. Eighty percent of the remaining mutations showed reduced thermostability, the exceptions being the two later-onset mutations which exhibited increased thermostability. Comparison of CSG with RSI detected a highly significant correlation (r(2) = 0.74, P = 0.002). We report the largest case series of homozygous GCK mutations to date and demonstrate that they can cause childhood-onset diabetes, with protein instability being the major determinant of mutation severity.

IMPORTANCE: Glycemic targets in diabetes have been developed to minimize complication risk. Patients with heterozygous, inactivating glucokinase (GCK) mutations have mild fasting hyperglycemia from birth, resulting in an elevated glycated hemoglobin (HbA1c) level that mimics recommended levels for type 1 and type 2 diabetes. OBJECTIVE: to assess the association between chronic, mild hyperglycemia and complication prevalence and severity in patients with GCK mutations. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional study in the United Kingdom between August 2008 and December 2010. Assessment of microvascular and macrovascular complications in participants 35 years or older was conducted in 99 GCK mutation carriers (median age, 48.6 years), 91 nondiabetic, familial, nonmutation carriers (control) (median age, 52.2 years), and 83 individuals with young-onset type 2 diabetes (YT2D), diagnosed at age 45 years or younger (median age, 54.7 years). MAIN OUTCOMES AND MEASURES: Prevalence and severity of nephropathy, retinopathy, peripheral neuropathy, peripheral vascular disease, and cardiovascular disease. RESULTS: Median HbA1c was 6.9% in patients with the GCK mutation, 5.8% in controls, and 7.8% in patients with YT2D. Patients with GCK had a low prevalence of clinically significant microvascular complications (1% [95% CI, 0%-5%]) that was not significantly different from controls (2% [95% CI, 0.3%-8%], P=.52) and lower than in patients with YT2D (36% [95% CI, 25%-47%], P

Wolcott–Rallison syndrome has been reported to be associated with early-onset diabetes, epiphyseal dysplasia, hepatic and renal dysfunction, mental retardation, severe growth retardation, neutropenia, exocrine pancreatic dysfunction, and central hypothyroidism. We report on primary hypothyroidism, which has not been previously described, of a patient with Wolcott–Rallison syndrome due to novel mutation (W521X), who showed improved growth after thyroid hormone treatment.

The contribution of cis-regulatory mutations to human disease remains poorly understood. Whole-genome sequencing can identify all noncoding variants, yet the discrimination of causal regulatory mutations represents a formidable challenge. We used epigenomic annotation in human embryonic stem cell (hESC)-derived pancreatic progenitor cells to guide the interpretation of whole-genome sequences from individuals with isolated pancreatic agenesis. This analysis uncovered six different recessive mutations in a previously uncharacterized ~400-bp sequence located 25 kb downstream of PTF1A (encoding pancreas-specific transcription factor 1a) in ten families with pancreatic agenesis. We show that this region acts as a developmental enhancer of PTF1A and that the mutations abolish enhancer activity. These mutations are the most common cause of isolated pancreatic agenesis. Integrating genome sequencing and epigenomic annotation in a disease-relevant cell type can thus uncover new noncoding elements underlying human development and disease.

BACKGROUND: Maturity-onset diabetes of the young (MODY) is uncommon; however, accurate diagnosis facilitates personalized management and informs prognosis in probands and relatives. OBJECTIVE: the objective of the study was to highlight that the appropriate use of genetic and nongenetic investigations leads to the correct classification of diabetes etiology. CASE DISCUSSION: a 30-year-old European female was diagnosed with insulin-treated gestational diabetes. She discontinued insulin after delivery; however, her fasting hyperglycemia persisted. β-Cell antibodies were negative and C-peptide was 0.79 nmol/L. Glucokinase (GCK)-MODY was suspected and confirmed by the identification of a GCK mutation (p.T206M). METHODS: Systematic clinical and biochemical characterization and GCK mutational analysis were implemented to determine the diabetes etiology in five relatives. Functional characterization of GCK mutations was performed. RESULTS: Identification of the p.T206M mutation in the proband's sister confirmed a diagnosis of GCK-MODY. Her daughter was diagnosed at 16 weeks with permanent neonatal diabetes (PNDM). Mutation analysis identified two GCK mutations that were inherited in trans-p. [(R43P);(T206M)], confirming a diagnosis of GCK-PNDM. Both mutations were shown to be kinetically inactivating. The proband's mother, other sister, and daughter all had a clinical diagnosis of type 1 diabetes, confirmed by undetectable C-peptide levels and β-cell antibody positivity. GCK mutations were not detected. CONCLUSIONS: Two previously misclassified family members were shown to have GCK-MODY, whereas another was shown to have GCK-PNDM. A diagnosis of type 1 diabetes was confirmed in three relatives. This family exemplifies the importance of careful phenotyping and systematic evaluation of relatives after discovering monogenic diabetes in an individual.

Hyperinsulinemic hypoglycemia is the most common cause of severe, persistent neonatal hypoglycemia. The treatment of hyperinsulinemic hypoglycemia that is unresponsive to diazoxide is subtotal pancreatectomy. We examined the effectiveness of the mammalian target of rapamycin (mTOR) inhibitor sirolimus in four infants with severe hyperinsulinemic hypoglycemia that had been unresponsive to maximal doses of diazoxide (20 mg per kilogram of body weight per day) and octreotide (35 μg per kilogram per day). All the patients had a clear glycemic response to sirolimus, although one patient required a small dose of octreotide to maintain normoglycemia. There were no major adverse events during 1 year of follow-up.

Increasing technological advances have resulted in the recognition of a range of genetic conditions not traditionally seen by clinical genetics teams. This has implications for the education of other healthcare professionals who may have insufficient knowledge to identify or support families with these conditions. The national genetic diabetes nurse (GDN) project, which trains diabetes specialist nurses (DSNs), was started in 2002 to increase awareness of monogenic diabetes among healthcare professionals across the UK. This paper describes the development and evaluation of the first 10 years of this project, indicating that GDNs have increased diagnostic referral rates and supported local families through diagnosis and treatment changes across the UK. The GDN project has proved an effective, innovative means of disseminating new genetic information from a centre of excellence and is suggested as a model for the successful and rapid dissemination of genetic information into routine clinical care in other conditions.

OBJECTIVE: Identifying glucokinase monogenic diabetes (GCK-MODY) in pregnancy is important, as management is different from management for other forms of gestational diabetes mellitus (GDM) and there is no increased maternal risk of type 2 diabetes. We calculated the population prevalence of GCK-MODY in pregnancy and determined the clinical characteristics that differentiate pregnant women with GCK-MODY from those with GDM. RESEARCH DESIGN AND METHODS: We calculated the population prevalence of GCK-MODY in pregnancy by testing a subset of patients from the population-based Atlantic Diabetes in Pregnancy (Atlantic DIP) study (n = 5,500). We sequenced for GCK mutations in 247 women with a fasting glucose ≥5.1 mmol/L and 109 randomly selected control subjects with normal fasting glucose. Using data from the cases found and 40 previously identified GCK-MODY pregnancies, we analyzed whether clinical criteria could be used to differentiate GCK-MODY from GDM. RESULTS: Four women with fasting glucose ≥5.1 mmol/L were diagnosed with GCK-MODY. No cases were identified with normal fasting glucose. The population prevalence of GCK-MODY is 1.1 in 1,000 (95% CI 0.3-2.9 in 1,000) and prevalence in GDM is 0.9% (95% CI 0.3-2.3). Fasting glucose and BMI significantly differentiate GCK-MODY from GDM (P < 0.0001). Combined criteria of BMI

BACKGROUND: Mutation specific effects in monogenic disorders are rare. We describe atypical Fanconi syndrome caused by a specific heterozygous mutation in HNF4A. Heterozygous HNF4A mutations cause a beta cell phenotype of neonatal hyperinsulinism with macrosomia and young onset diabetes. Autosomal dominant idiopathic Fanconi syndrome (a renal proximal tubulopathy) is described but no genetic cause has been defined. METHODS AND RESULTS: We report six patients heterozygous for the p.R76W HNF4A mutation who have Fanconi syndrome and nephrocalcinosis in addition to neonatal hyperinsulinism and macrosomia. All six displayed a novel phenotype of proximal tubulopathy, characterised by generalised aminoaciduria, low molecular weight proteinuria, glycosuria, hyperphosphaturia and hypouricaemia, and additional features not seen in Fanconi syndrome: nephrocalcinosis, renal impairment, hypercalciuria with relative hypocalcaemia, and hypermagnesaemia. This was mutation specific, with the renal phenotype not being seen in patients with other HNF4A mutations. In silico modelling shows the R76 residue is directly involved in DNA binding and the R76W mutation reduces DNA binding affinity. The target(s) selectively affected by altered DNA binding of R76W that results in Fanconi syndrome is not known. CONCLUSIONS: the HNF4A R76W mutation is an unusual example of a mutation specific phenotype, with autosomal dominant atypical Fanconi syndrome in addition to the established beta cell phenotype.

Background: Hepatocyte nuclear factor 4 alpha (HNF4A) gene mutations have a well-recognized role in maturity-onset diabetes of the young and have recently been described in congenital hyperinsulinism. A biphasic phenotype has been postulated, with macrosomia and congenital hyperinsulinism in infancy, and diabetes in young adulthood. In this case series, we report three children with HNF4A mutations (two de novo) and diazoxide-responsive congenital hyperinsulinism, highlighting the potential for ongoing diazoxide requirement and the importance of screening for these mutations even in the absence of family history. Case reports: all patients presented with macrosomia (mean birthweight 4.26 kg) and hyperinsulinaemic hypoglycaemia soon after birth (median age 1 day). All three (age range 7 months to 11 years 10 months) remain on diazoxide therapy, with dose requirements increasing in one patient. There was no prior family history of diabetes, neonatal hypoglycaemia or macrosomia. Parents were screened for HNF4A mutations post-diagnosis and one father was subsequently found to have maturity-onset diabetes of the young. Conclusions: This case series follows the evolving course of three patients with confirmed HNF4A-mediated congenital hyperinsulinism, highlighting (1) the variable natural history of these mutations, (2) the potential for prolonged diazoxide requirement, even into adolescence, and (3) the need for screening, regardless of family history. © 2013 Diabetes UK.

EGFR mutation testing of tumor samples is routinely performed to predict sensitivity to treatment with tyrosine kinase inhibitors for patients with non-small cell lung cancer. At least 9 different methodologies are employed in UK laboratories, and the aim of this study was to compare the sensitivity of different methods for the detection of EGFR mutations. Participating laboratories were sent coded samples with varying mutation loads (from 0% to 15%) to be tested for the p.Leu858Arg (p.L858R) missense mutation and c.2235_2249del exon 19 deletion. The p.L858R mutation and deletions within exon 19 of the EGFR gene account for ∼90% of mutation-positive cases. The 11 laboratories used their standard testing method(s) and submitted 15 sets of results for the p.L858R samples and 10 for the exon 19 deletion. The p.Leu858Arg (p.L858R) mutation was detected at levels between 1% and 7.5% by Sanger sequencing, pyrosequencing, real-time polymerase chain reaction (PCR), amplification refractory mutation system, and capillary electrophoresis single-strand conformation analysis. The c.2235_2249del mutation was detected at 1% to 5% by fragment size analysis, Sanger sequencing or real-time PCR. A mutation was detected in 24/25 (96%) of the samples tested which contained 5% mutated DNA. The 1% sensitivity claimed for commercial real-time PCR-targeted EGFR tests was achieved and our results show greater sensitivity for the Sanger sequencing and pyrosequencing screening methods compared to the 10% to 20% detection levels cited on clinical diagnostic reports. We conclude that multiple methodologies are suitable for the detection of acquired EGFR mutations.

Permanent neonatal diabetes mellitus is a rare condition mostly due to heterozygous mutations in the KCNJ11, ABCC8 and INS genes. Neonatal diabetes due to pancreatic agenesis is extremely rare. Mutations in PDX1, PTF1A, HNF1B, EIF2AK3, RFX6 and GATA6 genes have been shown to result in pancreatic agenesis or hypoplasia. This report describes a 40-day-old male infant diagnosed with permanent neonatal diabetes associated with atrial septal defect, pulmonary stenosis, patent ductus arteriosus and a novel de novo heterozygous missense mutation (p.N466S) in the GATA6 gene with no evidence of exocrine pancreas insufficiency. In addition to permanent neonatal diabetes, the patient had transient idiopathic neonatal cholestasis and hypoglycaemic episodes unrelated to insulin treatment, features that are rarely described in children with permanent neonatal diabetes. © 2013 Elsevier Masson SAS.

DNA polymerase δ, whose catalytic subunit is encoded by POLD1, is responsible for lagging-strand DNA synthesis during DNA replication. It carries out this synthesis with high fidelity owing to its intrinsic 3'- to 5'-exonuclease activity, which confers proofreading ability. Missense mutations affecting the exonuclease domain of POLD1 have recently been shown to predispose to colorectal and endometrial cancers. Here we report a recurring heterozygous single-codon deletion in POLD1 affecting the polymerase active site that abolishes DNA polymerase activity but only mildly impairs 3'- to 5'-exonuclease activity. This mutation causes a distinct multisystem disorder that includes subcutaneous lipodystrophy, deafness, mandibular hypoplasia and hypogonadism in males. This discovery suggests that perturbing the function of the ubiquitously expressed POLD1 polymerase has unexpectedly tissue-specific effects in humans and argues for an important role for POLD1 function in adipose tissue homeostasis. © 2013 Nature America, Inc. All rights reserved.

DNA polymerase δ, whose catalytic subunit is encoded by POLD1, is responsible for lagging-strand DNA synthesis during DNA replication. It carries out this synthesis with high fidelity owing to its intrinsic 3'- to 5'-exonuclease activity, which confers proofreading ability. Missense mutations affecting the exonuclease domain of POLD1 have recently been shown to predispose to colorectal and endometrial cancers. Here we report a recurring heterozygous single-codon deletion in POLD1 affecting the polymerase active site that abolishes DNA polymerase activity but only mildly impairs 3'- to 5'-exonuclease activity. This mutation causes a distinct multisystem disorder that includes subcutaneous lipodystrophy, deafness, mandibular hypoplasia and hypogonadism in males. This discovery suggests that perturbing the function of the ubiquitously expressed POLD1 polymerase has unexpectedly tissue-specific effects in humans and argues for an important role for POLD1 function in adipose tissue homeostasis.

BACKGROUND: Maturity-onset diabetes of the young (MODY) caused by heterozygous mutations in the glucokinase (GCK) gene typically presents with lifelong, stable, mild fasting hyperglycaemia. With the exception of pregnancy, patients with GCK-MODY usually do not require pharmacological therapy. We report two unrelated patients whose initial genetic test results indicated a deletion of GCK exon 10, but whose clinical phenotypes were not typical of GCK-MODY. CASE REPORTS: in case 1, the patient was hyperglycaemic at diagnosis (glucose > 30 mmol/l) and elevated glucose levels > 10 mmol/l persisted after withdrawal of insulin therapy. The patient in case 2 was also hyperglycaemic at diagnosis [HbA1c > 86 mmol/mol (10%)], which improved with the introduction of oral hypoglycaemic agents. These clinical features were not consistent with GCK-MODY. Both patients had a single nucleotide variant that prevented multiplex ligation-dependent probe analysis, which generated a false positive result of a GCK exon 10 deletion. CONCLUSION: False positive genetic results in these two unrelated cases were attributable to the presence of a rare single nucleotide variant that prevented ligation of the probe in the multiplex ligation-dependent probe analysis kit used and falsely indicated deletion of exon 10 within GCK. Both cases had clinical features that did not tally with the typical GCK-MODY phenotype. These cases emphasize the need to interpret the results of definitive genetic tests within the specific clinical context. Increased medical sequencing is likely to lead to more reports of novel mutations of uncertain significance. If genetic investigations do not agree with the clinical picture, clinicians should exercise caution when making therapeutic changes based on these results.

AIMS: Recessive PDX1 (IPF1) mutations are a rare cause of pancreatic agenesis, with three cases reported worldwide. A recent report described two cousins with a homozygous hypomorphic PDX1 mutation causing permanent neonatal diabetes with subclinical exocrine insufficiency. The aim of our study was to investigate the possibility of hypomorphic PDX1 mutations in a large cohort of patients with permanent neonatal diabetes and no reported pancreatic hypoplasia or exocrine insufficiency. METHODS: PDX1 was sequenced in 103 probands with isolated permanent neonatal diabetes in whom ABCC8, KCNJ11 and INS mutations had been excluded. RESULTS: Sequencing analysis identified biallelic PDX1 mutations in three of the 103 probands with permanent neonatal diabetes (2.9%). One proband and his affected brother were compound heterozygotes for a frameshift and a novel missense mutation (p.A34fsX191; c.98dupC and p.P87L; c.260C>T). The other two probands were homozygous for novel PDX1 missense mutations (p.A152G; c.455C>G and p.R176Q; c.527G>A). Both mutations affect highly conserved residues located within the homeobox domain. None of the four cases showed any evidence of exocrine pancreatic insufficiency, either clinically, or, where data were available, biochemically. In addition a heterozygous nonsense mutation (p.C18X; c.54C>A) was identified in a fourth case. CONCLUSIONS: This study demonstrates that recessive PDX1 mutations are a rare but important cause of isolated permanent neonatal diabetes in patients without pancreatic hypoplasia/agenesis. Inclusion of the PDX1 gene in mutation screening for permanent neonatal diabetes is recommended as a genetic diagnosis reveals the mode of inheritance, allows accurate estimation of recurrence risks and confirms the requirement for insulin treatment.

Background: Congenital hyperinsulinism (CHI) is a clinically heterogeneous condition. Mutations in eight genes (ABCC8, KCNJ11, GLUD1, GCK, HADH, SLC16A1, HNF4A and HNF1A) are known to cause CHI. Aim: to characterise the clinical and molecular aspects of a large cohort of patients with CHI. Methodology: Three hundred patients were recruited and clinical information was collected before genotyping. ABCC8 and KCNJ11 genes were analysed in all patients. Mutations in GLUD1, HADH, GCK and HNF4A genes were sought in patients with diazoxide-responsive CHI with hyperammonaemia (GLUD1), raised 3-hydroxybutyrylcarnitine and/or consanguinity (HADH), positive family history (GCK) or when CHI was diagnosed within the first week of life (HNF4A). Results: Mutations were identified in 136/300 patients (45.3%). Mutations in ABCC8/KCNJ11 were the commonest genetic cause identified (n=109, 36.3%). Among diazoxide-unresponsive patients (n=105), mutations in ABCC8/KCNJ11 were identified in 92 (87.6%) patients, of whom 63 patients had recessively inherited mutations while four patients had dominantly inherited mutations. A paternal mutation in the ABCC8/KCNJ11 genes was identified in 23 diazoxide-unresponsive patients, of whom six had diffuse disease. Among the diazoxide-responsive patients (n=183), mutations were identified in 41 patients (22.4%). These include mutations in ABCC8/KCNJ11 (n=15), HNF4A (n=7), GLUD1 (n=16) and HADH (n=3). Conclusions: a genetic diagnosis was made for 45.3% of patients in this large series. Mutations in the ABCC8 gene were the commonest identifiable cause. The vast majority of patients with diazoxideresponsive CHI (77.6%) had no identifiable mutations, suggesting other genetic and/or environmental mechanisms. © 2013 European Society of Endocrinology.

Next-generation sequencing (NGS) has significantly contributed to the transformation of genomic research by providing access to the genome for analysis, by significantly decreasing the sequencing costs and increasing the throughput. The next goal is to exploit this powerful technology in the clinic, namely for diagnostics and therapeutics. The 2013 annual meeting of the Human Genome Variation Society, held in Paris, France, provided a forum to discuss possible clinical applications of NGS, the potential of some of the current NGS systems to transition to the clinic, the identification of causative mutations for rare genetic disorders through whole-genome or targeted genome resequencing, the application of NGS for family genomics, and NGS data analysis tools. © 2013 WILEY PERIODICALS, INC.

Next-generation sequencing (NGS) has significantly contributed to the transformation of genomic research by providing access to the genome for analysis, by significantly decreasing the sequencing costs and increasing the throughput. The next goal is to exploit this powerful technology in the clinic, namely for diagnostics and therapeutics. The 2013 annual meeting of the Human Genome Variation Society, held in Paris, France, provided a forum to discuss possible clinical applications of NGS, the potential of some of the current NGS systems to transition to the clinic, the identification of causative mutations for rare genetic disorders through whole-genome or targeted genome resequencing, the application of NGS for family genomics, and NGS data analysis tools.

Congenital hyperinsulinism (CHI) is a common cause of hypoglycemia in infants. We report three cases of CHI with differing clinical, biochemical, and molecular genetic spectra. One patient was unresponsive to medical treatment and died after subtotal pancreatectomy because of complications due to the surgery. Two patients have been followed successfully with medical treatment. Early diagnosis and appropriate treatment of CHI are essential to prevent morbidity and mortality.

Over the last decade, we have witnessed major advances in the understanding of the molecular basis of neonatal and infancy-onset diabetes. It is now widely accepted that diabetes presenting before 6 months of age is unlikely to be autoimmune type 1 diabetes. The vast majority of such patients will have a monogenic disorder responsible for the disease and, in some of them, also for a number of other associated extrapancreatic clinical features. Reaching a molecular diagnosis will have immediate clinical consequences for about half of affected patients, as identification of a mutation in either of the two genes encoding the ATP-sensitive potassium channel allows switching from insulin injections to oral sulphonylureas. It also facilitates genetic counselling within the affected families and predicts clinical prognosis. Importantly, monogenic diabetes seems not to be limited to the first 6 months but extends to some extent into the second half of the first year of life, when type 1 diabetes is the more common cause of diabetes. From a scientific perspective, the identification of novel genetic aetiologies has provided important new knowledge regarding the development and function of the human pancreas. Copyright © 2013 S. Karger AG, Basel.

Monogenic diabetes due to mutations in the transcription factor genes hepatocyte nuclear factor 1A (HNF1A) and HNF4A is characterized by islet cell antibody negative, familial diabetes with residual insulin secretion. We report two sisters with childhood onset diabetes who are both heterozygous for the most common mutation in each of two transcription factors, HNF1A, and HNF4A. The proband was diagnosed with diabetes at 7 yr of age and treated with insulin for 4 yr. Her genetic diagnosis resulted in transition to sulfonylureas for one and a half years before insulin therapy was re-initiated due to declining glycemic control. Her sister was diagnosed with diabetes at 14 yr of age, treated initially with insulin but has been well controlled on oral sulfonylurea therapy for over 2 yr. Both sisters inherited the HNF4A gene mutation R127W from their mother and the HNF1A gene mutation P291fsinsC (c.872dup) from their father. The father was diagnosed with diabetes at 45 yr of age. Their brother is heterozygous for the HNF4A R127W mutation. Both the brother and mother have normal glucose tolerance at the ages of 16 and 46 yr, respectively. Digenic inheritance of HNF1A and HNF4A mutations is very rare and has only been reported in two families where conclusive evidence for the pathogenicity of their mutations was lacking. Follow-up studies in this family co-segregating the two most commonly reported HNF1A/HNF4A mutations will be informative for understanding the effect of digenic inheritance upon phenotypic severity and response to sulfonylurea therapy. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

We recently reported de novo GATA6 mutations as the most common cause of pancreatic agenesis, accounting for 15 of 27 (56%) patients with insulin-treated neonatal diabetes and exocrine pancreatic insufficiency requiring enzyme replacement therapy. We investigated the role of GATA6 mutations in 171 subjects with neonatal diabetes of unknown genetic etiology from a cohort of 795 patients with neonatal diabetes. Mutations in known genes had been confirmed in 624 patients (including 15 GATA6 mutations). Sequencing of the remaining 171 patients identified nine new case subjects (24 of 795, 3%). Pancreatic agenesis was present in 21 case subjects (six new); two patients had permanent neonatal diabetes with no enzyme supplementation and one had transient neonatal diabetes. Four parents with heterozygous GATA6 mutations were diagnosed with diabetes outside the neonatal period (12-46 years). Subclinical exocrine insufficiency was demonstrated by low fecal elastase in three of four diabetic patients who did not receive enzyme supplementation. One parent with a mosaic mutation was not diabetic but had a heart malformation. Extrapancreatic features were observed in all 24 probands and three parents, with congenital heart defects most frequent (83%). Heterozygous GATA6 mutations cause a wide spectrum of diabetes manifestations, ranging from pancreatic agenesis to adult-onset diabetes with subclinical or no exocrine insufficiency.

AIMS: Hepatocyte nuclear factor 1β (HNF1B) mutations cause a syndrome of renal cysts and diabetes, with whole gene deletions accounting for approximately 50% of cases. The severity of the renal phenotype is variable, from enlarged cystic kidneys incompatible with life to normal renal development and function. We investigated the prevalence of HNF1B deletions in patients with diabetes but no known renal disease. METHODS: We tested 461 patients with familial diabetes diagnosed before 45 years, including 258 probands who met clinical criteria for maturity-onset diabetes of the young (two generations affected and at least one family member diagnosed under 25 years). A fluorescent polymerase chain reaction assay was used to analyse two intragenic polymorphic HNF1B markers and identify heterozygous patients who therefore did not have whole gene deletions. Those patients homozygous for both markers were then tested for an HNF1B deletion using multiplex ligation-dependent probe amplification. RESULTS: Heterozygous HNF1B intragenic polymorphisms were identified in 337/461 subjects. Multiplex ligation-dependent probe amplification analysis showed an HNF1B gene deletion in three of the remaining 124 probands, all of whom met the criteria for maturity-onset diabetes of the young. Testing of their relatives identified three additional deletion carriers and ultrasound scanning showed renal developmental abnormalities in three of these six patients. CONCLUSIONS: We estimate that HNF1B mutations account for < 1% of cases of maturity-onset diabetes of the young. Although HNF1B mutations are a rare cause of diabetes in the absence of known renal disease, a genetic diagnosis of renal cysts and diabetes syndrome is important as it raises the possibility of subclinical renal disease and the 50% risk of renal cysts and diabetes syndrome in the patient's offspring.

Hepatoblastoma is a tumour of early childhood occurring in association with genetic syndromes including Beckwith-Wiedemann Syndrome (BWS) which results from dominance of paternally-inherited genes on chromosome 11p15. We report a child without clinical BWS, neonatally diagnosed with focal congenital hyperinsulinism resulting from a paternally-inherited recessively-acting mutation of ABCC8 and pancreatic paternal uniparental disomy (UPD) for chromosome 11p15, who subsequently developed hepatoblastoma. Genetic testing showed UPD 11p15 in the pancreas and liver but not systemically, allowing the expression of mutated ABCC8 in both tissues. Infants with large or multifocal forms of focal congenital hyperinsulinism may be at risk of BWS-like tumours due to mosaic UPD despite negative whole-blood and buccal DNA testing and tumour surveillance should be considered for this minority. © 2013 Elsevier Masson SAS.

Thiamine-responsive megaloblastic anemia (TRMA) is an autosomal recessive syndrome characterized by early-onset anemia, diabetes, and hearing loss caused by mutations in the SLC19A2 gene. We studied the genetic cause and clinical features of this condition in patients from the Persian population. A clinical and molecular investigation was performed in four patients from three families and their healthy family members. All had the typical diagnostic criteria. The onset of hearing loss in three patients was at birth and one patient also had a stroke and seizure disorder. Thiamine treatment effectively corrected the anemia in all of our patients but did not prevent hearing loss. Diabetes was improved in one patient who presented at the age of 8. months with anemia and diabetes after 2. months of starting thiamine. The coding regions of SLC19A2 were sequenced in all patients. The identified mutation was tested in all members of the families. Molecular analyses identified a homozygous nonsense mutation c.697C. >. T (p.Gln233*) as the cause of the disease in all families. This mutation was previously reported in a Turkish patient with TRMA and is likely to be a founder mutation in the Persian population. © 2013 Elsevier B.V.

Aims/hypothesis: Current genetic tests for diagnosing monogenic diabetes rely on selection of the appropriate gene for analysis according to the patient's phenotype. Next-generation sequencing enables the simultaneous analysis of multiple genes in a single test. Our aim was to develop a targeted next-generation sequencing assay to detect mutations in all known MODY and neonatal diabetes genes. Methods: We selected 29 genes in which mutations have been reported to cause neonatal diabetes, MODY, maternally inherited diabetes and deafness (MIDD) or familial partial lipodystrophy (FPLD). An exon-capture assay was designed to include coding regions and splice sites. A total of 114 patient samples were tested - 32 with known mutations and 82 previously tested for MODY (n = 33) or neonatal diabetes (n = 49) but in whom a mutation had not been found. Sequence data were analysed for the presence of base substitutions, small insertions or deletions (indels) and exonic deletions or duplications. Results: in the 32 positive controls we detected all previously identified variants (34 mutations and 36 polymorphisms), including 55 base substitutions, ten small insertions or deletions and five partial/whole gene deletions/duplications. Previously unidentified mutations were found in five patients with MODY (15%) and nine with neonatal diabetes (18%). Most of these patients (12/14) had mutations in genes that had not previously been tested. Conclusions/interpretation: Our novel targeted next-generation sequencing assay provides a highly sensitive method for simultaneous analysis of all monogenic diabetes genes. This single test can detect mutations previously identified by Sanger sequencing or multiplex ligation-dependent probe amplification dosage analysis. The increased number of genes tested led to a higher mutation detection rate. © 2013 the Author(s).

AIMS/HYPOTHESIS: Current genetic tests for diagnosing monogenic diabetes rely on selection of the appropriate gene for analysis according to the patient's phenotype. Next-generation sequencing enables the simultaneous analysis of multiple genes in a single test. Our aim was to develop a targeted next-generation sequencing assay to detect mutations in all known MODY and neonatal diabetes genes. METHODS: We selected 29 genes in which mutations have been reported to cause neonatal diabetes, MODY, maternally inherited diabetes and deafness (MIDD) or familial partial lipodystrophy (FPLD). An exon-capture assay was designed to include coding regions and splice sites. A total of 114 patient samples were tested--32 with known mutations and 82 previously tested for MODY (n = 33) or neonatal diabetes (n = 49) but in whom a mutation had not been found. Sequence data were analysed for the presence of base substitutions, small insertions or deletions (indels) and exonic deletions or duplications. RESULTS: in the 32 positive controls we detected all previously identified variants (34 mutations and 36 polymorphisms), including 55 base substitutions, ten small insertions or deletions and five partial/whole gene deletions/duplications. Previously unidentified mutations were found in five patients with MODY (15%) and nine with neonatal diabetes (18%). Most of these patients (12/14) had mutations in genes that had not previously been tested. CONCLUSIONS/INTERPRETATION: Our novel targeted next-generation sequencing assay provides a highly sensitive method for simultaneous analysis of all monogenic diabetes genes. This single test can detect mutations previously identified by Sanger sequencing or multiplex ligation-dependent probe amplification dosage analysis. The increased number of genes tested led to a higher mutation detection rate.

Maturity-onset diabetes of the young (MODY) is a monogenic disorder that results in a familial, young-onset non-insulin dependent form of diabetes, typically presenting in lean young adults before 25 years. Approximately 1% of diabetes has a monogenic cause but this is frequently misdiagnosed as Type 1 or Type 2 diabetes. A correct genetic diagnosis is important as it often leads to improved treatment for those affected with diabetes and enables predictive genetic testing for their asymptomatic relatives. An early diagnosis together with appropriate treatment is essential for reducing the risk of diabetic complications in later life. Mutations in the GCK and HNF1A/4 A genes account for up to 80% of all MODY cases. Mutations in the GCK gene cause a mild, asymptomatic and non-progressive fasting hyperglycaemia from birth usually requiring no treatment. In contrast, mutations in the genes encoding the transcription factors HNF1A and HNF4A cause a progressive insulin secretory defect and hyperglycaemia that can lead to vascular complications. The diabetes in these patients is usually well controlled with sulphonylurea tablets although insulin treatment may be required in later life. In this review, we outline the key clinical and laboratory characteristics of the common and rarer causes of MODY with the aim of raising awareness of this condition amongst health-care scientists.

Large scale genetic studies such as genome-wide association studies (GWAS) in Parkinson's disease (PD) have revealed genetic susceptibility factors and continue to offer new insights both into the genetics of sporadic disease and its pathogenesis, with the potential for identification of an at-risk population and novel therapeutic targets. However, the methodology importantly requires larger data sets for replication of novel findings.

Spondylocostal dysotosis (SCD) is a rare developmental congenital abnormality of the axial skeleton. Mutation of genes in the Notch signaling pathway cause SCD types 1-5. Dextrocardia with situs inversus is a rare congenital malformation in which the thoracic and abdominal organs are mirror images of normal. Such laterality defects are associated with gene mutations in the Nodal signaling pathway or cilia assembly or function. We investigated two distantly related individuals with a rare combination of severe segmental defects of the vertebrae (SDV) and dextrocardia with situs inversus. We found that both individuals were homozygous for the same mutation in HES7, and that this mutation caused a significant reduction of HES7 protein function; HES7 mutation causes SCD4. Two other individuals with SDV from two unrelated families were found to be homozygous for the same mutation. Interestingly, although the penetrance of the vertebral defects was complete, only 3/7 had dextrocardia with situs inversus, suggesting randomization of left-right patterning. Two of the affected individuals presented with neural tube malformations including myelomeningocele, spina bifida occulta and/or Chiari II malformation. Such neural tube phenotypes are shared with the originally identified SCD4 patient, but have not been reported in the other forms of SCD. In conclusion, it appears that mutation of HES7 is uniquely associated with defects in vertebral, heart and neural tube formation, and this observation will help provide a discriminatory diagnostic guide in patients with SCD, as well as inform molecular genetic testing. © 2013 Wiley Periodicals, Inc.

Maturity-onset diabetes of the young (MODY) is a monogenic disorder characterized by autosomal dominant inheritance of young-onset (typically

Maturity-onset diabetes of the young (MODY) is a monogenic disorder characterized by autosomal dominant inheritance of young-onset (typically

Next-generation sequencing (NGS) enables analysis of the human genome on a scale previously unachievable by Sanger sequencing. Exome sequencing of the coding regions and conserved splice sites has been very successful in the identification of disease-causing mutations, and targeting of these regions has extended clinical diagnostic testing from analysis of fewer than ten genes per phenotype to more than 100. Noncoding mutations have been less extensively studied despite evidence from mRNA analysis for the existence of deep intronic mutations in >20 genes. We investigated individuals with hyperinsulinaemic hypoglycaemia and biochemical or genetic evidence to suggest noncoding mutations by using NGS to analyze the entire genomic regions of ABCC8 (117 kb) and HADH (94 kb) from overlapping ~10 kb PCR amplicons. Two deep intronic mutations, c.1333-1013A>G in ABCC8 and c.636+471G>T HADH, were identified. Both are predicted to create a cryptic splice donor site and an out-of-frame pseudoexon. Sequence analysis of mRNA from affected individuals' fibroblasts or lymphoblastoid cells confirmed mutant transcripts with pseudoexon inclusion and premature termination codons. Testing of additional individuals showed that these are founder mutations in the Irish and Turkish populations, accounting for 14% of focal hyperinsulinism cases and 32% of subjects with HADH mutations in our cohort. The identification of deep intronic mutations has previously focused on the detection of aberrant mRNA transcripts in a subset of disorders for which RNA is readily obtained from the target tissue or ectopically expressed at sufficient levels. Our approach of using NGS to analyze the entire genomic DNA sequence is applicable to any disease.

BACKGROUND: Congenital hyperinsulinism (CHI) is a rare genetic disorder characterised by inappropriate insulin secretion in the face of severe hypoglycaemia. There are two histological subtypes of CHI namely diffuse and focal. Diffuse CHI is most common due to recessive mutations in ABCC8/KCNJ11 (which encode the SUR/KIR6.2 components of the pancreatic β-cell KATP channel) whereas focal CHI is due to a paternally inherited ABCC8/KCNJ11 mutation and somatic loss of heterozygosity for the 11p allele inside the focal lesion. Fluorine-18-l-dihydroxyphenylalanine positron emission tomography/computed tomography ((18)F-DOPA-PET/CT) is used in the pre-operative localisation of focal lesions prior to surgery. Diffuse CHI if medically unresponsive will require a near total pancreatectomy whereas focal CHI will only require a limited lesionectomy, thus curing the patient from the hypoglycaemia. AIMS: to report the first case of genetically confirmed CHI in Singapore from a heterozygous paternally inherited ABCC8 mutation. METHODS/RESULTS: a term male infant presented with severe hyperinsulinaemic hypoglycaemia (HH) after birth and failed medical treatment with diazoxide and octreotide. Genetic testing (paternally inherited mutation in ABCC8/p.D1472N) suggested focal disease, but due to the unavailability of (18)F-DOPA-PET/CT to confirm focal disease, a partial pancreatectomy was performed. Interestingly, histology of the resected pancreatic tissue showed changes typical of diffuse disease. CONCLUSION: Heterozygous paternally inherited ABCC8/KCNJ11 mutations can lead to diffuse or focal CHI. LEARNING POINTS: HH is a cause of severe hypoglycaemia in the newborn period.Paternal mutations in ABCC8/KCNJ11 can lead to diffuse or focal disease.(18)F-DOPA-PET/CT scan is the current imaging of choice for localising focal lesions.Gallium-68 tetra-aza-cyclododecane-N N'N″N-‴-tetra-acetate octreotate PET scan is not a useful imaging tool for localising focal lesions.The molecular mechanism by which a heterozygous ABCC8 mutation leads to diffuse disease is currently unclear.Focal lesions are curable by lesionectomy and so genetic studies in patients with HH must be followed by imaging using (18)F-DOPA-PET/CT scan.

Background: Neonatal diabetes mellitus (NDM) is defined as diabetes with onset before 6months of age. Nearly half of individuals with NDM are affected by permanent neonatal diabetes mellitus (PNDM). Mutations in KATP channel genes (KCNJ11, ABCC8) and the insulin gene (INS) are the most common causes of PNDM. Objective: to estimate the prevalence of PNDM among SEARCH for Diabetes in Youth (SEARCH) study participants (2001-2008) and to identify the genetic mutations causing PNDM. Methods: SEARCH is a multicenter population-based study of diabetes in youth

Congenital hyperinsulinism (CHI) is a rare cause of hyperinsulinaemic hypoglycaemia (HH) and is due to an inappropriate secretion of insulin by the pancreatic β-cells. Genetic defects in key genes lead to dysregulated insulin secretion and consequent hypoglycaemia. Mutations in the genes ABCC8/KCNJ11, encoding SUR1/Kir6.2 components of the KATP channels, respectively, are the commonest cause of CHI. A 33+6 week gestation male infant weighing 3.38 kg (above 90th centile) presented with severe neonatal symptomatic hypoglycaemia. He required a glucose infusion rate of 20 mg/kg/min to maintain normoglycaemia (blood glucose levels at >3.5 mmol/l). Investigations established the diagnosis of HH (blood glucose 2.2 mmol/l with simultaneous insulin of 97.4 mU/l). Subsequent molecular genetic studies identified a heterozygous pathogenic ABCC8 missense mutation, p.R1353H (c.4058G>A), inherited from an unaffected mother. His HH was diazoxide responsive and resolved within 3 months of life. Copyright 2013 BMJ Publishing Group. All rights reserved.

AIMS: Our study aims were to determine the frequency of MODY mutations (HNF1A, HNF4A, glucokinase) in a diverse population of youth with diabetes and to assess how well clinical features identify youth with maturity-onset diabetes of the young (MODY). METHODS: the SEARCH for Diabetes in Youth study is a US multicenter, population-based study of youth with diabetes diagnosed at age younger than 20 years. We sequenced genomic DNA for mutations in the HNF1A, HNF4A, and glucokinase genes in 586 participants enrolled in SEARCH between 2001 and 2006. Selection criteria included diabetes autoantibody negativity and fasting C-peptide levels of 0.8 ng/mL or greater. RESULTS: We identified a mutation in one of three MODY genes in 47 participants, or 8.0% of the tested sample, for a prevalence of at least 1.2% in the pediatric diabetes population. of these, only 3 had a clinical diagnosis of MODY, and the majority was treated with insulin. Compared with the MODY-negative group, MODY-positive participants had lower FCP levels (2.2 ± 1.4 vs 3.2 ± 2.1 ng/mL, P <. 01) and fewer type 2 diabetes-like metabolic features. Parental history of diabetes did not significantly differ between the 2 groups. CONCLUSIONS/INTERPRETATION: in this systematic study of MODY in a large pediatric US diabetes cohort, unselected by referral pattern or family history, MODY was usually misdiagnosed and incorrectly treated with insulin. Although many type 2 diabetes-like metabolic features were less common in the mutation-positive group, no single characteristic identified all patients with mutations. Clinicians should be alert to the possibility of MODY diagnosis, particularly in antibody-negative youth with diabetes.

Thiamine responsive megaloblastic anemia (TRMA) is an autosomal recessive disease caused by loss of function mutations in the SLC19A2 gene. TRMA is characterized by anemia, deafness, and diabetes. In some cases, optic atrophy or more rarely retinitis pigmentosa is noted. We now report two sisters, the eldest of which presented to a different hospital during childhood with sensorineural deafness, which was treated with a hearing prosthesis, insulin requiring diabetes, retinitis pigmentosa, optic atrophy, and macrocytic anemia. These features initially suggested a clinical diagnosis of Wolfram syndrome (WS). Therapy with thiamine was initiated which resulted in the resolution of the anemia. The younger sister, who was affected with sensorineural deafness, was referred to our hospital for non-autoimmune diabetes. She was found to have macrocytosis and ocular abnormalities. Because a diagnosis of TRMA was suspected, therapy with insulin and thiamine was started. Sequencing analysis of the SLC19A2 gene identified a compound heterozygous mutation p.Y81X/p.L457X (c.242insA/c.1370delT) in both sisters. Non-autoimmune diabetes associated with deafness and macrocytosis, without anemia, suggests a diagnosis of TRMA. Patients clinically diagnosed with WS with anemia and/or macrocytosis should be reevaluated for TRMA. © 2012 John Wiley & Sons A/S.

AIMS: HaemoglobinA1c (HbA1c) is recommended for diabetes diagnosis but fasting plasma glucose (FPG) has been useful for identifying patients with glucokinase (GCK) mutations which cause lifelong persistent fasting hyperglycaemia. We aimed to derive age-related HbA1c reference ranges for these patients to determine how well HbA1c can discriminate patients with a GCK mutation from unaffected family members and young-onset type 1 (T1D) and type 2 diabetes (T2D) and to investigate the proportion of GCK mutation carriers diagnosed with diabetes using HbA1c and/or FPG diagnostic criteria. METHODS: Individuals with inactivating GCK mutations (n = 129), familial controls (n = 100), T1D (n = 278) and T2D (n = 319) aged ≥18years were recruited. Receiver Operating Characteristic (ROC) analysis determined effectiveness of HbA1c and FPG to discriminate between groups. RESULTS: HbA1c reference ranges in subjects with GCK mutations were: 38-56 mmol/mol (5.6-7.3%) if aged ≤40years; 41-60 mmol/mol (5.9-7.6%) if >40years. All patients (123/123) with a GCK mutation were above the lower limit of the HbA1c age-appropriate reference ranges. 69% (31/99) of controls were below these lower limits. HbA1c was also effective in discriminating those with a GCK mutation from those with T1D/T2D. Using the upper limit of the age-appropriate reference ranges to discriminate those with a mutation from those with T1D/T2D correctly identified 97% of subjects with a mutation. The majority (438/597 (73%)) with other types of young-onset diabetes had an HbA1c above the upper limit of the age-appropriate GCK reference range. HbA1c ≥48 mmol/mol classified more people with GCK mutations as having diabetes than FPG ≥7 mmol/l (68% vs. 48%, p = 0.0009). CONCLUSIONS: Current HbA1c diagnostic criteria increase diabetes diagnosis in patients with a GCK mutation. We have derived age-related HbA1c reference ranges that can be used for discriminating hyperglycaemia likely to be caused by a GCK mutation and aid identification of probands and family members for genetic testing.

Wolcott-Rallison syndrome (WRS) is a rare condition characterized by permanent neonatal diabetes (PND), skeletal dysplasia, and recurrent hepatitis. Other features, including central hypothyroidism, have been reported. We compared the phenotype of five patients from two families with WRS caused by the same EIF2AK3 mutation who have been followed up since diagnosis. Direct sequencing of the EIF2AK3 gene identified a homozygous frameshift mutation (c.1259delA) in all patients that has been reported only in these families. All patients presented with PND and four experienced recurrent hepatitis. A 3.5-year-old girl has isolated PND, whereas her younger sister has typical WRS features. Two children developed skeletal abnormalities and two had transient central hypothyroidism. Other reported features of WRS were not detected. The EIF2AK3 c.1259delA mutation results in a variable phenotype, ranging from isolated PND to typical WRS. Thyroid dysfunction in WRS is a transient phenomenon refl ecting euthyroid sickness. © 2013 by Walter de Gruyter.

We describe a new syndrome of young onset diabetes, short stature and microcephaly with intellectual disability in a large consanguineous family with three affected children. Linkage analysis and whole exome sequencing were used to identify the causal nonsense mutation, which changed an arginine codon into a stop at position 127 of the tRNA methyltransferase homolog gene TRMT10A (also called RG9MTD2). TRMT10A mRNA and protein were absent in lymphoblasts from the affected siblings. TRMT10A is ubiquitously expressed but enriched in brain and pancreatic islets, consistent with the tissues affected in this syndrome. In situ hybridization studies showed that TRMT10A is expressed in human embryonic and fetal brain. TRMT10A is the mammalian ortholog of S. cerevisiae TRM10, previously shown to catalyze the methylation of guanine 9 (m(1)G9) in several tRNAs. Consistent with this putative function, in silico topology prediction indicated that TRMT10A has predominant nuclear localization, which we experimentally confirmed by immunofluorescence and confocal microscopy. TRMT10A localizes to the nucleolus of β- and non-β-cells, where tRNA modifications occur. TRMT10A silencing induces rat and human β-cell apoptosis. Taken together, we propose that TRMT10A deficiency negatively affects β-cell mass and the pool of neurons in the developing brain. This is the first study describing the impact of TRMT10A deficiency in mammals, highlighting a role in the pathogenesis of microcephaly and early onset diabetes. In light of the recent report that the type 2 diabetes candidate gene CDKAL1 is a tRNA methylthiotransferase, the findings in this family suggest broader relevance of tRNA methyltransferases in the pathogenesis of type 2 diabetes.

Alagille syndrome (ALGS), also known as arteriohepatic dysplasia, is a multisystem disorder due to defects in components of the Notch signalling pathway, most commonly due to mutation in JAG1 (ALGS type 1), but in a small proportion of cases mutation in NOTCH2 (ALGS type 2). The main clinical and pathological features are chronic cholestasis due to paucity of intrahepatic bile ducts, peripheral pulmonary artery stenosis, minor vertebral segmentation anomalies, characteristic facies, posterior embryotoxon/anterior segment abnormalities, pigmentary retinopathy, and dysplastic kidneys. It follows autosomal dominant inheritance, but reduced penetrance and variable expression are common in this disorder, and somatic/germline mosaicism may also be relatively frequent. This review discusses the clinical features of ALGS, including long-term complications, the clinical and molecular diagnosis, and management.

BACKGROUND: Mutations in the KCNJ11 gene, which encodes the Kir6.2 subunit of the β-cell K(ATP) channel, are a common cause of neonatal diabetes. The diabetes may be permanent neonatal diabetes mellitus (PNDM) or transient neonatal diabetes mellitus (TNDM), and in ≈ 20% of patients, neurological features are observed. A correlation between the position of the mutation in the protein and the clinical phenotype has previously been described; however, recently, this association has become less distinct with different mutations at the same residues now reported in patients with different diabetic and/or neurological phenotypes. METHODS: We identified from the literature, and our unpublished series, KCNJ11 mutations that affected residues harbouring various amino acid substitutions (AAS) causing differences in diabetic or neurological status. Using the Grantham amino acid scoring system, we investigated whether the difference in properties between the wild-type and the different AAS at the same residue could predict phenotypic severity. RESULTS: Pair-wise analysis demonstrated higher Grantham scores for mutations causing PNDM or diabetes with neurological features when compared with mutations affecting the same residue that causes TNDM (P=0.013) or diabetes without neurological features (P=0.016) respectively. In just five of the 25 pair-wise analyses, a lower Grantham score was observed for the more severe phenotype. In each case, the wild-type residue was glycine, the simplest amino acid. CONCLUSION: This study demonstrates the importance of the specific AAS in determining phenotype and highlights the potential utility of the Grantham score for predicting phenotypic severity for novel KCNJ11 mutations affecting previously mutated residues.

OBJECTIVE: in women with hyperglycemia due to heterozygous glucokinase (GCK) mutations, the fetal genotype determines its growth. If the fetus inherits the mutation, birth weight is normal when maternal hyperglycemia is not treated, whereas intensive treatment may adversely reduce fetal growth. However, fetal genotype is not usually known antenatally, making treatment decisions difficult. HISTORY AND EXAMINATION: We report two women with gestational diabetes mellitus resulting from GCK mutations with hyperglycemia sufficient to merit treatment. INVESTIGATION: in both women, DNA from chorionic villus sampling, performed after high-risk aneuploidy screening, showed the fetus had inherited the GCK mutation. Therefore, maternal hyperglycemia was not treated. Both offspring had a normal birth weight and no peripartum complications. CONCLUSIONS: in pregnancies where the mother has hyperglycemia due to a GCK mutation, knowing the fetal GCK genotype guides the management of maternal hyperglycemia. Fetal genotyping should be performed when fetal DNA is available from invasive prenatal diagnostic testing.

AIM: to describe the clinical and radiological features of four new families with a childhood presentation of COL4A1 mutation. METHOD: We retrospectively reviewed the clinical presentation. Investigations included radiological findings and COL4A1 mutation analysis of the four cases. Affected family members were identified. COL4A1 mutation analysis was performed in all index cases and, where possible, in affected family members. RESULTS: the three male and one female index cases presented with recurrent childhood-onset stroke, infantile hemiplegia/spastic quadriplegia, and infantile spasms. Additional features such as congenital cataracts and anterior segment dysgenesis were present. Microcephaly and developmental delay/learning difficulties were present in three cases. Three cases had one or more family member affected in multiple generations, with a total of 11 such individuals identified. The clinical features showed a wide intrafamilial variation. Magnetic resonance imaging (MRI) showed bilateral white matter change in all cases, except in one mutation-positive family member. Unilateral or bilateral porencephaly was present in cases with infantile hemiplegia, and a diagnosis of clinical stroke was supported by the presence of intracerebral haemorrhage. The age at diagnosis was between 1 year and 6 years for the children with presentation in infancy and 12 months after stroke in a 14-year-old male. Three new pathogenic mutations were identified in the COL4A1 gene. INTERPRETATION: COL4A1 mutations can present in children with infantile hemiplegia/quadriplegia, stroke or epilepsy, and a motor disorder. The presence of eye features and white matter change on MRI in childhood can help point towards the diagnosis. Once the diagnosis is made, a careful search can identify affected family members.

We report a series of patients with transient neonatal diabetes mellitus (TNDM). Paternal uniparental isodisomy of chromosome 6 and heterozygous KCNJ11 and ABC88 mutation were the mutations found. This first reported series of Brazilian patients expands the geographical data on TNDM contributing to better understanding of its pathophysiology. © 2012.

We describe a case of neonatal diabetes due to a homozygous mutation (c.3 G>T) at the INS gene, leading to lack of insulin expression and severe hyperglycemia from day one of life requiring permanent insulin replacement therapy. The genetic loss of endogenous insulin production likely led to lack of immune tolerance to insulin, with resultant autoantibody production against exogenous insulin and progressive immune-mediated lipoatrophy at injection sites. © 2011 John Wiley & Sons A/S.

We describe a case of neonatal diabetes due to a homozygous mutation (c.3 G>T) at the INS gene, leading to lack of insulin expression and severe hyperglycemia from day one of life requiring permanent insulin replacement therapy. The genetic loss of endogenous insulin production likely led to lack of immune tolerance to insulin, with resultant autoantibody production against exogenous insulin and progressive immune-mediated lipoatrophy at injection sites.

BACKGROUND: Galactokinase catalyses the first committed step in galactose metabolism, the conversion of galactose to galactose-1-phosphate. Galactokinase deficiency is an extremely rare form of galactosaemia, and the most frequent complication reported is cataracts. Congenital hyperinsulinism (CHI) is a cause of severe hypoglycaemia in the newborn period. Galactosaemia has not previously been reported in a neonate with concomitant CHI. AIMS: to report the first case of a patient with CHI and galactokinase deficiency, and to describe the diagnostic pitfalls with bedside blood glucose testing in a neonate with combined galactokinase deficiency and CHI. PATIENTS/METHODS: a 3-day-old baby girl from consanguineous parents presented with poor feeding, irritability and seizures. Capillary blood glucose testing using bedside test strips and glucometer showed a glucose level of 18 mmol/L, but the actual laboratory blood glucose level was only 1.8 mmol/L. After discontinuation of oral feeding (stopping provision of dietary galactose), the bedside capillary blood glucose correlated with laboratory glucose concentrations. RESULTS: Biochemically the patient had CHI (blood glucose level 2.3 mmol/L with simultaneous serum insulin level of 30 mU/L) and galactokinase deficiency (elevated serum galactose level 0.62 μmol/L). Homozygous loss of function mutations in ABCC8 and GALK1 were found, which explained the patient's CHI and galactokinase deficiency, respectively. CONCLUSION: This is the first reported case of CHI and galactokinase deficiency occurring in the same patient. Severe hypoglycaemia in neonates with CHI may go undetected with bedside blood glucose meters in patients with galactokinase deficiency.

We report on an adult woman with rare coexistence of acromegaly, pheochromocytoma (PHEO), gastrointestinal stromal tumor (GIST), intestinal polyposis, and thyroid follicular adenoma. At the age of 56, she was diagnosed with acromegaly caused by a pituitary macroadenoma, treated by transsphenoidal surgery, radiotherapy, and octreotide. During routine colonoscopy, multiple polyps were identified as tubular adenomas with high-grade dysplasia on histology. Years later, an abdominal mass of 8.0 x 6.2 cm was detected by routine ultrasound. Surgical exploration revealed an adrenal mass and another tumor adhered to the lesser gastric curvature, which were removed. Pathology confirmed the diagnosis of PHEO and GIST. PHEO immunohistochemistry was negative for GHRH. During follow-up, nodular goiter was found with normal levels of calcitonin and inconclusive cytology. Near-total thyroidectomy was performed, revealing a follicular adenoma. Her family history was negative for all of these tumor types. Genetic analysis for PHEO/paraganglioma genes (SDH A-D, SDHAF2, RET, VHL, TMEM127, and MAX), and pituitary-related genes (AIP, MEN1, and p27) were negative. Though the finding of PHEO and acromegaly with multiple other tumors could be a fortuitous coexistence, we suggest that this case may represent a new variant of MEN syndrome with a de novo germline mutation in a not yet identified gene. © ABEM todos os direitos reservados.

AIMS/HYPOTHESIS: the ABCC8 gene encodes the sulfonylurea receptor 1 (SUR1) subunit of the pancreatic beta cell ATP-sensitive potassium (K(ATP)) channel. Inactivating mutations cause congenital hyperinsulinism (CHI) and activating mutations cause transient neonatal diabetes (TNDM) or permanent neonatal diabetes (PNDM) that can usually be treated with sulfonylureas. Sulfonylurea sensitivity is also a feature of HNF1A and HNF4A MODY, but patients referred for genetic testing with clinical features of these types of diabetes do not always have mutations in the HNF1A/4A genes. Our aim was to establish whether mutations in the ABCC8 gene cause MODY that is responsive to sulfonylurea therapy. METHODS: We sequenced the ABCC8 gene in 85 patients with a BMI

Background: Permanent neonatal diabetes mellitus (PNDM) in European population has an incidence of at least 1 in 260 000 live births and is most commonly due to mutations in KCNJ11 and ABCC8. However, data on this condition in other populations are limited. Objective: to define the incidence, genetic aetiology, and clinical phenotype of PNDM in Al-Madinah region, northwest Saudi Arabia. Methods: Patients with PNDM diagnosed between 2001 and 2010 were identified and clinically phenotyped. Sequencing of KCNJ11, ABCC8, and INS were performed initially on all subjects, and EIF2AK3, GLIS3, SLC2A2, SLC19A2, GCK, IPF1, and NEUROD1 genes were sequenced according to the clinical phenotype. Results: in total, 17 patients from 11 consanguineous families were diagnosed with PNDM and the incidence was 1 in 21 196 live births. Six different mutations in four genes were identified, of which two GLIS3 and one SLC2A2 were novel and no patient had KCNJ11, ABCC8, or INS mutations. Fourteen (82.4%) patients had identifiable genetic aetiology and their PNDM was part of known autosomal-recessive syndromes including Wolcott Rallison (41.1%), neonatal diabetes and hypothyroidism (29.4%), Fanconi-Bickel (5.8%), and thiamine-responsive megaloblastic anaemia (5.8%). Two patients with isolated PNDM and one with intermediate developmental delay, epilepsy and neonatal diabetes had no identifiable cause. Conclusions: Al-Madinah region has the highest reported incidence of PNDM worldwide. In this region with high consanguinity, PNDM has different genetic aetiology and in the majority of cases presents as a part of rare familial autosomal-recessive syndrome rather than in isolation. © 2011 John Wiley & Sons A/S.

BACKGROUND: Permanent neonatal diabetes mellitus (PNDM) in European population has an incidence of at least 1 in 260 000 live births and is most commonly due to mutations in KCNJ11 and ABCC8. However, data on this condition in other populations are limited. OBJECTIVE: to define the incidence, genetic aetiology, and clinical phenotype of PNDM in Al-Madinah region, northwest Saudi Arabia. METHODS: Patients with PNDM diagnosed between 2001 and 2010 were identified and clinically phenotyped. Sequencing of KCNJ11, ABCC8, and INS were performed initially on all subjects, and EIF2AK3, GLIS3, SLC2A2, SLC19A2, GCK, IPF1, and NEUROD1 genes were sequenced according to the clinical phenotype. RESULTS: in total, 17 patients from 11 consanguineous families were diagnosed with PNDM and the incidence was 1 in 21 196 live births. Six different mutations in four genes were identified, of which two GLIS3 and one SLC2A2 were novel and no patient had KCNJ11, ABCC8, or INS mutations. Fourteen (82.4%) patients had identifiable genetic aetiology and their PNDM was part of known autosomal-recessive syndromes including Wolcott Rallison (41.1%), neonatal diabetes and hypothyroidism (29.4%), Fanconi-Bickel (5.8%), and thiamine-responsive megaloblastic anaemia (5.8%). Two patients with isolated PNDM and one with intermediate developmental delay, epilepsy and neonatal diabetes had no identifiable cause. CONCLUSIONS: Al-Madinah region has the highest reported incidence of PNDM worldwide. In this region with high consanguinity, PNDM has different genetic aetiology and in the majority of cases presents as a part of rare familial autosomal-recessive syndrome rather than in isolation.

OBJECTIVE - to demonstrate the importance of using a combined genetic and functional approach to correctly interpret a genetic test for monogenic diabetes. RESEARCH DESIGN AND METHODS - We identified three probands with a phenotype consistent with maturity-onset diabetes of the young (MODY) subtype GCK-MODY, in whom two potential pathogenic mutations were identified: [R43H/G68D], [E248 K/I225M], or [G261R/D217N]. Allele-specific PCR and cosegregation were used to determine phase. Single and double mutations were kinetically characterized. RESULTS - the mutations occurred in cis (double mutants) in two probands and in trans in one proband. Functional studies of all double mutants revealed inactivating kinetics. The previously reported GCK-MODY mutations R43H and G68D were inherited from an affected father and unaffected mother, respectively. Both our functional and genetic studies support R43H as the cause of GCK-MODY and G68D as a neutral rare variant. CONCLUSIONS - These data highlight the need for family/functional studies, even for previously reported pathogenic mutations. © 2012 by the American Diabetes Association.

Alagille syndrome is a multisystem disorder characterized by highly variable expressivity, most frequently caused by heterozygous JAG1 gene mutations. Classic diagnostic criteria combine the presence of bile duct paucity on liver biopsy with three of five systems affected; liver, heart, skeleton, eye and dysmorphic facies. The aim of this study was to determine the prevalence and distribution of JAG1 mutations in patients referred for routine clinical diagnostic testing. Clinical data were available for 241 patients from 135 families. The index cases were grouped according to the number of systems affected (heart, liver, skeletal, eye and facies) and the mutation frequency calculated for each group. JAG1 mutations were identified in 59/135 (44%) probands. The highest mutation detection rates were observed in patients with the most frequent presenting features of Alagille syndrome; ranging from 20% (one system) to 86% (five systems). The overall mutation pick-up rate in a clinical diagnostic setting was lower than in previous research studies. Identification of a JAG1 gene mutation is particularly useful for those patients with atypical or mild Alagille syndrome who do not meet classic diagnostic criteria as it provides a definite molecular diagnosis and allows accurate genetic counselling for the family. © 2011 John Wiley & Sons A/S.

Alagille syndrome is a multisystem disorder characterized by highly variable expressivity, most frequently caused by heterozygous JAG1 gene mutations. Classic diagnostic criteria combine the presence of bile duct paucity on liver biopsy with three of five systems affected; liver, heart, skeleton, eye and dysmorphic facies. The aim of this study was to determine the prevalence and distribution of JAG1 mutations in patients referred for routine clinical diagnostic testing. Clinical data were available for 241 patients from 135 families. The index cases were grouped according to the number of systems affected (heart, liver, skeletal, eye and facies) and the mutation frequency calculated for each group. JAG1 mutations were identified in 59/135 (44%) probands. The highest mutation detection rates were observed in patients with the most frequent presenting features of Alagille syndrome; ranging from 20% (one system) to 86% (five systems). The overall mutation pick-up rate in a clinical diagnostic setting was lower than in previous research studies. Identification of a JAG1 gene mutation is particularly useful for those patients with atypical or mild Alagille syndrome who do not meet classic diagnostic criteria as it provides a definite molecular diagnosis and allows accurate genetic counselling for the family.

Background/Objective: Mutations in the KATP channel genes are the commonest cause of permanent neonatal diabetes. Most patients obtain optimal glycemic control on sulfonylurea treatment. Genetic testing is currently recommended for all infants diagnosed before 6 months of age. We aimed to explore the prevalence of KATP channel diabetes in infants presenting between 6 and 12 months. Methods: the KCNJ11 and ABCC8 genes were sequenced in 115 infants with permanent diabetes diagnosed between 6 and 12 months and in 405 patients presenting before 6 months. Results: Mutations in either gene were identified in 197 patients diagnosed before 6 months (48.6%), three infants diagnosed between 6 and 9 months (4.2%) and none of those diagnosed after 9 months. Two patients diagnosed after 6 months were successfully transferred from insulin to sulfonylureas. Conclusion: KATP channel mutations are an uncommon cause of diabetes in infants presenting after 6 months. However, given the potential clinical benefit from identifying a KATP channel mutation, we recommend that KATP mutation testing should be routinely extended to infants diagnosed up to 9 months. © 2011 John Wiley & Sons A/S.

BACKGROUND/OBJECTIVE: Mutations in the K(ATP). channel genes are the commonest cause of permanent neonatal diabetes. Most patients obtain optimal glycemic control on sulfonylurea treatment. Genetic testing is currently recommended for all infants diagnosed before 6 months of age. We aimed to explore the prevalence of K(ATP). channel diabetes in infants presenting between 6 and 12 months. METHODS: the KCNJ11 and ABCC8 genes were sequenced in 115 infants with permanent diabetes diagnosed between 6 and 12 months and in 405 patients presenting before 6 months. RESULTS: Mutations in either gene were identified in 197 patients diagnosed before 6 months (48.6%), three infants diagnosed between 6 and 9 months (4.2%) and none of those diagnosed after 9 months. Two patients diagnosed after 6 months were successfully transferred from insulin to sulfonylureas. CONCLUSION: K(ATP). channel mutations are an uncommon cause of diabetes in infants presenting after 6 months. However, given the potential clinical benefit from identifying a K(ATP). channel mutation, we recommend that K(ATP). mutation testing should be routinely extended to infants diagnosed up to 9 months.

Background: Loss of function mutations in 3-Hydroxyacyl-CoA Dehydrogenase (HADH) cause protein sensitive hyperinsulinaemic hypoglycaemia (HH). HADH encodes short chain 3-hydroxacyl-CoA dehydrogenase, an enzyme that catalyses the penultimate reaction in mitochondrial β-oxidation of fatty acids. Mutations in GLUD1 encoding glutamate dehydrogenase, also cause protein sensitive HH (due to leucine sensitivity). Reports suggest a protein-protein interaction between HADH and GDH. This study was undertaken in order to understand the mechanism of protein sensitivity in patients with HADH mutations. Methods: an oral leucine tolerance test was conducted in controls and nine patients with HADH mutations. Basal GDH activity and the effect of GTP were determined in lymphoblast homogenates from 4 patients and 3 controls. Immunoprecipitation was conducted in patient and control lymphoblasts to investigate protein interactions. Results: Patients demonstrated severe HH (glucose range 1.73.2 mmol/l; insulin range 4.8-63.8 mU/l) in response to the oral leucine load, this HH was not observed in control patients subjected to the same leucine load. Basal GDH activity and half maximal inhibitory concentration of GTP was similar in patients and controls. HADH protein could be co-immunoprecipitated with GDH protein in control samples but not in patient samples. Conclusions: We conclude that GDH and HADH have a direct protein-protein interaction, which is lost in patients with HADH mutations causing leucine induced HH. This is not associated with loss of inhibitory effect of GTP on GDH (as in patients with GLUD1 mutations). © 2012 Heslegrave et al.; licensee BioMed Central Ltd.

Introduction: the young-onset diabetes seen in HNF1A-MODY is often misdiagnosed as Type 2 diabetes. Type 2 diabetes, unlike HNF1A-MODY, is associated with insulin resistance and a characteristic dyslipidaemia.We aimed to compare the lipid profiles in HNF1A-MODY, Type 2 diabetes and control subjects and to determine if lipids can be used to aid the differential diagnosis of diabetes sub-type. Methods: 1)14 subjects in each group (HNF1A-MODY, Type 2 diabetes and controls) were matched for gender and BMI. Fasting lipid profiles and HDL lipid constituents were compared in the 3 groups.2)HDL-cholesterol was assessed in a further 267 patients with HNF1A-MODY and 297 patients with a diagnosis of Type 2 diabetes to determine its discriminative value. Results: 1)In HNF1A-MODY subjects, plasma-triglycerides were lower (1.36 vs. 1.93mmol/l, p=0.07) and plasma-HDL-cholesterol was higher than in subjects with Type 2 diabetes (1.47 vs. 1.15mmol/l, p=0.0008), but was similar to controls. Furthermore, in the isolated HDL; HDL-phospholipid and HDL-cholesterol ester content were higher in HNF1A-MODY, than in Type 2 diabetes (1.59 vs. 1.33mmol/L, p=0.04 and 1.10 vs. 0.83mmol/L, p=0.019, respectively), but were similar to controls (1.59 vs. 1.45mmol/L, p=0.35 and 1.10 vs. 1.21mmol/L, p=0.19, respectively).2)A plasma-HDL-cholesterol >1.12mmol/L was 75% sensitive and 64% specific (ROC AUC=0.76) at discriminating HNF1A-MODY from Type 2 diabetes. Conclusion: the plasma-lipid profiles of HNF1A-MODY and the lipid constituents of HDL are similar to non-diabetic controls. However, HDL-cholesterol was higher in HNF1A-MODY than in Type 2 diabetes and could be used as a biomarker to aid in the identification of patients with HNF1A-MODY. © 2012 Elsevier B.V.

INTRODUCTION: the young-onset diabetes seen in HNF1A-MODY is often misdiagnosed as Type 2 diabetes. Type 2 diabetes, unlike HNF1A-MODY, is associated with insulin resistance and a characteristic dyslipidaemia. We aimed to compare the lipid profiles in HNF1A-MODY, Type 2 diabetes and control subjects and to determine if lipids can be used to aid the differential diagnosis of diabetes sub-type. METHODS: 1) 14 subjects in each group (HNF1A-MODY, Type 2 diabetes and controls) were matched for gender and BMI. Fasting lipid profiles and HDL lipid constituents were compared in the 3 groups. 2) HDL-cholesterol was assessed in a further 267 patients with HNF1A-MODY and 297 patients with a diagnosis of Type 2 diabetes to determine its discriminative value. RESULTS: 1) in HNF1A-MODY subjects, plasma-triglycerides were lower (1.36 vs. 1.93 mmol/l, p = 0.07) and plasma-HDL-cholesterol was higher than in subjects with Type 2 diabetes (1.47 vs. 1.15 mmol/l, p = 0.0008), but was similar to controls. Furthermore, in the isolated HDL; HDL-phospholipid and HDL-cholesterol ester content were higher in HNF1A-MODY, than in Type 2 diabetes (1.59 vs. 1.33 mmol/L, p = 0.04 and 1.10 vs. 0.83 mmol/L, p = 0.019, respectively), but were similar to controls (1.59 vs. 1.45 mmol/L, p = 0.35 and 1.10 vs. 1.21 mmol/L, p = 0.19, respectively). 2) a plasma-HDL-cholesterol > 1.12 mmol/L was 75% sensitive and 64% specific (ROC AUC = 0.76) at discriminating HNF1A-MODY from Type 2 diabetes. CONCLUSION: the plasma-lipid profiles of HNF1A-MODY and the lipid constituents of HDL are similar to non-diabetic controls. However, HDL-cholesterol was higher in HNF1A-MODY than in Type 2 diabetes and could be used as a biomarker to aid in the identification of patients with HNF1A-MODY.

Inactivating mutations in the pancreatic beta cell ATP-sensitive potassium (K(ATP) ) channel genes are identified by sequencing in approximately 80% of patients with diazoxide-unresponsive hyperinsulinaemic hypoglycaemia (HH). Genetic testing is clinically important as the mode of inheritance of a K(ATP) channel mutation(s) provides information on the histological subtype. For example in patients with a single paternally inherited mutation a focal lesion is possible and once confirmed, the patient can undergo a curative lesionectomy. By contrast, recessive inheritance indicates diffuse disease, which requires near-total pancreatectomy, if medical management is unsuccessful. We investigated ABCC8 and KCNJ11 gene dosage in 29 probands from a cohort of 125 with diazoxide-unresponsive HH where sequencing did not provide a genetic diagnosis. We identified heterozygous partial ABCC8 deletions in four probands. In two cases with focal pancreatic disease, a paternally inherited deletion was found. Two other probands with diffuse pancreatic disease were compound heterozygotes for a deletion and a recessively acting mutation that had been identified by sequencing. Family member studies confirmed compound heterozygosity for the deletion and the missense mutation in two affected siblings of one proband. Heterozygous deletions of the ABCC8 gene are a rare, but important cause of diazoxide-unresponsive HH. Dosage analysis should be undertaken in all patients when sequencing analysis does not confirm the genetic diagnosis as confirmation of the mode of inheritance can guide clinical management and will provide important information regarding recurrence risk.

Most cases of permanent form of neonatal diabetes mellitus (PNDM) are due to dominant heterozygous gain of function (activating) mutations in either KCNJ11 or ABCC8 genes, that code for Kir 6.2 and SUR1 subunits, respectively of the pancreatic b cell KATP channel. We describe the interesting case of an infant with PNDM, in whom a compound heterozygous activating/ inactivating mutation was found with clinically unaffected parents, each carrying a heterozygous mutation in ABCC8, one predicting gain of function (neonatal diabetes) and the other a loss of function (hyperinsulinemia).

Permanent neonatal diabetes mellitus (PNDM) is a rare form of diabetes diagnosed within the first 6 months of life. Heterozygous activation mutations in KCNJ11, encoding the Kir6.2 subunit of the ATP-sensitive potassium (K ATP) channel, which acts as a key role in insulin secretion regulation, account for about half of the cases of PNDM. The majority of the patients represent isolated cases resulting from de novo mutations. Approximately 20% have associated neurologic features: the most severe form, which includes epilepsy and developmental delay, is called developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome and the milder form, with less severe developmental delay and without epilepsy, is designated intermediate DEND syndrome. Individuals with KCNJ11 mutations have been successfully transitioned from insulin to sulfonylurea (SU) therapy. Furthermore, there have been cases reported with variable improvement in neurological function following a successful switching. We describe a 12-year-old Portuguese girl with PNDM due to the previously reported R201C mutation in the KCNJ11 gene. Her medical history includes prematurity and moderate developmental delay. The mutation was inherited from her mother who has isolated PNDM. The patient was successfully transferred from insulin to SU, whereas her mother showed SU resistance. Despite good glycemic control, no improvements in the cognitive performance were verified. We present our experience in switching treatment from insulin to oral SUs in this family, and also discuss whether or not the girl's developmental delay is related with the Kir6.2 mutation. To our knowledge, this is the first Portuguese patient reported with successful transition to SU treatment. © 2012 by Walter de Gruyter Berlin Boston.

Objective: Mutations in the KCNJ11 and ABCC8 genes that encode the pancreatic K ATP channel are the commonest cause of permanent neonatal diabetes mellitus (PNDM). The authors aimed to define the genetic causes of PNDM in a large cohort of Arab patients and compare them with a British cohort tested in the same laboratory. Design: Retrospective observational study. Setting: International genetics centre. Patients: Arab and British subjects with PNDM who were referred for genetic testing over the same period. Intervention: Comparison of genotypes and phenotypes between the two cohorts. Main outcome measures: the aetiology and phenotype of PNDM in an Arab compared to a British cohort. Results: 88 Arab and 77 British probands were referred between 2006 and 2011, inclusive. Consanguinity was higher among Arabs (63.6% vs 10.4%) and a higher percentage had a genetic diagnosis compared to the British cohort (63.6% vs 41.6%). Recessive EIF2AK3 gene mutations were the commonest cause of PNDM in the Arab cohort (22.7%) followed by INS (12.5%), and KCNJ11 and GCK (5.7% each), whereas K ATP channel mutations were the commonest cause (29.9%) in the British cohort. In 37.5% of Arab patients PNDM was part of a genetic syndrome compared to 7.8% of the British cohort. Conclusion: PNDM in the Arab population has a different genetic spectrum compared to British patients where KATP channel mutations are the commonest cause, similar to other European populations. In Arabs, PNDM is more likely to be part of a recessively inherited syndrome, possibly due to the higher rate of consanguinity.

Permanent neonatal diabetes mellitus (PNDM) is diagnosed within the first 6 months of life, and is usually monogenic in origin. Heterozygous mutations in ABCC8, KCNJ11, and INS genes account for around half of cases of PNDM; mutations in 10 further genes account for a further 10%, and the remaining 40% of cases are currently without a molecular genetic diagnosis. Thiamine-responsive megaloblastic anaemia (TRMA), due to mutations in the thiamine transporter SLC19A2, is associated with the classical clinical triad of diabetes, deafness, and megaloblastic anaemia. Diabetes in this condition is well described in infancy but has only very rarely been reported in association with neonatal diabetes. We used a combination of homozygosity mapping and evaluation of clinical information to identify cases of TRMA from our cohort of patients with PNDM. Homozygous mutations in SLC19A2 were identified in three cases in which diabetes presented in the first 6 months of life, and a further two cases in which diabetes presented between 6 and 12 months of age. We noted the presence of a significant neurological disorder in four of the five cases in our series, prompting us to examine the incidence of these and other non-classical clinical features in TRMA. From 30 cases reported in the literature, we found significant neurological deficit (stroke, focal, or generalized epilepsy) in 27%, visual system disturbance in 43%, and cardiac abnormalities in 27% of cases. TRMA should be considered in the differential diagnosis of diabetes presenting in the neonatal period. © 2012 John Wiley & Sons A/S.

Aims: the gene SLC2A2 encodes GLUT2, which is found predominantly in pancreas, liver, kidney and intestine. In mice, GLUT2 is the major glucose transporter into pancreatic beta cells, and biallelic Slc2a2 inactivation causes lethal neonatal diabetes. The role of GLUT2 in human beta cells is controversial, and biallelic SLC2A2 mutations cause Fanconi-Bickel syndrome (FBS), with diabetes rarely reported. We investigated the potential role of GLUT2 in the neonatal period by testing whether SLC2A2 mutations can present with neonatal diabetes before the clinical features of FBS appear. Methods: We studied SLC2A2 in patients with transient neonatal diabetes mellitus (TNDM; n = 25) or permanent neonatal diabetes mellitus (PNDM; n = 79) in whom we had excluded the common genetic causes of neonatal diabetes, using a combined approach of sequencing and homozygosity mapping. Results: of 104 patients, five (5%) were found to have homozygous SLC2A2 mutations, including four novel mutations (S203R, M376R, c.963+1G>A, F114LfsX16). Four out of five patients with SLC2A2 mutations presented with isolated diabetes and later developed features of FBS. Four out of five patients had TNDM (16% of our TNDM cohort of unknown aetiology). One patient with PNDM remains on insulin at 28 months. Conclusions: SLC2A2 mutations are an autosomal recessive cause of neonatal diabetes that should be considered in consanguineous families or those with TNDM, after excluding common causes, even in the absence of features of FBS. The finding that patients with homozygous SLC2A2 mutations can have neonatal diabetes supports a role for GLUT2 in the human beta cell. © 2012 Springer-Verlag.

AIMS: the gene SLC2A2 encodes GLUT2, which is found predominantly in pancreas, liver, kidney and intestine. In mice, GLUT2 is the major glucose transporter into pancreatic beta cells, and biallelic Slc2a2 inactivation causes lethal neonatal diabetes. The role of GLUT2 in human beta cells is controversial, and biallelic SLC2A2 mutations cause Fanconi-Bickel syndrome (FBS), with diabetes rarely reported. We investigated the potential role of GLUT2 in the neonatal period by testing whether SLC2A2 mutations can present with neonatal diabetes before the clinical features of FBS appear. METHODS: We studied SLC2A2 in patients with transient neonatal diabetes mellitus (TNDM; n = 25) or permanent neonatal diabetes mellitus (PNDM; n = 79) in whom we had excluded the common genetic causes of neonatal diabetes, using a combined approach of sequencing and homozygosity mapping. RESULTS: of 104 patients, five (5%) were found to have homozygous SLC2A2 mutations, including four novel mutations (S203R, M376R, c.963+1G>A, F114LfsX16). Four out of five patients with SLC2A2 mutations presented with isolated diabetes and later developed features of FBS. Four out of five patients had TNDM (16% of our TNDM cohort of unknown aetiology). One patient with PNDM remains on insulin at 28 months. CONCLUSIONS: SLC2A2 mutations are an autosomal recessive cause of neonatal diabetes that should be considered in consanguineous families or those with TNDM, after excluding common causes, even in the absence of features of FBS. The finding that patients with homozygous SLC2A2 mutations can have neonatal diabetes supports a role for GLUT2 in the human beta cell.

Neonatal/infancy-onset diabetes mellitus is a monogenic form of diabetes with onset within 6 months of age. Two distinct types of neonatal diabetes mellitus have been recognized: permanent and transient. Mutations within the K ATP channel and insulin genes are found in most patients with permanent diabetes mellitus. There have been several reports of the successful transition from insulin to sulfonylurea agents in patients with permanent diabetes mellitus caused by mutations in the KCNJ11 gene. We report on a term female neonate with a novel missense mutation, p.P1199L, in the ABCC8 gene that encodes the sulfonylurea receptor 1 whose treatment was successfully converted from insulin to sulfonylurea. © 2012 Nature America, Inc. All rights reserved.

OBJECTIVE - Misdiagnosis of maturity-onset diabetes of the young (MODY) remains widespread, despite the benefits of optimized management. This cross-sectional study examined diagnostic misclassification of MODY in subjects with clinically labeled young adult-onset type 1 and type 2 diabetes by extending genetic testing beyond current guidelines. RESEARCH DESIGN AND METHODS - Individuals were selected for diagnostic sequencing if they displayed features atypical for their diagnostic label. From 247 case subjects with clinically labeled type 1 diabetes, we sequenced hepatocyte nuclear factor 1 α (HNF1A) and hepatocyte nuclear factor 4 α (HNF4A) in 20 with residual β-cell function ≥3 years from diagnosis (random or glucagon-stimulated C-peptide ≥0.2 nmol/L). From 322 with clinically labeled type 2 diabetes, we sequenced HNF1A and HNF4A in 80 with diabetes diagnosed ≤30 years and/or diabetes diagnosed ≤45 years without metabolic syndrome. We also sequenced the glucokinase (GCK) in 40 subjects with mild fasting hyperglycemia. RESULTS - in the type 1 diabetic group, two HNF1A mutations were found (0.8% prevalence). In type 2 diabetic subjects, 10 HNF1A, two HNF4A, and one GCK mutation were identified (4.0%). Only 47% of MODY case subjects identified met current guidelines for diagnostic sequencing. Follow-up revealed a further 12 mutation carriers among relatives. Twenty-seven percent of newly identified MODY subjects changed treatment, all with improved glycemic control (HbA 1c 8.8 vs. 7.3% at 3 months; P = 0.02). CONCLUSIONS - the systematic use of widened diagnostic testing criteria doubled the numbers of MODY case subjects identified compared with current clinical practice. The yield was greatest in young adult-onset type 2 diabetes. We recommend that all patients diagnosed before age 30 and with presence of C-peptide at 3 years' duration are considered for molecular diagnostic analysis. © 2012 by the American Diabetes Association.

Objective: Ventricular hypertrophy (VH) has been observed in children with congenital hyperinsulinism (CHI), a condition of hypoglycaemia characterised by dysregulated insulin secretion, but the prevalence is not known. Patients and methods: Cardiac assessment was performed in children (n=49) with CHI at diagnosis and follow-up. Two dimensional and Doppler echocardiography studies were used to assess cardiac structures, while M-mode study was used to measure left ventricular (LV) dimensions, subsequently converted to Z scores. Where possible, LV hypertrophy was confirmed by LV mass index (g/m2.7) >95th centile. Results: Cardiac structural lesions were found in 14 (28%) children. At initial echocardiography, VH was present in 31 (65%) children with median (range) LV posterior wall dimension in diastole Z scores of +1.6 (-2.4 to +5.8) and interventricular septal wall dimension in end diastole Z scores of +1.9 (-1.7 to +17.2). At follow-up echocardiography, performed after an interval of 178 (45-390) days, VH persisted in 16 (33%) children. In regression analysis, the presence of VH (odds ratio (95% confidence intervals) 1.1 (1.0-1.2), P = 0.04) at initial echocardiography was correlated with maximum glucose requirement at diagnosis, indicating that severity of CHI at presentation may play a role in the pathogenesis of VH. Conclusions: a significant proportion of children with CHI have cardiac structural lesions. A majority also have VH, which may be associated with the severity of CHI at diagnosis. VH may persist in some children, which requires careful long-term cardiac review. © 2012 European Society of Endocrinology.

AIMS/HYPOTHESIS: Diagnosing MODY is difficult. To date, selection for molecular genetic testing for MODY has used discrete cut-offs of limited clinical characteristics with varying sensitivity and specificity. We aimed to use multiple, weighted, clinical criteria to determine an individual's probability of having MODY, as a crucial tool for rational genetic testing. METHODS: We developed prediction models using logistic regression on data from 1,191 patients with MODY (n = 594), type 1 diabetes (n = 278) and type 2 diabetes (n = 319). Model performance was assessed by receiver operating characteristic (ROC) curves, cross-validation and validation in a further 350 patients. RESULTS: the models defined an overall probability of MODY using a weighted combination of the most discriminative characteristics. For MODY, compared with type 1 diabetes, these were: lower HbA(1c), parent with diabetes, female sex and older age at diagnosis. MODY was discriminated from type 2 diabetes by: lower BMI, younger age at diagnosis, female sex, lower HbA(1c), parent with diabetes, and not being treated with oral hypoglycaemic agents or insulin. Both models showed excellent discrimination (c-statistic = 0.95 and 0.98, respectively), low rates of cross-validated misclassification (9.2% and 5.3%), and good performance on the external test dataset (c-statistic = 0.95 and 0.94). Using the optimal cut-offs, the probability models improved the sensitivity (91% vs 72%) and specificity (94% vs 91%) for identifying MODY compared with standard criteria of diagnosis

BACKGROUND: Congenital hyperinsulinism (CHI) is a cause of persistent hypoglycemia due to unregulated insulin secretion from pancreatic β-cells. Histologically, there are two major subgroups, focal and diffuse. Focal CHI is typically unresponsive to diazoxide and can be cured with surgical removal of the focal lesion. AIMS: We report on three patients with focal CHI to illustrate the marked clinical, genetic, radiological, and histological heterogeneity. METHODS AND RESULTS: the first two patients had focal CHI due to a paternal (c.3992-9G→A) ABCC8 mutation. One of these patients was fully responsive to a small dose (5 mg/kg · d) of diazoxide, whereas the other patient was medically unresponsive. In both patients, the focal lesions were accurately localized preoperatively by [(18)F]dihydroxyphenylalanine (DOPA) positron emission tomography (PET) and surgically resected. The third patient had a paternally inherited ABCC8 (A1493T) mutation, and the initial [(18)F]DOPA PET scan indicated extensive uptake of DOPA in the body and tail of the pancreas. However, despite surgical resection of the body and tail, this patient continued to have severe CHI. A subsequent [(18)F]DOPA PET scan now showed markedly increased DOPA uptake in the remaining body and head of the pancreas. This focal lesion occupied virtually the whole of the pancreas. conclusions: These three cases illustrate that focal lesions even with the same genotype (c.3992-9G→A) may have a different clinical presentation and that [(18)F]DOPA PET scans in very large focal lesions may be difficult to interpret.

Homozygous mutations in the glucokinase gene (GCK) result in a complete deficiency of the GCK enzyme, which leads to permanent neonatal diabetes mellitus. Whilst there has been one report of a patient (with a homozygous p.T168A) who was diagnosed with diabetes at the age of 2 months, all other cases were diagnosed with diabetes within the first 2 weeks of life. We now report a second unrelated patient with the same p.T168A GCK mutation who was diagnosed with diabetes at the age of 9 months. We conclude that the specific GCK mutation, as yet unidentified genetic modifiers, and/or environmental factors might have different effects on pancreatic β-cell functions, causing variability in the age at diagnosis of diabetes.

Wolcott-Rallison syndrome (WRS) is a rare autosomal recessive disorder characterized by early-onset diabetes, spondyloepiphyseal dysplasia, tendency to skeletal fractures secondary to osteopenia, and growth retardation. Mutations in the eukaryotic translation initiation factor 2α kinase (EIF2AK3) gene are responsible for this disorder. Here, we describe a boy with neonatal diabetes, diagnosed at 2 months of age, who developed severe growth retardation and a skeletal fracture during the follow-up period. The patient's skeletal X-ray revealed findings of skeletal dysplasia. A clinical diagnosis of WRS was confirmed by the identification of a novel homozygous nonsense mutation (R491X) in exon 9 of the EIF2AK3 gene. The aim of this report is to raise the awareness for Wolcott-Rallison syndrome in cases presenting with isolated neonatal diabetes. This patient demonstrates that the other findings of this syndrome might be obscured by a diagnosis of isolated neonatal diabetes. ©Journal of Clinical Research in Pediatric Endocrinology, Published by Galenos Publishing.

We identified a novel heterozygous mutation, W68R, in the Kir6.2 subunit of the ATP-sensitive potassium (K ATP) channel, in a patient with transient neonatal diabetes. This tryptophan is absolutely conserved in mammalian Kir channels. The functional effects of mutations at residue 68 of Kir6.2 were studied by heterologous expression in Xenopus oocytes, and by homology modelling. We found the Kir6.2-W68R mutation causes a small reduction in ATP inhibition in the heterozygous state and an increase in the whole-cell K ATP current. This can explain the clinical phenotype of the patient. The effect of the mutation was not charge or size dependent, the order of potency for ATP inhibition being W

Permanent neonatal diabetes was previously assumed to require insulin injection or infusion for life. Recently, permanent neonatal diabetes resulting from mutations in the two protein subunits of the adenosine triphosphate- sensitive potassium channel (Kir6.2 and SUR1) has proven to be successfully treatable with high doses of sulfonylureas rather than insulin. Many patients with these mutations first develop hyperglycemia in the nursery or intensive care unit. The awareness of the neonatolgist of this entity can have dramatic effects on the long-term care and quality of life of these patients and their families. In this study, we present the experience of our center, highlighting aspects relevant to neonatal diagnosis and treatment. © 2011 Nature America, Inc. All rights reserved.

Hyperinsulinism-hyperammonemia syndrome (HI/HA) (OMIM 606762), the second most common form of congenital hyperinsulinism (CHI) is associated with activating missense mutations in the GLUD1 gene, which encodes the mitochondrial matrix enzyme, glutamate dehydrogenase (GDH). Patients present with recurrent symptomatic postprandial hypoglycemia following protein-rich meals (leucine-sensitive hypoglycemia) as well as fasting hypoglycemia accompanied by asymptomatic elevations of plasma ammonia. In contrast to other forms of CHI, the phenotype is reported to be milder thus escaping recognition for the first few months of life. Early diagnosis and appropriate management are essential to avoid the neurodevelopmental consequences including epilepsy and learning disabilities which are prevalent in this disorder. We report an infant presenting with afebrile seizures secondary to hyperinsulinemic hypoglycemia resulting from a novel de novo mutation of the GLUD1 gene. © 2011 by Walter de Gruyter Berlin Boston.

OBJECTIVE: Congenital hyperinsulinism (CHI) is characterized by persistent hypoglycemia due to the inappropriate insulin secretion. Inactivating mutations in the ABCC8 and KCNJ11 genes, which encode the sulfonylurea receptor 1 and Kir6.2 subunits of the ATP-sensitive K(+) (K(ATP)) channel in pancreatic β-cell, are the most common cause of CHI. We studied the genetic etiology and phenotypes of CHI in Korean patients. METHODS: ABCC8 and KCNJ11 mutational analysis was performed in 17 patients with CHI. Medical records were retrospectively reviewed to identify phenotypes. RESULTS: Mutations (12 ABCC8 and three KCNJ11) were identified in 82% (14/17) of patients. of these, nine ABCC8 mutations (E100X, W430X, c.1630+1G>C, D813N, Q923X, E1087_A1094delinsDKSDT, Q1134H, H1135W, and E1209Rfs) and one KCNJ11 mutation (W91X) were novel. of the 14 patients, four had confirming recessively inherited CHI. The remaining ten patients had single heterozygous mutations. The majority (12/17) of patients were medically responsive. of the five diazoxide-responsive patients, four had an ABCC8 mutation. The five patients unresponsive to medical management and one diazoxide-responsive patient underwent pancreatectomy and had diffuse histology. of the operated six patients, two had recessively inherited mutations; three patients had a single heterozygous mutation (one maternally and two paternally inherited); and one patient had no identifiable K(ATP) channel mutation. CONCLUSIONS: This is the first study to report genotype and phenotype correlations among Korean patients with CHI. Mutations in ABCC8 and KCNJ11 are the most common causes of CHI in Korean patients. Similar to other studies, there is marked genetic heterogeneity and no clear genotype-phenotype correlation.

Congenital hyperinsulinism is the most frequent cause of severe, persistent hypoglycemia in infancy and childhood. We report a 2.5 year old girl with severe congenital hyperinsulinism. Mutation analysis showed that the child is a compound heterozygote for two missense mutations in the ABCC8 gene.

Objective: to present an Iranian patient with a nonclassic form of multiple endocrine neoplasia type 1 (MEN 1) who presented with ectopic Cushing syndrome (CS) secondary to a corticotropin (ACTH)-producing thymic neuroendocrine tumor (NET), recurrent renal stones, and a giant cell granuloma of the jaw due to primary hyperparathyroidism (PHPT) without involvement of the pituitary or pancreas.Methods: Relevant imaging and hormonal evaluations were performed. The patient was operated on 2 occasions for a thymic NET and on 3 occasions for PHPT. DNA from a peripheral blood sample was extracted for sequencing of the MEN1 gene.Result: Histopathologic evaluation of the thymic tumor removed during the first surgery showed an atypical carcinoid tumor with a Ki-67 labeling index of 5%. Evaluation after the second surgery revealed an invasive carcinoid tumor with a Ki-67 labeling index of 30%. Parathyroid pathology was suggestive of glandular hyperplasia. Menin gene sequencing revealed a novel frameshift mutation c.1642-1648dup in exon 10.Conclusion: This case of MEN 1 is unusual because most thymic NETs in MEN 1 are nonfunctional, and secretion of ACTH or other ectopic hormones rarely occurs. In patients presenting with thymic NETs, the possibility of MEN 1 should be considered, especially in the presence of hyperparathyroidism. This case also demonstrates that the behavior of thymic NETs can change over time from slow-growing tumors to highly invasive neoplasia, and that ectopic ACTH can be produced by these tumors in the context of MEN 1. Copyright © 2011 AACE.

Type 2 diabetes is a global problem, and current ineffective therapeutic strategies pave the way for novel treatments like small molecular activators targeting glucokinase (GCK). GCK activity is fundamental to beta cell and hepatocyte glucose metabolism, and heterozygous activating and inactivating GCK mutations cause hyperinsulinemic hypoglycemia (HH) and maturity onset diabetes of the young (MODY) respectively. Over 600 naturally occurring inactivating mutations have been reported, whereas only 13 activating mutations are documented to date. We report two novel GCK HH mutations (V389L and T103S) at residues where MODY mutations also occur (V389D and T103I). Using recombinant proteins with in vitro assays, we demonstrated that both HH mutants had a greater relative activity index than wild type (6.0 for V389L, 8.4 for T103S, and 1.0 for wild type). This was driven by an increased affinity for glucose (S0.5, 3.3 ± 0.1 and 3.5 ± 0.1 mM, respectively) versus wild type (7.5 ± 0.1 mM). Correspondingly, the V389D and T103I MODY mutants had markedly reduced relative activity indexes (

Recessive inactivating mutations in the ABCC8 and KCNJ11 genes encoding the adenosine triphosphate-sensitive potassium (K(ATP)) channel subunit sulphonylurea receptor 1 (SUR1) and inwardly rectifying potassium channel subunit (Kir6.2) are the most common cause of hyperinsulinaemic hypoglycaemia (HH). Most of these patients do not respond to treatment with the (K(ATP)) channel agonist diazoxide. Dominant inactivating ABCC8 and KCNJ11 mutations are less frequent, but are usually associated with a milder form of hypoglycaemia that is responsive to diazoxide therapy. We studied five patients from four families with HH who were unresponsive to diazoxide and required a near total pancreatectomy. Mutations in KCNJ11 and ABCC8 were sought by sequencing and dosage analysis. Three novel heterozygous ABCC8 mis-sense mutations (G1485E, D1506E and M1514K) were identified in four probands. All the mutations affect residues located within the Nucleotide Binding Domain 2 of the SUR1 subunit. Testing of family members showed that the mutations had arisen de novo with dominant inheritance in one pedigree. This study extends the clinical phenotype associated with dominant (K(ATP)) channel mutations to include severe congenital HH requiring near total pancreatectomy in addition to a milder form of diazoxide responsive hypoglycaemia. The identification of dominant vs recessive mutations does not predict clinical course but it is important for estimating the risk of HH in future siblings and offspring.

Charcot-Marie-Tooth disease is characterized by length-dependent axonal degeneration with distal sensory loss and weakness, deep-tendon-reflex abnormalities, and skeletal deformities. It is caused by mutations in more than 40 genes. We investigated a four-generation family with 23 members affected by the axonal form (type 2), for which the common causes had been excluded by Sanger sequencing. Exome sequencing of three affected individuals separated by eight meioses identified a single shared novel heterozygous variant, c.917A>G, in DYNC1H1, which encodes the cytoplasmic dynein heavy chain 1 (here, novel refers to a variant that has not been seen in dbSNP131or the August 2010 release of the 1000 Genomes project). Testing of six additional affected family members showed cosegregation and a maximum LOD score of 3.6. The shared DYNC1H1 gene variant is a missense substitution, p.His306Arg, at a highly conserved residue within the homodimerization domain. Three mouse models with different mutations within this domain have previously been reported with age-related progressive loss of muscle bulk and locomotor ability. Cytoplasmic dynein is a large multisubunit motor protein complex and has a key role in retrograde axonal transport in neurons. Our results highlight the importance of dynein and retrograde axonal transport in neuronal function in humans.

BACKGROUND: Congenital hyperinsulinism (CHI) is a cause of persistent hypoglycemia. Histologically, there are two subgroups, diffuse and focal. Focal CHI is a consequence of two independent events, inheritance of a paternal mutation in ABCC8/KCNJ11 and paternal uniparental isodisomy of chromosome 11p15 within the embryonic pancreas, leading to an imbalance in the expression of imprinted genes. The probability of both events occurring within siblings is rare. AIM: We describe the first familial form of focal CHI in two siblings. PATIENTS AND METHODS: the proband presented with medically unresponsive CHI. He underwent pancreatic venous sampling and Fluorine-18-L-dihydroxyphenylalanine positron emission tomography scan, which localized a 5-mm focal lesion in the isthmus of the pancreas. The sibling presented 8 yr later also with medically unresponsive CHI. An Fluorine-18-L-dihydroxyphenylalanine positron emission-computerised tomography scan showed a 7-mm focal lesion in the posterior section of the head of the pancreas. Both siblings were found to be heterozygous for two paternally inherited ABCC8 mutations, A355T and R1494W. Surgical removal of the focal lesions in both siblings cured the Hyperinsulinaemic hypoglycaemia. CONCLUSION: This is the first report of focal CHI occurring in siblings. Genetic counseling for families of patients with focal CHI should be recommended, despite the rare risk of recurrence of this disease.

Understanding the regulation of pancreatic development is key for efforts to develop new regenerative therapeutic approaches for diabetes. Rare mutations in PDX1 and PTF1A can cause pancreatic agenesis, however, most instances of this disorder are of unknown origin. We report de novo heterozygous inactivating mutations in GATA6 in 15/27 (56%) individuals with pancreatic agenesis. These findings define the most common cause of human pancreatic agenesis and establish a key role for the transcription factor GATA6 in human pancreatic development.

Background: While massively parallel DNA sequencing methods continue to evolve rapidly, the benchmark technique for detection and verification of rare (particularly disease-causing) sequence variants remains four-colour dye-terminator sequencing by capillary electrophoresis. The high throughput and long read lengths currently available have shifted the bottleneck in mutation detection away from data generation to data analysis. While excellent computational methods have been developed for quantifying sequence accuracy and detecting variants, either during de novo sequence assembly or for single-nucleotide polymorphism detection, the identification, verification and annotation of very rare sequence variants remains a rather labour-intensive process for which few software aids exist. Aim: to provide a freely available, intuitive software application for highly efficient mutation screening of large sequence batches. Methods and results: the authors developed GeneScreen, a desktop program that analyses capillary electropherograms and compares their sequences with a known reference for identification of mutations. The detected sequence variants are then made available for rapid assessment and annotation via a graphical user interface, allowing chosen variants to be exported for reporting and archiving. The program was validated using more than 16 000 diagnostic laboratory sequence traces. Conclusion: Using GeneScreen, a single user requires only a few minutes to identify rare mutations in hundreds of sequence traces, with comparable sensitivity to expensive commercial products.

CONTEXT AND OBJECTIVE: Recessive mutations in the hydroxyacyl-CoA dehydrogenase (HADH) gene encoding the enzyme 3-hydroxyacyl-CoA dehydrogenase are a rare cause of diazoxide-responsive hyperinsulinemic hypoglycemia (HH) with just five probands reported to date. HADH deficiency in the first three identified patients was associated with detectable urinary 3-hydroxyglutarate and raised plasma 3-hydroxybutyryl-carnitine levels, but two recent cases did not have abnormal urine organic acids or acylcarnitines. RESEARCH DESIGN AND METHODS: We studied 115 patients with diazoxide-responsive HH in whom the common genetic causes of HH had been excluded. No patients were reported to have abnormal acylcarnitines or urinary organic acids. Homozygosity mapping was undertaken in probands from 13 consanguineous pedigrees to search for regions harboring mutations that are identical by descent. RESULTS: HADH sequencing was performed after genome-wide single nucleotide polymorphism analysis revealed a large shared region of homozygosity spanning the HADH locus in six unrelated probands. Homozygous mutations were identified in three of these patients and in a further two probands from consanguineous families. HADH analysis in the remainder of the cohort identified mutations in a further six probands for whom consanguinity was not reported, but who originated from countries with high rates of consanguinity. Six different HADH mutations were identified in 11/115 (10%) patients tested. CONCLUSION: HADH mutations are a relatively common cause of diazoxide-responsive HH with a frequency similar to that of GLUD1 and HNF4A mutations. We recommend that HADH sequence analysis is considered in all patients with diazoxide-responsive HH when recessive inheritance is suspected.

OBJECTIVE: Maturity-onset diabetes of the young (MODY) as a result of mutations in hepatocyte nuclear factor 1-α (HNF1A) is often misdiagnosed as type 1 diabetes or type 2 diabetes. Recent work has shown that high-sensitivity C-reactive protein (hs-CRP) levels are lower in HNF1A-MODY than type 1 diabetes, type 2 diabetes, or glucokinase (GCK)-MODY. We aim to replicate these findings in larger numbers and other MODY subtypes. RESEARCH DESIGN AND METHODS: hs-CRP levels were assessed in 750 patients (220 HNF1A, 245 GCK, 54 HNF4-α [HNF4A], 21 HNF1-β (HNF1B), 53 type 1 diabetes, and 157 type 2 diabetes). RESULTS: hs-CRP was lower in HNF1A-MODY (median [IQR] 0.3 [0.1-0.6] mg/L) than type 2 diabetes (1.40 [0.60-3.45] mg/L; P < 0.001) and type 1 diabetes (1.10 [0.50-1.85] mg/L; P < 0.001), HNF4A-MODY (1.45 [0.46-2.88] mg/L; P < 0.001), GCK-MODY (0.60 [0.30-1.80] mg/L; P < 0.001), and HNF1B-MODY (0.60 [0.10-2.8] mg/L; P = 0.07). hs-CRP discriminated HNF1A-MODY from type 2 diabetes with hs-CRP

Aims/hypothesis: Dominantly acting loss-of-function mutations in the ABCC8/KCNJ11 genes can cause mild medically responsive hyperinsulinaemic hypoglycaemia (HH). As controversy exists over whether these mutations predispose to diabetes in adulthood we investigated the prevalence of diabetes in families with dominantly inherited ATP-sensitive potassium (KATP) channel mutations causing HH in the proband. Methods: We studied the phenotype of 30 mutation carriers (14 children and 16 adults) from nine families with dominant ABCC8/KCNJ11 mutations. Functional consequences of six novel missense mutations were examined by reconstituting the KATP channel in human embryonic kidney 293 (HEK293) cells and evaluating the effect of drugs and metabolic poisoning on the channels using the 86Rb flux assay. Results: the mutant channels all showed a lack of 86Rb efflux on exposure to the channel agonist diazoxide or metabolic inhibition. In the families, dominant ABCC8/KCNJ11 mutations were associated with increased birthweight (median + 1.56 SD score [SDS]). Fourteen children had HH and five adults were reported with HH or hypoglycaemic episodes (63%). Progression from hypoglycaemia to diabetes mellitus occurred in two individuals. Eight adults had a history of gestational diabetes in multiple pregnancies or were diabetic (diagnosed at a median age of 31 years). Within these families, none of the 19 adults who were not carriers of the ABCC8/KCNJ11 mutation was known to be diabetic. Conclusions/interpretation: the phenotype associated with dominant ABCC8/KCNJ11 mutations ranges from asymptomatic macrosomia to persistent HH in childhood. In adults, it may also be an important cause of dominantly inherited early-onset diabetes mellitus. © 2011 the Author(s).

Aim: the prevalence of hepatocyte nuclear factor (HNF)- 1A and HNF4A mutations, and the clinical implications following the genetic diagnosis of maturity-onset diabetes of the young (MODY) in the Irish population, remain unknown. The aim of this study was to establish the occurrence of HNF1A and HNF4A mutations in subjects classified clinically as MODY to identify novel mutations, and to determine the phenotypic features and response to therapy. Methods: a total of 36 unrelated index cases with a clinical diagnosis of MODY were analyzed for HNF1A/HNF4A mutations. OGTT was performed to determine the degree of glucose tolerance and insulin secretory response. Also, 38 relatives underwent OGTT and were tested for the relevant known mutations. HNF1A-/HNF4A-MODY subjects were compared with nine HNF1A mutation-negative relatives and 20 type 2 diabetic (T2DM) patients. Results: Seven different HNF1A mutations were identified in 11/36 (30.5%) index cases, two of which were novel (S352fsdelG and F426X), as well as two novel HNF4A mutations (M1? and R290C; 6%). Family screening revealed 20 subjects with HNF1A and seven with HNF4A mutations. Only 51.6% of HNF1A mutation carriers were diagnosed with diabetes by age 25 years; 11 of the mutation carriers were overweight and four were obese. Insulin secretory response to glucose was significantly lower in HNF1A-MODY subjects than in T2DM patients and HNF1A mutation-negative relatives (P= 0.01). Therapeutic changes occurred in 48% of mutation carriers following genetic diagnosis. Conclusion: There was an HNF1A-MODY pick-up rate of 30.5% and an HNF4A-MODY pick-up rate of 6% in Irish MODY families. Genetically confirmed MODY has significant therapeutic implications. © 2011 Elsevier Masson SAS.

Permanent neonatal diabetes (PND) - a rare form of monogenic diabetes - is diagnosed within the first 6 months of life. PND may be caused by mutations in the genes encoding the Kir6.2 (KCNJ11) or SUR1 (ABCC8) subunits of the adenosine triphosphate-sensitive potassium (KATP) channels in pancreatic beta-cells. Sulphonylureas can bind to the KATP channels allowing the release of insulin from these cells. In this article, the authors present the case of an individual who was diagnosed with type 1 diabetes at the age of 6 weeks and immediately commenced insulin treatment, but switched to sulphonylurea therapy at the age of 26 years, following genetic testing confirming mutation in the KCNJ11 gene. His HbA1c level improved from 10.1% (87 mmol/mol) prior to treatment transfer to 6.5% (48 mmol/mol) at 3 months following transfer; this level remained constant at 12 months.

OBJECTIVE - Congenital hyperinsulinism in infancy (CHI) is characterized by unregulated insulin secretion from pancreatic β-cells; severe forms are associated with defects in ABCC8 and KCNJ11 genes encoding sulfonylurea receptor 1 (SUR1) and Kir6.2 subunits, which form ATP-sensitive K+ (K ATP) channels in β-cells. Diazoxide therapy often fails in the treatment of CHI and may be a result of reduced cell surface expression of KATP channels. We hypothesized that conditions known to facilitate trafficking of cystic fibrosis transmembrane regulator (CFTR) and other proteins in recombinant expression systems might increase surface expression of K ATP channels in native CHI β-cells. RESEARCH DESIGN AND METHODS - Tissue was isolated during pancreatectomy from eight patients with CHI and from adult cadaver organ donors. Patients were screened for mutations in ABCC8 and KCNJ11. Isolated β-cells were maintained at 37°C or 25°C and in the presence of 1) phorbol myristic acid, forskolin and 3-isobutyl-1- methylxanthine, 2) BPDZ 154, or 3) 4-phenylbutyrate. Surface expression of functional channels was assessed by patch-clamp electrophysiology. RESULTS - Mutations in ABCC8 were detected for all patients tested (n = 7/8) and included three novel mutations. In five of eight patients, no changes in KATP channel activity were observed under different cell culture conditions. However, in three patients, in vitro recovery of functional KATP channels occurred. Here, we report the first cases of recovery of defective K ATP channels in human β-cells using modified cell culture conditions. CONCLUSIONS - Our study establishes the principle that chemical modification of KATP channel subunit trafficking could be of benefit for the future treatment of CHI. © 2011 by the American Diabetes Association.

AIM: Maturity-onset diabetes of the young is a monogenic form of familial, young-onset diabetes. It is rare (∼1% diabetes) and may be misdiagnosed as Type 1 diabetes and inappropriately treated with insulin. Type 1 diabetes is characterized by the presence of islet autoantibodies, including glutamate decarboxylase (GAD) and islet antigen-2 (IA-2) antibodies. The prevalence of islet autoantibodies is unknown in maturity-onset diabetes of the young and may have the potential to differentiate this form of diabetes from Type 1 diabetes. The aim of this study was to determine the prevalence of GAD and IA-2 antibodies in patients with maturity-onset diabetes of the young and Type 1 diabetes. METHODS: We measured plasma GAD and IA-2 antibodies in 508 patients with the most common forms of maturity-onset diabetes of the young (GCK: n = 227; HNF1A: n = 229; HNF4A: n = 52) and 98 patients with newly diagnosed Type 1 diabetes (diagnosed < 6 months). Autoantibodies were considered positive if ≥ 99th centile of 500 adult control subjects. RESULTS: GAD and/or IA-2 antibodies were present in 80/98 (82%) patients with Type 1 diabetes and 5/508 (< 1%) patients with maturity-onset diabetes of the young. In the cohort with Type 1 diabetes, both GAD and IA-2 antibodies were detected in 37.8% of patients, GAD only in 24.5% and IA-2 only in 19.4%. All five patients with maturity-onset diabetes of the young with detectable antibodies had GAD antibodies and none had detectable IA-2 antibodies. CONCLUSION: the prevalence of GAD and IA-2 antibodies in maturity-onset diabetes of the young is the same as in control subjects (< 1%). The finding of islet autoantibodies, especially IA-2 antibodies, makes the diagnosis of maturity-onset diabetes of the young very unlikely and genetic testing should only be performed if other clinical characteristics strongly suggest this form of diabetes rather than Type 1 diabetes. This supports routine islet autoantibody testing before proceeding to more expensive molecular genetic testing.

Heterozygous mutations of the KCNJ11 gene encoding the Kir6.2 subunit of the ATP-sensitive potassium channel (K(ATP) channel) of the pancreatic β-cell cause diabetes in about 30-60% of all permanent neonatal diabetes mellitus cases diagnosed before 6 months of age. The K(ATP) channel plays an essential role in the regulation of the electrical status of the membrane through which the secretion of insulin is activated. Transient neonatal diabetes mellitus due to KCNJ11 mutations is less frequent than abnormalities affecting the imprinted region of chromosome 6q24. We studied the genetic basis of two Cypriot patients who developed diabetes before 6 months of age. They both carried mutations of the KCNJ11 gene. The R201H mutation was identified in a patient who developed hyperglycemia and ketoacidosis at the age of 40 d and was successfully transferred to sulphonylureas which activate the channel through an ATP independent route. The R50Q mutation was identified in a child diagnosed at day 45 after birth with remission of his diabetes at 9 months of age. The same defect was identified both in his asymptomatic mother and the recently diagnosed 'type 2' diabetic maternal grandmother. The remission-relapse mechanism in cases of transient neonatal diabetes is not known. Nevertheless, it is possible that the residue of the mutation within the Kir6.2 molecule is associated with the sensitivity to ATP reflecting to the severity of the diabetic phenotype.

Advances in high-throughput sequencing techniques had presented a significant challenge to the processing capabilities of genetic laboratories. However, recent developments in the field of semi-automated mutation detection have revolutionised the task of mutation detection.This chapter provides user information for one commercially available program, Mutation Surveyor. The software is manufactured by SoftGenetics (Pennsylvania, USA) and provides an accurate and efficient program for detecting sequence variants. The chapter focuses on the methodology of setting up GenBank files as reference files and provides information on analysis parameters and data processing.

OBJECTIVE - Two novel mutations (E1506D, E1506G) in the nucleotide-binding domain 2 (NBD2) of the ATP-sensitive K+ channel (KATP channel) sulfonylurea receptor 1 (SUR1) subunit were detected heterozygously in patients with neonatal diabetes. A mutation at the same residue (E1506K) was previously shown to cause congenital hyperinsulinemia. We sought to understand why mutations at the same residue can cause either neonatal diabetes or hyperinsulinemia. RESEARCH DESIGN AND METHODS - Neonatal diabetic patients were sequenced for mutations in ABCC8 (SUR1) and KCNJ11 (Kir6.2). Wild-type and mutant KATP channels were expressed in Xenopus laevis oocytes and studied with electrophysiological methods. RESULTS - Oocytes expressing neonatal diabetes mutant channels had larger resting whole-cell KATP currents than wild-type, consistent with the patients' diabetes. Conversely, no E1506K currents were recorded at rest or after metabolic inhibition, as expected for a mutation causing hyperinsulinemia. KATP channels are activated by Mg-nucleotides (via SUR1) and blocked by ATP (via Kir6.2). All mutations decreased channel activation by MgADP but had little effect on MgATP activation, as assessed using an ATP-insensitive Kir6.2 subunit. Importantly, using wild-type Kir6.2, a 30-s preconditioning exposure to physiological MgATP concentrations (>300 μmol/L) caused a marked reduction in the ATP sensitivity of neonatal diabetic channels, a small decrease in that of wild-type channels, and no change for E1506K channels. This difference in MgATP inhibition may explain the difference in resting whole-cell currents found for the neonatal diabetes and hyperinsulinemia mutations. CONCLUSIONS - Mutations in the same residue can cause either hyperinsulinemia or neonatal diabetes. Differentially altered nucleotide regulation by NBD2 of SUR1 can explain the respective clinical phenotypes. © 2011 by the American Diabetes Association.

INTRODUCTION: Mutations in the GLI-similar 3 (GLIS3) gene encoding the transcription factor GLIS3 are a rare cause of neonatal diabetes and congenital hypothyroidism with six affected cases from three families reported to date. Additional features, described previously, include congenital glaucoma, hepatic fibrosis, polycystic kidneys, developmental delay and facial dysmorphism. SUBJECTS: We report two new cases from unrelated families with distinct novel homozygous partial GLIS3 deletions. Both patients presented with neonatal diabetes mellitus, severe resistant hypothyroidism in the presence of elevated thyroglobulin and normal thyroid anatomy, degenerative liver disease, cystic renal dysplasia, recurrent infections and facial dysmorphism. These novel mutations have also resulted in osteopenia, bilateral sensorineural deafness and pancreatic exocrine insufficiency, features that have not previously been associated with GLIS3 mutations. Gene dosage analysis showed that the parents were carriers of a deletion encompassing exons 1-2 (case 1) or exons 1-4 (case 2) of the 11 exon gene. Genome-wide SNP analysis did not reveal a common ancestral GLIS3 haplotype in patient 2. CONCLUSIONS: Our results confirm partial gene deletions as the most common type of GLIS3 mutations, accounting for four of five families identified to date. We propose that mutations in GLIS3 lead to a wider clinical phenotype than previously recognised. We also report the first case of a recessive GLIS3 mutation causing neonatal diabetes and congenital hypothyroidism in a child from a non-consanguineous pedigree, highlighting the importance of molecular genetic testing in any patient with this phenotype.

BACKGROUND: Loss of function mutations in the genes encoding the pancreatic β-cell ATP-sensitive potassium (KATP) channel are identified in approximately 80% of patients with diazoxide unresponsive hyperinsulinemic hypoglycemia (HH). For a small number of patients HH can occur as part of a multisystem disease such as Beckwith-Wiedemann syndrome (BWS). In approximately 20% of patients, BWS results from chromosome 11 paternal uniparental disomy (UPD), which causes dysregulation of imprinted growth regulation genes at 11p15.5. There is a considerable range in the clinical features and phenotypic severity associated with BWS which is likely to be due to somatic mosaicism. The cause of HH in these patients is not known. RESEARCH DESIGN AND METHODS: We undertook microsatellite analysis of 12 markers spanning chromosome 11p in two patients with severe HH and diffuse disease requiring a pancreatectomy. In both patients mutations in the K(ATP) channel genes had not been identified. RESULTS: We identified segmental paternal UPD in DNA extracted from pancreatic tissue in both patients. UPD was not observed in DNA extracted from the patient's leukocytes or buccal samples. In both cases the UPD encompassed the differentially methylated region at chromosome 11p15.5. Despite this neither patient had any further features of BWS. CONCLUSION: Paternal UPD of the chromosome 11p15.5 differentially methylated region limited to the pancreatic tissue may represent a novel cause of isolated diazoxide unresponsive HH. Loss of heterozygosity studies should therefore be considered in all patients with severe HH who have undergone pancreatic surgery when K(ATP) channel mutation(s) have not been identified.

OBJECTIVE: NEUROG3 plays a central role in the development of both pancreatic islets and enteroendocrine cells. Homozygous hypomorphic missense mutations in NEUROG3 have been recently associated with a rare form of congenital malabsorptive diarrhea secondary to enteroendocrine cell dysgenesis. Interestingly, the patients did not develop neonatal diabetes but childhood-onset diabetes. We hypothesized that null mutations in NEUROG3 might be responsible for the disease in a patient with permanent neonatal diabetes and severe congenital malabsorptive diarrhea. RESEARCH DESIGN AND METHODS: the single coding exon of NEUROG3 was amplified and sequenced from genomic DNA. The mutant protein isoforms were functionally characterized by measuring their ability to bind to an E-box element in the NEUROD1 promoter in vitro and to induce ectopic endocrine cell formation and cell delamination after in ovo chicken endoderm electroporation. RESULTS: Two different heterozygous point mutations in NEUROG3 were identified in the proband [c.82G>T (p.E28X) and c.404T>C (p.L135P)], each being inherited from an unaffected parent. Both in vitro and in vivo functional studies indicated that the mutant isoforms are biologically inactive. In keeping with this, no enteroendocrine cells were detected in intestinal biopsy samples from the patient. CONCLUSIONS: Severe deficiency of neurogenin 3 causes a rare novel subtype of permanent neonatal diabetes. This finding confirms the essential role of NEUROG3 in islet development and function in humans.

Background: Autoantibody-negative children diagnosed with type 1 diabetes might have unrecognized monogenic or type 2 diabetes. Research design and methods: at diagnosis of type 1 diabetes (between ages 0.5 and 16.3 yr, n = 470), autoantibodies [glutamic acid decarboxylase (GAD), insulinoma-associated protein 2 (IA2), insulin autoantibodies (IAA), and/or islet cell antibody (ICA)] were positive (ab+) in 330 and negative in 37 (unknown in 103). Autoantibody-negative patients were retested at median diabetes duration of 3.2 yr (range 0.9-16.2) for autoantibodies (GAD, IA2, ZnT8), human leukocyte antigen (HLA) typing, non-fasting C-peptide, and sequencing of HNF4A, HNF1A, KCNJ11, and INS. Results: Nineteen (5% of 367) remained persistently autoantibody negative (PAN), 17 were positive on repeat testing (PORT), and 1 refused retesting. No mutations were found in PORT. One PAN was heterozygous for P112L mutation in HNF1A and transferred from insulin to oral gliclazide. Another PAN transferred to metformin and the diagnosis was revised to type 2 diabetes. The remaining 17 PAN were indistinguishable from the ab+ group by clinical characteristics. HLA genotype was at high risk for type 1 diabetes in 82% of remaining PAN and 100% of PORT. After excluding patients with diabetes duration

BACKGROUND: Autoantibody-negative children diagnosed with type 1 diabetes might have unrecognized monogenic or type 2 diabetes. RESEARCH DESIGN AND METHODS: at diagnosis of type 1 diabetes (between ages 0.5 and 16.3 yr, n = 470), autoantibodies [glutamic acid decarboxylase (GAD), insulinoma-associated protein 2 (IA2), insulin autoantibodies (IAA), and/or islet cell antibody (ICA)] were positive (ab+) in 330 and negative in 37 (unknown in 103). Autoantibody-negative patients were retested at median diabetes duration of 3.2 yr (range 0.9-16.2) for autoantibodies (GAD, IA2, ZnT8), human leukocyte antigen (HLA) typing, non-fasting C-peptide, and sequencing of HNF4A, HNF1A, KCNJ11, and INS. RESULTS: Nineteen (5% of 367) remained persistently autoantibody negative (PAN), 17 were positive on repeat testing (PORT), and 1 refused retesting. No mutations were found in PORT. One PAN was heterozygous for P112L mutation in HNF1A and transferred from insulin to oral gliclazide. Another PAN transferred to metformin and the diagnosis was revised to type 2 diabetes. The remaining 17 PAN were indistinguishable from the ab+ group by clinical characteristics. HLA genotype was at high risk for type 1 diabetes in 82% of remaining PAN and 100% of PORT. After excluding patients with diabetes duration

AIM: Genome-wide association studies have identified >30 common variants associated with Type 2 diabetes (>5% minor allele frequency). These variants have small effects on individual risk and do not account for a large proportion of the heritable component of the disease. Monogenic forms of diabetes are caused by mutations that occur in 1%. We found no difference in carrier frequency between patients (5.7%) and control subjects (5.0%) (P=0.46). There were also no differences between patients and control subjects when analyses were limited to subsets of variants. The strongest subset were those variants in the DNA binding domain where all five variants identified were only found in patients (P=0.06). CONCLUSION: Approximately 5% of UK individuals carry a PDX1 variant, but there is no evidence that these variants, either individually or cumulatively, predispose to Type 2 diabetes. Further studies will need to consider strategies to assess the role of multiple variants that occur in

OBJECTIVE: in children with congenital hyperinsulinism (CHI), K(ATP) channel genes (ABCC8 and KCNJ11) can be screened rapidly for potential pathogenic mutations. We aimed to assess the contribution of rapid genetic testing to the clinical management of CHI. DESIGN: Follow-up observational study at two CHI referral hospitals. METHODS: Clinical outcomes such as subtotal pancreatectomy, (18)F-Dopa positron emission tomography-computed tomography (PET-CT) scanning, stability on medical treatment and remission were assessed in a cohort of 101 children with CHI. RESULTS: in total, 32 (32%) children had pathogenic mutations in K(ATP) channel genes (27 in ABCC8 and five in KCNJ11), of which 11 (34%) were novel. In those negative at initial screening, other mutations (GLUD1, GCK, and HNF4A) were identified in three children. Those with homozygous/compound heterozygous ABCC8/KCNJ11 mutations were more likely to require a subtotal pancreatectomy CHI (7/10, 70%). Those with paternal heterozygous mutations were investigated with (18)F-Dopa PET-CT scanning and 7/13 (54%) had a focal lesionectomy, whereas four (31%) required subtotal pancreatectomy for diffuse CHI. Those with maternal heterozygous mutations were most likely to achieve remission (5/5, 100%). In 66 with no identified mutation, 43 (65%) achieved remission, 22 (33%) were stable on medical treatment and only one child required a subtotal pancreatectomy. CONCLUSIONS: Rapid genetic analysis is important in the management pathway of CHI; it provides aetiological confirmation of the diagnosis, indicates the likely need for a subtotal pancreatectomy and identifies those who require (18)F-Dopa PET-CT scanning. In the absence of a mutation, reassurance of a favourable outcome can be given early in the course of CHI.

OBJECTIVE: Hepatocyte nuclear factor 1-α (HNF1A)/hepatocyte nuclear factor 4-α (HNF4A) maturity-onset diabetes of the young (MODY) is frequently misdiagnosed as type 1 diabetes, and patients are inappropriately treated with insulin. Blood C-peptide can aid in the diagnosis of MODY, but practical reasons limit its widespread use. Urinary C-peptide creatinine ratio (UCPCR), a stable measure of endogenous insulin secretion, is a noninvasive alternative. We aimed to compare stimulated UCPCR in adults with HNF1A/4A MODY, type 1 diabetes, and type 2 diabetes. RESEARCH DESIGN AND METHODS: Adults with diabetes for ≥ 5 years, without renal impairment, were studied (HNF1A MODY [n = 54], HNF4A MODY [n = 23], glucokinase MODY [n = 20], type 1 diabetes [n = 69], and type 2 diabetes [n = 54]). The UCPCR was collected in boric acid 120 min after the largest meal of the day and mailed for analysis. Receiver operating characteristic (ROC) curves were used to identify optimal UCPCR cutoffs to differentiate HNF1A/4A MODY from type 1 and type 2 diabetes. RESULTS: UCPCR was lower in type 1 diabetes than HNF1A/4A MODY (median [interquartile range]) (

Familial isolated pituitary adenoma (FIPA) is an autosomal dominant condition with variable genetic background and incomplete penetrance. Germline mutations of the aryl hydrocarbon receptor interacting protein (AIP) gene have been reported in 15-40% of FIPA patients. Limited data are available on the functional consequences of the mutations or regarding the regulation of the AIP gene. We describe a large cohort of FIPA families and characterize missense and silent mutations using minigene constructs, luciferase and β-galactosidase assays, as well as in silico predictions. Patients with AIP mutations had a lower mean age at diagnosis (23.6±11.2 years) than AIP mutation-negative patients (40.4±14.5 years). A promoter mutation showed reduced in vitro activity corresponding to lower mRNA expression in patient samples. Stimulation of the protein kinase A-pathway positively regulates the AIP promoter. Silent mutations led to abnormal splicing resulting in truncated protein or reduced AIP expression. A two-hybrid assay of protein-protein interaction of all missense variants showed variable disruption of AIP-phosphodiesterase-4A5 binding. In summary, exonic, promoter, splice-site, and large deletion mutations in AIP are implicated in 31% of families in our FIPA cohort. Functional characterization of AIP changes is important to identify the functional impact of gene sequence variants. © 2010 Wiley-Liss, Inc.

Background: Recessive inactivating mutations in ABCC8 and KCNJ11 (which encode the two subunits of the adenosine triphosphate-sensitive potassium (KATP) channels in β-cells) are the most common cause of medically unresponsive congenital hyperinsulinism (CHI) which requires a near-total pancreatectomy. Methods/Results: a patient born at term with marked macrosomia (5,900 g) presented at the age of 2 h with severe hyperinsulinaemic hypoglycaemia. He failed to respond to treatment with the KATP agonist, diazoxide. An 18FDOPA-PET scan showed intense diffuse uptake of 18FDOPA (consistent with diffuse disease) and genetic analysis of the ABCC8 gene confirmed a compound heterozygote missense ABCC8 mutation (R168C/S606T). However, unexpectedly in this patient the hyperinsulinaemic hypoglycaemia started to improve spontaneously at 7 weeks of age prior to planned pancreatic surgery. Conclusions: This is the first report of a patient with clinically severe autosomal-recessive diffuse CHI due to a compound heterozygous ABCC8 mutation that has resulted in spontaneous resolution at such an early age. Compound heterozygote ABCC8 mutations result in complex interactions, and it is possible that this interaction may modify the potential disease pathogenesis. It is important that physicians are aware of this unusual outcome in order to avoid unnecessary early pancreatic surgery with potential life-long implications. Copyright © 2010 S. Karger AG, Basel.

OBJECTIVE: the phenotype associated with heterozygous HNF4A gene mutations has recently been extended to include diazoxide responsive neonatal hypoglycemia in addition to maturity-onset diabetes of the young (MODY). To date, mutation screening has been limited to patients with a family history consistent with MODY. In this study, we investigated the prevalence of HNF4A mutations in a large cohort of patients with diazoxide responsive hyperinsulinemic hypoglycemia (HH). SUBJECTS AND METHODS: We sequenced the ABCC8, KCNJ11, GCK, GLUD1, and/or HNF4A genes in 220 patients with HH responsive to diazoxide. The order of genetic testing was dependent upon the clinical phenotype. RESULTS: a genetic diagnosis was possible for 59/220 (27%) patients. K(ATP) channel mutations were most common (15%) followed by GLUD1 mutations causing hyperinsulinism with hyperammonemia (5.9%), and HNF4A mutations (5%). Seven of the 11 probands with a heterozygous HNF4A mutation did not have a parent affected with diabetes, and four de novo mutations were confirmed. These patients were diagnosed with HI within the first week of life (median age 1 day), and they had increased birth weight (median +2.4 SDS). The duration of diazoxide treatment ranged from 3 months to ongoing at 8 years. CONCLUSIONS: in this large series, HNF4A mutations are the third most common cause of diazoxide responsive HH. We recommend that HNF4A sequencing is considered in all patients with diazoxide responsive HH diagnosed in the first week of life irrespective of a family history of diabetes, once K(ATP) channel mutations have been excluded.

Maturity-onset diabetes of the young (MODY) is initially misdiagnosed in 90% of cases, resulting in inappropriate advice and treatment. Lack of familiarity with the key characteristics of MODY - autosomal dominant inheritance, young age of diagnosis (

Aims the aim of this study was to elucidate the entities and the frequency of neonatal diabetes mellitus (NDM) in a large representative database for paediatric diabetes patients in Germany and Austria. Methods Based on the continuous diabetes data acquisition system for prospective surveillance (DPV), which includes 51 587 patients with onset of diabetes before the age of 18 years from 299 centres in Germany and Austria, we searched for patients with onset of diabetes mellitus in the first 6 months of life. Results Ninety patients were identified, comprising 0.17% of all paediatric cases in the DPV registry. This represented an incidence of approximately one case in 89 000 live births in Germany. A monogenic basis for NDM was established in 30 subjects (seven UPD6, 10 KCNJ11, seven ABCC8, two FOXP3, two PDX1, one INS, one EIF2AK3). Pancreatic hypoplasia or agenesis was reported in 10 patients and seven subjects were classified as having Type 1 diabetes by their centres. Transient neonatal diabetes (TNDM) accounted for approximately 10% of all cases with NDM. No aetiology was defined in 41 subjects, which may reflect incomplete genetic testing or novel genetic aetiologies. Conclusion Based on a large database, we identified a higher rate of NDM in Germany than has been reported previously. Full molecular genetic testing should be performed in all patients diagnosed before 6 months of age. © 2010 Diabetes UK.

Background. An infant diagnosed as having hypopituitarism and on adequate hydrocortisone replacement therapy was referred to a tertiary endocrine unit at 5 weeks of age with persistent hypoglycemia that required a high rate of intravenous glucose infusion (up to 18 mg/kg·min-1) to maintain euglycemia.Investigations. A controlled hypoglycemia screen was performed to measure levels of plasma glucose, insulin, C-peptide and 3-β 2-hydroxybutyrate concentrations. The pancreas was analyzed by fluorine-18-L-3,4-dihydroxyphenylalanine (18F-DOPA) PET scan. Genetic analyses were performed on the peripheral blood leukocytes, and loss of heterozygosity within the resected focal lesion of the pancreas was investigated by microsatellite analysis. A glucagon stimulation test helped determine pituitary function, and an MRI of the brain and pituitary gland was performed to define the anatomy of the intracranial structures and the pituitary gland.Diagnosis. Focal form of congenital hyperinsulinism localized to the head of the pancreas, septo-optic dysplasia and pituitary hormone deficiencies.Management. Resection of the focal lesion from the head of the pancreas and hormonal replacement therapy for hypopituitarism. © 2010 Macmillan Publishers Limited. All rights reserved.

This chapter reviews genetic test for diabetes mellitus and provides guidelines for clinical diagnosis of monogenic forms of diabetes mellitus. Monogenic diabetes is stated to be a diverse group of disorders with different clinical presentation and genetic causes. Mutations in the same gene is mentioned to have dramatic effect on clinical presentation, with mutations in the insulin gene causing either mild hyperglycemia presenting later in life or as severe insulin deficiency presenting as permanent neonatal diabetes. Monogenic forms of diabetes may be familial, whereas in others it may be sporadic as a result of de novo mutation and this will be familial in subsequent generations. The genetic testing for diabetes involves sequencing the gene of interest including the promoter, protein coding regions, splice acceptor and donor sites, and sites of RNA processing. Besides sequencing, analyses to detect deletions of the gene or parts of the gene may be necessary. The different forms of monogenic diabetes are discussed with their clinical features, the specific gene to be tested and therapy. These monogenic forms include glucokinase related familial fasting hyperglycemia, transcription factors associated familial early-onset diabetes and transient and permanent neonatal diabetes. © 2010 Elsevier Inc. All rights reserved.

OBJECTIVE: NEUROD1 is expressed in both developing and mature beta-cells. Studies in mice suggest that this basic helix-loop-helix transcription factor is critical in the development of endocrine cell lineage. Heterozygous mutations have previously been identified as a rare cause of maturity-onset diabetes of the young (MODY). We aimed to explore the potential contribution of NEUROD1 mutations in patients with permanent neonatal diabetes. RESEARCH DESIGN AND METHODS: We sequenced the NEUROD1 gene in 44 unrelated patients with permanent neonatal diabetes of unknown genetic etiology. RESULTS: Two homozygous mutations in NEUROD1 (c.427_ 428del and c.364dupG) were identified in two patients. Both mutations introduced a frameshift that would be predicted to generate a truncated protein completely lacking the activating domain. Both patients had permanent diabetes diagnosed in the first 2 months of life with no evidence of exocrine pancreatic dysfunction and a morphologically normal pancreas on abdominal imaging. In addition to diabetes, they had learning difficulties, severe cerebellar hypoplasia, profound sensorineural deafness, and visual impairment due to severe myopia and retinal dystrophy. CONCLUSIONS: We describe a novel clinical syndrome that results from homozygous loss of function mutations in NEUROD1. It is characterized by permanent neonatal diabetes and a consistent pattern of neurological abnormalities including cerebellar hypoplasia, learning difficulties, sensorineural deafness, and visual impairment. This syndrome highlights the critical role of NEUROD1 in both the development of the endocrine pancreas and the central nervous system in humans.

Neurogenin 3 (NGN3) commits pancreatic progenitors to an islet cell fate. We have induced NGN3 expression and identified upregulation of the gene encoding the Ras-associated small molecular mass GTP-binding protein, RAB3B. RAB3B localised to the cytoplasm of human β-cells, both during the foetal period and post natally. Genes encoding alternative RAB3 proteins and RAB27A were unaltered by NGN3 expression and in human adult islets their transcripts were many fold less prevalent than those of RAB3B. The regulation of insulin exocytosis in rodent β-cells and responsiveness to incretins are reliant on Rab family members, notably Rab3a and Rab27a, but not Rab3b. Our results support an important inter-species difference in regulating insulin exocytosis where RAB3B is the most expressed isoform in human islets. © 2010 Society for Endocrinology.

BACKGROUND: Neonatal diabetes mellitus (NDM) is a rare monogenic form of diabetes which is diagnosed in the first 6 months of life. Several studies in the last few years provide information on genetic causes for NDM. OBJECTIVE: the aim of this study was to identify all patients with diabetes in the first 6 months of life through the Austrian Diabetes Register, which is available since 1989. A retrospective data analyses was performed to calculate the current incidence of NDM. SUBJECTS AND METHODS: Ten patients were registered with diabetes onset within the first 6 months of life in the Austrian Diabetes Register. Evaluation of detailed clinical data was performed by sending a questionnaire to all diabetes centers. RESULTS: Ten patients from nine different families with NDM were diagnosed in Austria from 1989 until September 2007. Seven patients (one male, six females) had transient NDM (TNDM), three (two males, one female) showed a permanent course [permanent neonatal diabetes mellitus (PNDM)]. One had immunodeficiency, polyendocrinopathy and enteropathy X-linked (IPEX) syndrome and another showed aplasia of the pancreas; no genetic etiology was found in the third case. In three out of seven patients with a transient course of NDM a genetic diagnosis was possible. Two female siblings had activating point mutations in the ABCC8 gene, although one patient had paternal uniparental isodisomy of chromosome 6q24. One patient's family did not consent to genetic testing. CONCLUSIONS: the incidence of NDM in Austria is 1/160 949, with an incidence of 1/ 536 499 for PNDM and 1/229 928 for TNDM.

AIMS: to investigate all-cause and cardiovascular mortality in subjects with diabetes caused by a mutation in the hepatocyte nuclear factor 1alpha gene (HNF1A). METHODS: We identified 39 British families with HNF1A mutations. Consenting individuals were asked details of age and cause of death of parents and siblings. Copies of death certificates were requested from the family or were obtained via the Offices for National Statistics. RESULTS: Data were collated on 241 control subjects and 153 mutation carriers. of those who died, 66% of mutation carriers died from a cardiovascular-related illness compared with 43% of control subjects (P = 0.02). Family members with HNF1A mutations died at a younger age than familial control subjects [all-cause hazard ratio, adjusting for sex and smoking status: 1.9 (95% confidence interval 1.2, 2.9, P = 0.006; cardiovascular hazard ratio: 2.3, confidence interval 1.3, 4.2, P = 0.006)]. CONCLUSIONS: We have shown that individuals known to have diabetes caused by a mutation in the HNF1A gene have an increased risk of cardiovascular mortality compared with their unaffected family members. As with other forms of diabetes, consideration should be given to early statin therapy despite a seemingly protective lipid profile.

ATP-sensitive potassium (KATP) channels regulate insulin secretion from pancreatic beta-cells. Gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of this channel cause neonatal diabetes. We report two novel mutations on the same haplotype (cis), F60Y and V64L, in the slide helix of Kir6.2 in a patient with neonatal diabetes, developmental delay and epilepsy. Functional analysis revealed the F60Y mutation increases the intrinsic channel open probability (Po(0)), thereby indirectly producing a marked decrease in channel inhibition by ATP and an increase in whole-cell KATP currents. When expressed alone, the V64L mutation caused a small reduction in apparent ATP inhibition, by enhancing the ability of MgATP to stimulate channel activity. The V64L mutation also ameliorated the deleterious effects on the F60Y mutation when it was expressed on the same (but not a different) subunit. These data indicate that F60Y is the pathogenic mutation and reveal that interactions between slide helix residues can influence KATP channel gating. © the Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org.

Objective. To identify the genetic cause of transient neonatal diabetes mellitus in three siblings from an Indian family. Methods. Case reports with clinical and molecular evaluation of an activating mutation in the KCNJ11 gene are presented. We describe an Indian family with two asymptomatic parents with 3 children presenting with hyperglycemia at 6, 1.5 and 1 month of age respectively. Blood glucose levels at presentation were 22.2, 18.3 and 20 mmol/L and the diabetes remitted in all three children by 5 years of age. None of the affected siblings had dysmorphism or neurological abnormalities. Diabetes relapsed in the oldest sibling at 9.4 years of age and she is now euglycemic on 1mg/Kg of Glibenclamide twice a day. Results. A novel heterozygous missense mutation (G53V) in the KCNJ11 gene was identified in all 3 affected children and the father. Conclusions. The report suggests that screening for KCNJ11 mutations is appropriate in patients diagnosed with neonatal diabetes as it provides valuable information concerning possible course of the disease and choice of treatment.

Neonatal diabetes mellitus is a rare metabolic disorder with an estimated incidence of 1:300.000 to 400.000 newborns, and less than 50% of the neonates have permanent neonatal diabetes mellitus (PNDM). Recently, activating mutation in the KCNJ11 gene encoding Kir6.2 subunit of the adenosin triphosphate- sensitive potassium (KATP) channel has been described as the most frequent cause of PNDM. Under physiological circumstances KATP channel closure plays a central role in glucose-stimulated insulin secretion from pancreatic beta cells. Sulphonylurea drugs stimulate insulin secretion by binding to and closing KATP channels and thus bypassing beta cell metabolism stimulate the same chain of reactions as glucose. We describe a boy diagnosed with PNDM at the age of 3 months when insulin therapy was started, and at the age of 4.5 years KCNJ11 gene was sequenced and found that the boy carried a de novo activating R201H mutation. Insulin therapy was successfully switched to low doses of oral glibenclamide. Accordingly, it is important to emphasize that every person diagnosed with diabetes before six months of life, however old they actually are, should be tested for KATP mutations which is offered via the website www.diabetesgenes.org.

AIMS/HYPOTHESIS: Maturity-onset diabetes of the young is frequently misdiagnosed as type 1 or type 2 diabetes. A correct diagnosis of MODY is important for determining treatment, but can only be confirmed by molecular genetic testing. We aimed to compare the regional distribution of confirmed MODY cases in the UK and to estimate the minimum prevalence. METHODS: UK referrals for genetic testing in 2,072 probands and 1,280 relatives between 1996 and 2009 were examined by region, country and test result. Referral rate and prevalence were calculated using UK Census 2001 figures. RESULTS: MODY was confirmed in 1,177 (35%) patients, with HNF1A (52%) and GCK mutations (32%) being most frequent in probands confirmed with MODY. There was considerable regional variation in proband referral rates (from 50 per million for South West England and Scotland) and patients diagnosed with MODY (5.3 per million in Northern Ireland, 48.9 per million in South West England). Referral rates and confirmed cases were highly correlated (r = 0.96, p < 0.0001). The minimum prevalence of MODY was estimated to be 108 cases per million. CONCLUSIONS/INTERPRETATION: Assuming this minimal prevalence throughout the UK then >80% of MODY is not diagnosed by molecular testing. The marked regional variation in the prevalence of confirmed MODY directly results from differences in referral rates. This could reflect variation in awareness of MODY or unequal access to genetic testing. Increased referral for diagnostic testing is required if the majority of MODY patients are to have the genetic diagnosis necessary for optimal treatment.

We have previously shown that heterozygous single-base deletions in the carboxyl-ester lipase (CEL) gene cause exocrine and endocrine pancreatic dysfunction in two multigenerational families. These deletions were found in the first and fourth repeats of a variable number of tandem repeats (VNTR), which has proven challenging to sequence due to high GC-content and considerable length variation. We have therefore developed a screening method consisting of a multiplex PCR followed by fragment analysis. The method detected putative disease-causing insertions and deletions in the proximal repeats of the VNTR, and determined the VNTR-length of each allele. When blindly testing 56 members of the two families with known single-base deletions in the CEL VNTR, the method correctly assessed the mutation carriers. Screening of 241 probands from suspected maturity-onset diabetes of the young (MODY) families negative for mutations in known MODY genes (95 individuals from Denmark and 146 individuals from UK) revealed no deletions in the proximal repeats of the CEL VNTR. However, we found one Danish patient with a short, novel CEL allele containing only three VNTR repeats (normal range 7-23 in healthy controls). This allele co-segregated with diabetes or impaired glucose tolerance in the patient's family as six of seven mutation carriers were affected. We also identified individuals who had three copies of a complete CEL VNTR. In conclusion, the CEL gene is highly polymorphic, but mutations in CEL are likely to be a rare cause of monogenic diabetes.

OBJECTIVE: Congenital uterine abnormalities are common and may be associated with developmental renal abnormalities. Mutations of the hepatocyte nuclear factor-1β (HNF1B) gene are associated with renal and uterine abnormalities. We aimed to study the role of HNF1B mutations in a cohort with congenital uterine abnormalities. STUDY DESIGN: We tested 108 probands with uterine abnormalities for HNF1B mutations. We collected clinical information from patient records. RESULTS: Nine of 108 women (8%) had a mutation or deletion in the HNF1B gene. Abnormal HNF1B was found in 18% of the 50 probands who had both uterine and renal abnormalities but in none of the 58 women with isolated uterine abnormalities. CONCLUSION: Mutations of the HNF1B gene are found in women with both uterine and renal abnormalities but are rare in isolated uterine abnormalities. We suggest that HNF1B testing should be performed in patients with both renal and uterine abnormalities, but not in patients with isolated uterine abnormalities.

The ATP-sensitive potassium (K(ATP)) channel is composed of two subunits SUR1 and Kir6.2. The channel is key for glucose stimulated insulin release from the pancreatic beta cell. Activating mutations have been identified in the genes encoding these subunits, ABCC8 and KCNJ11, and account for approximately 40% of permanent neonatal diabetes cases. The majority of patients with a K(ATP) mutation present with isolated diabetes however some have presented with the Developmental delay, Epilepsy and Neonatal Diabetes syndrome. This review focuses on mutations in the K(ATP) channel which result in permanent neonatal diabetes, we review the clinical and functional effects as well as the implications for treatment.

OBJECTIVE: Mutations in the HNF1A gene are the most common cause of maturity-onset diabetes of the young (MODY). There is a substantial variation in the age at diabetes diagnosis, even within families where diabetes is caused by the same mutation. We investigated the hypothesis that common polygenic variants that predispose to type 2 diabetes might account for the difference in age at diagnosis. RESEARCH DESIGN AND METHODS: Fifteen robustly associated type 2 diabetes variants were successfully genotyped in 410 individuals from 203 HNF1A-MODY families, from two study centers in the U.K. and Norway. We assessed their effect on the age at diagnosis both individually and in a combined genetic score by summing the number of type 2 diabetes risk alleles carried by each patient. RESULTS: We confirmed the effects of environmental and genetic factors known to modify the age at HNF1A-MODY diagnosis, namely intrauterine hyperglycemia (-5.1 years if present, P = 1.6 x 10(-10)) and HNF1A mutation position (-5.2 years if at least two isoforms affected, P = 1.8 x 10(-2)). Additionally, our data showed strong effects of sex (females diagnosed 3.0 years earlier, P = 6.0 x 10(-4)) and age at study (0.3 years later diagnosis per year increase in age, P = 4.7 x 10(-38)). There were no strong individual single nucleotide polymorphism effects; however, in the combined genetic score model, each additional risk allele was associated with 0.35 years earlier diabetes diagnosis (P = 5.1 x 10(-3)). CONCLUSIONS: We show that type 2 diabetes risk variants of modest effect sizes reduce the age at diagnosis in HNF1A-MODY. This is one of the first studies to demonstrate that clinical characteristics of a monogenic disease can be modified by common polygenic variants.

Heterozygous coding mutations in the INS gene that encodes preproinsulin were recently shown to be an important cause of permanent neonatal diabetes. These dominantly acting mutations prevent normal folding of proinsulin, which leads to beta-cell death through endoplasmic reticulum stress and apoptosis. We now report 10 different recessive INS mutations in 15 probands with neonatal diabetes. Functional studies showed that recessive mutations resulted in diabetes because of decreased insulin biosynthesis through distinct mechanisms, including gene deletion, lack of the translation initiation signal, andalteredmRNAstability because of the disruption of a polyadenylation signal. A subset of recessive mutations caused abnormal INS transcription, including the deletion of the C1 and E1 cis regulatory elements, or three different single base-pair substitutions in a CC dinucleotide sequence located between E1 and A1 elements. In keeping with an earlier and more severe beta-cell defect, patients with recessive INS mutations had a lower birth weight (-3.2 SD score vs.-2.0 SD score) and were diagnosed earlier (median 1 week vs. 10 weeks) compared to those with dominant INS mutations. Mutations in the insulin gene can therefore result in neonatal diabetes as a result of two contrasting pathogenic mechanisms. Moreover, the recessively inherited mutations provide a genetic demonstration of the essential role of multiple sequence elements that regulate the biosynthesis of insulin in man.

CONTEXT: Approximately 39% of cases with permanent neonatal diabetes (PNDM) and about 11% with maturity onset diabetes of the young (MODY) have an unknown genetic aetiology. Many of the known genes causing MODY and PNDM were identified as being critical for beta cell function before their identification as a cause of monogenic diabetes. OBJECTIVE: We used nominations from the EU beta cell consortium EURODIA project partners to guide gene candidacy. SUBJECTS: Seventeen cases with permanent neonatal diabetes and 8 cases with maturity onset diabetes of the young. MAIN OUTCOME MEASURES: the beta cell experts within the EURODIA consortium were asked to nominate 3 "gold", 3 "silver" and 4 "bronze" genes based on biological or genetic grounds. We sequenced twelve candidate genes from the list based on evidence for candidacy. RESULTS: Sequencing ISL1, LMX1A, MAFA, NGN3, NKX2.2, NKX6.1, PAX4, PAX6, SOX2, SREBF1, SYT9 and UCP2 did not identify any pathogenic mutations. CONCLUSION: Further work is needed to identify novel causes of permanent neonatal diabetes and maturity onset diabetes of the young utilising genetic approaches as well as further candidate genes.

The authors describe a 14-yr-old boy who presented with non-ketotic hyperglycemia, elevated serum creatinine levels and deranged liver function. There was no microalbuminuria or proteinuria. He also had mild mental retardation with learning difficulties. Ultrasonography of the abdomen revealed multiple renal cysts of varying sizes in both the kidneys. Dosage analysis of the hepatocyte nuclear factor (HNF)-1β gene by multiplex ligation-dependent probe amplification (MLPA) detected a heterozygous whole gene deletion (p.Met1Trp557del). This finding is consistent with the diagnosis of renal cysts and diabetes (RCAD) syndrome. This is the first case of the RCAD syndrome reported in an Indian patient. Pediatricians need to be aware of this entity whenever renal disease is seen in a diabetic child in the absence of microalbuminuria or proteinuria. © Dr. K C Chaudhuri Foundation 2010.

CONTEXT: the interpretation of novel missense variants is a challenge with increasing numbers of such variants being identified and a responsibility to report the findings in the context of all available scientific evidence. Various in silico bioinformatic tools have been developed that predict the likely pathogenicity of missense variants; however, their utility within the diagnostic setting requires further investigation. AIM: the aim of our study was to test the predictive value of two of these tools, sorting intolerant from tolerant (SIFT) and polymorphism phenotyping (PolyPhen), in a set of 141 missense variants (131 pathogenic, 8 benign) identified in the ABCC8, GCK, and KCNJ11 genes. METHODS: Sixty-six of the mutations caused a gain of protein function, while 67 were loss-of-function mutations. The evolutionary conservation at each residue was also investigated using multiple sequence alignments from the UCSC genome browser. RESULTS: the sensitivity of SIFT and PolyPhen was reasonably high (69% and 68%, respectively), but their specificity was low (13% and 16%). Both programs were significantly better at predicting loss-of-function mutations than gain-of-function mutations (SIFT, p = 0.001; PolyPhen, p < or = 0.0001). The most reliable method for assessing the likely pathogenicity of a missense variant was to investigate the degree of conservation at the affected residue. Eighty-eight percent of the mutations affected highly conserved residues, while all of the benign variants occurred at residues that were polymorphic across multiple species. CONCLUSIONS: Although SIFT and PolyPhen may be useful in prioritizing changes that are likely to cause a loss of protein function, their low specificity means that their predictions should be interpreted with caution and further evidence to support/refute pathogenicity should be sought before reporting novel missense changes.

BACKGROUND: HADH encodes for the enzyme 3-hydroxyacyl-coenzyme a dehydrogenase (HADH) and catalyses the penultimate reaction in the beta-oxidation of fatty acids. All previously reported patients with mutations in HADH gene and hyperinsulinemic hypoglycemia (HH) showed raised plasma hydroxybutyrylcarnitine and urinary 3-hydroxyglutarate. AIMS: the aims of the study were: 1) to report a novel HADH gene mutation not associated with abnormal acylcarnitine or urinary organic acid profile; and 2) to report the novel observation of severe protein-sensitive HH in three patients with HADH gene mutations. RESEARCH DESIGN AND METHODS: the index case presented at 4 months of age with hypoglycemic seizures. Her HH responded to diazoxide, but she continued to have episodes of hypoglycemia even on diazoxide, especially when consuming high-protein foods. RESULTS: Investigations confirmed HH (blood glucose level of 1.8 mmol/liter with simultaneous serum insulin level of 58 mU/liter) with normal acylcarnitines and urine organic acids. Sequencing of the HADH gene identified a homozygous missense mutation (c.562A>G; p.Met188Val). Hydroxyacyl-coenzyme a dehydrogenase activity was significantly decreased compared with controls (index patient, mean +/- sem, 26.8 +/- 4.8 mU/mg protein; controls, 48.0 +/- 8.1 mU/mg protein; P = 0.029) in skin fibroblasts. This patient was severely protein sensitive. Two other children with HH due to HADH gene mutations also demonstrated marked protein sensitivity. CONCLUSIONS: Mutations in the HADH gene are associated with protein-induced HH, and patients with HH due to HADH gene mutations may have normal acylcarnitines and urine organic acids.

BACKGROUND AND AIMS: Hepatocyte nuclear factor-1 alpha (HNF1A) gene mutations are the commonest cause of monogenic diabetes, but patients are often misdiagnosed as having Type 1 diabetes and started on insulin treatment. Patients with HNF1A diabetes are particularly sensitive to the glucose-lowering effect of sulphonylureas, which are the pharmacological treatment of choice. We aimed to assess if patients do change from insulin to sulphonylurea treatment when HNF1A diabetes is confirmed and the impact of this treatment change on long-term glycaemic control. METHODS: We investigated the clinical course of 43 patients who were insulin treated from diagnosis for a median 4 years (range 1-14) before an HNF1A gene mutation was identified. RESULTS: Thirty-four patients (79%) stopped insulin following genetic testing and transferred to sulphonylureas. Twenty-four of them (71%) remained off insulin at a median 39 months (range 17-90) post-transfer. The 10 patients who recommenced insulin had a trend towards a longer duration of diabetes (18 vs. 7 years, P = 0.066) compared with those remaining on tablets. The median glycated haemoglobin (HbA(1c)) was good (6.9%; interquartile range 6.3-8.0%) in the patients who remained off insulin and 19/24 patients (79%) achieved HbA(1c) < 7.5% or improved their pre-genetic diagnosis HbA(1c) by > 1.0%. Transfer off insulin was not attempted in eight patients: one of these was planning pregnancy and two chose to remain on insulin. CONCLUSION: in this observational study we found that a molecular genetic diagnosis of HNF1A diabetes does alter treatment in clinical practice, with 79% attempting transfer to sulphonylureas. Transfer to sulphonylureas was successful in the majority of patients without deterioration in glycaemic control.

CONTEXT: Activating mutations in genes encoding the Kir6.2 (KCNJ11) and SUR1 (ABCC8) subunits of the pancreatic ATP-sensitive K(+) channel are a common cause of permanent neonatal diabetes (PNDM). All Kir6.2 mutations identified to date are missense mutations. We describe here a novel in-frame deletion (residues 28-32) in Kir6.2 in a heterozygous patient with PNDM without neurological problems that are detectable by standard evaluation. OBJECTIVE: the aim of the study was to identify the mutation responsible for neonatal diabetes in this patient and characterize its functional effects. DESIGN: Wild-type and mutant Kir6.2/SUR1 channels were examined by heterologous expression in Xenopus oocytes. RESULTS: the Kir6.2-28Delta32 mutation produced a significant decrease in ATP inhibition and an increase in whole-cell K(ATP) currents, explaining the diabetes of the patient. Tolbutamide block was only slightly reduced in the simulated heterozygous state, suggesting that the patient should respond to sulfonylurea therapy. The mutation decreased ATP inhibition indirectly, by increasing the intrinsic (unliganded) channel open probability. Neither effect was observed when Kir6.2 was expressed in the absence of SUR1, suggesting that the mutation impairs coupling between SUR1 and Kir6.2. Coimmunoprecipitation studies further revealed that the mutation disrupted a physical interaction between Kir6.2 and residues 1-288 (but not residues 1-196) of SUR1. CONCLUSIONS: We report a novel KCNJ11 mutation causing PNDM. Our results show that residues 28-32 in the N terminus of Kir6.2 interact both physically and functionally with SUR1 and suggest that residues 196-288 of SUR1 are important in this interaction.

OBJECTIVE: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is caused by FOXP3 mutations. We aimed to determine the prevalence, genetics, and clinical phenotype of FOXP3 mutations in a large cohort with permanent neonatal diabetes (PNDM). RESEARCH DESIGN AND METHODS: the 11 coding exons and the polyadenylation region of FOXP3 were sequenced in 26 male subjects with diabetes diagnosed before 6 months of age in whom common genetic causes of PNDM had been excluded. Ten subjects had at least one additional immune-related disorder, and the remaining 16 had isolated diabetes. RESULTS: We identified four hemizygous FOXP3 mutations in 6 of 10 patients with associated immune-related disorders and in 0 of 16 patients with isolated diabetes (P = 0.002). Three patients with two novel mutations (R337Q and P339A) and the previously reported L76QfsX53 developed classic IPEX syndrome and died within the first 13 months. The novel mutation V408M was found in three patients from two unrelated families and had a mild phenotype with hypothyroidism and autoimmune enteropathy (n = 2) or nephrotic syndrome (n = 1) and survival to 12-15 years. CONCLUSIONS: FOXP3 mutations result in approximately 4% of cases of male patients with permanent diabetes diagnosed before 6 months. Patients not only have classic IPEX syndrome but, unexpectedly, may have a more benign phenotype. FOXP3 sequencing should be performed in any male patient with the diagnosis of diabetes in the first 6 months who develops other possible autoimmune-associated conditions, even in the absence of full IPEX syndrome.

BACKGROUND: Many different techniques have been designed for the quantification of JAK2V617F allelic burden, sometimes producing discrepant results. DESIGN AND METHODS: JAK2V617F quantification techniques were compared among 16 centers using 11 assays based on quantitative polymerase chain reaction (with mutation-specific primers or probes, or fluorescent resonance energy transfer/melting curve analysis), allele-specific polymerase chain reaction, conventional sequencing or pyrosequencing. RESULTS: a first series of blinded samples (granulocyte DNA, n=29) was analyzed. Seven assays (12 centers) reported values inside the mean +/- 2SD; the mean coefficient of variation was 31%. Sequencing techniques lacked sensitivity, and strong discrepancies were observed with four techniques, which could be attributed to inadequate standards or to different modes of expression of results. Indeed, quantification of JAK2V617F in relation to another control gene produced higher than expected values, suggesting the possibility of more than two JAK2 copies/cell. After calibration of assays with common 1% to 100% JAK2V617F standards (dilutions of UKE-1 cells in normal leukocytes), 14 centers tested ten new samples. JAK2V617F allelic burdens greater or equal than 1% were then reliably quantified by five techniques -- one allele specific-polymerase chain reaction and four TaqMan allele-specific quantitative polymerase chain reaction assays, including one previously giving results outside the mean +/- 2SD -- with a lower mean coefficient of variation (21%). of these, only the two TaqMan allele-specific quantitative polymerase chain reaction assays with primer-based specificity could detect 0.2% JAK2V617F. CONCLUSIONS: Techniques expressing the allelic burden as JAK2V617F/total JAK2 and using a common set of standards produced similar quantification results but with variable sensitivity. Calibration to a reference standard improved reproducibility.

Chromosome 13q deletions are common in multiple myeloma and other cancers, demonstrating the importance of this region in tumorigenesis. We used a novel single nucleotide polymorphism (SNP)-based technique, digital SNP (dSNP), to identify loss of heterozygosity (LOH) at chromosome 13q in paraffin-embedded bone marrow biopsies from 22 patients with multiple myeloma. We analyzed heterozygous SNPs at 13q for the presence of allelic imbalances and examined the results by sequential probability ratio analysis. Where possible, dSNP results were confirmed by fluorescence in situ hybridization. Using dSNP, we identified 13q LOH in 16/18 (89%) (95% Confidence Interval; 65%, 99%) patients without the need for neoplastic cell enrichment. In 8/16 (50%) cases, either partial or interstitial patterns of LOH were observed. Both fluorescence in situ hybridization and dSNP data proved concordant in just 3/9 cases. Five of the six discrepancies showed LOH by dSNP occurring beyond the boundaries of the fluorescence in situ hybridization probes. Our findings show that dSNP represents a useful technique for the analysis of LOH in archival tissue with minimal infiltration of neoplastic cells. The high-resolution screening afforded by the dSNP technology allowed for the identification of complex chromosomal rearrangements, resulting in either partial or interstitial LOH. Digital SNP represents an attractive approach for the investigation of tumors not suitable for genomic-array analysis.

Deletions of chromosome 13q14 are common in chronic lymphocytic leukemia and other cancers, demonstrating the importance of this region in tumorigenesis. We report the use of two single-nucleotide polymorphism (SNP)-based techniques to determine 13q loss of heterozygosity (LOH) status in 15 patients with CLL: (i) digital SNP (dSNP), where analysis of heterozygous SNPs detects allelic imbalances, and (ii) DNA sequencing, where LOH is identified by comparison of allelic peak heights in normal and neoplastic cells. The SNP-based techniques were compared with established molecular techniques, fluorescence in situ hybridization and multiplex ligation-dependent probe amplification, to determine their utility and relative sensitivity. dSNP proved to be the most sensitive technique, identifying 13q14 LOH in 11 of 13 (85%) patients (95% CI: 55%, 98%) without the need for neoplastic cell enrichment. Three cases showed evidence of LOH by dSNP that was not apparent by other techniques. In 8 of 13 (62%) cases, partial or interstitial patterns of LOH were observed by dSNP. Our findings demonstrate that dSNP represents a useful, sensitive technique for the analysis of chromosomal aberrations that result in LOH. It may have applications for the analysis of other malignancies that are difficult to assess by conventional molecular techniques.

Mutations in hepatocyte nuclear factor 1B (HNF1B), which is a transcription factor expressed in tissues including renal epithelia, associate with abnormal renal development. While studying renal phenotypes of children with HNF1B mutations, we identified a teenager who presented with tetany and hypomagnesemia. We retrospectively reviewed radiographic and laboratory data for all patients from a single center who had been screened for an HNF1B mutation. We found heterozygous mutations in 21 (23%) of 91 cases of renal malformation. All mutation carriers had abnormal fetal renal ultrasonography. Plasma magnesium levels were available for 66 patients with chronic kidney disease (stages 1 to 3). Striking, 44% (eight of 18) of mutation carriers had hypomagnesemia (

Hyperinsulinaemic hypoglycaemia (HH) occurs as a consequence of unregulated insulin secretion from pancreatic beta cells. In the newborn period it is the most common cause of severe and persistent hypoglycaemia. As HH is a major risk factor for brain injury and subsequent neurodevelopment handicap, the identification, rapid diagnosis and prompt management of patients with HH is essential if brain damage is to be avoided. Advances in molecular genetics, radiological imaging techniques (such as fluorine-18 L-3, 4-dihydroxyphenylalanine positron emission tomography ((18F)DOPA-PET) scanning) and laparoscopic surgery have completely changed the clinical approach to infants with the severe congenital forms of HH. This review gives an outline of the clinical presentation, the diagnostic cascade, the underlying molecular mechanisms and the management of HH with a particular focus on congenital forms of hyperinsulinism.

Hyperinsulinemic hypoglycemia (HH) is one of the most common reasons for persisting hypoglycemia in newborns infants and children. Its incidence is 1 in 30 000 - 50 000 live births. The clinical manifestation is varying and the prognosis of life depends on the early diagnosis and the efficacy of treatment. Our aim is to present the clinical manifestation and the effect of the treatment in the first three patients in Bulgaria with identified mutations in the ABCC8 gene encoding SUR1 subunit of the KATP channel, causing HH. The first two patients are sisters from a family with consanguinity with manifestation of the disease after birth. They carry one and the same homozygous mutation of ABCC8 gene: R1215W/R1215W (c.c.3643C>T; p.Arg1215Trp), confirming an autosomal recessive form of HH. Both parents are heterozygous carriers of the R1215W mutation. The third patient is a boy who presented his hypoglycemia at the age of 3 months. The gene analysis showed heterozygous state of a novel missense mutation in exon 7 of the ABCC8 gene. The A355T mutation (c.c.1036G>A; p.Ala355Thr) affects a residue conserved from human to chicken and substitutes an uncharged polar amino acid (threonine) for a nonpolar amino acid (alanine). The same heterozygous missense mutation (A355T/N) has been identified in the mother, who is clinically unaffected. The mother is thought to be a carrier of congenital hyperinsulinism and the child is thought to have a recessive form of hyperinsulinism although a paternal mutation has not been identified. The three patients show slight deficiency in the intellectual development and need for special care. The possibility of DNA analysis for mutations of ABCC8 gene gives the opportunity for more precise diagnosis of any child with persisting hypoglycemia.

BACKGROUND: Activating mutations in the GLUD1 gene (which encodes for the intra-mitochondrial enzyme glutamate dehydrogenase, GDH) cause the hyperinsulinism-hyperammonaemia (HI/HA) syndrome. Patients present with HA and leucine-sensitive hypoglycaemia. GDH is regulated by another intra-mitochondrial enzyme sirtuin 4 (SIRT4). Sirt4 knockout mice demonstrate activation of GDH with increased amino acid-stimulated insulin secretion. OBJECTIVES: to study the genotype-phenotype correlations in patients with GLUD1 mutations. To report the phenotype and functional analysis of a novel mutation (P436L) in the GLUD1 gene associated with the absence of HA. Patients and methods Twenty patients with HI from 16 families had mutational analysis of the GLUD1 gene in view of HA (n=19) or leucine sensitivity (n=1). Patients negative for a GLUD1 mutation had sequence analysis of the SIRT4 gene. Functional analysis of the novel P436L GLUD1 mutation was performed. RESULTS: Heterozygous missense mutations were detected in 15 patients with HI/HA, 2 of which are novel (N410D and D451V). In addition, a patient with a normal serum ammonia concentration (21 micromol/l) was heterozygous for a novel missense mutation P436L. Functional analysis of this mutation confirms that it is associated with a loss of GTP inhibition. Seizure disorder was common (43%) in our cohort of patients with a GLUD1 mutation. No mutations in the SIRT4 gene were identified. CONCLUSION: Patients with HI due to mutations in the GLUD1 gene may have normal serum ammonia concentrations. Hence, GLUD1 mutational analysis may be indicated in patients with leucine sensitivity; even in the absence of HA. A high frequency of epilepsy (43%) was observed in our patients with GLUD1 mutations.

OBJECTIVE: Inactivating mutations in glucokinase (GCK) cause mild fasting hyperglycemia. Identification of a GCK mutation has implications for treatment and prognosis; therefore, it is important to identify these individuals. A significant number of patients have a phenotype suggesting a defect in glucokinase but no abnormality of GCK. We hypothesized that the GCK beta-cell promoter region, which currently is not routinely screened, could contain pathogenic mutations; therefore, we sequenced this region in 60 such probands. RESEARCH DESIGN AND METHODS: the beta-cell GCK promoter was sequenced in patient DNA. The effect of the identified novel mutation on GCK promoter activity was assessed using a luciferase reporter gene expression system. Electrophoretic mobility shift assays (EMSAs) were used to determine the impact of the mutation on Sp1 binding. RESULTS: a novel -71G>C mutation was identified in a nonconserved region of the human promoter sequence in six apparently unrelated probands. Family testing established cosegregation with fasting hyperglycemia (> or = 5.5 mmol/l) in 39 affected individuals. Haplotype analysis in the U.K. family and four of the Slovakian families demonstrated that the mutation had arisen independently. The mutation maps to a potential transcriptional activator binding site for Sp1. Reporter assays demonstrated that the mutation reduces promoter activity by up to fourfold. EMSAs demonstrated a dramatic reduction in Sp1 binding to the promoter sequence corresponding to the mutant allele. CONCLUSIONS: a novel beta-cell GCK promoter mutation was identified that significantly reduces gene expression in vitro through loss of regulation by Sp1. To ensure correct diagnosis of potential GCK-MODY (maturity-onset diabetes of the young) cases, analysis of the beta-cell GCK promoter should be included.

OBJECTIVE: Mutations in the ABCC8 gene encoding the SUR1 subunits of the beta-cell K-ATP channel cause neonatal diabetes (ND) mellitus. We aimed to determine the contribution of ABCC8 gene to ND in Poland, to describe the clinical phenotype associated with its mutations and to examine potential modifying factors. PATIENTS: the Nationwide Registry of ND in Poland includes patients diagnosed before 6 months of age. In total 16 Kir6.2 negative patients with ND, 14 permanent and 2 relapsed transient, were examined. MEASUREMENTS: ABCC8 gene mutations were detected by direct sequencing. Mutation carriers' characteristics included clinical data and biochemical parameters. In addition, we performed the hyperinsulinaemic euglycaemic clamp and tested for islet-specific antibodies in diabetic subjects. RESULTS: We identified two probands with permanent ND (one heterozygous F132V mutation carrier and one compound heterozygote with N23H and R826W mutations) and two others with relapsed transient ND (heterozygotes for R826W and V86A substitutions, respectively). One subject, a heterozygous relative with the R826W mutation, had adult onset diabetes. There were striking differences in the clinical picture of the mutation carriers as the carrier of two mutations, N23H and R826W, was controlled on diet alone with HbA(1c) of 7.3%, whereas the F132V mutation carrier was on 0.66 IU/kg/day of insulin with HbA(1c) of 11.7%. The C-peptide level varied from 0.1 ng/ml (F132V) to 0.75 ng/ml (V86A). We also observed a variable insulin resistance, from moderate (M = 5.5 and 5.6 mg/kg/min, respectively, in the two R826W mutation carriers) to severe (M = 2.6 mg/kg/min in the F132V mutation carrier). We were able to transfer two patients off insulin to sulphonylurea (SU) and to reduce insulin dose in one other patient. Interestingly, there was no response to SU in the most insulin resistant F132V mutation carrier despite high dose of glibenclamide. All examined auto-antibodies were present in one of the subjects, the V86A mutation carrier, although this did not seem to influence the clinical picture, as we were able to transfer this girl off insulin. CONCLUSION: Mutations in SUR1 are the cause of about 15% of Kir6.2 negative permanent ND in Poland. The clinical phenotype of SUR1 diabetic mutation carriers is heterogeneous and it appears to be modified by variable sensitivity to insulin.

Recent research has demonstrated that mutations of the hepatocyte nuclear factor 4-alpha (HNF4A) gene are associated with neonatal hyperinsulinaemic hypoglycaemia. Mutations of this gene also cause one of the subtypes of monogenic diabetes, a form of diabetes formerly known as maturity-onset diabetes of the young. This article describes a family discovered to have a novel frame-shift mutation of the HNF4A gene in the setting of early-onset maternal diabetes and severe neonatal hyperinsulinaemic hypoglycaemia. The implications of a diagnosis of HNF4A gene mutation for obstetric and paediatric practice are discussed. © 2009 the Royal Australian and New Zealand College of Obstetricians and Gynaecologists.

AIM: to assess determinants of fetal growth in the offspring of pregnant women with hyperglycaemia due to a heterozygous glucokinase (GCK) gene mutation. METHODS: Details of gestational age at delivery, fetal birth weight and maternal antenatal treatment were collected from patients and retrospective case note review of 82 offspring born to 42 women with GCK gene mutations and 31 offspring born to 13 unaffected normoglycaemic women with an affected partner. Fetal genotype was determined using direct sequencing from either a mouth swab or a blood sample. RESULTS: in mothers with GCK mutations, non-mutation-carrying offspring were heavier than mutation-carrying offspring (corrected birth weight 3.9 +/- 0.6 vs. 3.2 +/- 0.8 kg; P < 0.001) and more likely to be macrosomic (> 4.0 kg; 39% vs. 7%, P = 0.001). There was no difference in corrected birth weight between offspring of insulin- and diet-treated women (3.7 +/- 0.7 vs. 3.8 +/- 0.6 kg; P = 0.1), although insulin-treated mothers delivered earlier (37.5 +/- 1.7 vs. 38.9 +/- 2.3 weeks; P < 0.001) due to increased obstetric intervention. CONCLUSIONS: Offspring of women with GCK mutations are at increased risk of macrosomia and its obstetric consequences. Fetal birth weight is predominantly altered by fetal genotype and not treatment of maternal hyperglycaemia with insulin. This probably reflects the large effect of a fetal GCK mutation on fetal insulin secretion and the difficulty in reducing the regulated maternal glycaemia caused by a glucose sensing defect in people with GCK mutations.

BACKGROUND: the past 10 years have seen an improvement in sequence data quality due to the introduction of capillary sequencers and new sequencing chemistries. In parallel, new software programs for automated mutation detection have been developed. We evaluated the sensitivity of semiautomated unidirectional sequence analysis for the detection of heterozygous base substitutions using the Mutation Surveyor software package. METHODS: Detection rates for heterozygous base substitutions in 29 genes by automated and visual inspection were compared. Examples of heterozygous bases not detected in one direction during bidirectional analysis were also sought through a national survey of United Kingdom (UK) genetics laboratories. Sequence quality was assessed in a consecutive cohort of 50 patients for whom the 39 exons of the ABCC8 gene had been sequenced in one direction. RESULTS: a total of 701 different heterozygous base substitutions were detected by the software with no false negatives (sensitivity >or=99.57%). Four examples of heterozygous bases missed in one direction during bidirectional analysis were reported. Two were detected using unidirectional analysis settings, and the other two bases had low-quality scores. of the 1950 amplicons examined, 97.2% had a quality score >or=30 and an average PHRED-like score >or=50 for the defined region of interest, and 98.1% of the 323,650 bases had a PHRED score >40. CONCLUSIONS: We found no evidence to support a requirement for bidirectional sequencing. Semiautomated analysis of good quality unidirectional sequence data has high sensitivity and is suitable for heterozygote mutation scanning in clinical diagnostic laboratories. Further work is required to determine minimum quality parameters for semiautomated analysis.

OBJECTIVE: Digenic causes of human disease are rarely reported. Insulin via its receptor, which is encoded by INSR, plays a key role in both metabolic and growth signaling pathways. Heterozygous INSR mutations are the most common cause of monogenic insulin resistance. However, growth retardation is only reported with homozygous or compound heterozygous mutations. We describe a novel translocation [t(7,19)(p15.2;p13.2)] cosegregating with insulin resistance and pre- and postnatal growth deficiency. Chromosome translocations present a unique opportunity to identify modifying loci; therefore, our objective was to determine the mutational mechanism resulting in this complex phenotype. RESEARCH DESIGN AND METHODS: Breakpoint mapping was performed by fluorescence in situ hybridization (FISH) on patient chromosomes. Sequencing and gene expression studies of disrupted and adjacent genes were performed on patient-derived tissues. RESULTS Affected individuals had increased insulin, C-peptide, insulin-to-C-peptide ratio, and adiponectin levels consistent with an insulin receptoropathy. FISH mapping established that the translocation breakpoints disrupt INSR on chromosome 19p15.2 and CHN2 on chromosome 7p13.2. Sequencing demonstrated INSR haploinsufficiency accounting for elevated insulin levels and dysglycemia. CHN2 encoding beta-2 chimerin was shown to be expressed in insulin-sensitive tissues, and its disruption was shown to result in decreased gene expression in patient-derived adipose tissue. CONCLUSIONS: We present a likely digenic cause of insulin resistance and growth deficiency resulting from the combined heterozygous disruption of INSR and CHN2, implicating CHN2 for the first time as a key element of proximal insulin signaling in vivo.

Spondylocostal dysostosis (SCD) is a genetic disorder characterized by vertebral segmentation and formation defects associated with changes of the ribs. Autosomal dominant and recessive modes of inheritance have been reported. Methylmalonic aciduria (MMA) is an inborn error of propionate or cobalamin metabolism. It is an autosomal recessive disorder and one of the most frequent forms of branched-chain organic acidurias. Here we report on a case of a Brazilian boy with both diseases. As we know, it is the first case in the literature with the occurrence of both SCD and MMA--the first a skeletal disease and the latter an inborn error of metabolism.

The beta-cell ATP-sensitive potassium (K(ATP)) channel is a key component of stimulus-secretion coupling in the pancreatic beta-cell. The channel couples metabolism to membrane electrical events bringing about insulin secretion. Given the critical role of this channel in glucose homeostasis it is therefore not surprising that mutations in the genes encoding for the two essential subunits of the channel can result in both hypo- and hyperglycemia. The channel consists of four subunits of the inwardly rectifying potassium channel Kir6.2 and four subunits of the sulfonylurea receptor 1 (SUR1). It has been known for some time that loss of function mutations in KCNJ11, which encodes for Kir6.2, and ABCC8, which encodes for SUR1, can cause oversecretion of insulin and result in hyperinsulinism of infancy, while activating mutations in KCNJ11 and ABCC8 have recently been described that result in the opposite phenotype of diabetes. This review focuses on reported mutations in both genes, the spectrum of phenotypes, and the implications for treatment on diagnosing patients with mutations in these genes.

Glucokinase is a key regulatory enzyme in the pancreatic beta-cell. It plays a crucial role in the regulation of insulin secretion and has been termed the glucose sensor in pancreatic beta-cells. Given its central role in the regulation of insulin release it is understandable that mutations in the gene encoding glucokinase (GCK) can cause both hyper- and hypoglycemia. Heterozygous inactivating mutations in GCK cause maturity-onset diabetes of the young (MODY) subtype glucokinase (GCK), characterized by mild fasting hyperglycemia, which is present at birth but often only detected later in life during screening for other purposes. Homozygous inactivating GCK mutations result in a more severe phenotype presenting at birth as permanent neonatal diabetes mellitus (PNDM). A growing number of heterozygous activating GCK mutations that cause hypoglycemia have also been reported. A total of 620 mutations in the GCK gene have been described in a total of 1,441 families. There are no common mutations, and the mutations are distributed throughout the gene. The majority of activating mutations cluster in a discrete region of the protein termed the allosteric activator site. The identification of a GCK mutation in patients with both hyper- and hypoglycemia has implications for the clinical course and clinical management of their disorder. © 2009 Wiley-Liss, Inc.

CONTEXT AND OBJECTIVE: Mutations in EIF2AK3 cause Wolcott-Rallison syndrome (WRS), a rare recessive disorder characterized by early-onset diabetes, skeletal abnormalities, and liver dysfunction. Although early diagnosis is important for clinical management, genetic testing is generally performed after the full clinical picture develops. We aimed to identify patients with WRS before any other abnormalities apart from diabetes are present and study the overall frequency of WRS among patients with permanent neonatal diabetes. RESEARCH DESIGN AND METHODS: the coding regions of EIF2AK3 were sequenced in 34 probands with infancy-onset diabetes with a clinical phenotype suggestive of WRS (n = 28) or homozygosity at the WRS locus (n = 6). RESULTS: Twenty-five probands (73.5%) were homozygous or compound heterozygous for mutations in EIF2AK3. Twenty of the 26 mutations identified were novel. Whereas a diagnosis of WRS was suspected before genetic testing in 22 probands, three patients with apparently isolated diabetes were diagnosed after identifying a large homozygous region encompassing EIF2AK3. In contrast to nonconsanguineous pedigrees, mutations in EIF2AK3 are the most common known genetic cause of diabetes among patients born to consanguineous parents (24 vs. < 2%). Age at diabetes onset and birth weight might be used to prioritize genetic testing in the latter group. CONCLUSIONS: WRS is the most common cause of permanent neonatal diabetes mellitus in consanguineous pedigrees. In addition to testing patients with a definite clinical diagnosis, EIF2AK3 should be tested in patients with isolated neonatal diabetes diagnosed after 3 wk of age from known consanguineous families, isolated populations, or countries in which inbreeding is frequent.

AIMS/HYPOTHESIS: Heterozygous activating mutations in the pancreatic ATP-sensitive K+ channel cause permanent neonatal diabetes mellitus (PNDM). This results from a decrease in the ability of ATP to close the channel, which thereby suppresses insulin secretion. PNDM mutations that cause a severe reduction in ATP inhibition may produce additional symptoms such as developmental delay and epilepsy. We identified a heterozygous mutation (L164P) in the pore-forming (Kir6.2) subunit of the channel in three unrelated patients and examined its functional effects. METHODS: the patients (currently aged 2, 8 and 20 years) developed diabetes shortly after birth. The two younger patients attempted transfer to sulfonylurea therapy but were unsuccessful (up to 1.1 mg kg(-1) day(-1)). They remain insulin dependent. None of the patients displayed neurological symptoms. Functional properties of wild-type and mutant channels were examined by electrophysiology in Xenopus oocytes. RESULTS: Heterozygous (het) and homozygous L164P K(ATP) channels showed a marked reduction in channel inhibition by ATP. Consistent with its predicted location within the pore, L164P enhanced the channel open state, which explains the reduction in ATP sensitivity. HetL164P currents exhibited greatly increased whole-cell currents that were unaffected by sulfonylureas. This explains the inability of sulfonylureas to ameliorate the diabetes of affected patients. CONCLUSIONS/INTERPRETATION: Our results provide the first demonstration that mutations such as L164P, which produce a severe reduction in ATP sensitivity, do not inevitably cause developmental delay or neurological problems. However, the neonatal diabetes of these patients is unresponsive to sulfonylurea therapy. Functional analysis of PNDM mutations can predict the sulfonylurea response.

Autosomal dominant nonautoimmune hyperthyroidism (ADNAH) is caused by gain of function mutations in the TSH receptor (TSHr) gene and characterized by toxic thyroid hyperplasia with a variable age of onset in the absence of thyroid antibodies and clinical symptoms of autoimmune thyroid disease in at least two generations. We report here a Turkish family with a novel TSHr gene mutation with distinct features all consistent with ADNAH. Thyroid function tests of the proband were as follows: free T3: 13.1 pg/ml (N: 1.8-4.6); free T4: 5.1 ng/dl (N: 0.9-1.7); TSH: 0.01 microIU/ml (N: 0.2-4.2); and TSH receptor antibody: 2 IU/ml (N: 0-10). A heterozygous missense mutation in exon 10 of the TSHr gene (c.1454C>T) resulting in the substitution of valine for alanine at codon 485 (p.Ala485Val) was found in the father and his son and daughter. This mutation had arisen de novo in the father. Functional studies of the novel TSHr germline mutation demonstrated a higher constitutive activation of adenyl cyclase than wild type without any effect on phospholipase C activity. In conclusion, our data indicate that gain of function germline mutations in the TSHr gene should be investigated in families with members suffering from thyrotoxicosis and progressive growth of goiter, but without clinical and biochemical evidence of autoimmune thyroid disease. In addition, patients harboring the same mutation of the TSHr gene may show wide phenotypic variability with respect to the age at onset, and severity of hyperthyroidism and thyroid growth.

We report a patient born to consanguineous parents as a further example of a recently described phenotype comprising neonatal diabetes, intestinal atresias and gall bladder agenesis. Other reports have described cases with overlapping patterns including malrotation, biliary atresia and pancreatic hypoplasia (e.g. as described by Martínez-Frías). We propose that these cases may represent variations of the same syndrome. It is likely that this disorder is inherited as an autosomal recessive trait. Our case is the first to have neonatal diabetes without a demonstrable structural pancreatic abnormality, showing that a deficit in pancreatic function is involved. We sequenced genes with a recognized role in monogenic forms of diabetes, including KCNJ11, ABCC8, GCK, IPF1, HNF1β, NeuroD1 and TCF7L2, as well as a novel candidate gene, HNF6, known to be involved in hepatobiliary and pancreatic development, but did not identify mutations. © 2008 Wiley-Liss, Inc.

We report a patient born to consanguineous parents as a further example of a recently described phenotype comprising neonatal diabetes, intestinal atresias and gall bladder agenesis. Other reports have described cases with overlapping patterns including malrotation, biliary atresia and pancreatic hypoplasia (e.g. as described by Martínez-Frías). We propose that these cases may represent variations of the same syndrome. It is likely that this disorder is inherited as an autosomal recessive trait. Our case is the first to have neonatal diabetes without a demonstrable structural pancreatic abnormality, showing that a deficit in pancreatic function is involved. We sequenced genes with a recognized role in monogenic forms of diabetes, including KCNJ11, ABCC8, GCK, IPF1, HNF1beta, NeuroD1 and TCF7L2, as well as a novel candidate gene, HNF6, known to be involved in hepatobiliary and pancreatic development, but did not identify mutations.

OBJECTIVE: Activating glucokinase (GCK) mutations are a rarely reported cause of congenital hyperinsulinism (CHI), but the prevalence of GCK mutations is not known. METHODS: from a pooled cohort of 201 non-syndromic children with CHI from three European referral centres (Denmark, n=141; Norway, n=26; UK, n=34), 108 children had no K(ATP)-channel (ABCC8/KCNJ11) gene abnormalities and were screened for GCK mutations. Novel GCK mutations were kinetically characterised. RESULTS: in five patients, four heterozygous GCK mutations (S64Y, T65I, W99R and A456V) were identified, out of which S64Y was novel. Two of the mutations arose de novo, three were dominantly inherited. All the five patients were medically responsive. In the combined Danish and Norwegian cohort, the prevalence of GCK-CHI was estimated to be 1.2% (2/167, 95% confidence interval (CI) 0-2.8%) of all the CHI patients. In the three centre combined cohort of 72 medically responsive children without K(ATP)-channel mutations, the prevalence estimate was 6.9% (5/72, 95% CI 1.1-12.8%). All activating GCK mutations mapped to the allosteric activator site. The novel S64Y mutation resulted in an increased affinity for the substrate glucose (S(0.5) 1.49+/-0.08 and 7.39+/-0.05 mmol/l in mutant and wild-type proteins respectively), extrapolating to a relative activity index of approximately 22 compared with the wild type. CONCLUSION: in the largest study performed to date on GCK in children with CHI, GCK mutations were found only in medically responsive children who were negative for ABCC8 and KCNJ11 mutations. The estimated prevalence (approximately 7%) suggests that screening for activating GCK mutations is warranted in those patients.

OBJECTIVE: Congenital hyperinsulinism (CHI) may be due to diffuse or focal pancreatic disease. The diffuse form is associated with an increase in the size of beta-cell nuclei throughout the whole of the pancreas and most commonly results from recessive ATP-sensitive K(+) channel (K(ATP) channel) mutations. Focal lesions are the consequence of somatic uniparental disomy for a paternally inherited K(ATP) channel mutation with enlargement of the beta-cell nuclei confined to the focal lesion. Some "atypical" cases defy classification and show pancreatic beta-cell nuclear enlargement confined to discrete regions of the pancreas. We investigated an atypical case with normal morphology within the tail of the pancreas but occasional enlarged endocrine nuclei in parts of the body and head. RESEARCH DESIGN AND METHODS: the KCNJ11 and ABCC8 genes encoding the K(ATP) channel subunits and microsatellite markers on chromosome 11 were analyzed in DNA samples from the patient and her parents. RESULTS: a mosaic ABCC8 nonsense mutation (Q54X) was identified in the proband. The paternally inherited mutation was present at 90% in lymphocytes and 50% in normal pancreatic sections but between 64 and 74% in abnormal sections. Microsatellite analysis showed mosaic interstitial paternal uniparental isodisomy (UPD) for chromosome 11p15.1. CONCLUSIONS: We report a novel genetic mechanism to explain atypical histological diffuse forms of CHI due to mosaic UPD in patients with dominantly inherited ABCC8 (or KCNJ11) gene mutations.

BACKGROUND: Mutation analysis with direct DNA sequencing is commonly used for the molecular diagnosis of multiple endocrine neoplasia type 1 (MEN1). However, a significant number of patients, despite clinical features of MEN1, do not show MEN1 mutations on direct DNA sequencing. Some of these patients may have gross gene deletions not detected by direct DNA sequencing or mutations in the noncoding regions of the gene not examined routinely. OBJECTIVE: to determine the prevalence of gross deletions in MEN1 in a large cohort of MEN1 patients. PATIENTS AND METHODS: During 1997-2006, we screened MEN1 mutations by direct DNA sequencing in 368 probands referred to our diagnostic molecular genetic laboratory. of these, 101 probands (23 familial, 78 sporadic) fulfilled the clinical criteria for MEN1 (presence of at least two of the parathyroid, pancreatic or pituitary tumours) but were negative for mutations on DNA sequencing. Their DNA samples were examined for gross deletions of one or more exons of MEN1 by using multiple ligation-dependent probe amplification (MLPA) and long-range polymerase chain reaction (PCR) amplification. We also sequenced the minimal promoter region of MEN1 for mutations in the familial cases. RESULTS: We identified a gross deletion involving exons 5 and 6 of MEN1 in one proband (prevalence rate 1%). The sequencing of the minimal promoter region in the familial cases revealed no mutations. CONCLUSION: Gross deletion in the MEN1 gene is an uncommon cause of MEN1 and should be tested for in patients with a high clinical suspicion but without mutations on direct DNA sequencing.

AIMS/HYPOTHESIS: Mutations in the GCK and HNF1A genes are the most common cause of the monogenic forms of diabetes known as 'maturity-onset diabetes of the young'. GCK encodes the glucokinase enzyme, which acts as the pancreatic glucose sensor, and mutations result in stable, mild fasting hyperglycaemia. A progressive insulin secretory defect is seen in patients with mutations in the HNF1A and HNF4A genes encoding the transcription factors hepatocyte nuclear factor-1 alpha and -4 alpha. A molecular genetic diagnosis often changes management, since patients with GCK mutations rarely require pharmacological treatment and HNF1A/4A mutation carriers are sensitive to sulfonylureas. These monogenic forms of diabetes are often misdiagnosed as type 1 or 2 diabetes. Best practice guidelines for genetic testing were developed to guide testing and reporting of results. METHODS: a workshop was held to discuss clinical criteria for testing and the interpretation of molecular genetic test results. The participants included 22 clinicians and scientists from 13 countries. Draft best practice guidelines were formulated and edited using an online tool (http://www.coventi.com). RESULTS: an agreed set of clinical criteria were defined for the testing of babies, children and adults for GCK, HNF1A and HNF4A mutations. Reporting scenarios were discussed and consensus statements produced. CONCLUSIONS/INTERPRETATION: Best practice guidelines have been established for monogenic forms of diabetes caused by mutations in the GCK, HNF1A and HNF4A genes. The guidelines include both diagnostic and predictive genetic tests and interpretation of the results.

Monogenic diabetes resulting from mutations that primarily reduce beta-cell function accounts for 1-2% of diabetes cases, although it is often misdiagnosed as either type 1 or type 2 diabetes. Knowledge of the genetic etiology of diabetes enables more-appropriate treatment, better prediction of disease progression, screening of family members and genetic counseling. We propose that the old clinical classifications of maturity-onset diabetes of the young and neonatal diabetes are obsolete and that specific genetic etiologies should be sought in four broad clinical situations because of their specific treatment implications. Firstly, diabetes diagnosed before 6 months of age frequently results from mutation of genes that encode Kir6.2 (ATP-sensitive inward rectifier potassium channel) or sulfonylurea receptor 1 subunits of an ATP-sensitive potassium channel, and improved glycemic control can be achieved by treatment with high-dose sulfonylureas rather than insulin. Secondly, patients with stable, mild fasting hyperglycemia detected particularly when they are young could have a glucokinase mutation and might not require specific treatment. Thirdly, individuals with familial, young-onset diabetes that does not fit with either type 1 or type 2 diabetes might have mutations in the transcription factors HNF-1alpha (hepatocyte nuclear factor 1-alpha) or HNF-4alpha, and can be treated with low-dose sulfonylureas. Finally, extrapancreatic features, such as renal disease (caused by mutations in HNF-1beta) or deafness (caused by a mitochondrial m.3243A>G mutation), usually require early treatment with insulin.

OBJECTIVE: Neonatal diabetes is a heterogeneous group of disorders with diabetes manifestation in the first 6 months of life. The most common etiology in permanent neonatal diabetes is mutations of the ATP-sensitive K(+) channel subunits; in transient neonatal diabetes, chromosome 6q24 abnormalities are the most common cause. RESEARCH DESIGN AND METHODS: We report a sporadic case of diabetes without ketoacidosis diagnosed on the fourth day of life. RESULTS: Analysis of the KCNJ11 gene found a novel R365H mutation in the proband and her unaffected father. The functional analysis did not support pathogenicity of this variant. When the patient's diabetes remitted in the seventh month of life, the 6q24 region was analyzed and a paternally inherited duplication was identified. CONCLUSIONS: Our case reports a coincidental novel KCNJ11 variant in a patient with transient neonatal diabetes due to a 6q24 duplication, illustrating the difficulty in testing neonates before the clinical course of neonatal diabetes is known.

Objective: the G319S HNF1A variant is associated with an increased risk of type 2 diabetes in the Canadian Oji-Cree population. We hypothesised that the variant site at the 3' end of exon 4 might influence splicing and characterised mRNA transcripts to investigate the mutational mechanism underlying this susceptibility to diabetes. Research design and methods: We established lymphoblastoid cell lines from a G319S homozygote and controls. HNF1A transcripts were characterised in the cell lines and pancreatic tissue by sequence analysis of RT-PCR products and quantification using real-time PCR. Susceptibility to mRNA surveillance was investigated using cycloheximide. Results: Full-length G319S mRNA accounted for 24% of mRNA transcripts in the homozygous G319S cell line. A novel isoform lacking the terminal 12 bases of exon 4 was up-regulated (55% of mRNA transcripts) compared to control cell lines (33%) and human pancreatic tissue (17%). Two abnormal transcripts present only in the G319S cell line included premature termination codons as a result of the inclusion of 7 nucleotides from intron 4 or the deletion of exon 8. Cycloheximide treatment increased the levels of both transcripts. Conclusions: the G319S variant results in the production of two abnormal transcripts and an alteration in the relative balance of normal splicing products. This is predicted to lead to a reduction in total HNF1A transcript levels but residual HNF-1alpha protein activity in G319S homozygotes may still reach up to 66% of normal levels. A combination of abnormal splicing and reduced activity of the G319S protein may explain the diabetes susceptibility.

OBJECTIVE-The G319S HNF1A variant is associated with an increased risk of type 2 diabetes in the Canadian Oji-Cree population. We hypothesized that the variant site at the 3' end of exon 4 might influence splicing and characterized mRNA transcripts to investigate the mutational mechanism underlying this susceptibility to diabetes. RESEARCH DESIGN AND METHODS-We established lym- phoblastoid cell lines from a G319S homozygote and controls. HNF1A transcripts were characterized in the cell lines and pancreatic tissue by sequence analysis of RT-PCR products and quantification using real-time PCR. Susceptibility to mRNA surveillance was investigated using cycloheximide. RESULTS-Full-length G319S mRNA accounted for 24% of mRNA transcripts in the homozygous G319S cell line. A novel isoform lacking the terminal 12 bases of exon 4 was upregulated (55% of mRNA transcripts) compared with control cell lines (33%) and human pancreatic tissue (17%). Two abnormal transcripts present only in the G319S cell line included premature termination codons as a result of the inclusion of seven nucleotides from intron 4 or the deletion of exon 8. Cycloheximide treatment increased the levels of both transcripts. CONCLUSIONS-The G319S variant results in the production of two abnormal transcripts and an alteration in the relative balance of normal splicing products. This is predicted to lead to a reduction in total HNF1A transcript levels, but residual hepa- tocyte nuclear factor-1α protein activity in G319S homozygotes may still reach up to 66% of normal levels. A combination of abnormal splicing and reduced activity of the G319S protein may explain the diabetes susceptibility. © 2008 by the American Diabetes Association.

OBJECTIVE: Neonatal diabetes can result from mutations in the Kir6.2 or sulfonylurea receptor 1 (SUR1) subunits of the ATP-sensitive K(+) channel. Transfer from insulin to oral sulfonylureas in patients with neonatal diabetes due to Kir6.2 mutations is well described, but less is known about changing therapy in patients with SUR1 mutations. We aimed to describe the response to sulfonylurea therapy in patients with SUR1 mutations and to compare it with Kir6.2 mutations. RESEARCH DESIGN AND METHODS: We followed 27 patients with SUR1 mutations for at least 2 months after attempted transfer to sulfonylureas. Information was collected on clinical features, treatment before and after transfer, and the transfer protocol used. We compared successful and unsuccessful transfer patients, glycemic control before and after transfer, and treatment requirements in patients with SUR1 and Kir6.2 mutations. RESULTS: Twenty-three patients (85%) successfully transferred onto sulfonylureas without significant side effects or increased hypoglycemia and did not need insulin injections. In these patients, median A1C fell from 7.2% (interquartile range 6.6-8.2%) on insulin to 5.5% (5.3-6.2%) on sulfonylureas (P = 0.01). When compared with Kir6.2 patients, SUR1 patients needed lower doses of both insulin before transfer (0.4 vs. 0.7 units x kg(-1) x day(-1); P = 0.002) and sulfonylureas after transfer (0.26 vs. 0.45 mg x kg(-1) x day(-1); P = 0.005). CONCLUSIONS: Oral sulfonylurea therapy is safe and effective in the short term in most patients with diabetes due to SUR1 mutations and may successfully replace treatment with insulin injections. A different treatment protocol needs to be developed for this group because they require lower doses of sulfonylureas than required by Kir6.2 patients.

Mutations in the LMNA gene result in diverse phenotypes including Emery Dreifuss muscular dystrophy, limb girdle muscular dystrophy, dilated cardiomyopathy with conduction system disease, Dunnigan type familial partial lipodystrophy, mandibulo acral dysplasia, Hutchinson Gilford progeria syndrome, restrictive dermopathy and autosomal recessive Charcot Marie Tooth type 2. The c.1930C > T (R644C) missense mutation has previously been reported in eight unrelated patients with variable features including left ventricular hypertrophy, limb girdle muscle weakness, dilated cardiomyopathy and atypical progeria. Here we report on the details of nine additional patients in eight families with this mutation. Patients 1 and 2 presented with lipodystrophy and insulin resistance, Patient 1 having in addition focal segmental glomerulosclerosis. Patient 3 presented with motor neuropathy, Patient 4 with arthrogryposis and dilated cardiomyopathy with left ventricular non-compaction, Patient 5 with severe scoliosis and contractures, Patient 6 with limb girdle weakness and Patient 7 with hepatic steatosis and insulin resistance. Patients 8 and 9 are brothers with proximal weakness and contractures. Nonpenetrance was observed frequently in first degree relatives. This report provides further evidence of the extreme phenotypic diversity and low penetrance associated with the R644C mutation. Possible explanations for these observations are discussed. © 2008 Wiley-Liss, Inc.

Mutations in the LMNA gene result in diverse phenotypes including Emery Dreifuss muscular dystrophy, limb girdle muscular dystrophy, dilated cardiomyopathy with conduction system disease, Dunnigan type familial partial lipodystrophy, mandibulo acral dysplasia, Hutchinson Gilford progeria syndrome, restrictive dermopathy and autosomal recessive Charcot Marie Tooth type 2. The c.1930C > T (R644C) missense mutation has previously been reported in eight unrelated patients with variable features including left ventricular hypertrophy, limb girdle muscle weakness, dilated cardiomyopathy and atypical progeria. Here we report on the details of nine additional patients in eight families with this mutation. Patients 1 and 2 presented with lipodystrophy and insulin resistance, Patient 1 having in addition focal segmental glomerulosclerosis. Patient 3 presented with motor neuropathy, Patient 4 with arthrogryposis and dilated cardiomyopathy with left ventricular non-compaction, Patient 5 with severe scoliosis and contractures, Patient 6 with limb girdle weakness and Patient 7 with hepatic steatosis and insulin resistance. Patients 8 and 9 are brothers with proximal weakness and contractures. Nonpenetrance was observed frequently in first degree relatives. This report provides further evidence of the extreme phenotypic diversity and low penetrance associated with the R644C mutation. Possible explanations for these observations are discussed.

Heterozygous activating mutations of KCNJ11 (Kir6.2) are the most common cause of permanent neonatal diabetes mellitus (PNDM) and several cases have been successfully treated with oral sulfonylureas. We report on the attempted transfer of insulin therapy to glibenclamide in a 4-year old child with PNDM and DEND syndrome, bearing a C166Y mutation in KCNJ11. An inpatient transition from subcutaneous NPH insulin (0.2 units/kg/d) to oral glibenclamide (1 mg/kg/d and 1.5 mg/kg/d) was performed. Glucose and C-peptide responses stimulated by oral glucose tolerance test (OGTT), hemoglobin A1c levels, the 8-point self-measured blood glucose (SMBG) profile and the frequency of hypoglycemia episodes were analyzed, before and during treatment with glibenclamide. Neither diabetes control nor neurological improvements were observed. We concluded that C166Y mutation was associated with a form of PNDM insensitive to glibenclamide.

BACKGROUND: Hepatocyte nuclear factor-1beta (HNF-1beta) is a critical transcription factor in pancreatic and renal development. Our previous report identified HNF-1beta mutations in 23/160 patients with unexplained renal disease. The most common phenotype is renal cysts, which is frequently associated with early-onset diabetes in the renal cysts and diabetes (RCAD) syndrome. HNF-1beta gene deletions have recently been shown to cause renal malformations and early-onset diabetes. METHODS: We developed a multiplex ligation-dependent probe amplification (MLPA) assay for HNF-1beta gene dosage analysis and tested patients with unexplained renal disease in whom mutations had not been found by sequencing. RESULTS: Whole HNF-1beta gene deletions were detected in 15/133 probands. Renal cysts were present in 13/15, including three with glomerulocystic kidney disease and one with cystic renal dysplasia. Renal function ranged from normal to transplantation aged 3 years. Ten probands had diabetes (nine having RCAD). In addition, four had abnormal liver function tests, two showed pancreatic atrophy and 3/10 female probands had uterine malformations. Whole HNF-1beta gene deletions are a common cause of developmental renal disease, particularly renal cystic disease with or without diabetes. CONCLUSIONS: the phenotype associated with deletions or coding region/splicing mutations is very similar suggesting that haploinsufficiency is the underlying mechanism. Patients with features suggestive of the HNF-1beta clinical phenotype should be tested for mutations both by sequence and dosage analysis.

Duane's retraction syndrome (DRS) is a complex congenital eye movement disorder caused by aberrant innervation of the extraocular muscles by axons of brainstem motor neurons. Studying families with a variant form of the disorder (DURS2-DRS), we have identified causative heterozygous missense mutations in CHN1, a gene on chromosome 2q31 that encodes alpha2-chimaerin, a Rac guanosine triphosphatase-activating protein (RacGAP) signaling protein previously implicated in the pathfinding of corticospinal axons in mice. We found that these are gain-of-function mutations that increase alpha2-chimaerin RacGAP activity in vitro. Several of the mutations appeared to enhance alpha2-chimaerin translocation to the cell membrane or enhance its ability to self-associate. Expression of mutant alpha2-chimaerin constructs in chick embryos resulted in failure of oculomotor axons to innervate their target extraocular muscles. We conclude that alpha2-chimaerin has a critical developmental function in ocular motor axon pathfinding.

We have previously described individuals presenting with transient neonatal diabetes and showing a variable pattern of DNA hypomethylation at imprinted loci throughout the genome. We now report mutations in ZFP57, which encodes a zinc-finger transcription factor expressed in early development, in seven pedigrees with a shared pattern of mosaic hypomethylation and a conserved range of clinical features. This is the first description of a heritable global imprinting disorder that is compatible with life.

OBJECTIVE: Insulin gene (INS) mutations have recently been described as a cause of permanent neonatal diabetes (PND). We aimed to determine the prevalence, genetics, and clinical phenotype of INS mutations in large cohorts of patients with neonatal diabetes and permanent diabetes diagnosed in infancy, childhood, or adulthood. RESEARCH DESIGN AND METHODS: the INS gene was sequenced in 285 patients with diabetes diagnosed before 2 years of age, 296 probands with maturity-onset diabetes of the young (MODY), and 463 patients with young-onset type 2 diabetes (nonobese, diagnosed

OBJECTIVE: Activating mutations in the KCNJ11 and ABCC8 genes encoding the Kir6.2 and SUR1 subunits of the pancreatic ATP-sensitive K(+) channel are the most common cause of permanent neonatal diabetes. In contrast to KCNJ11, where only dominant heterozygous mutations have been identified, recessively acting ABCC8 mutations have recently been found in some patients with neonatal diabetes. These genes are co-located on chromosome 11p15.1, centromeric to the imprinted Beckwith-Wiedemann syndrome (BWS) locus at 11p15.5. We investigated a male with hemihypertrophy, a condition classically associated with neonatal hyperinsulinemia and hypoglycemia, who developed neonatal diabetes at age 5 weeks. RESEARCH DESIGN AND METHODS: the KCNJ11 and ABCC8 genes and microsatellite markers on chromosome 11 were analyzed in DNA samples from the patient and his parents. RESULTS: a paternally inherited activating mutation (N72S) in the ABCC8 gene was identified in the proband. The mutation was present at 70% in the patient's leukocytes and 50% in buccal cells. Microsatellite analysis demonstrated mosaic segmental paternal uniparental isodisomy (UPD) of 11pter-11p14 in the proband that encompassed the ABCC8 gene and the BWS locus. CONCLUSIONS: We report a patient with neonatal diabetes, hemihypertrophy, and relatively high birth weight resulting from telomeric segmental paternal UPD of chromosome 11, which unmasks a recessively acting gain-of-function mutation in the ABCC8 gene and causes deregulation of imprinted genes at the BWS locus on 11p15.5.

Spondylothoracic dysostosis (STD), also known as Jarcho-Levin syndrome (JLS), is an autosomal-recessive disorder characterized by abnormal vertebral segmentation and defects affecting spine formation, with complete bilateral fusion of the ribs at the costovertebral junction producing a "crab-like" configuration of the thorax. The shortened spine and trunk can severely affect respiratory function during early childhood. The condition is prevalent in the Puerto Rican population, although it is a panethnic disorder. By sequencing a set of candidate genes involved in mouse segmentation, we identified a recessive E103X nonsense mutation in the mesoderm posterior 2 homolog (MESP2) gene in a patient, of Puerto Rican origin and from the Boston area, who had been diagnosed with STD/JLS. We then analyzed 12 Puerto Rican families with STD probands for the MESP2 E103X mutation. Ten patients were homozygous for the E103X mutation, three patients were compound heterozygous for a second nonsense mutation, E230X, or a missense mutation, L125V, which affects a conserved leucine residue within the bHLH region. Thus, all affected probands harbored the E103X mutation. Our findings suggest a founder-effect mutation in the MESP2 gene as a major cause of the classical Puerto Rican form of STD/JLS.

OBJECTIVE: in human pregnancy, placental weight is strongly associated with birth weight. It is uncertain whether there is regulation of the placenta by the fetus or vice versa. We aimed to test the hypothesis that placental growth is mediated, either directly or indirectly, by fetal insulin. RESEARCH DESIGN AND METHODS: Birth weight and placental weight were measured in 43 offspring of 21 parents with mutations in the glucokinase (GCK) gene (25 had inherited the mutation and 18 had not), which results in reduced fetal insulin secretion. Birth weight, placental weight, umbilical cord insulin, and maternal glucose and insulin concentrations were measured in 573 nondiabetic, healthy, term pregnancies. RESULTS: GCK mutation carriers were lighter and also had smaller placentas (610 vs. 720 g, P = 0.042). This difference was also seen in 17 discordant sibling pairs (600 vs. 720 g, P = 0.003). GCK mRNA was not detected in the placenta by RT-PCR. In the normal pregnancies, placental weight was strongly correlated with birth weight (r = 0.61, P < 0.001). Cord insulin concentrations were directly related to placental weight (r = 0.28) and birth weight (r = 0.36) (P < 0.001 for both). CONCLUSIONS: These results suggest that insulin, directly or indirectly, plays a role in placental growth, especially as a mutation in the GCK gene, which is known to only alter fetal insulin secretion, results in altered placental weight. This finding is consistent with the preferential localization of the insulin receptors in the fetal endothelium of the placenta in the last trimester of pregnancy.

OBJECTIVE: to evaluate the clinical response to sulphonylurea treatment in a child with a homozygous T168A GCK (glucokinase) mutation, causing permanent neonatal diabetes mellitus (PNDM). STUDY DESIGN: Oral glibenclamide was given for 3 months. Pancreatic beta cell function was assessed by a glucagon stimulation test. Mutant and wild-type (WT) GCK were characterized. RESULTS: Sulphonylurea treatment resulted in a 12-fold increase in basal and stimulated C-peptide levels. HbA1c levels were reduced from 9.4% to 8.1% on a reduced insulin dose (0.85 to 0.60 U/kg/day). Mutant T168A-GST-GCK showed reduced kinetic activity (0.02 fold) compared to WT. CONCLUSIONS: Sulphonylureas can close the adenosine triphosphate (ATP)-sensitive potassium channel and elicit insulin secretion, but the ATP generated from metabolism is insufficient to fully restore insulin secretory capacity. Nonetheless, sulphonylurea treatment should be tried in patients with GCK-PNDM, particularly those with mutations resulting in less severe kinetic defects, in whom improved glycemic control may be obtained with lower doses of insulin.

CONTEXT: Neonatal diabetes is a rare disorder with an incidence of 1 in 215,000-500,000 live births with 50% of them having permanent neonatal diabetes mellitus. CASE REPORT: We present a case of permanent neonatal diabetes mellitus due to a C96Y (c.287G>A; p.Cys96Tyr) heterozygous mutation in the insulin (INS) gene. Both the patient and his father (who had childhood-onset insulin-requiring diabetes) were found to be carriers of a heterozygous missense mutation C96Y in exon 3 of the INS gene. It has been hypothesized that these mutations disrupt the folding of the proinsulin molecule and result in a misfolded protein or retention of the protein in the endoplasmic reticulum, resulting in endoplasmic reticulum stress and beta cell apoptosis. Subjects with this form of diabetes will need lifelong insulin therapy. CONCLUSION: Insulin gene mutations appear to be an important cause of neonatal diabetes worldwide. This is the first report of a case from the Indian subcontinent. It is important to carry out genetic tests for mutations linked to pancreatic beta cell dysfunction in all patients with persistent neonatal diabetes mellitus in order to decide on therapy.

OBJECTIVE: Mutations in the human HNF4A gene encoding the hepatocyte nuclear factor (HNF)-4alpha are known to cause maturity-onset diabetes of the young (MODY), which is characterized by autosomal-dominant inheritance and impaired glucose-stimulated insulin secretion from pancreatic beta-cells. HNF-4alpha has a key role in regulating the multiple transcriptional factor networks in the islet. Recently, heterozygous mutations in the HNF4A gene were reported to cause transient hyperinsulinemic hypoglycemia associated with macrosomia. RESEARCH DESIGN AND METHODS: Three infants presented with macrosomia and severe hypoglycemia with a positive family history of MODY. The hypoglycemia was confirmed to be due to hyperinsulinism, and all three patients required diazoxide therapy to maintain normoglycemia. Two of the three infants are still requiring diazoxide therapy at 8 and 18 months, whereas one of them had resolution of hyperinsulinemic hypoglycemia at 32 months of age. RESULTS: Sequencing of the HNF4A gene identified heterozygous mutations in all three families. In family 1, a frameshift mutation L330fsdel17ins9 (c.987 1003del17ins9; p.Leu330fs) was present in the proband; a mutation affecting the conserved a nucleotide of the intron 2 branch site (c.264-21A>G) was identified in the proband of family 2; and finally a nonsense mutation, Y16X (c.48C>G, p.Tyr16X), was found in the proband of family 3. CONCLUSIONS: Heterozygous HNF4A mutations can therefore cause both transient and persistent hyperinsulinemic hypoglycemia associated with macrosomia. We recommend that macrosomic infants with transient or persistent hyperinsulinemic hypoglycemia should be screened for HNF4A mutations if there is a family history of youth-onset diabetes.

OBJECTIVE: to determine the pathogenicity of a novel conserved missense mutation, p.Ser98Phe, in the emopamil binding protein (EBP) gene in order to perform a prenatal diagnostic test for X-linked dominant chondrodysplasia punctata (CDPX2) in a male foetus at 50% risk. METHODS: Family members were tested for p.Ser98Phe using PCR and sequence analysis of leucocyte or buccal cell DNA. Haplotype analysis was employed to identify the grandparental chromosome on which p.Ser98Phe had arisen. In silico protein analyses were used to predict whether p.Ser98Phe significantly altered the EBP protein structure. RESULTS: the mutation was detected in the proband and her affected mother but not in the maternal grandmother, who was thought to be mildly affected. However, haplotype analysis showed that p.Ser98Phe had arisen de novo on the grandpaternal X chromosome. Protein secondary structure predictions suggested that p.Ser98Phe alters the properties of the helix within which it is located. CONCLUSION: We concluded that p.Ser98Phe is likely to be pathogenic and proceeded with prenatal testing. The male foetus had not inherited p.Ser98Phe and his unaffected status was confirmed at birth. This family demonstrates some of the difficulties in interpreting the significance of novel missense mutations, particularly when results are needed urgently for prenatal diagnosis.

A 10-year-old Arabic boy of consanguineous parents has suffered eight episodes of acute liver failure with haemolysis triggered by intercurrent febrile illnesses. The first crisis occurred at 9 months of age, after which diabetes mellitus developed. By the age of 6 years, short stature, mild myopathy and later skeletal epiphyseal dysplasia also became evident. His psychosocial development and educational achievements have remained within normal limits. While there were no clear biochemical indicators of a mitochondrial disorder, an almost complete deficiency of complex I of the respiratory chain was demonstrated in liver but not in fibroblast or muscle samples. Molecular analysis of the eukaryotic translation initiation factor 2alpha kinase gene (EIF2AK3) demonstrated a homozygous mutation, compatible with a diagnosis of Wolcott-Rallison syndrome (WRS). This patient's course adds a new perspective to the presentation of WRS caused by mutations in the EIF2AK3 gene linking it to mitochondrial disorders: recoverable and recurrent acute liver failure. The findings also illustrate the diagnostic difficulty of mitochondrial disease as it cannot be excluded by muscle or skin biopsy in patients presenting with liver disease. The case also further complicates the decision-making process for liver transplantation in cases of acute liver failure in the context of a possible mitochondrial disorder. Such patients may be more likely to recover spontaneously if a mitochondrial disorder underlies the liver failure, yet without neurological features liver transplantation remains an option.

CONTEXT: IGF-II is an imprinted gene (predominantly transcribed from the paternally inherited allele), which has an important role in fetal growth in mice. IGF2 gene expression is regulated by a complex system of enhancers and promoters that determine tissue-specific and development-specific transcription. In mice, enhancers of the IGF2 gene are located up to 260 kb telomeric to the gene. The role of IGF-II in humans is unclear. OBJECTIVE: a woman of short adult stature (1.46 m, -3 sd score) born with severe intrauterine growth retardation (1.25 kg at term, -5.4 SD score) and atypical diabetes diagnosed at the age of 23 yr had a balanced chromosomal translocation t(1;11) (p36.22; p15.5). We hypothesized that her phenotype resulted from disruption of her paternally derived IGF2 gene because her daughter who inherited the identical translocation had normal birth weight. DESIGN: Both chromosomal break points were identified using fluorescent in situ hybridization. Sequence, methylation, and expression of the IGF2 gene was examined. Hyperinsulinemic, euglycemic clamp with glucose tracers and magnetic resonance imaging of the thorax, abdomen, and pelvis were performed. RESULTS: the 11p15.5 break point mapped 184 kb telomeric of the IGF2 gene. Microsatellite markers confirmed paternal origin of this chromosome. IGF2 gene sequence and methylation was normal. IGF2 gene expression was reduced in lymphoblasts. Clamp studies showed marked hepatic and total insulin resistance. Massive excess sc fat was seen on magnetic resonance imaging despite slim body mass index (21.1 kg/m2). CONCLUSIONS: a break point 184 kb upstream of the paternally derived IGF2 gene, separating it from some telomeric enhancers, resulted in reduced expression in some mesoderm-derived adult tissues causing intrauterine growth retardation, short stature, lactation failure, and insulin resistance with altered fat distribution.

We describe a woman who first presented with gestational diabetes at 26 weeks gestation and was managed with insulin. Following delivery of a healthy baby she had an abnormal OGTT (oral glucose tolerance test) 6 weeks post partum and was managed with diet. In her second pregnancy she was diagnosed with gestational diabetes at 10 weeks and required insulin. Following delivery she was again managed on diet alone. Four years later, during her third pregnancy, she was managed with insulin from the outset. She remained on insulin post partum and for several years. Later her two younger children, aged 11 years and 7 years, were found to have GCK mutation causing MODY (Maturity Onset Diabetes of the Young) subtype glucokinase. Following this she underwent molecular genetic testing and was also shown to have the GCK mutation. She was gradually taken off insulin and is now managed on diet alone with excellent glycaemic control. Her two children are under regular follow up care and on no medication for diabetes. © 2008 Versita Warsaw and Springer-Verlag.

OBJECTIVE: Mutations in the alternatively spliced HNF4A gene cause maturity-onset diabetes of the young (MODY). We characterized the spatial and developmental expression patterns of HNF4A transcripts in human tissues and investigated their role as potential moderators of the MODY phenotype. RESEARCH DESIGN AND METHODS: We measured the expression of HNF4A isoforms in human adult tissues and gestationally staged fetal pancreas by isoform-specific real-time PCR. The correlation between mutation position and age of diagnosis or age-related penetrance was assessed in a cohort of 190 patients with HNF4A mutations. RESULTS: HNF4A was expressed exclusively from the P2 promoter in adult pancreas, but from 9 weeks until at least 26 weeks after conception, up to 23% of expression in fetal pancreas was of P1 origin. HNF4A4-6 transcripts were not detected in any tissue. In whole pancreas, HNF4A9 expression was greater than in islets isolated from the endocrine pancreas (relative level 22 vs. 7%). Patients with mutations in exons 9 and 10 (absent from HNF4A3, HNF4A6, and HNF4A9 isoforms) developed diabetes later than those with mutations in exons 2-8, where all isoforms were affected (40 vs. 24 years; P = 0.029). Exon 9/10 mutations were also associated with a reduced age-related penetrance (53 vs. 10% without diabetes at age 55 years; P < 0.00001). CONCLUSIONS: We conclude that isoforms derived from the HNF4A P1 promoter are expressed in human fetal, but not adult, pancreas, and that their presence during pancreatic development may moderate the diabetic phenotype in individuals with mutations in the HNF4A gene.

Heterozygous mutations of the tissue-specific transcription factor hepatocyte nuclear factor (HNF)1beta, cause maturity onset diabetes of the young (MODY5) and kidney anomalies including agenesis, hypoplasia, dysplasia and cysts. Because of these renal anomalies, HNF1beta is classified as a CAKUT (congenital anomalies of the kidney and urinary tract) gene. We searched for human fetal kidney proteins interacting with the N-terminal region of HNF1beta using a bacterial two-hybrid system and identified five novel proteins along with the known partner DCoH. The interactions were confirmed for four of these proteins by GST pull-down assays. Overexpression of two proteins, E4F1 and ZFP36L1, in Xenopus embryos interfered with pronephros formation. Further, in situ hybridization showed overlapping expression of HNF1beta, E4F1 and ZFP36L1 in the developing pronephros. HNF1beta is present largely in the nucleus where it colocalized with E4F1. However, ZFP36L1 was located predominantly in the cytoplasm. A nuclear function for ZFP36L1 was shown as it was able to reduce HNF1beta transactivation in a luciferase reporter system. Our studies show novel proteins may cooperate with HNF1beta in human metanephric development and propose that E4F1 and ZFP36L1 are CAKUT genes. We searched for mutations in the open reading frame of the ZFP36L1 gene in 58 patients with renal anomalies but found none.

Obesity is a serious international health problem that increases the risk of several common diseases. The genetic factors predisposing to obesity are poorly understood. A genome-wide search for type 2 diabetes-susceptibility genes identified a common variant in the FTO (fat mass and obesity associated) gene that predisposes to diabetes through an effect on body mass index (BMI). An additive association of the variant with BMI was replicated in 13 cohorts with 38,759 participants. The 16% of adults who are homozygous for the risk allele weighed about 3 kilograms more and had 1.67-fold increased odds of obesity when compared with those not inheriting a risk allele. This association was observed from age 7 years upward and reflects a specific increase in fat mass.

OBJECTIVES: Activating mutations in the human KCNJ11 gene, encoding the pore-forming subunit (Kir6.2) of the ATP-sensitive potassium (K(ATP)) channel, are one cause of neonatal diabetes mellitus. In a few patients, KCNJ11 mutations cause a triad of developmental delay, epilepsy, and neonatal diabetes (DEND syndrome). The aim of this study was to determine the clinical effects, functional cause, and sensitivity to sulfonylurea treatment of a novel KCNJ11 mutation producing DEND syndrome. METHODS: We screened the DNA of a 3-year-old patient with neonatal diabetes, severe developmental delay, and therapy-resistant epilepsy for mutations in KCNJ11. We carried out electrophysiologic analysis of wild-type and mutant K(ATP) channels heterologously expressed in Xenopus oocytes. RESULTS: We identified a novel Kir6.2 mutation (I167L) causing DEND syndrome. Functional analysis showed both homomeric and heterozygous mutant channels were less inhibited by MgATP leading to an increase in whole-cell K(ATP) currents. This effect was due to an increase in the intrinsic open probability. Heterozygous channels were strongly inhibited by the sulfonylurea tolbutamide. Treatment of the patient with the sulfonylurea glibenclamide not only enabled insulin therapy to be stopped, but also resulted in improvement in epilepsy and psychomotor abilities. CONCLUSIONS: We report a case of developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome that shows neurologic improvement with sulfonylurea therapy. Early recognition of patients with DEND syndrome may have considerable therapeutic benefit for the patient.

Achieving a specific diagnosis of polycythemia vera (PV) and other myeloproliferative disorders (MPDs) is often costly and complex. However, the recent identification of a V617F mutation in the JH2 domain of the JAK2 gene in a high proportion of patients suffering from MPDs may provide confirmation of a diagnosis. This is an acquired mutation and, as such, may only be present in a small number of cells within a sample. There is therefore a clinical need for highly sensitive detection techniques. We have developed a sensitive real-time polymerase chain reaction (PCR)-based approach for both detection and quantification of the JAK2 V671F mutation load, which allows determination of mutation status without the need for prior purification of granulocytes. We have performed a comparison of this assay with two previously published detection methods. Although an amplification refractory mutation system (ARMS) was shown to be slightly superior in terms of sensitivity, our real-time PCR method provides the potential for quantification of the JAK2 V617F mutation, having potential future applications in the monitoring of minimal residual disease or predicting outcome of disease severity.

BACKGROUND/AIM: Glucokinase (GCK)-activating mutations cause persistent hyperinsulinaemic hypoglycaemia of infancy (PHHI). GCK-PHHI patients have regulated insulin secretion and can usually be treated with diazoxide. The six reported cases suggest that the severity of the mutation predicts the clinical phenotype. The aim of this study was to relate genotype to phenotype [clinical phenotype, glucose-stimulated insulin release (GSIR) and GCK functional analysis] in a large pedigree with eight affected individuals. METHODS: the genes encoding B-cell GCK and the K(ATP) channel subunits (ABCC8 and KCNJ11) were sequenced to identify mutations for functional analysis. Genetic variants influencing B-cell function were genotyped in affected individuals. Islet secretory capacity was determined by oral glucose tolerance test RESULTS: a novel GCK mutation (G68V) co-segregating with hypoglycaemia was identified in eight family members. Kinetic analysis revealed that G68V-GCK activity is ~16 times more than wild-type-GCK with an increased affinity for glucose [concentration at half maximal activation (S(0.5)) 1.94 +/- 0.16 vs. 7.43 +/- 0.12, mutant vs. wild type, mean +/- sem]. Mathematical modelling predicted a threshold for GSIR of 1.9 mmol/l in the mutant. Oral glucose tolerance tests showed regulated insulin secretion. The severity of hypoglycaemia and related symptoms in affected subjects were heterogeneous. Clinical presentations were asymptomatic (n = 1), extreme hunger (n = 3), seizures (n = 2) and loss of consciousness (n = 2); 7/8 were managed with diet but the proband was treated with diazoxide and octreotide. Phenotypic modification by a second mutation in the K(ATP) channel genes (ABCC8, KCNJ11) or by common genetic variants in KCNJ11, GCK and TCF7L2 was excluded. CONCLUSION: the novel activating GCK mutation G68V is associated with variable phenotypic severity, supporting modification of GSIR by genetic and/or environmental factors.

Gain-of-function mutations in the genes encoding the ATP-sensitive potassium (K(ATP)) channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) are a common cause of neonatal diabetes mellitus. Here we investigate the molecular mechanism by which two heterozygous mutations in the second nucleotide-binding domain (NBD2) of SUR1 (R1380L and R1380C) separately cause neonatal diabetes. SUR1 is a channel regulator that modulates the gating of the pore formed by Kir6.2. K(ATP) channel activity is inhibited by ATP binding to Kir6.2 but is stimulated by MgADP binding, or by MgATP binding and hydrolysis, at the NBDs of SUR1. Functional analysis of purified NBD2 showed that each mutation enhances MgATP hydrolysis by purified isolated fusion proteins of maltose-binding protein and NBD2. Inhibition of ATP hydrolysis by MgADP was unaffected by mutation of R1380, but inhibition by beryllium fluoride (which traps the ATPase cycle in the prehydrolytic state) was reduced. MgADP-dependent activation of K(ATP) channel activity was unaffected. These data suggest that the R1380L and R1380C mutations enhance the off-rate of P(i), thereby enhancing the hydrolytic rate. Molecular modeling studies supported this idea. Because mutant channels were inhibited less strongly by MgATP, this would increase K(ATP) currents in pancreatic beta cells, thus reducing insulin secretion and producing diabetes.

We report 10 heterozygous mutations in the human insulin gene in 16 probands with neonatal diabetes. A combination of linkage and a candidate gene approach in a family with four diabetic members led to the identification of the initial INS gene mutation. The mutations are inherited in an autosomal dominant manner in this and two other small families whereas the mutations in the other 13 patients are de novo. Diabetes presented in probands at a median age of 9 weeks, usually with diabetic ketoacidosis or marked hyperglycemia, was not associated with beta cell autoantibodies, and was treated from diagnosis with insulin. The mutations are in critical regions of the preproinsulin molecule, and we predict that they prevent normal folding and progression of proinsulin in the insulin secretory pathway. The abnormally folded proinsulin molecule may induce the unfolded protein response and undergo degradation in the endoplasmic reticulum, leading to severe endoplasmic reticulum stress and potentially beta cell death by apoptosis. This process has been described in both the Akita and Munich mouse models that have dominant-acting missense mutations in the Ins2 gene, leading to loss of beta cell function and mass. One of the human mutations we report here is identical to that in the Akita mouse. The identification of insulin mutations as a cause of neonatal diabetes will facilitate the diagnosis and possibly, in time, treatment of this disorder.

BACKGROUND: Macrosomia is associated with considerable neonatal and maternal morbidity. Factors that predict macrosomia are poorly understood. The increased rate of macrosomia in the offspring of pregnant women with diabetes and in congenital hyperinsulinaemia is mediated by increased foetal insulin secretion. We assessed the in utero and neonatal role of two key regulators of pancreatic insulin secretion by studying birthweight and the incidence of neonatal hypoglycaemia in patients with heterozygous mutations in the maturity-onset diabetes of the young (MODY) genes HNF4A (encoding HNF-4alpha) and HNF1A/TCF1 (encoding HNF-1alpha), and the effect of pancreatic deletion of Hnf4a on foetal and neonatal insulin secretion in mice. METHODS AND FINDINGS: We examined birthweight and hypoglycaemia in 108 patients from families with diabetes due to HNF4A mutations, and 134 patients from families with HNF1A mutations. Birthweight was increased by a median of 790 g in HNF4A-mutation carriers compared to non-mutation family members (p < 0.001); 56% (30/54) of HNF4A-mutation carriers were macrosomic compared with 13% (7/54) of non-mutation family members (p < 0.001). Transient hypoglycaemia was reported in 8/54 infants with heterozygous HNF4A mutations, but was reported in none of 54 non-mutation carriers (p = 0.003). There was documented hyperinsulinaemia in three cases. Birthweight and prevalence of neonatal hypoglycaemia were not increased in HNF1A-mutation carriers. Mice with pancreatic beta-cell deletion of Hnf4a had hyperinsulinaemia in utero and hyperinsulinaemic hypoglycaemia at birth. CONCLUSIONS: HNF4A mutations are associated with a considerable increase in birthweight and macrosomia, and are a novel cause of neonatal hypoglycaemia. This study establishes a key role for HNF4A in determining foetal birthweight, and uncovers an unanticipated feature of the natural history of HNF4A-deficient diabetes, with hyperinsulinaemia at birth evolving to decreased insulin secretion and diabetes later in life.

OBJECTIVES: 'Persistent cloaca' is a severe malformation affecting females in which the urinary, genital and alimentary tracts share a single conduit. Previously, a Uroplakin IIIA (UPIIIA) mutation was reported in one individual with persistent cloaca, and UPIIIA, Sonic Hedgehog (SHH), Ephrin B2 (EFNB2) and Hepatocyte Nuclear Factor 1beta (HNF1beta) are expressed during the normal development of organs that are affected in this condition. HNF1beta mutations have been associated with uterine malformations in humans, and mutations of genes homologous to human SHH or EFNB2 cause persistent cloaca in mice. PATIENTS AND METHODS: We sought mutations of coding regions of UPIIIA, SHH, EFNB2 and HNF1beta genes by direct sequencing in a group of 20 patients with persistent cloaca. Most had associated malformations of the upper renal tract and over half had impaired renal excretory function. The majority of patients had congenital anomalies outside the renal/genital tracts and two had the VACTERL association. RESULTS: Apart from a previously described index case, we failed to find UPIIIA mutations, and no patient had a SHH, EFNB2 or HNF1beta mutation. CONCLUSION: Persistent cloaca is only rarely associated with UPIIIA mutation. Despite the fact that SHH and EFNB2 are appealing candidate genes, based on their expression patterns and mutant mice phenotypes, they were not mutated in these humans with persistent cloaca. Although HNF1beta mutations can perturb paramesonephric duct fusion in humans, HNF1beta was not mutated in persistent cloaca.

Transient neonatal diabetes mellitus (TNDM) is diagnosed in the first 6 months of life, with remission in infancy or early childhood. For approximately 50% of patients, their diabetes will relapse in later life. The majority of cases result from anomalies of the imprinted region on chromosome 6q24, and 14 patients with ATP-sensitive K+ channel (K(ATP) channel) gene mutations have been reported. We determined the 6q24 status in 97 patients with TNDM. In patients in whom no abnormality was identified, the KCNJ11 gene and/or ABCC8 gene, which encode the Kir6.2 and SUR1 subunits of the pancreatic beta-cell K(ATP) channel, were sequenced. K(ATP) channel mutations were found in 25 of 97 (26%) TNDM probands (12 KCNJ11 and 13 ABCC8), while 69 of 97 (71%) had chromosome 6q24 abnormalities. The phenotype associated with KCNJ11 and ABCC8 mutations was similar but markedly different from 6q24 patients who had a lower birth weight and who were diagnosed and remitted earlier (all P < 0.001). K(ATP) channel mutations were identified in 26 additional family members, 17 of whom had diabetes. of 42 diabetic patients, 91% diagnosed before 6 months remitted, but those diagnosed after 6 months had permanent diabetes (P < 0.0001). K(ATP) channel mutations account for 89% of patients with non-6q24 TNDM and result in a discrete clinical subtype that includes biphasic diabetes that can be treated with sulfonylureas. Remitting neonatal diabetes was observed in two of three mutation carriers, and permanent diabetes occurred after 6 months of age in subjects without an initial diagnosis of neonatal diabetes.

AIMS/HYPOTHESIS: Heterozygous mutations of glucokinase (GCK) and hepatocyte nuclear factor-1 alpha (HNF1A; also known as hepatic transcription factor 1 [TCF1]) genes are the most common cause of MODY. Genomic deletions of the HNF1B (also known as TCF2) gene have recently been shown to account for one third of mutations causing renal cysts and diabetes syndrome. We investigated the prevalence of partial and whole gene deletions in UK patients meeting clinical criteria for GCK or HNF-1alpha/-4alpha MODY and in whom no mutation had been identified by sequence analysis. METHODS: a multiplex ligation-dependent probe amplification (MLPA) assay was developed using synthetic oligonucleotide probes for 30 exons of the GCK, HNF1A and HNF4A genes. RESULTS: Partial or whole gene deletions were identified in 1/29 (3.5%) probands using the GCK MLPA assay and 4/60 (6.7%) of probands using the HNF1A/-4A MLPA assay. Four different deletions were detected: GCK exon 2, HNF1A exon 1, HNF1A exons 2 to 10 and HNF1A exons 1 to 10. An additional Danish pedigree with evidence of linkage to HNF1A had a deletion of exons 2 to 10. Testing other family members confirmed co-segregation of the deletion mutations with diabetes in the pedigrees. CONCLUSIONS/INTERPRETATION: Large deletions encompassing whole exons can cause GCK or HNF-1alpha MODY and will not be detected by sequencing. Gene dosage assays, such as MLPA, are a useful adjunct to sequence analysis when a diagnosis of MODY is strongly suspected.

BACKGROUND: UK and US policy initiatives have suggested that, in the future, patients and clinicians in mainstream medicine could use genetic information to prevent common illnesses. There are no studies on patients' experience and understanding of the process of testing for common genetic susceptibilities in mainstream medicine. METHODS: Qualitative interviews with 42 individuals who had undergone testing for a genetic susceptibility for deep vein thrombosis in primary and secondary care in the UK. RESULTS: Some participants, often from higher social classes, had a good understanding of the test and its implications. They had often sought additional information on thrombophilia from relatives and from the Internet. Others, often from less privileged backgrounds, had a poorer understanding of the test--seven individuals were unaware of having had the genetic test. Features of genetic information led to misunderstandings: (i) at referral, (ii) when communicating results, and (iii) when making sense of the implications of testing. Participants' accounts indicated that non-specialist doctors may feel obliged to refer a patient for a genetic test they know little about, because a patient requests it after a relative had tested positive. Sometimes a referral for a genetic test was lost under information overload when multiple tests and issues were considered. The inconsistent and informal ways of communicating test results--for example by phone--in mainstream medicine also led to confusion. Participants did not generally overestimate their risk, but some were uncertain about whether they were taking the right preventive actions and/or whether their children were at risk. Information about genetic susceptibilities was difficult to make sense of, as it related to ambiguous risks for participants and family members, complicated and unfamiliar terminology and multiple genes and preventive strategies. CONCLUSION: Policy visions of clinicians and patients in mainstream medicine seeking and using genetic information at their own initiative may not be realistic. Patients need more direct support in making sense of genetic information, if this information is to bring the anticipated health benefits, and not fuel health inequalities or create ethical problems. Clinicians in secondary and primary care need guidance to help them introduce genetic tests, communicate their results and explain their implications.

CONTEXT: Mutations in the KCNJ11 and ABCC8 genes encoding the pancreatic beta-cell K(ATP) channel have recently been shown to be the most common cause of permanent neonatal diabetes mellitus (PNDM). Information regarding the frequency of PNDM has been based mainly on nonpopulation or short-term collections only. Thus, the aim of this study was to identify the incidence of PNDM in Slovakia and to switch patients to sulfonylurea (SU) where applicable. DESIGN: We searched for PNDM patients in the Slovak Children Diabetes Registry. In insulin-treated patients who matched the clinical criteria for PNDM, the KCNJ11 or ABCC8 genes were sequenced, and mutation carriers were invited for replacement of insulin with SU. RESULTS: Eight patients with diabetes onset before the sixth month of life without remission were identified since 1981, which corresponds to the PNDM incidence in Slovakia of one case in 215,417 live births. In four patients, three different KCNJ11 mutations were found (R201H, H46Y, and L164P). Three patients with the KCNJ11 mutations (R201H and H46Y) were switched from insulin to SU, decreasing their glycosylated hemoglobin from 9.3-11.0% on insulin to 5.7-6.6% on SU treatment. One patient has a novel V86A mutation in the ABCC8 gene and was also substituted with SU. CONCLUSIONS: PNDM frequency in Slovakia is much higher (one in 215,417 live births) than previously suggested from international estimates (about one in 800,000). We identified one ABCC8 and four KCNJ11 mutation carriers, of whom four were successfully transferred to SU, dramatically improving their diabetes control and quality of life.

The molecular mechanisms involved in the development of type 2 diabetes are poorly understood. Starting from genome-wide genotype data for 1924 diabetic cases and 2938 population controls generated by the Wellcome Trust Case Control Consortium, we set out to detect replicated diabetes association signals through analysis of 3757 additional cases and 5346 controls and by integration of our findings with equivalent data from other international consortia. We detected diabetes susceptibility loci in and around the genes CDKAL1, CDKN2A/CDKN2B, and IGF2BP2 and confirmed the recently described associations at HHEX/IDE and SLC30A8. Our findings provide insight into the genetic architecture of type 2 diabetes, emphasizing the contribution of multiple variants of modest effect. The regions identified underscore the importance of pathways influencing pancreatic beta cell development and function in the etiology of type 2 diabetes.

BACKGROUND: Congenital fibrosis of the extraocular muscles types 1 and 3 (CFEOM1/CFEOM3) are autosomal dominant strabismus disorders that appear to result from maldevelopment of ocular nuclei and nerves. We previously reported that most individuals with CFEOM1 and rare individuals with CFEOM3 harbor heterozygous mutations in KIF21A. KIF21A encodes a kinesin motor involved in anterograde axonal transport, and the familial and de novo mutations reported to date predictably alter one of only a few KIF21A amino acids--three within the third coiled-coil region of the stalk and one in the distal motor domain, suggesting they result in altered KIF21A function. To further define the spectrum of KIF21A mutations in CFEOM we have now identified all CFEOM probands newly enrolled in our study and determined if they harbor mutations in KIF21A. RESULTS: Sixteen CFEOM1 and 29 CFEOM3 probands were studied. Three previously unreported de novo KIF21A mutations were identified in three CFEOM1 probands, all located in the same coiled-coil region of the stalk that contains all but one of the previously reported mutations. Eight additional CFEOM1 probands harbored three of the mutations previously reported in KIF21A; seven had one of the two most common mutations, while one harbored the mutation in the distal motor domain. No mutation was detected in 5 CFEOM1 or any CFEOM3 probands. CONCLUSION: Analysis of sixteen CFEOM1 probands revealed three novel KIF21A mutations and confirmed three reported mutations, bringing the total number of reported KIF21A mutations in CFEOM1 to 11 mutations among 70 mutation positive probands. All three new mutations alter amino acids in heptad repeats within the third coiled-coil region of the KIF21A stalk, further highlighting the importance of alterations in this domain in the etiology of CFEOM1.

Apolipoprotein AV (ApoAV) gene variant, -1131T>C, is associated with increased triglyceride concentrations in all ethnic groups studied. An MseI based RFLP analysis is the most commonly used method for genotyping this SNP. We genotyped a large cohort comprising 1185 Asian Indians and 173 UK Caucasians for -1131T>C using an ARMS-PCR based tetra-primer method. For quality control, we re-genotyped approximately 10% random samples from this cohort utilizing the MseI RFLP, which showed a 2.9% (3/102) genotyping error rate between the two methods. To investigate further, we sequenced the 900 bp region around the -1131T>C polymorphism in 25 Asian Indians and 15 UK Caucasians and found a number of polymorphisms including the -987C>T polymorphism. Further analysis of the -987C>T SNP showed a higher rare allele frequency of 0.23 in Asian Indians (n = 158) compared to 0.09 in the UK Caucasians (n = 157). This SNP is located 4 bp from the 3' end of the RFLP forward primer and is in weak linkage disequilibrium with -1131T>C variant (r2 = 0.084 and D' = 1). Repeated RFLP analysis of seven subjects heterozygous for -987C>T (seven times), showed discordant results with the sequence at -1131T>C SNP nearly one third (15/49) of the time. We conclude that presence of -987C>T polymorphism in the forward primer of the MseI RFLP assay may lead to allelic drop-out and generate unforeseen errors in genotyping the -1131T>C polymorphism. Our results also emphasise the need for careful quality control in all molecular genetic studies, particularly while transferring genotyping methods between various ethnic groups.

AIMS: Maturity onset diabetes of the young (MODY) is a monogenic form of diabetes where correct diagnosis alters treatment, prognosis and genetic counselling. The first UK survey of childhood MODY identified 20 White, but no Asian children with MODY. We hypothesized that MODY causes diabetes in UK Asians, but is underdiagnosed. METHODS: Children with dominant family histories of diabetes were recruited. Direct sequencing for mutations in the two most common MODY genes; HNF1A (TCF1) and GCK was performed in autoantibody-negative probands. We also compared MODY testing data for Asian and White cases from the Exeter MODY database, to 2001 UK census data. RESULTS: We recruited 30 families and identified three Asian families with MODY gene mutations (two HNF1A, one GCK) and three White UK families (two HNF1A, one GCK). Heterozygous MODY phenotypes were similar in Asians and Whites. Only eight (0.5%) of 1369 UK referrals for MODY testing were known to be Asian, but in 2001 Asians represented 4% of the English/Welsh population and have a higher prevalence of diabetes. CONCLUSIONS: We identified three cases of childhood MODY in UK Asians and demonstrated reduced rates of MODY testing in Asians, which has negative implications for treatment. It is unclear why this is. MODY should be considered in autoantibody-negative Asian diabetes patients lacking evidence of insulin resistance.

A molecular genetic diagnosis is now possible for > 80% of patients with monogenic diabetes. This not only provides accurate information regarding inheritance and prognosis, but can inform treatment decisions and improve clinical outcome. Mild fasting hyperglycaemia caused by heterozygous GCK mutations rarely requires pharmacological intervention, whereas patients with mutations in the genes encoding the transcription factors HNF-1alpha and HNF-4alpha respond well to low doses of sulphonylureas. The recent discovery that mutations in the KCNJ11 gene (encoding the Kir6.2 subunit of the K(ATP) channel) are the most common cause of permanent neonatal diabetes, has enabled children to stop insulin injections and achieve improved glycaemic control with high doses of sulphonylurea tablets. Molecular genetic testing is an essential prerequisite for the pharmacogenetic treatment of monogenic diabetes.

Children with permanent diabetes are usually assumed to have type 1 diabetes. It has recently been shown that there are genetic subgroups of diabetes that are often diagnosed during the neonatal period but may present later. A recent Italian study proposed that type 1 diabetes is rare before 6 months of age. We aimed to examine genetic susceptibility to type 1 diabetes in patients diagnosed with diabetes before the age of 2 years. We analyzed HLA class II genotypes, markers of autoimmune diabetes, in 187 children with permanent diabetes diagnosed at

AIM: the transcription factor hepatocyte nuclear factor-1beta (HNF-1beta) is expressed in rodent pancreatic progenitor cells, where it is an important member of the genetic hierarchy that regulates the generation of pancreatic endocrine and exocrine cells. The recent description of an HNF-1beta mutation in a patient with neonatal diabetes suggests that HNF-1beta may also play a key role in human pancreatic B-cell development. We aimed to investigate the role of HNF-1beta mutations in neonatal diabetes and also the impact of HNF-1beta mutations on fetal growth. METHODS: We sequenced the HNF-1beta gene in 27 patients with neonatal diabetes in whom other known genetic aetiologies had been excluded. Birth weight was investigated in 21 patients with HNF-1beta mutations. RESULTS: a heterozygous HNF-1beta mutation, S148L, was identified in one patient with neonatal diabetes diagnosed at 17 days, which rapidly resolved only to relapse at 8 years. This patient had pancreatic atrophy, mild exocrine insufficiency and low birth weight (1.83 kg at 40 weeks' gestation). Intrauterine growth was markedly reduced in patients born to unaffected mothers with a median birth weight of 2.4 kg (range 1.8-3.3) (P = 0.006), median centile weight 3 (0.008-38), and 69% were small for gestational age. CONCLUSION: HNF-1beta mutations are a rare cause of neonatal diabetes as well as pancreatic exocrine and endocrine dysfunction. Low birth weight is a common feature of patients with HNF-1beta mutations and is consistent with reduced insulin secretion in utero. These findings support a key role for HNF-1beta in early pancreatic progenitor cells in man.

The generation of multiple transcripts by mRNA processing has the potential to moderate differences in gene expression both between tissues and at different stages of development. Where gene function is compromised by mutation, the presence of multiple isoforms may influence the resulting phenotype. Heterozygous mutations in the transcription factor hepatocyte nuclear factor-1 alpha (HNF1A or TCF1 gene) result in early-onset diabetes as a result of pancreatic beta-cell dysfunction. We investigated the expression of the three alternatively processed isoforms of the HNF1A gene and their impact on the phenotype associated with mutations. Real-time PCR demonstrated variation in tissue expression of HNF1A isomers: HNF1A(A), with the lowest transactivation activity compared with the truncated isoforms HNF1A(B) and HNF1A(C), is the major isomer in liver (54%) and kidney (67%) but not in adult pancreas (24%) and islets (26%). However, in fetal pancreas HNF1A(A) is the major transcript (84%), which supports developmental regulation of isomer expression. We examined whether the isomers affected by the mutation altered the diabetes phenotype in 564 subjects with 123 mutations in HNF1A. Mutations that affected only isomer HNF1A(A) (exons 8-10) were diagnosed later (25.5 years) than mutations affecting all three isomers (exons 1-6) (18.0 years) (P=0.006). This first genotype/phenotype relationship described for patients with HNF1A mutations is explained by isomer structure and not by either mutation type or functional domain. We conclude that all three isomers may be critical for beta-cell function and could play a role in both the developing and mature beta cell.

Heterozygous activating mutations in the gene encoding for the ATP-sensitive potassium channel subunit Kir6.2 (KCNJ11) have recently been shown to be a common cause of permanent neonatal diabetes. Kir6.2 is expressed in muscle, neuron and brain as well as the pancreatic beta-cell, so patients with KCNJ11 mutations could have a neurological phenotype in addition to their diabetes. It is proposed that some patients with KCNJ11 mutations have neurological features that are part of a discrete neurological syndrome termed developmental Delay, Epilepsy and Neonatal Diabetes (DEND), but there are also neurological consequences of chronic or acute diabetes. We identified KCNJ11 mutations in four of 10 probands with permanent neonatal diabetes and one affected parent; this included the novel C166F mutation and the previously described V59M and R201H. Four of the five patients with mutations had neurological features: the patient with the C166F mutation had marked developmental delay, severe generalised epilepsy, hypotonia and muscle weakness; mild developmental delay was present in the patient with the V59M mutation; one patient with the R201H mutation had acute and chronic neurological consequences of cerebral oedema and another had diabetic neuropathy from chronic hyperglycaemia. In conclusion, the clinical features in these patients support the existence of a discrete neurological syndrome with KCNJ11 mutations. The severe DEND syndrome was seen with the novel C166F mutation and mild developmental delay with the V59M mutation. These features differ markedly from the neurological consequences of acute or chronic diabetes.

Polymerase chain reaction is commonly used to detect t(11;14)(q13;q32) and t(14;18)(q32;q21) chromosomal translocations associated with mantle cell lymphoma and follicular lymphoma. We tested a total of 482 samples from patients with suspected non-Hodgkin's lymphoma and sequenced unusual-sized t(11;14)(q13;q32) and t(14;18)(q32;q21) products from 33 of these patients. BCL-1 or BCL-2 gene rearrangements were confirmed in 23 of 33 patients (70%). Considerable size variation was observed using t(11;14) primers, with MTCA and MTCB t(11;14) products ranging from 234 to 934 bp and 143 to 560 bp respectively. Less variability was observed for t(14;18) Major Breakpoint Region (MBR) products (100-252 bp) but Minor Cluster Region (MCR) products ranged from 217 to 498 bp. We demonstrate the utility of sequence analysis to confirm unusual-sized translocation products and reduce false-positive results because of nonspecific amplification.

BACKGROUND: Trastuzumab-based chemotherapy can improve median survival in the metastatic setting when used in patients with cancer that overexpresses HER2/neu. In addition to HER2 expression, other molecular markers are needed to better predict outcomes after the initiation of trastuzumab-based chemotherapy and to elucidate potential mechanisms of resistance to trastuzumab. PATIENTS AND METHODS: Patients with clinical documentation of HER2/neu-overexpressing metastatic breast cancer treated with trastuzumab between 1998 and 2004 were identified from the British Columbia Provincial Pharmacy Database. Tissues were obtained for microarray analysis of 153 of 306 patients who fit our clinical criteria. Tissue microarrays were constructed for the analysis of multiple molecular factors, and results were correlated to clinical outcomes. Immunohistochemistry was performed on the microarray specimens, and results were correlated with survival and time to progression. RESULTS: Factors commonly associated with poor prognosis in the metastatic setting in general, including short disease-free intervals, high tumor grade, and low estrogen receptor status, were all associated with poor survival in this population with HER2 overexpression. Overexpression of HER3 was observed in 9% of specimens and was associated with a trend toward worse overall survival (P = 0.1). HER3 expression was not correlated with a significant difference in time to progression but did trend to predict for worse survival after progression (P = 0.06). In multivariate analysis, tumor grade and HER3 expression were significantly predictive of overall survival. Phosphatase and tensin homologue status did not appear to correlate with response or survival. CONCLUSION: Our findings suggest that prognosis after initiation of trastuzumab-based chemotherapy depends, in part, on coexpression of HER3.

The spondylocostal dysostoses (SCDs) are a heterogeneous group of vertebral malsegmentation disorders that arise during embryonic development by a disruption of somitogenesis. Previously, we had identified two genes that cause a subset of autosomal recessive forms of this disease: DLL3 (SCD1) and MESP2 (SCD2). These genes are important components of the Notch signaling pathway, which has multiple roles in development and disease. Here, we have used a candidate-gene approach to identify a mutation in a third Notch pathway gene, LUNATIC FRINGE (LFNG), in a family with autosomal recessive SCD. LFNG encodes a glycosyltransferase that modifies the Notch family of cell-surface receptors, a key step in the regulation of this signaling pathway. A missense mutation was identified in a highly conserved phenylalanine close to the active site of the enzyme. Functional analysis revealed that the mutant LFNG was not localized to the correct compartment of the cell, was unable to modulate Notch signaling in a cell-based assay, and was enzymatically inactive. This represents the first known mutation in the human LFNG gene and reinforces the hypothesis that proper regulation of the Notch signaling pathway is an absolute requirement for the correct patterning of the axial skeleton.

Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the pore-forming subunit of the ATP-sensitive K(+) channel (K(ATP) channel), are a common cause of neonatal diabetes. We identified a novel KCNJ11 mutation, R50Q, that causes permanent neonatal diabetes (PNDM) without neurological problems. We investigated the functional effects this mutation and another at the same residue (R50P) that led to PNDM in association with developmental delay. Wild-type or mutant Kir6.2/SUR1 channels were examined by heterologous expression in Xenopus oocytes. Both mutations increased resting whole-cell currents through homomeric and heterozygous K(ATP) channels by reducing channel inhibition by ATP, an effect that was larger in the presence of Mg(2+). However the magnitude of the reduction in ATP sensitivity (and the increase in the whole-cell current) was substantially larger for the R50P mutation. This is consistent with the more severe phenotype. Single-R50P channel kinetics (in the absence of ATP) did not differ from wild type, indicating that the mutation primarily affects ATP binding and/or transduction. This supports the idea that R50 lies in the ATP-binding site of Kir6.2. The sulfonylurea tolbutamide blocked heterozygous R50Q (89%) and R50P (84%) channels only slightly less than wild-type channels (98%), suggesting that sulfonylurea therapy may be of benefit for patients with either mutation.

AIMS/HYPOTHESIS: Heterozygous activating mutations in KCNJ11, which encodes the Kir6.2 subunit of the pancreatic ATP-sensitive potassium (K(ATP)) channel, cause both permanent and transient neonatal diabetes. A minority of patients also have neurological features. The identification of a KCNJ11 mutation has important therapeutic implications, as many patients can replace insulin injections with sulfonylurea tablets. We aimed to determine the age of presentation of patients with KCNJ11 mutations and to examine if there was a relationship between genotype and phenotype. SUBJECTS AND METHODS: KCNJ11 was sequenced in 239 unrelated patients from 21 countries, who were diagnosed with permanent diabetes before 2 years of age. RESULTS: Thirty-one of the 120 patients (26%) diagnosed in the first 26 weeks of life had a KCNJ11 mutation; no mutations were found in the 119 cases (0%) diagnosed after this age. Fourteen different heterozygous mutations were identified, with the majority resulting from de novo mutations. These include seven novel mutations: H46Y, R50Q, G53D C166Y, K170T, L164P and Y330S. All 11 probands with the most common mutation, R201H, had isolated diabetes. In contrast, developmental delay in addition to diabetes was seen in four of five probands with the V59M mutation and two of four with the R201C mutation. Five patients with developmental delay, epilepsy and neonatal diabetes (DEND) syndrome had unique mutations not associated with other phenotypes. CONCLUSIONS/INTERPRETATION: KCNJ11 mutations are a common cause of permanent diabetes diagnosed in the first 6 months and all patients diagnosed in this age group should be tested. There is a strong genotype-phenotype relationship with the mutation being an important determinant of associated neurological features.

BACKGROUND: Hepatocyte nuclear factor-1 beta (HNF-1beta) is a widely distributed transcription factor which plays a critical role in embryonic development of the kidney, pancreas, liver, and Mullerian duct. Thirty HNF-1beta mutations have been reported in patients with renal cysts and other renal developmental disorders, young-onset diabetes, pancreatic atrophy, abnormal liver function tests, and genital tract abnormalities. METHODS: We sequenced the HNF-1beta gene in 160 unrelated subjects with renal disease, 40% of whom had a personal/family history of diabetes. RESULTS: Twenty three different heterozygous HNF-1beta mutations were identified in 23/160 subjects (14%), including 10 novel mutations (V61G, V110G, S148L, K156E, Q176X, R276Q, S281fsinsC, R295P, H324fsdelCA, Q470X). Seven (30%) cases were proven to be due to de novo mutations. Renal cysts were found in 19/23 (83%) patients (four with glomerulocystic kidney disease, GCKD) and diabetes in 11/23 (48%, while three other families had a family history of diabetes. Only 26% of families met diagnostic criteria for maturity-onset diabetes of the young (MODY) but 39% had renal cysts and diabetes (RCAD). We found no clear genotype/phenotype relationships. CONCLUSION: We report the largest series to date of HNF-1beta mutations and confirm HNF-1beta mutations as an important cause of renal disease. Despite the original description of HNF-1beta as a MODY gene, a personal/family history of diabetes is often absent and the most common clinical manifestation is renal cysts. Molecular genetic testing for HNF-1beta mutations should be considered in patients with unexplained renal cysts (including GCKD), especially when associated with diabetes, early-onset gout, or uterine abnormalities.

The beta-cell ATP-sensitive potassium channel is a key component of stimulus-secretion coupling in the pancreatic beta-cell. The channel couples metabolism to membrane electrical events, bringing about insulin secretion. Given the critical role of this channel in glucose homeostasis, it is not surprising that mutations in the genes encoding for the two essential subunits of the channel can result in both hypo- and hyperglycemia. The channel consists of four subunits of the inwardly rectifying potassium channel Kir6.2 and four subunits of the sulfonylurea receptor 1. It has been known for some time that loss of function mutations in KCNJ11, which encodes for Kir6.2, and ABCC8, which encodes for SUR1, can cause oversecretion of insulin and result in hyperinsulinemia (HI) of infancy; however, heterozygous activating mutations in KCNJ11 that result in the opposite phenotype of diabetes have recently been described. This review focuses on reported mutations in both genes, the spectrum of phenotypes, and the implications for treatment when patients are diagnosed with mutations in these genes.

Maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes mellitus characterized by autosomal dominant inheritance, early age of onset (often

The multiple endocrine neoplasia (MEN) type 2B is an autosomal dominant condition characterized by aggressive medullary C-cell tumors, pheochromocytoma, and a discrete physical appearance (marfanoid habitus, prominent corneal nerve fibers, thick lips, and mucosal and intestinal neuromas). A specific point mutation in the RET proto-oncogene is present in 95% cases. Occasionally cases present with the characteristic physical appearance of MEN 2B but no identifiable germline mutation or endocrinopathy, and it has been suggested that these patients may represent a discrete subgroup termed pure mucosal neuroma syndrome (MNS). We present a patient with MNS, who had a thyroidectomy at age 14.5 years with normal thyroid histology. Direct sequencing of all 20 exons of the RET gene showed no mutation. This case supports the suggestion that pure MNS can exist in the absence of an identifiable RET gene mutation. We suggest that prophylactic thyroidectomy is unnecessary in these patients although they should still be screened for endocrinopathy on a regular basis.

Maternally inherited diabetes and deafness and mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes result from the 3243A>G mitochondrial point mutation. Current methods to detect the presence of the mutation have limited sensitivity and may lead to potential misclassification of patients with low levels of heteroplasmy. Here, we describe development and validation of a rapid real-time polymerase chain reaction (PCR) method for detection and quantification of levels of heteroplasmy in a single assay. Standard curve analysis indicated that the sensitivity of detection was less than 0.1%. Time from sample loading to data analysis was 110 minutes. We tested 293 samples including 23 known positives, 40 known negatives, and 230 samples from patients clinically classified as having type 2 diabetes. All positive samples were correctly detected, and of those samples previously quantified, heteroplasmy levels determined using the real-time assay correlated well (r(2) = 0.88 and 0.93) with results from fluorescently labeled PCR-restriction fragment length polymorphism and pyrosequencing methods. Screening of 230 patients classified as having type 2 diabetes revealed one patient with 0.6% heteroplasmy who had previously tested negative by PCR-restriction fragment length polymorphism. Real-time PCR provides rapid simultaneous detection and quantification of the 3243A>G mutation to a detection limit of less than 0.1%, without post-PCR manipulation.

BACKGROUND: Heterozygous activating mutations in KCNJ11, encoding the Kir6.2 subunit of the ATP-sensitive potassium (K(ATP)) channel, cause 30 to 58 percent of cases of diabetes diagnosed in patients under six months of age. Patients present with ketoacidosis or severe hyperglycemia and are treated with insulin. Diabetes results from impaired insulin secretion caused by a failure of the beta-cell K(ATP) channel to close in response to increased intracellular ATP. Sulfonylureas close the K(ATP) channel by an ATP-independent route. METHODS: We assessed glycemic control in 49 consecutive patients with Kir6.2 mutations who received appropriate doses of sulfonylureas and, in smaller subgroups, investigated the insulin secretory responses to intravenous and oral glucose, a mixed meal, and glucagon. The response of mutant K(ATP) channels to the sulfonylurea tolbutamide was assayed in xenopus oocytes. RESULTS: a total of 44 patients (90 percent) successfully discontinued insulin after receiving sulfonylureas. The extent of the tolbutamide blockade of K(ATP) channels in vitro reflected the response seen in patients. Glycated hemoglobin levels improved in all patients who switched to sulfonylurea therapy (from 8.1 percent before treatment to 6.4 percent after 12 weeks of treatment, P

The laminopathies are a diverse group of conditions caused by mutations in the LMNA gene (MIM*150330). LMNA encodes the nuclear envelope proteins lamin a and lamin C by utilization of an alternative splice site in exon 10. The human LMNA gene was identified in 1986 but it was another 13 years before it was found to be the causative gene for a disease, namely Emery Dreifuss muscular dystrophy. Since then, a further eight clearly defined phenotypes have been associated with LMNA mutations. The diversity of these phenotypes is striking with features such as premature ageing, axonal neuropathy, lipodystrophy and myopathy being seen. These phenotypes and the emerging genotype/phenotype correlations are the subject of this review.

Allogeneic bone marrow transplantation (BMT) with marrow ablative conditioning is the treatment of choice for haematopoietic malignancies. The use of nonmyeloablative stem cell transplants has allowed the treatment of patients previously ineligible for BMT because of age or other disease. These reduced conditioning regimes allow the persistence initially of some recipient cells in the blood and bone marrow (haematopoietic chimaerism). Monitoring of the relative proportion of donor and recipient cells is required to assess the success of the procedure, to predict subsequent rejection or impending relapse and to guide the use of donor lymphocyte infusions. We present a quantitative real-time PCR approach for the measurement of haematopoietic chimaerism using the TaqMan. This approach exploits the presence of single-nucleotide polymorphisms (SNPs) to distinguish cells of patient or donor origin. We have designed and validated a panel of seven allele-specific probes to quantify the contribution of patient and donor cells in the haematopoietic population from 12 patient and donor pairs. We have compared the performance of this approach with an existing method and proved it to be superior in both accuracy and sensitivity. The use of more sensitive and accurate techniques permits earlier intervention for improved clinical outcome.

OBJECTIVE: Patients with diabetes due to hepatocyte nuclear factor (HNF)-1alpha mutations have beta-cell deficiency, insulin sensitivity, altered proinsulin levels, and a low renal threshold for glucose. It is uncertain how many of these features precede the development of diabetes. The aim of our study was to test for these characteristics in young nondiabetic HNF-1alpha mutation carriers. RESEARCH DESIGN AND METHODS: a total of 47 offspring from 19 extended families underwent genetic testing, a standard oral glucose tolerance test, and urine testing. RESULTS: HNF-1alpha mutations were found in 20 offspring, 7 with diabetes and 13 without diabetes. The 13 nondiabetic mutation carriers were compared with 27 family control subjects, who were matched for age, sex, and BMI. There was marked beta-cell deficiency with reduced insulinogenic index (53.5 [31.5-90.9] vs. 226.0 [126.0-407.1], SD [range], P < 0.001) and area under the curve for insulin (P < 0.001). Insulin sensitivity was increased in mutation carriers (homeostatic model assessment of insulin sensitivity 144.6 [82.7-252.7] vs. 100 [66.9-149.4], P = 0.025). A total of 38% of mutation carriers had glycosuria at 2 h compared with 0% of control subjects (P = 0.0034). Those with glycosuria had peak glucose values that were higher than the mutations carriers without glycosuria (range 8.1-11.8 vs. 6.2-8.4 mmol/l, P = 0.002). The seven subjects with diabetes all showed glycosuria. CONCLUSIONS: We conclude that marked beta-cell deficiency, increased insulin sensitivity, and a low renal threshold are present in young nondiabetic HNF-1alpha mutation carriers. The presence of glycosuria post-glucose load could be used to screen children of mutation carriers as it occurs in all mutation carriers with a peak glucose in the oral glucose tolerance test >8.4 mmol/l.

OBJECTIVE: Diagnostic molecular genetic testing for multiple endocrine neoplasia type 1 (MEN1) has been available since the identification of the MEN1 gene in 1997. Mutation screening of the MEN1 gene has been recommended for patients who meet clinical criteria for MEN1 (at least two of the following: parathyroid hyperplasia, pancreatic endocrine tumour or pituitary adenoma) and those in whom a diagnosis of MEN1 is suspected. We examined the appropriateness of these clinical criteria. PATIENTS AND METHODS: a total of 292 patients were referred for diagnostic testing. The coding region of the MEN1 gene was sequenced in 186 index cases and mutation testing was requested for 106 subjects, including 83 asymptomatic relatives. RESULTS: MEN1 gene mutations were identified in 68/186 index cases (37%). Twenty-nine of the 60 MEN1 mutations reported are novel. The likelihood of finding a mutation was correlated with the number of MEN1-related tumours (mutation detection rate of 79%, 37% and 15% in patients with three, two and one main MEN1-related tumours; P < or = 0.00001) and increased in the presence of a family history (mutation detection rate of 91%, 69% and 29%vs. 69%, 23% and 0% in sporadic cases with three, two or one main MEN1-related tumours, respectively; P < or = 0.00001). The pick-up rate in the 83% of subjects who met proposed criteria for diagnostic testing was 42%, but in those who did not meet these criteria this fell to 0%. CONCLUSIONS: the likelihood of finding an MEN1 mutation depends on the clinical features of the patient and their family. This large series supports present referral criteria for diagnostic mutation screening, but suggests that patients with sporadic isolated tumours rarely have MEN1 mutations.

The differential diagnosis for children with diabetes includes a group of monogenic diabetic disorders known as maturity-onset diabetes of the young (MODY). So far, six underlying gene defects have been identified. The most common subtypes are caused by mutations in the genes encoding the transcription factor HNF-1a (MODY 3) and the glycolytic enzyme glucokinase (GCK) (MODY 2). MODY 2 is the most benign form of diabetes as the threshold for glucose sensing is elevated resulting in mild, regulated hyperglycemia. MODY 2 may usually be treated with diet alone without risk of microvascular complications. Patients with MODY usually present as children or young adults. Genetic testing for MODY in diabetic subjects is often not performed because of the costs and its unavailability in Switzerland. We describe the impact of the genetic analysis for MODY 2 on diabetes management and treatment costs in a five-year-old girl. The patient and her diabetic mother were both found to have a heterozygous missense mutation (V203A) in the glucokinase gene. The five-year-old girl was started on insulin therapy for her diabetes but because her HbA1c remained between 5.8-6.4% (reference 4.1-5.7%) and her clinical presentation suggested MODY insulin was discontinued. She is now well controlled on a carbohydrate controlled diet regimen only. Omission of insulin treatment made regular blood glucose monitoring unnecessary and removed her risk of hypoglycemia. Costs for the genetic analysis were 500 Euro. At our centre costs for diabetes care of a patient with type 1 diabetes are approximately 2050 Euro/year compared to 410 Euro/year for the care of a patient with MODY 2. In addition, a diagnosis of MODY 2 may reassure patients and their families, as microvascular complications are uncommon. Thus there are both health and financial benefits in diagnosing MODY 2. We recommend genetic testing for MODY 2 in clinically selected patients even though this analysis is currently not available in Switzerland and costs are not necessarily covered by the health insurances.

Glucokinase (GCK) serves as the pancreatic glucose sensor. Heterozygous inactivating GCK mutations cause hyperglycemia, whereas activating mutations cause hypoglycemia. We studied the GCK V62M mutation identified in two families and co-segregating with hyperglycemia to understand how this mutation resulted in reduced function. Structural modeling locates the mutation close to five naturally occurring activating mutations in the allosteric activator site of the enzyme. Recombinant glutathionyl S-transferase-V62M GCK is paradoxically activated rather than inactivated due to a decreased S0.5 for glucose compared with wild type (4.88 versus 7.55 mM). The recently described pharmacological activator (RO0281675) interacts with GCK at this site. V62M GCK does not respond to RO0281675, nor does it respond to the hepatic glucokinase regulatory protein (GKRP). The enzyme is also thermally unstable, but this lability is apparently less pronounced than in the proven instability mutant E300K. Functional and structural analysis of seven amino acid substitutions at residue Val62 has identified a non-linear relationship between activation by the pharmacological activator and the van der Waals interactions energies. Smaller energies allow a hydrophobic interaction between the activator and glucokinase, whereas larger energies prohibit the ligand from fitting into the binding pocket. We conclude that V62M may cause hyperglycemia by a complex defect of GCK regulation involving instability in combination with loss of control by a putative endogenous activator and/or GKRP. This study illustrates that mutations that cause hyperglycemia are not necessarily kinetically inactivating but may exert their effects by other complex mechanisms. Elucidating such mechanisms leads to a deeper understanding of the GCK glucose sensor and the biochemistry of beta-cells and hepatocytes.

AIMS/HYPOTHESIS: Heterozygous mutations in the gene of the transcription factor hepatocyte nuclear factor 4alpha (HNF-4alpha) are considered a rare cause of MODY with only 14 mutations reported to date. The description of the phenotype is limited to single families. We investigated the genetics and phenotype of HNF-4alpha mutations in a large European Caucasian collection. METHODS: HNF-4alpha was sequenced in 48 MODY probands, selected for a phenotype of HNF-1alpha MODY but negative for HNF-1alpha mutations. Clinical characteristics and biochemistry were compared between 54 HNF-4alpha mutation carriers and 32 familial controls from ten newly detected or previously described families. RESULTS: Mutations in HNF-4alpha were found in 14/48 (29%) probands negative for HNF-1alpha mutations. The mutations found included seven novel mutations: S34X, D206Y, E276D, L332P, I314F, L332insCTG and IVS5nt+1G>A. I314F is the first reported de novo HNF-4alpha mutation. The average age of diagnosis was 22.9 years with frequent clinical evidence of sensitivity to sulphonylureas. Beta cell function, but not insulin sensitivity, was reduced in diabetic mutation carriers compared to control subjects (homeostasis model assessment of beta cell function 29% p

We describe a novel homozygous missense glucokinase mutation (R397L) resulting in insulin-treated neonatal diabetes in an infant from a consanguineous Asian family. Both parents were heterozygous for R397L and had mild hyperglycemia. Glucokinase mutations should be considered in infants of all ethnic groups with neonatal diabetes and consanguinity.

Neonatal diabetes can either remit and hence be transient or else may be permanent. These two phenotypes were considered to be genetically distinct. Abnormalities of 6q24 are the commonest cause of transient neonatal diabetes (TNDM). Mutations in KCNJ11, which encodes Kir6.2, the pore-forming subunit of the ATP-sensitive potassium channel (K(ATP)), are the commonest cause of permanent neonatal diabetes (PNDM). In addition to diabetes, some KCNJ11 mutations also result in marked developmental delay and epilepsy. These mutations are more severe on functional characterization. We investigated whether mutations in KCNJ11 could also give rise to TNDM. We identified the three novel heterozygous mutations (G53S, G53R, I182V) in three of 11 probands with clinically defined TNDM, who did not have chromosome 6q24 abnormalities. The mutations co-segregated with diabetes within families and were not found in 100 controls. All probands had insulin-treated diabetes diagnosed in the first 4 months and went into remission by 7-14 months. Functional characterization of the TNDM associated mutations was performed by expressing the mutated Kir6.2 with SUR1 in Xenopus laevis oocytes. All three heterozygous mutations resulted in a reduction in the sensitivity to ATP when compared with wild-type (IC(50) approximately 30 versus approximately 7 microM, P-value for is all

The nonsense-mediated decay (NMD) pathway is an mRNA surveillance mechanism that detects and degrades transcripts containing premature termination codons. The position of a truncating mutation can govern the resulting phenotype as mutations in the last exon evade NMD. In this study we investigated the susceptibility to NMD of six truncating HNF-1beta mutations by allele-specific quantitative real-time PCR using transformed lymphoblastoid cell lines. Four of six mutations (R181X, Q243fs