Journal articles
Locke JM, da Silva Xavier G, Rutter, GA, Harries LW (In Press). An alternative polyadenylation signal in TCF7L2 generates isoforms that inhibit T cell factor/lymphoid-enhancer factor (TCF/LEF)-dependent target genes.
DiabetologiaAbstract:
An alternative polyadenylation signal in TCF7L2 generates isoforms that inhibit T cell factor/lymphoid-enhancer factor (TCF/LEF)-dependent target genes.
AIMS/HYPOTHESIS: Intronic single nucleotide polymorphisms within the transcription factor 7-like 2 (TCF7L2) gene are associated with risk of type 2 diabetes. It is widely hypothesised that the predisposing variation is involved in cis-regulation of TCF7L2 activity. The aim of this study was to seek evidence for the existence of novel TCF7L2 isoforms encoded within the type 2 diabetes-associated genomic region.
METHODS: We searched expressed sequence tag (EST) databases for novel TCF7L2 transcripts and sought to validate the function and integrity of any isoforms found using a combination of RT-PCR, western blotting and reporter gene techniques.
RESULTS: Analysis of EST databases suggested the presence of an alternative polyadenylation site located in intron 4 of TCF7L2. We used 3' rapid amplification of cDNA ends and real-time PCR to validate the integrity of this polyadenylation signal and show its wide use across human tissues. Western blotting results are consistent with the use of this polyadenylation signal to generate novel protein isoforms. The alternative polyadenylation signal results in the production of isoforms that retain the β-catenin binding domain but do not possess the high-mobility group box DNA-binding domain. Promoter-reporter gene assays suggest that these isoforms inhibit TCF7L2-dependent target genes by sequestering β-catenin.
CONCLUSIONS/INTERPRETATION: We have identified a novel polyadenylation signal within TCF7L2 that can result in the production of isoforms that act to repress TCF/LEF-dependent target genes. These findings may provide new insights into the association of TCF7L2 with susceptibility to type 2 diabetes.
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Manni E, Jeffery N, Chambers D, Slade L, Etheridge T, Harries LW (2023). An evaluation of the role of miR-361-5p in senescence and systemic ageing. Experimental Gerontology, 174, 112127-112127.
Harries LW (2023). Dysregulated RNA processing and metabolism: a new hallmark of ageing and provocation for cellular senescence.
FEBS J,
290(5), 1221-1234.
Abstract:
Dysregulated RNA processing and metabolism: a new hallmark of ageing and provocation for cellular senescence.
The human genome is capable of producing hundreds of thousands of different proteins and non-coding RNAs from
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Turner J, Pound P, Owen C, Hutchinson I, Hop M, Chau DYS, Barrios Silva LV, Coleman M, Dubourg A, Harries LW, et al (2023). Incorporating new approach methodologies into regulatory nonclinical pharmaceutical safety assessment.
ALTEX,
40(3), 519-533.
Abstract:
Incorporating new approach methodologies into regulatory nonclinical pharmaceutical safety assessment.
New approach methodologies (NAMs) based on human biology enable the assessment of adverse biological effects of pharmaceuticals and other chemicals. Currently, however, it is unclear how NAMs should be used during drug development to improve human safety evaluation. A series of 5 workshops with 13 international experts (regulators, preclinical scientists, and NAMs developers) was conducted to identify feasible NAMs and to discuss how to exploit them in specific safety assessment contexts. Participants generated four “maps” of how NAMs can be exploited in the safety assessment of the liver, respiratory, cardiovascular, and central nervous systems. Each map shows relevant endpoints measured and tools used (e.g. cells, assays, platforms), and highlights gaps where further development and validation of NAMs remains necessary. Each map addresses the fundamental scientific requirements for the safety assessment of that organ system, providing users with guidance on the selection of appropriate NAMs. In addition to generating the maps, participants offered suggestions for encouraging greater NAM adoption within drug development and their inclusion in regulatory guidelines. A specific recommendation was that pharmaceutical companies should be more transparent about how they use NAMs in-house. As well as giving guidance for the four organ systems, the maps provide a template that could be used for additional organ safety testing contexts. Moreover, their conversion to an interactive format would enable users to drill down to the detail necessary to answer specific scientific and regulatory questions.
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Scott E, Archer Goode E, Garnham R, Hodgson K, Orozco-Moreno M, Turner H, Livermore K, Putri Nangkana K, Frame FM, Bermudez A, et al (2023). ST6GAL1-mediated aberrant sialylation promotes prostate cancer progression.
J Pathol,
261(1), 71-84.
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ST6GAL1-mediated aberrant sialylation promotes prostate cancer progression.
Aberrant glycosylation is a universal feature of cancer cells, and cancer-associated glycans have been detected in virtually every cancer type. A common change in tumour cell glycosylation is an increase in α2,6 sialylation of N-glycans, a modification driven by the sialyltransferase ST6GAL1. ST6GAL1 is overexpressed in numerous cancer types, and sialylated glycans are fundamental for tumour growth, metastasis, immune evasion, and drug resistance, but the role of ST6GAL1 in prostate cancer is poorly understood. Here, we analyse matched cancer and normal tissue samples from 200 patients and verify that ST6GAL1 is upregulated in prostate cancer tissue. Using MALDI imaging mass spectrometry (MALDI-IMS), we identify larger branched α2,6 sialylated N-glycans that show specificity to prostate tumour tissue. We also monitored ST6GAL1 in plasma samples from >400 patients and reveal ST6GAL1 levels are significantly increased in the blood of men with prostate cancer. Using both in vitro and in vivo studies, we demonstrate that ST6GAL1 promotes prostate tumour growth and invasion. Our findings show ST6GAL1 introduces α2,6 sialylated N-glycans on prostate cancer cells and raise the possibility that prostate cancer cells can secrete active ST6GAL1 enzyme capable of remodelling glycans on the surface of other cells. Furthermore, we find α2,6 sialylated N-glycans expressed by prostate cancer cells can be targeted using the sialyltransferase inhibitor P-3FAX -Neu5Ac. Our study identifies an important role for ST6GAL1 and α2,6 sialylated N-glycans in prostate cancer progression and highlights the opportunity to inhibit abnormal sialylation for the development of new prostate cancer therapeutics. © 2023 the Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of the Pathological Society of Great Britain and Ireland.
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Bramwell LR, Harries LW (2023). Senescence, regulators of alternative splicing and effects of trametinib treatment in progeroid syndromes.
GeroScienceAbstract:
Senescence, regulators of alternative splicing and effects of trametinib treatment in progeroid syndromes
AbstractProgeroid syndromes such as Hutchinson Gilford Progeroid syndrome (HGPS), Werner syndrome (WS) and Cockayne syndrome (CS), result in severely reduced lifespans and premature ageing. Normal senescent cells show splicing factor dysregulation, which has not yet been investigated in syndromic senescent cells. We sought to investigate the senescence characteristics and splicing factor expression profiles of progeroid dermal fibroblasts. Natural cellular senescence can be reversed by application of the senomorphic drug, trametinib, so we also investigated its ability to reverse senescence characteristics in syndromic cells. We found that progeroid cultures had a higher senescence burden, but did not always have differences in levels of proliferation, DNA damage repair and apoptosis. Splicing factor gene expression appeared dysregulated across the three syndromes. 10 µM trametinib reduced senescent cell load and affected other aspects of the senescence phenotype (including splicing factor expression) in HGPS and Cockayne syndromes. Werner syndrome cells did not demonstrate changes in in senescence following treatment. Splicing factor dysregulation in progeroid cells provides further evidence to support this mechanism as a hallmark of cellular ageing and highlights the use of progeroid syndrome cells in the research of ageing and age-related disease. This study suggests that senomorphic drugs such as trametinib could be a useful adjunct to therapy for progeroid diseases.
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Scott E, Hodgson K, Calle B, Turner H, Cheung K, Bermudez A, Marques FJG, Pye H, Yo EC, Islam K, et al (2023). Upregulation of GALNT7 in prostate cancer modifies O-glycosylation and promotes tumour growth.
Oncogene,
42(12), 926-937.
Abstract:
Upregulation of GALNT7 in prostate cancer modifies O-glycosylation and promotes tumour growth.
Prostate cancer is the most common cancer in men and it is estimated that over 350,000 men worldwide die of prostate cancer every year. There remains an unmet clinical need to improve how clinically significant prostate cancer is diagnosed and develop new treatments for advanced disease. Aberrant glycosylation is a hallmark of cancer implicated in tumour growth, metastasis, and immune evasion. One of the key drivers of aberrant glycosylation is the dysregulated expression of glycosylation enzymes within the cancer cell. Here, we demonstrate using multiple independent clinical cohorts that the glycosyltransferase enzyme GALNT7 is upregulated in prostate cancer tissue. We show GALNT7 can identify men with prostate cancer, using urine and blood samples, with improved diagnostic accuracy than serum PSA alone. We also show that GALNT7 levels remain high in progression to castrate-resistant disease, and using in vitro and in vivo models, reveal that GALNT7 promotes prostate tumour growth. Mechanistically, GALNT7 can modify O-glycosylation in prostate cancer cells and correlates with cell cycle and immune signalling pathways. Our study provides a new biomarker to aid the diagnosis of clinically significant disease and cements GALNT7-mediated O-glycosylation as an important driver of prostate cancer progression.
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Kennedy NA, Janjua M, Chanchlani N, Lin S, Bewshea C, Nice R, McDonald TJ, Auckland C, Harries LW, Davies M, et al (2023). Vaccine escape, increased breakthrough and reinfection in infliximab-treated patients with IBD during the Omicron wave of the SARS-CoV-2 pandemic.
Gut,
72(2), 295-305.
Abstract:
Vaccine escape, increased breakthrough and reinfection in infliximab-treated patients with IBD during the Omicron wave of the SARS-CoV-2 pandemic.
OBJECTIVE: Antitumour necrosis factor (TNF) drugs impair serological responses following SARS-CoV-2 vaccination. We sought to assess if a third dose of a messenger RNA (mRNA)-based vaccine substantially boosted anti-SARS-CoV-2 antibody responses and protective immunity in infliximab-treated patients with IBD. DESIGN: Third dose vaccine induced anti-SARS-CoV-2 spike (anti-S) receptor-binding domain (RBD) antibody responses, breakthrough SARS-CoV-2 infection, reinfection and persistent oropharyngeal carriage in patients with IBD treated with infliximab were compared with a reference cohort treated with vedolizumab from the impaCt of bioLogic therApy on saRs-cov-2 Infection and immuniTY (CLARITY) IBD study. RESULTS: Geometric mean (SD) anti-S RBD antibody concentrations increased in both groups following a third dose of an mRNA-based vaccine. However, concentrations were lower in patients treated with infliximab than vedolizumab, irrespective of whether their first two primary vaccine doses were ChAdOx1 nCoV-19 (1856 U/mL (5.2) vs 10 728 U/mL (3.1), p
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Frankum R, Jameson TSO, Knight BA, Stephens FB, Wall BT, Donlon TA, Torigoe T, Willcox BJ, Willcox DC, Allsopp RC, et al (2022). Correction to: Extreme longevity variants at the FOXO3 locus may moderate FOXO3 isoform levels.
Geroscience,
44(2), 1169-1170.
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Mou Z, Spencer J, Knight B, John J, McCullagh P, McGrath JS, Harries LW (2022). Gene expression analysis reveals a 5-gene signature for progression-free survival in prostate cancer.
Frontiers in Oncology,
12Abstract:
Gene expression analysis reveals a 5-gene signature for progression-free survival in prostate cancer
Prostate cancer (PCa) is the second most common male cancer worldwide, but effective biomarkers for the presence or progression risk of disease are currently elusive. In a series of nine matched histologically confirmed PCa and benign samples, we carried out an integrated transcriptome-wide gene expression analysis, including differential gene expression analysis and weighted gene co-expression network analysis (WGCNA), which identified a set of potential gene markers highly associated with tumour status (malignant vs. benign). We then used these genes to establish a minimal progression-free survival (PFS)-associated gene signature (GS) (PCBP1, PABPN1, PTPRF, DANCR, and MYC) using least absolute shrinkage and selection operator (LASSO) and stepwise multivariate Cox regression analyses from the Cancer Genome Atlas prostate adenocarcinoma (TCGA-PRAD) dataset. Our signature was able to predict PFS over 1, 3, and 5 years in TCGA-PRAD dataset, with area under the curve (AUC) of 0.64–0.78, and our signature remained as a prognostic factor independent of age, Gleason score, and pathological T and N stages. A nomogram combining the signature and Gleason score demonstrated improved predictive capability for PFS (AUC: 0.71–0.85) and was superior to the Cambridge Prognostic Group (CPG) model alone and some conventionally used clinicopathological factors in predicting PFS. In conclusion, we have identified and validated a novel five-gene signature and established a nomogram that effectively predicted PFS in patients with PCa. Findings may improve current prognosis tools for PFS and contribute to clinical decision-making in PCa treatment.
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Davies M, Bramwell LR, Jeffery N, Bunce B, Lee BP, Knight B, Auckland C, Masoli JA, Harries LW (2022). Persistence of clinically relevant levels of SARS-CoV2 envelope gene subgenomic RNAs in non-immunocompromised individuals.
Int J Infect Dis,
116, 418-425.
Abstract:
Persistence of clinically relevant levels of SARS-CoV2 envelope gene subgenomic RNAs in non-immunocompromised individuals.
OBJECTIVES: This study aimed to evaluate the associations between COVID-19 severity and active viral load, and to characterize the dynamics of active SARS-CoV-2 clearance in a series of archival samples taken from patients in the first wave of COVID-19 infection in the South West of the UK. METHODS: Subgenomic RNA (sgRNA) and E-gene genomic sequences were measured in a retrospective collection of PCR-confirmed SARS-CoV-2-positive samples from 176 individuals, and related to disease severity. Viral clearance dynamics were then assessed in relation to symptom onset and last positive test. RESULTS: Whilst E-gene sgRNAs declined before E-gene genomic sequences, some individuals retained sgRNA positivity for up to 68 days. 13% of sgRNA-positive cases still exhibited clinically relevant levels of virus after 10 days, with no clinical features previously associated with prolonged viral clearance times. CONCLUSIONS: Our results suggest that potentially active virus can sometimes persist beyond a 10-day period, and could pose a potential risk of onward transmission. Where this would pose a serious public health threat, additional mitigation strategies may be necessary to reduce the risk of secondary cases in vulnerable settings.
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Ammous Z, Rawlins LE, Jones H, Leslie JS, Wenger O, Scott E, Deline J, Herr T, Evans R, Scheid A, et al (2021). A biallelic SNIP1 Amish founder variant causes a recognizable neurodevelopmental disorder.
PLOS Genetics,
17(9), e1009803-e1009803.
Abstract:
A biallelic SNIP1 Amish founder variant causes a recognizable neurodevelopmental disorder
SNIP1 (Smad nuclear interacting protein 1) is a widely expressed transcriptional suppressor of the TGF-β signal-transduction pathway which plays a key role in human spliceosome function. Here, we describe extensive genetic studies and clinical findings of a complex inherited neurodevelopmental disorder in 35 individuals associated with aSNIP1NM_024700.4:c.1097A>G, p.(Glu366Gly) variant, present at high frequency in the Amish community. The cardinal clinical features of the condition include hypotonia, global developmental delay, intellectual disability, seizures, and a characteristic craniofacial appearance. Our gene transcript studies in affected individuals define altered gene expression profiles of a number of molecules with well-defined neurodevelopmental and neuropathological roles, potentially explaining clinical outcomes. Together these data confirm thisSNIP1gene variant as a cause of an autosomal recessive complex neurodevelopmental disorder and provide important insight into the molecular roles of SNIP1, which likely explain the cardinal clinical outcomes in affected individuals, defining potential therapeutic avenues for future research.
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Jeffery N, Chambers D, Invergo BM, Ames RM, Harries LW (2021). Changes to the identity of EndoC-βH1 beta cells may be mediated by stress-induced depletion of HNRNPD.
Cell & Bioscience,
11(1).
Abstract:
Changes to the identity of EndoC-βH1 beta cells may be mediated by stress-induced depletion of HNRNPD
Abstract
. Background
. Beta cell identity changes occur in the islets of donors with diabetes, but the molecular basis of this remains unclear. Protecting residual functional beta cells from cell identity changes may be beneficial for patients with diabetes.
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. Results
. A somatostatin-positive cell population was induced in stressed clonal human EndoC-βH1 beta cells and was isolated using FACS. A transcriptomic characterisation of somatostatin-positive cells was then carried out. Gain of somatostatin-positivity was associated with marked dysregulation of the non-coding genome. Very few coding genes were differentially expressed. Potential candidate effector genes were assessed by targeted gene knockdown. Targeted knockdown of the HNRNPD gene induced the emergence of a somatostatin-positive cell population in clonal EndoC-βH1 beta cells comparable with that we have previously reported in stressed cells.
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. Conclusions
. We report here a role for the HNRNPD gene in determination of beta cell identity in response to cellular stress. These findings widen our understanding of the role of RNA binding proteins and RNA biology in determining cell identity and may be important for protecting remaining beta cell reserve in diabetes.
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Frankum R, Jameson TSO, Knight BA, Stephens FB, Wall BT, Donlon TA, Torigoe T, Willcox BJ, Willcox DC, Allsopp RC, et al (2021). Extreme longevity variants at the FOXO3 locus may moderate FOXO3 isoform levels.
GeroScience,
44(2), 1129-1140.
Abstract:
Extreme longevity variants at the FOXO3 locus may moderate FOXO3 isoform levels
AbstractThe rs2802292, rs2764264 and rs13217795 variants of FOXO3 have been associated with extreme longevity in multiple human populations, but the mechanisms underpinning this remain unclear. We aimed to characterise potential effects of longevity-associated variation on the expression and mRNA processing of the FOXO3 gene. We performed a comprehensive assessment of FOXO3 isoform usage across a wide variety of human tissues and carried out a bioinformatic analysis of the potential for longevity-associated variants to disrupt regulatory regions involved in isoform choice. We then related the expression of full length and 5′ truncated FOXO3 isoforms to rs13217795 genotype in peripheral blood and skeletal muscle from individuals of different rs13217795 genotypes. FOXO3 isoforms displayed considerable tissue specificity. We determined that rs13231195 and its tightly aligned proxy variant rs9400239 may lie in regulatory regions involved in isoform choice. The longevity allele at rs13217795 was associated with increased levels of full length FOXO3 isoforms in peripheral blood and a decrease in truncated FOXO3 isoforms in skeletal muscle RNA. We suggest that the longevity effect of FOXO3 SNPs may in part derive from a shift in isoform usage in skeletal muscle away from the production of 5′ truncated FOXO3 isoforms lacking a complete forkhead DNA binding domain, which may have compromised functionality.
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Lee BP, Harries LW (2021). Senotherapeutic Drugs: a New Avenue for Skincare?.
PLASTIC AND RECONSTRUCTIVE SURGERY,
148(6S), 21S-26S.
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Bramwell LR, Harries LW (2021). Targeting Alternative Splicing for Reversal of Cellular Senescence in the Context of Aesthetic Aging.
Plast Reconstr Surg,
147(1S-2), 25S-32S.
Abstract:
Targeting Alternative Splicing for Reversal of Cellular Senescence in the Context of Aesthetic Aging.
Cellular senescence is a state of stable cell cycle arrest that has increasingly been linked with cellular, tissue, and organismal aging; targeted removal of senescent cells brings healthspan and lifespan benefits in animal models. Newly emerging approaches to specifically ablate or rejuvenate senescent cells are now the subject of intense study to explore their utility to provide novel treatments for the aesthetic signs and diseases of aging in humans. Here, we discuss different strategies that are being trialed in vitro, and more recently in vivo, for the targeted removal or reversal of senescent cells. Finally, we describe the evidence for a newly emerging molecular mechanism that may underpin senescence; dysregulation of alternative splicing. We will explore the potential of restoring splicing regulation as a novel "senotherapeutic" approach and discuss strategies by which this could be integrated into the established portfolio of skin aging therapeutics.
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Deane CS, Willis CRG, Phillips BE, Atherton PJ, Harries LW, Ames RM, Szewczyk NJ, Etheridge T (2021). Transcriptomic meta-analysis of disuse muscle atrophy vs. resistance exercise-induced hypertrophy in young and older humans.
J Cachexia Sarcopenia Muscle,
12(3), 629-645.
Abstract:
Transcriptomic meta-analysis of disuse muscle atrophy vs. resistance exercise-induced hypertrophy in young and older humans.
BACKGROUND: Skeletal muscle atrophy manifests across numerous diseases; however, the extent of similarities/differences in causal mechanisms between atrophying conditions in unclear. Ageing and disuse represent two of the most prevalent and costly atrophic conditions, with resistance exercise training (RET) being the most effective lifestyle countermeasure. We employed gene-level and network-level meta-analyses to contrast transcriptomic signatures of disuse and RET, plus young and older RET to establish a consensus on the molecular features of, and therapeutic targets against, muscle atrophy in conditions of high socio-economic relevance. METHODS: Integrated gene-level and network-level meta-analysis was performed on publicly available microarray data sets generated from young (18-35 years) m. vastus lateralis muscle subjected to disuse (unilateral limb immobilization or bed rest) lasting ≥7 days or RET lasting ≥3 weeks, and resistance-trained older (≥60 years) muscle. RESULTS: Disuse and RET displayed predominantly separate transcriptional responses, and transcripts altered across conditions were mostly unidirectional. However, disuse and RET induced directly inverted expression profiles for mitochondrial function and translation regulation genes, with COX4I1, ENDOG, GOT2, MRPL12, and NDUFV2, the central hub components of altered mitochondrial networks, and ZMYND11, a hub gene of altered translation regulation. A substantial number of genes (n = 140) up-regulated post-RET in younger muscle were not similarly up-regulated in older muscle, with young muscle displaying a more pronounced extracellular matrix (ECM) and immune/inflammatory gene expression response. Both young and older muscle exhibited similar RET-induced ubiquitination/RNA processing gene signatures with associated PWP1, PSMB1, and RAF1 hub genes. CONCLUSIONS: Despite limited opposing gene profiles, transcriptional signatures of disuse are not simply the converse of RET. Thus, the mechanisms of unloading cannot be derived from studying muscle loading alone and provides a molecular basis for understanding why RET fails to target all transcriptional features of disuse. Loss of RET-induced ECM mechanotransduction and inflammatory profiles might also contribute to suboptimal ageing muscle adaptations to RET. Disuse and age-dependent molecular candidates further establish a framework for understanding and treating disuse/ageing atrophy.
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Kuo CL, Joaquim M, Kuchel GA, Ferrucci L, Harries LW, Pilling LC, Melzer D (2020). Erratum: the longevity-associated SH2B3 (LNK) genetic variant: selected aging phenotypes in 379,758 subjects (Journals of Gerontology - Series a Biological Sciences and Medical Sciences DOI: 10.1093/gerona/glz191).
Journals of Gerontology - Series a Biological Sciences and Medical Sciences,
75(9).
Abstract:
Erratum: the longevity-associated SH2B3 (LNK) genetic variant: selected aging phenotypes in 379,758 subjects (Journals of Gerontology - Series a Biological Sciences and Medical Sciences DOI: 10.1093/gerona/glz191)
In the article, the Longevity-Associated SH2B3 (LNK) Genetic Variant: Selected Aging Phenotypes in 379,758 Subjects, the first two sentences of the Funding section have been updated to the following: This study was partly funded by an award to D.M. by the UK Medical Research Council (MR/M023095/1) and an Intergovernmental Personnel Act agreement (20170526) by the Intramural research Program of the NIH, National institute on Aging.
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Haque S, Ames RM, Moore K, Lee BP, Jeffery N, Harries LW (2020). Islet-expressed circular RNAs are associated with type 2 diabetes status in human primary islets and in peripheral blood.
BMC Med Genomics,
13(1).
Abstract:
Islet-expressed circular RNAs are associated with type 2 diabetes status in human primary islets and in peripheral blood.
BACKGROUND: Circular RNAs are non-coding RNA molecules with gene regulatory potential that have been associated with several human diseases. They are stable and present in the circulation, making them excellent candidates for biomarkers of disease. Despite their promise as biomarkers or future therapeutic targets, information on their expression and functionality in human pancreatic islets is a relatively unexplored subject. METHODS: Here we aimed to produce an enriched circRNAome profile for human pancreatic islets by CircleSeq, and to explore the relationship between circRNA expression, diabetes status, genotype at T2D risk loci and measures of glycaemia (insulin secretory index; SI and HbA1c) in human islet preparations from healthy control donors and donors with type 2 diabetes using ANOVA or linear regression as appropriate. We also assessed the effect of elevated glucose, cytokine and lipid and hypoxia on circRNA expression in the human beta cell line EndoC-βH1. RESULTS: We identified over 2600 circRNAs present in human islets. of the five most abundant circRNAs in human islets, four (circCIRBP, circZKSCAN, circRPH3AL and circCAMSAP1) demonstrated marked associations with diabetes status. CircCIRBP demonstrated an association with insulin secretory index in isolated human islets and circCIRBP and circRPH3AL displayed altered expression with elevated fatty acid in treated EndoC-βH1 cells. CircCAMSAP1 was also noted to be associated with T2D status in human peripheral blood. No associations between circRNA expression and genotype at T2D risk loci were identified in our samples. CONCLUSIONS: Our data suggest that circRNAs are abundantly expressed in human islets, and that some are differentially regulated in the islets of donors with type 2 diabetes. Some islet circRNAs are also expressed in peripheral blood and the expression of one, circCAMSAP1, correlates with diabetes status. These findings highlight the potential of circRNAs as biomarkers for T2D.
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Lee BP, Smith M, Buffenstein R, Harries LW (2020). Negligible senescence in naked mole rats may be a consequence of well-maintained splicing regulation.
Geroscience,
42(2), 633-651.
Abstract:
Negligible senescence in naked mole rats may be a consequence of well-maintained splicing regulation.
Naked mole-rats (NMRs) have amongst the longest lifespans relative to body size of any known, non-volant mammalian species. They also display an enhanced stress resistance phenotype, negligible senescence and very rarely are they burdened with chronic age-related diseases. Alternative splicing (AS) dysregulation is emerging as a potential driver of senescence and ageing. We hypothesised that the expression of splicing factors, important regulators of patterns of AS, may differ in NMRs when compared to other species with relatively shorter lifespans. We designed assays specific to NMR splicing regulatory factors and also to a panel of pre-selected brain-expressed genes known to demonstrate senescence-related alterations in AS in other species, and measured age-related changes in the transcript expression levels of these using embryonic and neonatal developmental stages through to extreme old age in NMR brain samples. We also compared splicing factor expression in both young mouse and NMR spleen and brain samples. Both NMR tissues showed approximately double the expression levels observed in tissues from similarly sized mice. Furthermore, contrary to observations in other species, following a brief period of labile expression in early life stages, adult NMR splicing factors and patterns of AS for functionally relevant brain genes remained remarkably stable for at least two decades. These findings are consistent with a model whereby the conservation of splicing regulation and stable patterns of AS may contribute to better molecular stress responses and the avoidance of senescence in NMRs, contributing to their exceptional lifespan and prolonged healthspan.
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Kuo C-L, Joaquim M, Kuchel GA, Ferrucci L, Harries LW, Pilling LC, Melzer D (2020). The Longevity-Associated SH2B3 (LNK) Genetic Variant: Selected Aging Phenotypes in 379,758 Subjects.
J Gerontol a Biol Sci Med Sci,
75(9), 1656-1662.
Abstract:
The Longevity-Associated SH2B3 (LNK) Genetic Variant: Selected Aging Phenotypes in 379,758 Subjects.
Human SH2B3 is involved in growth factor and inflammation signaling. A SH2B3 missense variant (rs3184504) is associated with cardiovascular diseases plus breast, colorectal, and lung cancers, with highly correlated variants across the ATXN2/SH2B3/BRAP locus linked to parental age at death, suggesting a geroscience common mechanism of aging and disease. To better understand the SH2B3-related aging pathway and its potential as an intervention target, we undertook a phenotype-wide association study (PheWAS) of 52 aging traits. Data were obtained from 379,758 European-descent UK Biobank participants, aged 40-70 at baseline: 27% of participants were CC homozygotes and 23% TT at rs3184504. Parental extreme longevity (mothers aged ≥98 years, fathers aged ≥96 years) was more common in CC versus TT (odds ratio [OR] = 1.18, 95% confidence interval [CI]: 1.07 to 1.29) with an additive per allele effect. The C allele associated with better cognitive function and white blood cell counts were more likely to be normal. The C allele reduced risks of coronary heart disease (OR = 0.95, 95% CI: 0.93 to 0.96) but was also associated with a modestly higher cancer rate (OR = 1.03, 95% CI: 1.02 to 1.04), suggesting a trade-off across aging outcomes and limiting its potential as an anti-aging target.
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Calimport SRG, Bentley BL, Stewart CE, Pawelec G, Scuteri A, Vinciguerra M, Slack C, Chen D, Harries LW, Marchant G, et al (2020). The inherent challenges of classifying senescence−−Response. Science, 368(6491), 595-596.
Haque S, Ames RM, Moore K, Pilling LC, Peters LL, Bandinelli S, Ferrucci L, Harries LW (2020). circRNAs expressed in human peripheral blood are associated with human aging phenotypes, cellular senescence and mouse lifespan.
Geroscience,
42(1), 183-199.
Abstract:
circRNAs expressed in human peripheral blood are associated with human aging phenotypes, cellular senescence and mouse lifespan.
Circular RNAs (circRNAs) are an emerging class of non-coding RNA molecules that are thought to regulate gene expression and human disease. Despite the observation that circRNAs are known to accumulate in older organisms and have been reported in cellular senescence, their role in aging remains relatively unexplored. Here, we have assessed circRNA expression in aging human blood and followed up age-associated circRNA in relation to human aging phenotypes, mammalian longevity as measured by mouse median strain lifespan and cellular senescence in four different primary human cell types. We found that circRNAs circDEF6, circEP300, circFOXO3 and circFNDC3B demonstrate associations with parental longevity or hand grip strength in 306 subjects from the InCHIANTI study of aging, and furthermore, circFOXO3 and circEP300 also demonstrate differential expression in one or more human senescent cell types. Finally, four circRNAs tested showed evidence of conservation in mouse. Expression levels of one of these, circPlekhm1, was nominally associated with lifespan. These data suggest that circRNA may represent a novel class of regulatory RNA involved in the determination of aging phenotypes, which may show future promise as both biomarkers and future therapeutic targets for age-related disease.
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Galvis D, Walsh D, Harries LW, Latorre E, Rankin J (2019). A dynamical systems model for the measurement of cellular senescence.
Journal of the Royal Society Interface,
16(159).
Abstract:
A dynamical systems model for the measurement of cellular senescence
Senescent cells provide a good in vitro model to study ageing. However, cultures of 'senescent' cells consist of a mix of cell subtypes (proliferative, senescent, growth-arrested and apoptotic). Determining the proportion of senescent cells is crucial for studying ageing and developing new anti-degenerative therapies. Commonly used markers such as doubling population, senescence-associated β-galactosidase, Ki-67, γH2AX and TUNEL assays capture diverse and overlapping cellular populations and are not purely specific to senescence. A newly developed dynamical systems model follows the transition of an initial culture to senescence tracking population doubling, and the proportion of cells in proliferating, growth-arrested, apoptotic and senescent states. Our model provides a parsimonious description of transitions between these states accruing towards a predominantly senescent population. Using a genetic algorithm, these model parameters are well constrained by an in vitro human primary fibroblast dataset recording five markers at 16 time points. The computational model accurately fits to the data and translates these joint markers into the first complete description of the proportion of cells in different states over the lifetime. The high temporal resolution of the dataset demonstrates the efficacy of strategies for reconstructing the trajectory towards replicative senescence with a minimal number of experimental recordings.
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Latorre E, Mesonero JE, Harries LW (2019). Alternative splicing in serotonergic system: Implications in neuropsychiatric disorders.
J Psychopharmacol,
33(11), 1352-1363.
Abstract:
Alternative splicing in serotonergic system: Implications in neuropsychiatric disorders.
BACKGROUND: the serotonergic system is a key component of physiological brain function and is essential for proper neurological activity. Numerous neuropsychiatric disorders are associated with deregulation of the serotonergic system. Accordingly, many pharmacological treatments are focused on modulation of this system. While providing a promising line of therapeutic moderation, these approaches may be complicated due to the presence of alternative splicing events for key genes in this pathway. Alternative splicing is a co-transcriptional process by which different mRNA transcripts can be produced from the same gene. These different isoforms may have diverse activities and functions, and their relative balance is often critical for the maintenance of homeostasis. Alternative splicing greatly increases the production of proteins, augmenting cell plasticity, and provides an important control point for regulation of gene expression. AIM: the objective of this narrative review is to discuss the potential impact of alternative splicing of different components of the serotonergic system and speculate on their involvement in several neuropsychiatric disorders. CONCLUSIONS: the specific role of each isoform in disease and their relative activities in the signalling pathways involved are yet to be determined. We need to gain a better understanding of the basis of alternative isoforms of the serotonergic system in order to fully understand their impact and be able to develop new effective pharmacological isoform-specific targets.
Abstract.
Author URL.
Munkley J, Li L, Krishnan SRG, Hysenaj G, Scott E, Dalgliesh C, Oo HZ, Maia TM, Cheung K, Ehrmann I, et al (2019). Androgen-regulated transcription of ESRP2 drives alternative splicing patterns in prostate cancer.
Elife,
8Abstract:
Androgen-regulated transcription of ESRP2 drives alternative splicing patterns in prostate cancer.
Prostate is the most frequent cancer in men. Prostate cancer progression is driven by androgen steroid hormones, and delayed by androgen deprivation therapy (ADT). Androgens control transcription by stimulating androgen receptor (AR) activity, yet also control pre-mRNA splicing through less clear mechanisms. Here we find androgens regulate splicing through AR-mediated transcriptional control of the epithelial-specific splicing regulator ESRP2. Both ESRP2 and its close paralog ESRP1 are highly expressed in primary prostate cancer. Androgen stimulation induces splicing switches in many endogenous ESRP2-controlled mRNA isoforms, including splicing switches correlating with disease progression. ESRP2 expression in clinical prostate cancer is repressed by ADT, which may thus inadvertently dampen epithelial splice programmes. Supporting this, treatment with the AR antagonist bicalutamide (Casodex) induced mesenchymal splicing patterns of genes including FLNB and CTNND1. Our data reveals a new mechanism of splicing control in prostate cancer with important implications for disease progression.
Abstract.
Author URL.
Lye JJ, Latorre E, Lee BP, Bandinelli S, Holley JE, Gutowski NJ, Ferrucci L, Harries LW (2019). Astrocyte senescence may drive alterations in GFAPα, CDKN2A p14ARF, and TAU3 transcript expression and contribute to cognitive decline.
Geroscience,
41(5), 561-573.
Abstract:
Astrocyte senescence may drive alterations in GFAPα, CDKN2A p14ARF, and TAU3 transcript expression and contribute to cognitive decline.
The accumulation of senescent cells in tissues is causally linked to the development of several age-related diseases; the removal of senescent glial cells in animal models prevents Tau accumulation and cognitive decline. Senescent cells can arise through several distinct mechanisms; one such mechanism is dysregulation of alternative splicing. In this study, we characterised the senescent cell phenotype in primary human astrocytes in terms of SA-β-Gal staining and SASP secretion, and then assessed splicing factor expression and candidate gene splicing patterns. Finally, we assessed associations between expression of dysregulated isoforms and premature cognitive decline in 197 samples from the InCHIANTI study of ageing, where expression was present in both blood and brain. We demonstrate here that senescent astrocytes secrete a modified SASP characterised by increased IL8, MMP3, MMP10, and TIMP2 but decreased IL10 levels. We identified significant changes in splicing factor expression for 10/20 splicing factors tested in senescent astrocytes compared with early passage cells, as well as dysregulation of isoform levels for 8/13 brain or senescence genes tested. Finally, associations were identified between peripheral blood GFAPα, TAU3, and CDKN2A (P14ARF) isoform levels and mild or severe cognitive decline over a 3-7-year period. Our data are suggestive that some of the features of cognitive decline may arise from dysregulated splicing of important genes in senescent brain support cells, and that defects in alternative splicing or splicing regulator expression deserve exploration as points of therapeutic intervention in the future.
Abstract.
Author URL.
Jeffery N, Richardson S, Chambers D, Morgan NG, Harries LW (2019). Cellular stressors may alter islet hormone cell proportions by moderation of alternative splicing patterns.
Hum Mol Genet,
28(16), 2763-2774.
Abstract:
Cellular stressors may alter islet hormone cell proportions by moderation of alternative splicing patterns.
Changes to islet cell identity in response to type 2 diabetes (T2D) have been reported in rodent models, but are less well characterized in humans. We assessed the effects of aspects of the diabetic microenvironment on hormone staining, total gene expression, splicing regulation and the alternative splicing patterns of key genes in EndoC-βH1 human beta cells. Genes encoding islet hormones [somatostatin (SST), insulin (INS), Glucagon (GCG)], differentiation markers [Forkhead box O1 (FOXO1), Paired box 6, SRY box 9, NK6 Homeobox 1, NK6 Homeobox 2] and cell stress markers (DNA damage inducible transcript 3, FOXO1) were dysregulated in stressed EndoC-βH1 cells, as were some serine arginine rich splicing factor splicing activator and heterogeneous ribonucleoprotein particle inhibitor genes. Whole transcriptome analysis of primary T2D islets and matched controls demonstrated dysregulated splicing for ~25% of splicing events, of which genes themselves involved in messenger ribonucleic acid processing and regulation of gene expression comprised the largest group. Approximately 5% of EndoC-βH1 cells exposed to these factors gained SST positivity in vitro. An increased area of SST staining was also observed ex vivo in pancreas sections recovered at autopsy from donors with type 1 diabetes (T1D) or T2D (9.3% for T1D and 3% for T2D, respectively compared with 1% in controls). Removal of the stressful stimulus or treatment with the AKT Serine/Threonine kinase inhibitor SH-6 restored splicing factor expression and reversed both hormone staining effects and patterns of gene expression. This suggests that reversible changes in hormone expression may occur during exposure to diabetomimetic cellular stressors, which may be mediated by changes in splicing regulation.
Abstract.
Author URL.
Jeffery N, Harries LW (2019). Corrigendum to "miRNAs responsive to the diabetic microenvironment in the human beta cell line EndoC βH1 may target genes in the FOXO, HIPPO and Lysine degradation pathways" [Exp. Cell Res. 29 (2019) 111559].
Exp Cell Res,
385(1).
Author URL.
Lee BP, Mulvey L, Barr G, Garratt J, Goodman E, Selman C, Harries LW (2019). Dietary restriction in ILSXISS mice is associated with widespread changes in splicing regulatory factor expression levels.
Exp Gerontol,
128Abstract:
Dietary restriction in ILSXISS mice is associated with widespread changes in splicing regulatory factor expression levels.
Dietary restriction (DR) represents one of the most reproducible interventions to extend lifespan and improve health outcomes in a wide range of species, but substantial variability in DR response has been observed, both between and within species. The mechanisms underlying this variation in effect are still not well characterised. Splicing regulatory factors have been implicated in the pathways linked with DR-induced longevity in C. elegans and are associated with lifespan itself in mice and humans. We used qRT-PCR to measure the expression levels of a panel of 16 age- and lifespan-associated splicing regulatory factors in brain, heart and kidney derived from three recombinant inbred strains of mice with variable lifespan responses to short-term (2 months) or long-term (10 months) 40% DR to determine their relationship to DR-induced longevity. We identified 3 patterns of association; i) splicing factors associated with DR alone, ii) splicing factors associated with strain alone or iii) splicing factors associated with both DR and strain. Tissue specific variation was noted in response to short-term or long-term DR, with the majority of effects noted in brain following long-term DR in the positive responder strain TejJ89. Association in heart and kidney were less evident, and occurred following short-term DR. Splicing factors associated with both DR and strain may be mechanistically involved in strain-specific differences in response to DR. We provide here evidence concordant with a role for some splicing factors in the lifespan modulatory effects of DR across different mouse strains and in different tissues.
Abstract.
Author URL.
Latorre E, Ostler EL, Faragher RGA, Harries LW (2019). FOXO1 and ETV6 genes may represent novel regulators of splicing factor expression in cellular senescence.
FASEB J,
33(1), 1086-1097.
Abstract:
FOXO1 and ETV6 genes may represent novel regulators of splicing factor expression in cellular senescence.
Cellular plasticity is a key facet of cellular homeostasis requiring correct temporal and spatial patterns of alternative splicing. Splicing factors, which orchestrate this process, demonstrate age-related dysregulation of expression; they are emerging as potential influences on aging and longevity. The upstream drivers of these alterations are still unclear but may involve aberrant cellular signaling. We compared the phosphorylation status of proteins in multiple signaling pathways in early and late passage human primary fibroblasts. We then assessed the impact of chemical inhibition or targeted knockdown of direct downstream targets of the ERK and AKT pathways on splicing factor expression, cellular senescence, and proliferation kinetics in senescent primary human fibroblasts. Components of the ERK and AKT signaling pathways demonstrated altered activation during cellular aging. Inhibition of AKT and ERK pathways led to up-regulation of splicing factor expression, reduction in senescent cell load, and partial reversal of multiple cellular senescence phenotypes in a dose-dependent manner. Furthermore, targeted knockdown of the genes encoding the downstream targets FOXO1 or ETV6 was sufficient to mimic these observations. Our results suggest that age-associated dysregulation of splicing factor expression and cellular senescence may derive in part from altered activity of ERK and AKT signaling and may act in part through the ETV6 and FOXO1 transcription factors. Targeting the activity of downstream effectors of ERK and AKT may therefore represent promising targets for future therapeutic intervention.-Latorre, E. Ostler, E. L. Faragher, R. G. A. Harries, L. W. FOXO1 and ETV6 genes may represent novel regulators of splicing factor expression in cellular senescence.
Abstract.
Author URL.
Harries L (2019). It's time for scientists to shout about RNA therapies.
Nature,
574(7778).
Author URL.
Meerson A, Eliraz Y, Yehuda H, Knight B, Crundwell M, Ferguson D, Lee BP, Harries LW (2019). Obesity impacts the regulation of miR-10b and its targets in primary breast tumors.
BMC Cancer,
19(1).
Abstract:
Obesity impacts the regulation of miR-10b and its targets in primary breast tumors
Background: Obesity increases breast cancer (BC) risk in post-menopausal women by mostly unknown molecular mechanisms which may partly be regulated by microRNAs (miRNAs). Methods: We isolated RNA from paired benign and malignant biopsies from 83 BC patients and determined miRNA profiles in samples from 12 women at the extremes of the BMI distribution by RNA-seq. Candidates were validated in all samples. Associations between miR-10b expression and validated target transcript levels, and effects of targeted manipulation of miR-10b levels in a primary BC cell line on proliferation and invasion potential, were explored. Results: of the 148 miRNAs robustly expressed in breast tissues, the levels of miR-21, miR-10b, miR-451a, miR-30c, and miR-378d were significantly associated with presence of cancer. of these, miR-10b showed a stronger down-regulation in the tumors of the obese subjects, as opposed to the lean. In ductal but not lobular tumors, significant inverse correlations were observed between the tumor levels of miR-10b and miR-30c and the mRNA levels of cancer-relevant target genes SRSF1, PIEZO1, MAPRE1, CDKN2A, TP-53 and TRA2B, as well as tumor grade. Suppression of miR-10b levels in BT-549 primary BC-derived cells increased cell proliferation and invasive capacity, while exogenous miR-10b mimic decreased invasion. Manipulation of miR-10b levels also inversely affected the mRNA levels of miR-10b targets BCL2L11, PIEZO1 and NCOR2. Conclusions: Our findings suggest that miR-10b may be a mediator between obesity and cancer in post-menopausal women, regulating several known cancer-relevant genes. MiR-10b expression may have diagnostic and therapeutic implications for the incidence and prognosis of BC in obese women.
Abstract.
Harries LW (2019). RNA Biology Provides New Therapeutic Targets for Human Disease.
FRONTIERS IN GENETICS,
10 Author URL.
Lee BP, Pilling LC, Bandinelli S, Ferrucci L, Melzer D, Harries LW (2019). The transcript expression levels of HNRNPM, HNRNPA0 and AKAP17A splicing factors may be predictively associated with ageing phenotypes in human peripheral blood.
Biogerontology,
20(5), 649-663.
Abstract:
The transcript expression levels of HNRNPM, HNRNPA0 and AKAP17A splicing factors may be predictively associated with ageing phenotypes in human peripheral blood.
Dysregulation of splicing factor expression is emerging as a driver of human ageing; levels of transcripts encoding splicing regulators have previously been implicated in ageing and cellular senescence both in vitro and in vivo. We measured the expression levels of an a priori panel of 20 age- or senescence-associated splicing factors by qRT-PCR in peripheral blood samples from the InCHIANTI Study of Aging, and assessed longitudinal relationships with human ageing phenotypes (cognitive decline and physical ability) using multivariate linear regression. AKAP17A, HNRNPA0 and HNRNPM transcript levels were all predictively associated with severe decline in MMSE score (p = 0.007, 0.001 and 0.008 respectively). Further analyses also found expression of these genes was associated with a performance decline in two other cognitive measures; the Trail Making Test and the Purdue Pegboard Test. AKAP17A was nominally associated with a decline in mean hand-grip strength (p = 0.023), and further analyses found nominal associations with two other physical ability measures; the Epidemiologic Studies of the Elderly-Short Physical Performance Battery and calculated speed (m/s) during a timed 400 m fast walking test. These data add weight to the hypothesis that splicing dyregulation may contribute to the development of some ageing phenotypes in the human population.
Abstract.
Author URL.
Calimport SRG, Bentley BL, Stewart CE, Pawelec G, Scuteri A, Vinciguerra M, Slack C, Chen D, Harries LW, Marchant G, et al (2019). To help aging populations, classify organismal senescence. Science, 366(6465), 576-578.
Jeffery N, Harries LW (2019). miRNAs responsive to the diabetic microenvironment in the human beta cell line EndoC-βH1 may target genes in the FOXO, HIPPO and Lysine degradation pathways.
Exp Cell Res,
384(1).
Abstract:
miRNAs responsive to the diabetic microenvironment in the human beta cell line EndoC-βH1 may target genes in the FOXO, HIPPO and Lysine degradation pathways.
Altered expression of miRNAs is evident in the islets of diabetic human donors, but the effects of specific aspects of the diabetic microenvironment and identity of gene ontology pathways demonstrating target gene enrichment in response to each is understudied. We assessed changes in the miRNA milieu in response to high/low glucose, hypoxia, dyslipidaemia and inflammatory factors in a humanised EndoC-βH1 beta cell culture system and performed miRPath analysis for each treatment individually. The 10 miRNAs demonstrating the greatest dysregulation across treatments were then independently validated and Gene Set Enrichment Analysis to confirm targeted pathways undertaken. 171 of 392 miRNAs displayed altered expression in response to one or more cellular stressors. miRNA changes were treatment specific, but their target genes were enriched in conserved pathways. 5 miRNAs (miR-136-5p, miR299-5p, miR-454-5p, miR-152 and miR-185) were dysregulated in response to multiple stressors and survived validation in independent samples (p = 0.008, 0.002, 0.012, 0.005 and 0.024 respectively). Target genes of dysregulated miRNAs were clustered into FOXO1, HIPPO and Lysine degradation pathways (p = 0.02, p = 5.84 × 10-5 and p = 3.00 × 10-3 respectively). We provide evidence that the diabetic microenvironment may induce changes to the expression of miRNAs targeting genes enriched in pathways involved in cell stress response and cell survival.
Abstract.
Author URL.
Ferguson DCJ, Smerdon GR, Harries LW, Dodd NJF, Murphy MP, Curnow A, Winyard PG (2018). Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia.
Free Radic Biol Med,
126, 322-333.
Abstract:
Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia.
In vivo, mammalian cells reside in an environment of 0.5-10% O2 (depending on the tissue location within the body), whilst standard in vitro cell culture is carried out under room air. Little is known about the effects of this hyperoxic environment on treatment-induced oxidative stress, relative to a physiological oxygen environment. In the present study we investigated the effects of long-term culture under hyperoxia (air) on photodynamic treatment. Upon photodynamic irradiation, cells which had been cultured long-term under hyperoxia generated higher concentrations of mitochondrial reactive oxygen species, compared with cells in a physioxic (2% O2) environment. However, there was no significant difference in viability between hyperoxic and physioxic cells. The expression of genes encoding key redox homeostasis proteins and the activity of key antioxidant enzymes was significantly higher after the long-term culture of hyperoxic cells compared with physioxic cells. The induction of antioxidant genes and increased antioxidant enzyme activity appear to contribute to the development of a phenotype that is resistant to oxidative stress-induced cellular damage and death when using standard cell culture conditions. The results from experiments using selective inhibitors suggested that the thioredoxin antioxidant system contributes to this phenotype. To avoid artefactual results, in vitro cellular responses should be studied in mammalian cells that have been cultured under physioxia. This investigation provides new insights into the effects of physioxic cell culture on a model of a clinically relevant photodynamic treatment and the associated cellular pathways.
Abstract.
Author URL.
Galloway T, Baglin N, Harries LW, Lee BP, Kocur AL, Shepherd M, Steele A, BPA Schools Study Consortium (2018). An engaged research study to assess the effect of a ‘real-world’ dietary intervention on urinary bisphenol a (BPA) levels in teenagers. BMJ Open, 8, e018742-e018742.
Clissold RL, Harries LW, Ellard S, Bingham C, Hattersley AT (2018). Comment on Dubois-Laforgue et al. Diabetes, Associated Clinical Spectrum, Long-term Prognosis, and Genotype/Phenotype Correlations in 201 Adult Patients with Hepatocyte Nuclear Factor 1B (HNF1B) Molecular Defects. Diabetes Care 2017;40:1436-1443.
Diabetes Care,
41(1).
Author URL.
Latorre E, Torregrossa R, Wood ME, Whiteman M, Harries LW (2018). Mitochondria-targeted hydrogen sulfide attenuates endothelial senescence by selective induction of splicing factors HNRNPD and SRSF2.
Aging (Albany NY),
10(7), 1666-1681.
Abstract:
Mitochondria-targeted hydrogen sulfide attenuates endothelial senescence by selective induction of splicing factors HNRNPD and SRSF2.
Cellular senescence is a key driver of ageing, influenced by age-related changes to the regulation of alternative splicing. Hydrogen sulfide (H2S) has similarly been described to influence senescence, but the pathways by which it accomplishes this are unclear.We assessed the effects of the slow release H2S donor Na-GYY4137 (100 µg/ml), and three novel mitochondria-targeted H2S donors AP39, AP123 and RT01 (10 ng/ml) on splicing factor expression, cell proliferation, apoptosis, DNA replication, DNA damage, telomere length and senescence-related secretory complex (SASP) expression in senescent primary human endothelial cells.All H2S donors produced up to a 50% drop in senescent cell load assessed at the biochemical and molecular level. Some changes were noted in the composition of senescence-related secretory complex (SASP); IL8 levels increased by 24% but proliferation was not re-established in the culture as a whole. Telomere length, apoptotic index and the extent of DNA damage were unaffected. Differential effects on splicing factor expression were observed depending on the intracellular targeting of the H2S donors. Na-GYY4137 produced a general 1.9 - 3.2-fold upregulation of splicing factor expression, whereas the mitochondria-targeted donors produced a specific 2.5 and 3.1-fold upregulation of SRSF2 and HNRNPD splicing factors only. Knockdown of SRSF2 or HNRNPD genes in treated cells rendered the cells non-responsive to H2S, and increased levels of senescence by up to 25% in untreated cells.Our data suggest that SRSF2 and HNRNPD may be implicated in endothelial cell senescence, and can be targeted by exogenous H2S. These molecules may have potential as moderators of splicing factor expression and senescence phenotypes.
Abstract.
Author URL.
Locke JM, Saint-Martin C, Laver TW, Patel KA, Wood AR, Sharp SA, Ellard S, Bellanné-Chantelot C, Hattersley AT, Harries LW, et al (2018). The Common HNF1A Variant I27L is a Modifier of Age at Diabetes Diagnosis in Individuals with HNF1A-MODY.
Diabetes,
67(9), 1903-1907.
Abstract:
The Common HNF1A Variant I27L is a Modifier of Age at Diabetes Diagnosis in Individuals with HNF1A-MODY.
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.
Abstract.
Author URL.
Latorre E, Pilling LC, Lee BP, Bandinelli S, Melzer D, Ferrucci L, Harries LW (2018). The VEGFA156b isoform is dysregulated in senescent endothelial cells and may be associated with prevalent and incident coronary heart disease.
Clin Sci (Lond),
132(3), 313-325.
Abstract:
The VEGFA156b isoform is dysregulated in senescent endothelial cells and may be associated with prevalent and incident coronary heart disease.
Coronary heart disease (CHD) is a leading cause of morbidity in people over 65 years of age; >40% of all deaths are due to this condition. The association between increasing age and CHD is well documented; the accumulation of senescent cells in cardiac and vascular tissues may represent one factor underpinning this observation. We aimed to identify senescence-related expression changes in primary human senescent cardiomyocytes and endothelial cells and to relate transcript expression in peripheral blood leucocytes to prevalent and incident CHD in the InCHIANTI study of aging. We quantified splicing factor expression and splicing patterns of candidate transcripts in proliferative and senescent later passage endothelial cells and cardiomyocytes using qRTPCR. Senescence-associated isoforms also expressed in peripheral blood leucocytes were then examined for associations with CHD status in 134 pairs of age, sex and BMI-matched CHD cases and controls. Splicing factor expression was dysregulated in senescent cardiomyocytes, as previously reported for endothelial cells, as was the expression of alternatively expressed cardiac and vascular candidate genes in both cell types. We found nominal associations between the expression of VEGFA156b and FNI-EIIIIA isoforms in peripheral blood mRNA and CHD status. Dysregulated splicing factor expression is a key feature of senescent cardiomyocytes and endothelial cells. Altered splicing of key cardiac or endothelial genes may contribute to the risk of CHD in the human population.
Abstract.
Author URL.
Harries L, Goljanek-Whysall K (2018). The biology of ageing and the omics revolution. Biogerontology, 19(6), 435-436.
Flanagan SE, Dũng VC, Houghton JAL, De Franco E, Ngoc CTB, Damhuis A, Ashcroft FM, Harries LW, Ellard S (2017). An ABCC8 Nonsense Mutation Causing Neonatal Diabetes Through Altered Transcript Expression.
J Clin Res Pediatr Endocrinol,
9(3), 260-264.
Abstract:
An ABCC8 Nonsense Mutation Causing Neonatal Diabetes Through Altered Transcript Expression.
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.
Abstract.
Author URL.
Haque S, Harries LW (2017). Circular RNAs (circRNAs) in Health and Disease.
Genes (Basel),
8(12).
Abstract:
Circular RNAs (circRNAs) in Health and Disease.
Splicing events do not always produce a linear transcript. Circular RNAs (circRNAs) are a class of RNA that are emerging as key new members of the gene regulatory milieu, which are produced by back-splicing events within genes. In circRNA formation, rather than being spliced in a linear fashion, exons can be circularised by use of the 3' acceptor splice site of an upstream exon, leading to the formation of a circular RNA species. circRNAs have been demonstrated across species and have the potential to present genetic information in new orientations distinct from their parent transcript. The importance of these RNA players in gene regulation and normal cellular homeostasis is now beginning to be recognised. They have several potential modes of action, from serving as sponges for micro RNAs and RNA binding proteins, to acting as transcriptional regulators. In accordance with an important role in the normal biology of the cell, perturbations of circRNA expression are now being reported in association with disease. Furthermore, the inherent stability of circRNAs conferred by their circular structure and exonuclease resistance, and their expression in blood and other peripheral tissues in association with endosomes and microvesicles, renders them excellent candidates as disease biomarkers. In this review, we explore the state of knowledge on this exciting class of transcripts in regulating gene expression and discuss their emerging role in health and disease.
Abstract.
Author URL.
Pilling LC, Kuo C-L, Sicinski K, Tamosauskaite J, Kuchel GA, Harries LW, Herd P, Wallace R, Ferrucci L, Melzer D, et al (2017). Human longevity: 25 genetic loci associated in 389,166 UK biobank participants.
Aging (Albany NY),
9(12), 2504-2520.
Abstract:
Human longevity: 25 genetic loci associated in 389,166 UK biobank participants.
We undertook a genome-wide association study (GWAS) of parental longevity in European descent UK Biobank participants. For combined mothers' and fathers' attained age, 10 loci were associated (p
Abstract.
Author URL.
Lee BP, Burić I, George-Pandeth A, Flurkey K, Harrison DE, Yuan R, Peters LL, Kuchel GA, Melzer D, Harries LW, et al (2017). MicroRNAs miR-203-3p, miR-664-3p and miR-708-5p are associated with median strain lifespan in mice.
Sci Rep,
7Abstract:
MicroRNAs miR-203-3p, miR-664-3p and miR-708-5p are associated with median strain lifespan in mice.
MicroRNAs (miRNAs) are small non-coding RNA species that have been shown to have roles in multiple processes that occur in higher eukaryotes. They act by binding to specific sequences in the 3' untranslated region of their target genes and causing the transcripts to be degraded by the RNA-induced silencing complex (RISC). MicroRNAs have previously been reported to demonstrate altered expression in several aging phenotypes such as cellular senescence and age itself. Here, we have measured the expression levels of 521 small regulatory microRNAs (miRNAs) in spleen tissue from young and old animals of 6 mouse strains with different median strain lifespans by quantitative real-time PCR. Expression levels of 3 microRNAs were robustly associated with strain lifespan, after correction for multiple statistical testing (miR-203-3p [β-coefficient = -0.6447, p = 4.8 × 10-11], miR-664-3p [β-coefficient = 0.5552, p = 5.1 × 10-8] and miR-708-5p [β-coefficient = 0.4986, p = 1.6 × 10-6]). Pathway analysis of binding sites for these three microRNAs revealed enrichment of target genes involved in key aging and longevity pathways including mTOR, FOXO and MAPK, most of which also demonstrated associations with longevity. Our results suggests that miR-203-3p, miR-664-3p and miR-708-5p may be implicated in pathways determining lifespan in mammals.
Abstract.
Author URL.
Jeynes JCG, Geraki K, Jeynes C, Zhaohong M, Bettiol AA, Latorre E, Harries LW, Soeller C (2017). Nanoscale Properties of Human Telomeres Measured with a Dual Purpose X-ray Fluorescence and Super Resolution Microscopy Gold Nanoparticle Probe.
ACS Nano,
11(12), 12632-12640.
Abstract:
Nanoscale Properties of Human Telomeres Measured with a Dual Purpose X-ray Fluorescence and Super Resolution Microscopy Gold Nanoparticle Probe.
Techniques to analyze human telomeres are imperative in studying the molecular mechanism of aging and related diseases. Two important aspects of telomeres are their length in DNA base pairs (bps) and their biophysical nanometer dimensions. However, there are currently no techniques that can simultaneously measure these quantities in individual cell nuclei. Here, we develop and evaluate a telomere "dual" gold nanoparticle-fluorescent probe simultaneously compatible with both X-ray fluorescence (XRF) and super resolution microscopy. We used silver enhancement to independently visualize the spatial locations of gold nanoparticles inside the nuclei, comparing to a standard QFISH (quantitative fluorescence in situ hybridization) probe, and showed good specificity at ∼90%. For sensitivity, we calculated telomere length based on a DNA/gold binding ratio using XRF and compared to quantitative polymerase chain reaction (qPCR) measurements. The sensitivity was low (∼10%), probably because of steric interference prohibiting the relatively large 10 nm gold nanoparticles access to DNA space. We then measured the biophysical characteristics of individual telomeres using super resolution microscopy. Telomeres that have an average length of ∼10 kbps, have diameters ranging between ∼60-300 nm. Further, we treated cells with a telomere-shortening drug and showed there was a small but significant difference in telomere diameter in drug-treated vs control cells. We discuss our results in relation to the current debate surrounding telomere compaction.
Abstract.
Author URL.
Pilling LC, Atkins JL, Duff MO, Beaumont RN, Jones SE, Tyrrell J, Kuo C-L, Ruth KS, Tuke MA, Yaghootkar H, et al (2017). Red blood cell distribution width: Genetic evidence for aging pathways in 116,666 volunteers.
PLoS One,
12(9).
Abstract:
Red blood cell distribution width: Genetic evidence for aging pathways in 116,666 volunteers.
INTRODUCTION: Variability in red blood cell volumes (distribution width, RDW) increases with age and is strongly predictive of mortality, incident coronary heart disease and cancer. We investigated inherited genetic variation associated with RDW in 116,666 UK Biobank human volunteers. RESULTS: a large proportion RDW is explained by genetic variants (29%), especially in the older group (60+ year olds, 33.8%,
Abstract.
Author URL.
Latorre E, Birar VC, Sheerin AN, Jeynes JCC, Hooper A, Dawe HR, Melzer D, Cox LS, Faragher RGA, Ostler EL, et al (2017). Small molecule modulation of splicing factor expression is associated with rescue from cellular senescence.
BMC Cell Biol,
18(1).
Abstract:
Small molecule modulation of splicing factor expression is associated with rescue from cellular senescence.
BACKGROUND: Altered expression of mRNA splicing factors occurs with ageing in vivo and is thought to be an ageing mechanism. The accumulation of senescent cells also occurs in vivo with advancing age and causes much degenerative age-related pathology. However, the relationship between these two processes is opaque. Accordingly we developed a novel panel of small molecules based on resveratrol, previously suggested to alter mRNA splicing, to determine whether altered splicing factor expression had potential to influence features of replicative senescence. RESULTS: Treatment with resveralogues was associated with altered splicing factor expression and rescue of multiple features of senescence. This rescue was independent of cell cycle traverse and also independent of SIRT1, SASP modulation or senolysis. Under growth permissive conditions, cells demonstrating restored splicing factor expression also demonstrated increased telomere length, re-entered cell cycle and resumed proliferation. These phenomena were also influenced by ERK antagonists and agonists. CONCLUSIONS: This is the first demonstration that moderation of splicing factor levels is associated with reversal of cellular senescence in human primary fibroblasts. Small molecule modulators of such targets may therefore represent promising novel anti-degenerative therapies.
Abstract.
Author URL.
Latorre E, Harries LW (2017). Splicing regulatory factors, ageing and age-related disease.
Ageing Res Rev,
36, 165-170.
Abstract:
Splicing regulatory factors, ageing and age-related disease.
Alternative splicing is a co-transcriptional process, which allows for the production of multiple transcripts from a single gene and is emerging as an important control point for gene expression. Alternatively expressed isoforms often have antagonistic function and differential temporal or spatial expression patterns, yielding enormous plasticity and adaptability to cells and increasing their ability to respond to environmental challenge. The regulation of alternative splicing is critical for numerous cellular functions in both pathological and physiological conditions, and deregulated alternative splicing is a key feature of common chronic diseases. Isoform choice is controlled by a battery of splicing regulatory proteins, which include the serine arginine rich (SRSF) proteins and the heterogeneous ribonucleoprotein (hnRNP) classes of genes. These important splicing regulators have been implicated in age-related disease, and in the ageing process itself. This review will outline the important contribution of splicing regulator proteins to ageing and age-related disease.
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Author URL.
Munkley J, McClurg UL, Livermore KE, Ehrmann I, Knight B, Mccullagh P, Mcgrath J, Crundwell M, Harries LW, Leung HY, et al (2017). The cancer-associated cell migration protein TSPAN1 is under control of androgens and its upregulation increases prostate cancer cell migration.
Sci Rep,
7(1).
Abstract:
The cancer-associated cell migration protein TSPAN1 is under control of androgens and its upregulation increases prostate cancer cell migration.
Cell migration drives cell invasion and metastatic progression in prostate cancer and is a major cause of mortality and morbidity. However the mechanisms driving cell migration in prostate cancer patients are not fully understood. We previously identified the cancer-associated cell migration protein Tetraspanin 1 (TSPAN1) as a clinically relevant androgen regulated target in prostate cancer. Here we find that TSPAN1 is acutely induced by androgens, and is significantly upregulated in prostate cancer relative to both normal prostate tissue and benign prostate hyperplasia (BPH). We also show for the first time, that TSPAN1 expression in prostate cancer cells controls the expression of key proteins involved in cell migration. Stable upregulation of TSPAN1 in both DU145 and PC3 cells significantly increased cell migration and induced the expression of the mesenchymal markers SLUG and ARF6. Our data suggest TSPAN1 is an androgen-driven contributor to cell survival and motility in prostate cancer.
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Author URL.
Jeffery N, Richardson S, Beall C, Harries LW (2017). The species origin of the cellular microenvironment influences markers of beta cell fate and function in EndoC-βH1 cells. Experimental Cell Research, 361(2), 284-291.
Lee BP, Pilling LC, Emond F, Flurkey K, Harrison DE, Yuan R, Peters LL, Kuchel GA, Ferrucci L, Melzer D, et al (2016). Changes in the expression of splicing factor transcripts and variations in alternative splicing are associated with lifespan in mice and humans.
Aging Cell,
15(5), 903-913.
Abstract:
Changes in the expression of splicing factor transcripts and variations in alternative splicing are associated with lifespan in mice and humans.
Dysregulation of splicing factor expression and altered alternative splicing are associated with aging in humans and other species, and also with replicative senescence in cultured cells. Here, we assess whether expression changes of key splicing regulator genes and consequent effects on alternative splicing are also associated with strain longevity in old and young mice, across 6 different mouse strains with varying lifespan (A/J, NOD.B10Sn-H2(b) /J, PWD.Phj, 129S1/SvlmJ, C57BL/6J and WSB/EiJ). Splicing factor expression and changes to alternative splicing were associated with strain lifespan in spleen and to a lesser extent in muscle. These changes mainly involved hnRNP splicing inhibitor transcripts with most changes more marked in spleens of young animals from long-lived strains. Changes in spleen isoform expression were suggestive of reduced cellular senescence and retained cellular proliferative capacity in long-lived strains. Changes in muscle isoform expression were consistent with reduced pro-inflammatory signalling in longer-lived strains. Two splicing regulators, HNRNPA1 and HNRNPA2B1, were also associated with parental longevity in humans, in the InCHIANTI aging study. Splicing factors may represent a driver, mediator or early marker of lifespan in mouse, as expression differences were present in the young animals of long-lived strains. Changes to alternative splicing patterns of key senescence genes in spleen and key remodelling genes in muscle suggest that correct regulation of alternative splicing may enhance lifespan in mice. Expression of some splicing factors in humans was also associated with parental longevity, suggesting that splicing regulation may also influence lifespan in humans.
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Author URL.
Lee BP, Lloyd-Laney HO, Locke JM, McCulloch LJ, Knight B, Yaghootkar H, Cory G, Kos K, Frayling TM, Harries LW, et al (2016). Functional characterisation of ADIPOQ variants using individuals recruited by genotype.
Mol Cell Endocrinol,
428, 49-57.
Abstract:
Functional characterisation of ADIPOQ variants using individuals recruited by genotype.
Four non-coding GWAS variants in or near the ADIPOQ gene (rs17300539, rs17366653, rs3821799 and rs56354395) together explain 4% of the variation in circulating adiponectin. The functional basis for this is unknown. We tested the effect of these variants on ADIPOQ transcription, splicing and stability respectively in adipose tissue samples from participants recruited by rs17366653 genotype. Transcripts carrying rs17300539 demonstrated a 17% increase in expression (p = 0.001). Variant rs17366653 was associated with disruption of ADIPOQ splicing leading to a 7 fold increase in levels of a non-functional transcript (p = 0.002). Transcripts carrying rs56354395 demonstrated a 59% decrease in expression (p =
Abstract.
Author URL.
Pilling LC, Joehanes R, Kacprowski T, Peters M, Jansen R, Karasik D, Kiel DP, Harries LW, Teumer A, Powell J, et al (2016). Gene transcripts associated with muscle strength: a CHARGE meta-analysis of 7,781 persons.
Physiol Genomics,
48(1), 1-11.
Abstract:
Gene transcripts associated with muscle strength: a CHARGE meta-analysis of 7,781 persons.
Lower muscle strength in midlife predicts disability and mortality in later life. Blood-borne factors, including growth differentiation factor 11 (GDF11), have been linked to muscle regeneration in animal models. We aimed to identify gene transcripts associated with muscle strength in adults. Meta-analysis of whole blood gene expression (overall 17,534 unique genes measured by microarray) and hand-grip strength in four independent cohorts (n = 7,781, ages: 20-104 yr, weighted mean = 56), adjusted for age, sex, height, weight, and leukocyte subtypes. Separate analyses were performed in subsets (older/younger than 60, men/women). Expression levels of 221 genes were associated with strength after adjustment for cofactors and for multiple statistical testing, including ALAS2 (rate-limiting enzyme in heme synthesis), PRF1 (perforin, a cytotoxic protein associated with inflammation), IGF1R, and IGF2BP2 (both insulin like growth factor related). We identified statistical enrichment for hemoglobin biosynthesis, innate immune activation, and the stress response. Ten genes were associated only in younger individuals, four in men only and one in women only. For example, PIK3R2 (a negative regulator of PI3K/AKT growth pathway) was negatively associated with muscle strength in younger (
Abstract.
Author URL.
Munkley J, Vodak D, Livermore KE, James K, Wilson BT, Knight B, Mccullagh P, Mcgrath J, Crundwell M, Harries LW, et al (2016). Glycosylation is an Androgen-Regulated Process Essential for Prostate Cancer Cell Viability.
EBioMedicine,
8, 103-116.
Abstract:
Glycosylation is an Androgen-Regulated Process Essential for Prostate Cancer Cell Viability
Steroid androgen hormones play a key role in the progression and treatment of prostate cancer, with androgen deprivation therapy being the first-line treatment used to control cancer growth. Here we apply a novel search strategy to identify androgen-regulated cellular pathways that may be clinically important in prostate cancer. Using RNASeq data, we searched for genes that showed reciprocal changes in expression in response to acute androgen stimulation in culture, and androgen deprivation in patients with prostate cancer. Amongst 700 genes displaying reciprocal expression patterns we observed a significant enrichment in the cellular process glycosylation. of 31 reciprocally-regulated glycosylation enzymes, a set of 8 (GALNT7, ST6GalNAc1, GCNT1, UAP1, PGM3, CSGALNACT1, ST6GAL1 and EDEM3) were significantly up-regulated in clinical prostate carcinoma. Androgen exposure stimulated synthesis of glycan structures downstream of this core set of regulated enzymes including sialyl-Tn (sTn), sialyl LewisX (SLeX), O-GlcNAc and chondroitin sulphate, suggesting androgen regulation of the core set of enzymes controls key steps in glycan synthesis. Expression of each of these enzymes also contributed to prostate cancer cell viability. This study identifies glycosylation as a global target for androgen control, and suggests loss of specific glycosylation enzymes might contribute to tumour regression following androgen depletion therapy.
Abstract.
Pilling LC, Atkins JL, Bowman K, Jones SE, Tyrrell J, Beaumont RN, Ruth KS, Tuke MA, Yaghootkar H, Wood AR, et al (2016). Human longevity is influenced by many genetic variants: evidence from 75,000 UK Biobank participants.
Aging (Albany NY),
8(3), 547-560.
Abstract:
Human longevity is influenced by many genetic variants: evidence from 75,000 UK Biobank participants.
Variation in human lifespan is 20 to 30% heritable in twins but few genetic variants have been identified. We undertook a Genome Wide Association Study (GWAS) using age at death of parents of middle-aged UK Biobank participants of European decent (n=75,244 with father's and/or mother's data, excluding early deaths). Genetic risk scores for 19 phenotypes (n=777 proven variants) were also tested. In GWAS, a nicotine receptor locus(CHRNA3, previously associated with increased smoking and lung cancer) was associated with fathers' survival. Less common variants requiring further confirmation were also identified. Offspring of longer lived parents had more protective alleles for coronary artery disease, systolic blood pressure, body mass index, cholesterol and triglyceride levels, type-1 diabetes, inflammatory bowel disease and Alzheimer's disease. In candidate analyses, variants in the TOMM40/APOE locus were associated with longevity, but FOXO variants were not. Associations between extreme longevity (mother >=98 years, fathers >=95 years, n=1,339) and disease alleles were similar, with an additional association with HDL cholesterol (p=5.7x10-3). These results support a multiple protective factors model influencing lifespan and longevity (top 1% survival) in humans, with prominent roles for cardiovascular-related pathways. Several of these genetically influenced risks, including blood pressure and tobacco exposure, are potentially modifiable.
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Author URL.
Atkins JL, Pilling LC, Ble A, Dutta A, Harries LW, Murray A, Brayne C, Robine J-M, Kuchel GA, Ferrucci L, et al (2016). Longer-Lived Parents and Cardiovascular Outcomes: 8-Year Follow-Up in 186,000 U.K. Biobank Participants.
J Am Coll Cardiol,
68(8), 874-875.
Author URL.
Chen BH, Hivert M-F, Peters MJ, Pilling LC, Hogan JD, Pham LM, Harries LW, Fox CS, Bandinelli S, Dehghan A, et al (2016). Peripheral Blood Transcriptomic Signatures of Fasting Glucose and Insulin Concentrations.
Diabetes,
65(12), 3794-3804.
Abstract:
Peripheral Blood Transcriptomic Signatures of Fasting Glucose and Insulin Concentrations.
Genome-wide association studies (GWAS) have successfully identified genetic loci associated with glycemic traits. However, characterizing the functional significance of these loci has proven challenging. We sought to gain insights into the regulation of fasting insulin and fasting glucose through the use of gene expression microarray data from peripheral blood samples of participants without diabetes in the Framingham Heart Study (FHS) (n = 5,056), the Rotterdam Study (RS) (n = 723), and the InCHIANTI Study (Invecchiare in Chianti) (n = 595). Using a false discovery rate q
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Author URL.
Tugay K, Guay C, Marques AC, Allagnat F, Locke JM, Harries LW, Rutter GA, Regazzi R (2016). Role of microRNAs in the age-associated decline of pancreatic beta cell function in rat islets.
Diabetologia,
59(1), 161-169.
Abstract:
Role of microRNAs in the age-associated decline of pancreatic beta cell function in rat islets
Aims/hypothesis: Ageing can lead to reduced insulin sensitivity and loss of pancreatic beta cell function, predisposing individuals to the development of diabetes. The aim of this study was to assess the contribution of microRNAs (miRNAs) to age-associated beta cell dysfunction. Methods: the global mRNA and miRNA profiles of 3- and 12-month-old rat islets were collected by microarray. The functional impact of age-associated differences in miRNA expression was investigated by mimicking the observed changes in primary beta cells from young animals. Results: Beta cells from 12-month-old rats retained normal insulin content and secretion, but failed to proliferate in response to mitotic stimuli. The islets of these animals displayed modifications at the level of several miRNAs, including upregulation of miR-34a, miR-124a and miR-383, and downregulation of miR-130b and miR-181a. Computational analysis of the transcriptomic modifications observed in the islets of 12-month-old rats revealed that the differentially expressed genes were enriched for miR-34a and miR-181a targets. Indeed, the induction of miR-34a and reduction of miR-181a in the islets of young animals mimicked the impaired beta cell proliferation observed in old animals. mRNA coding for alpha-type platelet-derived growth factor receptor, which is critical for compensatory beta cell mass expansion, is directly inhibited by miR34a and is likely to be at least partly responsible for the effects of this miRNA. Conclusions/interpretation: Changes in the level of specific miRNAs that occur during ageing affect the proliferative capacity of beta cells. This might reduce their ability to expand under conditions of increased insulin demand, favouring the development of type 2 diabetes.
Abstract.
Tugay K, Guay C, Marques AC, Allagnat F, Locke JM, Harries LW, Rutter GA, Regazzi R (2016). Role of microRNAs in the age-associated decline of pancreatic beta cell function in rat islets.
Diabetologia,
59(1), 161-169.
Abstract:
Role of microRNAs in the age-associated decline of pancreatic beta cell function in rat islets.
AIMS/HYPOTHESIS: Ageing can lead to reduced insulin sensitivity and loss of pancreatic beta cell function, predisposing individuals to the development of diabetes. The aim of this study was to assess the contribution of microRNAs (miRNAs) to age-associated beta cell dysfunction. METHODS: the global mRNA and miRNA profiles of 3- and 12-month-old rat islets were collected by microarray. The functional impact of age-associated differences in miRNA expression was investigated by mimicking the observed changes in primary beta cells from young animals. RESULTS: Beta cells from 12-month-old rats retained normal insulin content and secretion, but failed to proliferate in response to mitotic stimuli. The islets of these animals displayed modifications at the level of several miRNAs, including upregulation of miR-34a, miR-124a and miR-383, and downregulation of miR-130b and miR-181a. Computational analysis of the transcriptomic modifications observed in the islets of 12-month-old rats revealed that the differentially expressed genes were enriched for miR-34a and miR-181a targets. Indeed, the induction of miR-34a and reduction of miR-181a in the islets of young animals mimicked the impaired beta cell proliferation observed in old animals. mRNA coding for alpha-type platelet-derived growth factor receptor, which is critical for compensatory beta cell mass expansion, is directly inhibited by miR34a and is likely to be at least partly responsible for the effects of this miRNA. CONCLUSIONS/INTERPRETATION: Changes in the level of specific miRNAs that occur during ageing affect the proliferative capacity of beta cells. This might reduce their ability to expand under conditions of increased insulin demand, favouring the development of type 2 diabetes.
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Kamenska A, Simpson C, Vindry C, Broomhead H, Bénard M, Ernoult-Lange M, Lee BP, Harries LW, Weil D, Standart N, et al (2016). The DDX6-4E-T interaction mediates translational repression and P-body assembly.
Nucleic Acids Res,
44(13), 6318-6334.
Abstract:
The DDX6-4E-T interaction mediates translational repression and P-body assembly.
4E-Transporter binds eIF4E via its consensus sequence YXXXXLΦ, shared with eIF4G, and is a nucleocytoplasmic shuttling protein found enriched in P-(rocessing) bodies. 4E-T inhibits general protein synthesis by reducing available eIF4E levels. Recently, we showed that 4E-T bound to mRNA however represses its translation in an eIF4E-independent manner, and contributes to silencing of mRNAs targeted by miRNAs. Here, we address further the mechanism of translational repression by 4E-T by first identifying and delineating the interacting sites of its major partners by mass spectrometry and western blotting, including DDX6, UNR, unrip, PAT1B, LSM14A and CNOT4. Furthermore, we document novel binding between 4E-T partners including UNR-CNOT4 and unrip-LSM14A, altogether suggesting 4E-T nucleates a complex network of RNA-binding protein interactions. In functional assays, we demonstrate that joint deletion of two short conserved motifs that bind UNR and DDX6 relieves repression of 4E-T-bound mRNA, in part reliant on the 4E-T-DDX6-CNOT1 axis. We also show that the DDX6-4E-T interaction mediates miRNA-dependent translational repression and de novo P-body assembly, implying that translational repression and formation of new P-bodies are coupled processes. Altogether these findings considerably extend our understanding of the role of 4E-T in gene regulation, important in development and neurogenesis.
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Author URL.
Jeffery N, Harries LW (2016). β-cell differentiation status in type 2 diabetes.
Diabetes Obes Metab,
18(12), 1167-1175.
Abstract:
β-cell differentiation status in type 2 diabetes.
Type 2 diabetes (T2D) affects 415 million people worldwide and is characterized by chronic hyperglycaemia and insulin resistance, progressing to insufficient insulin production, as a result of β-cell failure. Over time, chronic hyperglycaemia can ultimately lead to loss of β-cell function, leaving patients insulin-dependent. Until recently the loss of β-cell mass seen in T2D was considered to be the result of increased rates of apoptosis; however, it has been proposed that apoptosis alone cannot account for the extent of β-cell mass loss seen in the disease, and that a loss of function may also occur as a result of changes in β-cell differentiation status. In the present review, we consider current knowledge of determinants of β-cell fate in the context of understanding its relevance to disease process in T2D, and also the impact of a diabetogenic environment (hyperglycaemia, hypoxia, inflammation and dyslipidaemia) on the expression of genes involved in maintenance of β-cell identity. We describe current knowledge of the impact of the diabetic microenvironment on gene regulatory processes such alternative splicing, the expression of disallowed genes and epigenetic modifications. Elucidating the molecular mechanisms that underpin changes to β-cell differentiation status and the concomitant β-cell failure offers potential treatment targets for the future management of patients with T2D.
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Author URL.
Locke JM, Wei FY, Tomizawa K, Weedon MN, Harries LW (2015). A cautionary tale: the non-causal association between type 2 diabetes risk SNP, rs7756992, and levels of non-coding RNA, CDKAL1-v1.
Diabetologia,
58(4), 745-748.
Abstract:
A cautionary tale: the non-causal association between type 2 diabetes risk SNP, rs7756992, and levels of non-coding RNA, CDKAL1-v1
Aims/hypothesis: Intronic single nucleotide polymorphisms (SNPs) in the CDKAL1 gene are associated with risk of developing type 2 diabetes. A strong correlation between risk alleles and lower levels of the non-coding RNA, CDKAL1-v1, has recently been reported in whole blood extracted from Japanese individuals. We sought to replicate this association in two independent cohorts: one using whole blood from white UK-resident individuals, and one using a collection of human pancreatic islets, a more relevant tissue type to study with respect to the aetiology of diabetes. Methods: Levels of CDKAL1-v1 were measured by real-time PCR using RNA extracted from human whole blood (n = 70) and human pancreatic islets (n = 48). Expression with respect to genotype was then determined. Results: in a simple linear regression model, expression of CDKAL1-v1 was associated with the lead type 2 diabetes-associated SNP, rs7756992, in whole blood and islets. However, these associations were abolished or substantially reduced in multiple regression models taking into account rs9366357 genotype: a moderately linked SNP explaining a much larger amount of the variation in CDKAL1-v1 levels, but not strongly associated with risk of type 2 diabetes. Conclusions/interpretation: Contrary to previous findings, we provide evidence against a role for dysregulated expression of CDKAL1-v1 in mediating the association between intronic SNPs in CDKAL1 and susceptibility to type 2 diabetes. The results of this study illustrate how caution should be exercised when inferring causality from an association between disease-risk genotype and non-coding RNA expression.
Abstract.
Locke JM, Wei F-Y, Tomizawa K, Weedon MN, Harries LW (2015). A cautionary tale: the non-causal association between type 2 diabetes risk SNP, rs7756992, and levels of non-coding RNA, CDKAL1-v1.
Diabetologia,
58(4), 745-748.
Abstract:
A cautionary tale: the non-causal association between type 2 diabetes risk SNP, rs7756992, and levels of non-coding RNA, CDKAL1-v1.
AIMS/HYPOTHESIS: Intronic single nucleotide polymorphisms (SNPs) in the CDKAL1 gene are associated with risk of developing type 2 diabetes. A strong correlation between risk alleles and lower levels of the non-coding RNA, CDKAL1-v1, has recently been reported in whole blood extracted from Japanese individuals. We sought to replicate this association in two independent cohorts: one using whole blood from white UK-resident individuals, and one using a collection of human pancreatic islets, a more relevant tissue type to study with respect to the aetiology of diabetes. METHODS: Levels of CDKAL1-v1 were measured by real-time PCR using RNA extracted from human whole blood (n = 70) and human pancreatic islets (n = 48). Expression with respect to genotype was then determined. RESULTS: in a simple linear regression model, expression of CDKAL1-v1 was associated with the lead type 2 diabetes-associated SNP, rs7756992, in whole blood and islets. However, these associations were abolished or substantially reduced in multiple regression models taking into account rs9366357 genotype: a moderately linked SNP explaining a much larger amount of the variation in CDKAL1-v1 levels, but not strongly associated with risk of type 2 diabetes. CONCLUSIONS/INTERPRETATION: Contrary to previous findings, we provide evidence against a role for dysregulated expression of CDKAL1-v1 in mediating the association between intronic SNPs in CDKAL1 and susceptibility to type 2 diabetes. The results of this study illustrate how caution should be exercised when inferring causality from an association between disease-risk genotype and non-coding RNA expression.
Abstract.
Author URL.
Blackwell J, Harries LW, Pilling LC, Ferrucci L, Jones A, Melzer D (2015). Changes in CEBPB expression in circulating leukocytes following eccentric elbow-flexion exercise.
J Physiol Sci,
65(1), 145-150.
Abstract:
Changes in CEBPB expression in circulating leukocytes following eccentric elbow-flexion exercise.
In mouse models, CCAAT enhancer-binding protein beta (CEBPB) is necessary for M2 macrophage-mediated regeneration after muscle injury. In humans, CEBPB expression in blood was strongly associated with muscle strength. In this study we aimed to test whether CEBPB expression in blood in people is increased 2 days after exercise designed to induce muscle damage and subsequent repair. Sixteen healthy male volunteers undertook elbow flexor exercises designed to induce acute muscle micro-damage. Peripheral blood samples were collected at baseline and days 1, 2, 4 and 7 following exercise. Expression of CEBPB and related genes were analysed by qRT-PCR. Extent of muscle damage was determined by decline in maximal voluntary isometric torque and by plasma creatine kinase activity. Nine subjects had peak (day 4) creatine kinase activity exceeding 10,000 U/l. In this subgroup, CEBPB expression was elevated from baseline to 2 days post exercise (paired-samples t (1,8) = 3.72, p = 0.006). Related expression and selected cytokine changes after exercise did not reach significance. Muscle-damaging exercise in humans can be followed by induction of CEBPB transcript expression in peripheral blood. Associations between CEBPB expression in blood and muscle strength may be consistent with the CEBPB-dependent muscle repair process.
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Author URL.
Holly AC, Grellscheid S, van de Walle P, Dolan D, Pilling LC, Daniels DJ, von Zglinicki T, Ferrucci L, Melzer D, Harries LW, et al (2015). Comparison of senescence-associated miRNAs in primary skin and lung fibroblasts.
Biogerontology,
16(4), 423-434.
Abstract:
Comparison of senescence-associated miRNAs in primary skin and lung fibroblasts.
MicroRNAs are non-coding RNAs with roles in many cellular processes. Tissue-specific miRNA profiles associated with senescence have been described for several cell and tissue types. We aimed to characterise miRNAs involved in core, rather than tissue-specific, senescence pathways by assessment of common miRNA expression differences in two different cell types, with follow-up of predicted targets in human peripheral blood. MicroRNAs were profiled in early and late passage primary lung and skin fibroblasts to identify commonly-deregulated miRNAs. Expression changes of their bioinformatically-predicted mRNA targets were then assessed in both cell types and in human peripheral blood from elderly participants in the InCHIANTI study. 57/178 and 26/492 microRNAs were altered in late passage skin and lung cells respectively. Three miRNAs (miR-92a, miR-15b and miR-125a-3p) were altered in both tissues. 14 mRNA targets of the common miRNAs were expressed in lung and skin fibroblasts, of which two demonstrated up-regulation in late passage skin and lung cells (LYST; p = 0.02 [skin] and 0.02 [lung] INMT; p = 0.03 [skin] and 0.04 [lung]). ZMPSTE24 and LHFPL2 demonstrated altered expression in late passage skin cells only (p = 0.01 and 0.05 respectively). LHFPL2 was also positively correlated with age in peripheral blood (p value = 6.6 × 10(-5)). We find that the majority of senescence-associated miRNAs demonstrate tissue-specific effects. However, miRNAs showing common effects across tissue types may represent those associated with core, rather than tissue-specific senescence processes.
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Author URL.
Pilling LC, Joehanes R, Melzer D, Harries LW, Henley W, Dupuis J, Lin H, Mitchell M, Hernandez D, Ying S-X, et al (2015). Gene expression markers of age-related inflammation in two human cohorts.
Exp Gerontol,
70, 37-45.
Abstract:
Gene expression markers of age-related inflammation in two human cohorts.
INTRODUCTION: Chronically elevated circulating inflammatory markers are common in older persons but mechanisms are unclear. Many blood transcripts (>800 genes) are associated with interleukin-6 protein levels (IL6) independent of age. We aimed to identify gene transcripts statistically mediating, as drivers or responders, the increasing levels of IL6 protein in blood at older ages. METHODS: Blood derived in-vivo RNA from the Framingham Heart Study (FHS, n=2422, ages 40-92 yrs) and InCHIANTI study (n=694, ages 30-104 yrs), with Affymetrix and Illumina expression arrays respectively (>17,000 genes tested), were tested for statistical mediation of the age-IL6 association using resampling techniques, adjusted for confounders and multiple testing. RESULTS: in FHS, IL6 expression was not associated with IL6 protein levels in blood. 102 genes (0.6% of 17,324 expressed) statistically mediated the age-IL6 association of which 25 replicated in InCHIANTI (including 5 of the 10 largest effect genes). The largest effect gene (SLC4A10, coding for NCBE, a sodium bicarbonate transporter) mediated 19% (adjusted CI 8.9 to 34.1%) and replicated by PCR in InCHIANTI (n=194, 35.6% mediated, p=0.01). Other replicated mediators included PRF1 (perforin, a cytolytic protein in cytotoxic T lymphocytes and NK cells) and IL1B (Interleukin 1 beta): few other cytokines were significant mediators. CONCLUSIONS: This transcriptome-wide study on human blood identified a small distinct set of genes that statistically mediate the age-IL6 association. Findings are robust across two cohorts and different expression technologies. Raised IL6 levels may not derive from circulating white cells in age related inflammation.
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Galloway TS, Fletcher T, Thomas OJ, Lee BP, Pilling LC, Harries LW (2015). PFOA and PFOS are associated with reduced expression of the parathyroid hormone 2 receptor (PTH2R) gene in women.
Chemosphere,
120, 555-562.
Abstract:
PFOA and PFOS are associated with reduced expression of the parathyroid hormone 2 receptor (PTH2R) gene in women.
Little is known about interactions between environmental and genetic risk factors for osteoarthritis (OA). Genetic factors include variation or mutation in genes involved in parathyroid hormone signalling. Exposure to the endocrine disrupting chemicals perfluoro-octanoic acid (PFOA) or perfluorooctane sulfonate (PFOS) have been suggested as potential environmental contributors, although evidence to support this association is conflicting. Here we test the hypothesis that PFOA and PFOS may alter the mRNA expression of genes in the parathyroid signalling cascade to provide evidence on possible pathways between these chemicals and OA. We measured the relationship between PFOA or PFOS serum levels and the in vivo expression of the Parathyroid hormone 1 and 2 genes (PTH, PTH2), Parathyroid hormone 1 and 2 receptor genes (PTH1R, PTH2R) and the parathyroid hormone-like (PTHLH) gene in peripheral blood from a cross-sectional population study designed to assess the potential health effects of these chemicals. We used multivariate linear regression models and found that PFOA or PFOS was inversely correlated with parathyroid hormone 2 receptor (PTH2R) expression (coefficients=-0.43 and -0.32, p=p=0.017 and 0.006 for PFOA and PFOS respectively) in 189 female subjects. The levels of PTH2 transcripts encoding the ligand of PTH2r, were also found to be lower in women with OA (median 2.08) compared with controls (median 3.41, p=0.046). As the parathyroid signalling cascade is a known candidate for osteoarthritis risk and our findings raise the possibility that exposure to these chemicals may contribute to the pathogenesis of OA in some individuals.
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Author URL.
Locke JM, Hysenaj G, Wood AR, Weedon MN, Harries LW (2015). Targeted allelic expression profiling in human islets identifies cis-regulatory effects for multiple variants identified by type 2 diabetes genome-wide association studies.
Diabetes,
64(4), 1484-1491.
Abstract:
Targeted allelic expression profiling in human islets identifies cis-regulatory effects for multiple variants identified by type 2 diabetes genome-wide association studies.
Genome-wide association studies (GWAS) have identified variation at >65 genomic loci associated with susceptibility to type 2 diabetes, but little progress has been made in elucidating the molecular mechanisms behind most of these associations. Using samples heterozygous for transcribed single nucleotide polymorphisms (SNPs), allelic expression profiling is a powerful technique for identifying cis-regulatory variants controlling gene expression. In this study, exonic SNPs, suitable for measuring mature mRNA levels and in high linkage disequilibrium with 65 lead type 2 diabetes GWAS SNPs, were identified and allelic expression determined by real-time PCR using RNA and DNA isolated from islets of 36 white nondiabetic donors. A significant allelic expression imbalance (AEI) was identified for 7/14 (50%) genes tested (ANPEP, CAMK2B, HMG20A, KCNJ11, NOTCH2, SLC30A8, and WFS1), with significant AEI confirmed for five of these genes using other linked exonic SNPs. Lastly, results of a targeted islet expression quantitative trait loci experiment support the AEI findings for ANPEP, further implicating ANPEP as the causative gene at its locus. The results of this study support the hypothesis that changes to cis-regulation of gene expression are involved in a large proportion of SNP associations with type 2 diabetes susceptibility.
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Munkley J, Livermore KE, McClurg UL, Kalna G, Knight B, McCullagh P, McGrath J, Crundwell M, Leung HY, Robson CN, et al (2015). The PI3K regulatory subunit gene PIK3R1 is under direct control of androgens and repressed in prostate cancer cells.
Oncoscience,
2(9), 755-764.
Abstract:
The PI3K regulatory subunit gene PIK3R1 is under direct control of androgens and repressed in prostate cancer cells.
Androgen receptor (AR) signalling and the PI3K pathway mediate survival signals in prostate cancer, and have been shown to regulate each other by reciprocal negative feedback, such that inhibition of one activates the other. Understanding the reciprocal regulation of these pathways is important for disease management as tumour cells can adapt and survive when either single pathway is inhibited pharmacologically. We recently carried out genome-wide exon-specific profiling of prostate cancer cells to identify novel androgen-regulated transcriptional events. Here we interrogated this dataset for novel androgen-regulated genes associated with the PI3K pathway. We find that the PI3K regulatory subunits PIK3R1 (p85α) and PIK3R3 (p55γ) are direct targets of the AR which are rapidly repressed by androgens in LNCaP cells. Further characterisation revealed that the PIK3CA p110α catalytic subunit is also indirectly regulated by androgens at the protein level. We show that PIK3R1 mRNA is significantly under-expressed in prostate cancer (PCa) tissue, and provide data to suggest a context-dependent regulatory mechanism whereby repression of the p85α protein by the AR results in destabilisation of the PI3K p110α catalytic subunit and downstream PI3K pathway inhibition that functionally affects the properties of prostate cancer cells.
Abstract.
Author URL.
Munkley J, Oltean S, Vodák D, Wilson BT, Livermore KE, Zhou Y, Star E, Floros VI, Johannessen B, Knight B, et al (2015). The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer.
Oncotarget,
6(33), 34358-34374.
Abstract:
The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer.
Patterns of glycosylation are important in cancer, but the molecular mechanisms that drive changes are often poorly understood. The androgen receptor drives prostate cancer (PCa) development and progression to lethal metastatic castration-resistant disease. Here we used RNA-Seq coupled with bioinformatic analyses of androgen-receptor (AR) binding sites and clinical PCa expression array data to identify ST6GalNAc1 as a direct and rapidly activated target gene of the AR in PCa cells. ST6GalNAc1 encodes a sialytransferase that catalyses formation of the cancer-associated sialyl-Tn antigen (sTn), which we find is also induced by androgen exposure. Androgens induce expression of a novel splice variant of the ST6GalNAc1 protein in PCa cells. This splice variant encodes a shorter protein isoform that is still fully functional as a sialyltransferase and able to induce expression of the sTn-antigen. Surprisingly, given its high expression in tumours, stable expression of ST6GalNAc1 in PCa cells reduced formation of stable tumours in mice, reduced cell adhesion and induced a switch towards a more mesenchymal-like cell phenotype in vitro. ST6GalNAc1 has a dynamic expression pattern in clinical datasets, beingsignificantly up-regulated in primary prostate carcinoma but relatively down-regulated in established metastatic tissue. ST6GalNAc1 is frequently upregulated concurrently with another important glycosylation enzyme GCNT1 previously associated with prostate cancer progression and implicated in Sialyl Lewis X antigen synthesis. Together our data establishes an androgen-dependent mechanism for sTn antigen expression in PCa, and are consistent with a general role for the androgen receptor in driving important coordinate changes to the glycoproteome during PCa progression.
Abstract.
Author URL.
Locke JM, Lango Allen H, Harries LW (2014). A rare SNP in pre-miR-34a is associated with increased levels of miR-34a in pancreatic beta cells.
Acta Diabetologica,
51(2), 325-329.
Abstract:
A rare SNP in pre-miR-34a is associated with increased levels of miR-34a in pancreatic beta cells
Changes in the levels of specific microRNAs (miRNAs) can reduce glucose-stimulated insulin secretion and increase beta-cell apoptosis, two causes of islet dysfunction and progression to type 2 diabetes. Studies have shown that single nucleotide polymorphisms (SNPs) within miRNA genes can affect their expression. We sought to determine whether miRNAs, with a known role in beta-cell function, possess SNPs within the pre-miRNA structure which can affect their expression. Using published literature and dbSNP, we aimed to identify miRNAs with a role in beta-cell function that also possess SNPs within the region encoding its pre-miRNA. Following transfection of plasmids, encoding the pre-miRNA and each allele of the SNP, miRNA expression was measured. Two rare SNPs located within the pre-miRNA structure of two miRNA genes important to beta-cell function (miR-34a and miR-96) were identified. Transfection of INS-1 and MIN6 cells with plasmids encoding pre-miR-34a and the minor allele of rs72631823 resulted in significantly (p < 0.05) higher miR-34a expression, compared to cells transfected with plasmids encoding the corresponding major allele. Similarly, higher levels were also observed upon transfection of HeLa cells. Transfection of MIN6 cells with plasmids encoding pre-miR-96 and each allele of rs41274239 resulted in no significant differences in miR-96 expression. A rare SNP in pre-miR-34a is associated with increased levels of mature miR-34a. Given that small changes in miR-34a levels have been shown to cause increased levels of beta-cell apoptosis this finding may be of interest to studies looking at determining the effect of rare variants on type 2 diabetes susceptibility. © 2013 the Author(s).
Abstract.
Locke JM, Lango Allen H, Harries LW (2014). A rare SNP in pre-miR-34a is associated with increased levels of miR-34a in pancreatic beta cells.
Acta Diabetol,
51(2), 325-329.
Abstract:
A rare SNP in pre-miR-34a is associated with increased levels of miR-34a in pancreatic beta cells.
Changes in the levels of specific microRNAs (miRNAs) can reduce glucose-stimulated insulin secretion and increase beta-cell apoptosis, two causes of islet dysfunction and progression to type 2 diabetes. Studies have shown that single nucleotide polymorphisms (SNPs) within miRNA genes can affect their expression. We sought to determine whether miRNAs, with a known role in beta-cell function, possess SNPs within the pre-miRNA structure which can affect their expression. Using published literature and dbSNP, we aimed to identify miRNAs with a role in beta-cell function that also possess SNPs within the region encoding its pre-miRNA. Following transfection of plasmids, encoding the pre-miRNA and each allele of the SNP, miRNA expression was measured. Two rare SNPs located within the pre-miRNA structure of two miRNA genes important to beta-cell function (miR-34a and miR-96) were identified. Transfection of INS-1 and MIN6 cells with plasmids encoding pre-miR-34a and the minor allele of rs72631823 resulted in significantly (p < 0.05) higher miR-34a expression, compared to cells transfected with plasmids encoding the corresponding major allele. Similarly, higher levels were also observed upon transfection of HeLa cells. Transfection of MIN6 cells with plasmids encoding pre-miR-96 and each allele of rs41274239 resulted in no significant differences in miR-96 expression. A rare SNP in pre-miR-34a is associated with increased levels of mature miR-34a. Given that small changes in miR-34a levels have been shown to cause increased levels of beta-cell apoptosis this finding may be of interest to studies looking at determining the effect of rare variants on type 2 diabetes susceptibility.
Abstract.
Author URL.
Cipelli R, Harries L, Okuda K, Yoshihara S, Melzer D, Galloway T (2014). Bisphenol a modulates the metabolic regulator oestrogen-related receptor-α in T-cells.
Reproduction,
147(4), 419-426.
Abstract:
Bisphenol a modulates the metabolic regulator oestrogen-related receptor-α in T-cells.
Bisphenol a (BPA) is a widely used plastics constituent that has been associated with endocrine, immune and metabolic effects. Evidence for how BPA exerts significant biological effects at chronic low levels of exposure has remained elusive. In adult men, exposure to BPA has been associated with higher expression of two nuclear receptors, oestrogen receptor-β (ERβ) and oestrogen-related-receptor-α (ERRα), in peripheral white blood cells in vivo. In this study, we explore the expression of ESR2 (ERβ) and ESRRA (ERRα) in human leukaemic T-cell lymphoblasts (Jurkat cells) exposed to BPA in vitro. We show that exposure to BPA led to enhanced expression of ESRRA within 6 h of exposure (mean±s.e.m.: 1.43±0.08-fold increase compared with the control, P
Abstract.
Author URL.
Hogg DR, Harries LW (2014). Human genetic variation and its effect on miRNA biogenesis, activity and function.
Biochem Soc Trans,
42(4), 1184-1189.
Abstract:
Human genetic variation and its effect on miRNA biogenesis, activity and function.
miRNAs are small non-coding regulators of gene expression that are estimated to regulate over 60% of all human genes. Each miRNA can target multiple mRNA targets and as such, miRNAs are responsible for some of the 'fine tuning' of gene expression and are implicated in regulation of all cellular processes. miRNAs bind to target genes by sequence complementarity, resulting in target degradation or translational blocking and usually a reduction in target gene expression. Like mRNA, miRNAs are transcribed from genomic DNA and are processed in several steps that are heavily reliant on correct secondary and tertiary structure. Secondary structure is determined by RNA sequence, which is in turn determined by the sequence of the genome. The human genome, however, like most eukaryotes is variable. Large numbers of SNPs (single nucleotide polymorphisms), small insertions and deletions (indels) and CNVs (copy number variants) have been described in our genome. Should this genetic variation occur in regions critical for the correct secondary structure or target binding, it may interfere with normal gene regulation and cause disease. In this review, we outline the consequences of genetic variation involving different aspects of miRNA biosynthesis, processing and regulation, with selected examples of incidences when this has potential to affect human disease.
Abstract.
Author URL.
Locke JM, Da Silva Xavier G, Dawe HR, Rutter GA, Harries LW (2014). Increased expression of miR-187 in human islets from individuals with type 2 diabetes is associated with reduced glucose-stimulated insulin secretion.
Diabetologia,
57(1), 122-128.
Abstract:
Increased expression of miR-187 in human islets from individuals with type 2 diabetes is associated with reduced glucose-stimulated insulin secretion
Aims/hypothesis: Type 2 diabetes is characterised by progressive beta cell dysfunction, with changes in gene expression playing a crucial role in its development. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and therefore alterations in miRNA levels may be involved in the deterioration of beta cell function. Methods: Global TaqMan arrays and individual TaqMan assays were used to measure islet miRNA expression in discovery (n = 20) and replication (n = 20) cohorts from individuals with and without type 2 diabetes. The role of specific dysregulated miRNAs in regulating insulin secretion, content and apoptosis was subsequently investigated in primary rat islets and INS-1 cells. Identification of miRNA targets was assessed using luciferase assays and by measuring mRNA levels. Results: in the discovery and replication cohorts miR-187 expression was found to be significantly increased in islets from individuals with type 2 diabetes compared with matched controls. An inverse correlation between miR-187 levels and glucose-stimulated insulin secretion (GSIS) was observed in islets from normoglycaemic donors. This correlation paralleled findings in primary rat islets and INS-1 cells where overexpression of miR-187 markedly decreased GSIS without affecting insulin content or apoptotic index. Finally, the gene encoding homeodomain-interacting protein kinase-3 (HIPK3), a known regulator of insulin secretion, was identified as a direct target of miR-187 and displayed reduced expression in islets from individuals with type 2 diabetes. Conclusions/interpretation: Our findings suggest a role for miR-187 in the blunting of insulin secretion, potentially involving regulation of HIPK3, which occurs during the pathogenesis of type 2 diabetes. © 2013 the Author(s).
Abstract.
Lunnon K, Smith R, Hannon E, De Jager PL, Srivastava G, Volta M, Troakes C, Al-Sarraj S, Burrage J, Macdonald R, et al (2014). Methylomic profiling implicates cortical deregulation of ANK1 in Alzheimer's disease.
Nat Neurosci,
17(9), 1164-1170.
Abstract:
Methylomic profiling implicates cortical deregulation of ANK1 in Alzheimer's disease.
Alzheimer's disease (AD) is a chronic neurodegenerative disorder that is characterized by progressive neuropathology and cognitive decline. We performed a cross-tissue analysis of methylomic variation in AD using samples from four independent human post-mortem brain cohorts. We identified a differentially methylated region in the ankyrin 1 (ANK1) gene that was associated with neuropathology in the entorhinal cortex, a primary site of AD manifestation. This region was confirmed as being substantially hypermethylated in two other cortical regions (superior temporal gyrus and prefrontal cortex), but not in the cerebellum, a region largely protected from neurodegeneration in AD, or whole blood obtained pre-mortem from the same individuals. Neuropathology-associated ANK1 hypermethylation was subsequently confirmed in cortical samples from three independent brain cohorts. This study represents, to the best of our knowledge, the first epigenome-wide association study of AD employing a sequential replication design across multiple tissues and highlights the power of this approach for identifying methylomic variation associated with complex disease.
Abstract.
Author URL.
Harries LW (2014). MicroRNAs as Mediators of the Ageing Process.
Genes (Basel),
5(3), 656-670.
Abstract:
MicroRNAs as Mediators of the Ageing Process.
Human ageing is a complex and integrated gradual deterioration of cellular processes. There are nine major hallmarks of ageing, that include changes in DNA repair and DNA damage response, telomere shortening, changes in control over the expression and regulation of genes brought about by epigenetic and mRNA processing changes, loss of protein homeostasis, altered nutrient signaling, mitochondrial dysfunction, stem cell exhaustion, premature cellular senescence and altered intracellular communication. Like practically all other cellular processes, genes associated in features of ageing are regulated by miRNAs. In this review, I will outline each of the features of ageing, together with examples of specific miRNAs that have been demonstrated to be involved in each one. This will demonstrate the interconnected nature of the regulation of transcripts involved in human ageing, and the role of miRNAs in this process. Definition of the factors involved in degeneration of organismal, tissue and cellular homeostasis may provide biomarkers for healthy ageing and increase understanding of the processes that underpin the ageing process itself.
Abstract.
Author URL.
Holly AC, Pilling LC, Hernandez D, Lee BP, Singleton A, Ferrucci L, Melzer D, Harries LW (2014). Splicing factor 3B1 hypomethylation is associated with altered SF3B1 transcript expression in older humans.
Mech Ageing Dev,
135, 50-56.
Abstract:
Splicing factor 3B1 hypomethylation is associated with altered SF3B1 transcript expression in older humans.
Ageing in man is associated with changes to the splicing factor pool. A proportion of splicing factors are regulated during ageing by mechanisms involving the Ataxia Telangiectasia Mutated (ATM) gene, but the factors that determine the remaining proportion have yet to be identified. DNA methylation is known to be an important regulatory mechanism of gene expression. We assessed age-associated methylation and expression levels for 27 splicing factor genes, in peripheral blood samples from the InCHIANTI study. Examination of splicing patterns at specific loci was examined in a second cohort, the Exeter 10000 study. 27/502 methylation probes in 17 different genes were associated with age. Most changes were not associated with transcript expression levels or splicing patterns, but hypomethylation of the SF3B1 promoter region was found to mediate 53% of the relationship between age and transcript expression at this locus (p=0.02). DNA methylation does not appear to play a major role in regulation of the splicing factors, but changes in SF3B1 expression may be attributable to promoter hypomethylation at this locus. SF3B1 encodes a critical component of the U2 snRNP; altered expression of this gene may therefore contribute to the loss of regulated mRNA splicing that occurs with age.
Abstract.
Author URL.
Harries LW, McCulloch LJ, Holley JE, Rawling TJ, Welters HJ, Kos K (2013). A role for SPARC in the moderation of human insulin secretion.
PLoS One,
8(6).
Abstract:
A role for SPARC in the moderation of human insulin secretion.
AIMS/HYPOTHESIS: We have previously shown the implication of the multifunctional protein SPARC (Secreted protein acidic and rich in cysteine)/osteonectin in insulin resistance but potential effects on beta-cell function have not been assessed. We therefore aimed to characterise the effect of SPARC on beta-cell function and features of diabetes. METHODS: We measured SPARC expression by qRT-PCR in human primary pancreatic islets, adipose tissue, liver and muscle. We then examined the relation of SPARC with glucose stimulated insulin secretion (GSIS) in primary human islets and the effect of SPARC overexpression on GSIS in beta cell lines. RESULTS: SPARC was expressed at measurable levels in human islets, adipose tissue, liver and skeletal muscle, and demonstrated reduced expression in primary islets from subjects with diabetes compared with controls (p< = 0.05). SPARC levels were positively correlated with GSIS in islets from control donors (p< = 0.01). Overexpression of SPARC in cultured beta-cells resulted in a 2.4-fold increase in insulin secretion in high glucose conditions (p< = 0.01). CONCLUSIONS: Our data suggest that levels of SPARC are reduced in islets from donors with diabetes and that it has a role in insulin secretion, an effect which appears independent of SPARC's modulation of obesity-induced insulin resistance in adipose tissue.
Abstract.
Author URL.
Harries LW, Pilling LC, Lampron A, Rivest S, Melzer D (2013). Alzheimer’s pathology: should peripheral monocytes and CCR2 take center stage?. Neurodegenerative Disease Management, 3(1), 9-12.
Fletcher T, Galloway TS, Melzer D, Holcroft P, Cipelli R, Pilling LC, Mondal D, Luster M, Harries LW (2013). Associations between PFOA, PFOS and changes in the expression of genes involved in cholesterol metabolism in humans.
Environ Int,
57-58, 2-10.
Abstract:
Associations between PFOA, PFOS and changes in the expression of genes involved in cholesterol metabolism in humans.
Perfluorooctanoic acid (PFOA, 'C8') and perfluoroctane sulphonate (PFOS) are environmentally stable compounds with industrial and consumer uses and long half-lives in humans. Concern has been raised over chronic exposure effects to human health, especially in relation to cholesterol metabolism. Here, we explore the association between exposure to PFOA and PFOS and the in vivo expression of genes involved in cholesterol metabolism. We studied 290 individuals exposed to background levels of PFOS and elevated concentrations of PFOA through drinking water. Using adjusted linear regression models, we found inverse associations between serum PFOA levels and the whole blood expression level of genes involved in cholesterol transport (NR1H2, NPC1 and ABCG1; p=0.002, 0.026 and 0.014 respectively). A positive association was seen between PFOS and a transcript involved in cholesterol mobilisation (NCEH1; p=0.018), and a negative relationship with a transcript involved in cholesterol transport (NR1H3; p=0.044). When sexes were analysed separately, reductions in the levels of mRNAs involved in cholesterol transport were seen with PFOA in men (NPC1, ABCG1, and PPARA; p=0.025, 0.024 and 0.012 respectively) and in women (NR1H2 expression; p=0.019), whereas an increase in the levels of a cholesterol mobilisation transcript (NCEH1; p=0.036) was noted in women alone. PFOS was positively associated with expression of genes involved in both cholesterol mobilisation and transport in women (NCEH1 and PPARA; p=0.003 and 0.039 respectively), but no effects were evident in men. This is the first report of associations between the in vivo expression of genes involved in cholesterol metabolism and exposure to PFOA or PFOS, suggested that exposure to these compounds may promote a hypercholesterolaemic environment, with wider implications for human disease.
Abstract.
Author URL.
Holly AC, Melzer D, Pilling LC, Fellows AC, Tanaka T, Ferrucci L, Harries LW (2013). Changes in splicing factor expression are associated with advancing age in man.
Mech Ageing Dev,
134(9), 356-366.
Abstract:
Changes in splicing factor expression are associated with advancing age in man.
Human ageing is associated with decreased cellular plasticity and adaptability. Changes in alternative splicing with advancing age have been reported in man, which may arise from age-related alterations in splicing factor expression. We determined whether the mRNA expression of key splicing factors differed with age, by microarray analysis in blood from two human populations and by qRT-PCR in senescent primary fibroblasts and endothelial cells. Potential regulators of splicing factor expression were investigated by siRNA analysis. Approximately one third of splicing factors demonstrated age-related transcript expression changes in two human populations. Ataxia Telangiectasia Mutated (ATM) transcript expression correlated with splicing factor expression in human microarray data. Senescent primary fibroblasts and endothelial cells also demonstrated alterations in splicing factor expression, and changes in alternative splicing. Targeted knockdown of the ATM gene in primary fibroblasts resulted in up-regulation of some age-responsive splicing factor transcripts. We conclude that isoform ratios and splicing factor expression alters with age in vivo and in vitro, and that ATM may have an inhibitory role on the expression of some splicing factors. These findings suggest for the first time that ATM, a core element in the DNA damage response, is a key regulator of the splicing machinery in man.
Abstract.
Author URL.
Kendall AC, Whatmore JL, Harries LW, Winyard PG, Eggleton P, Smerdon GR (2013). Different oxygen treatment pressures alter inflammatory gene expression in human endothelial cells.
Undersea and Hyperbaric Medicine,
40(2), 115-123.
Abstract:
Different oxygen treatment pressures alter inflammatory gene expression in human endothelial cells.
Hyperbaric oxygen has proven to be a useful treatment for chronic wounds. However, therapeutic conditions vary between treatment centers, and we wished to investigate the effects of different treatment pressures on cells under inflammatory conditions. Endothelial cells were exposed to a chronic wound model comprising hypoxia (2% O2 at 1 atmosphere absolute (atm abs); PO2 ~ 2 kPa) in the presence of 0.5 μg/ml lipopolysaccharide and 1 ng/ml TNF-alpha for 24 hours, then treated with normobaric oxygen (NBO2; 95%O2/5%CO2 at 1.0 atm abs; PO2 ~ 96.3 kPa), hyperbaric oxygen (HBO2) at 1.5 atm abs (1.5HBO2; 96.7%O2/3.3%CO2 at 1.5 atm abs; PO2 ~ 147 kPa) and HBO2 at 2.4 atm abs (2.4 HBO2; 97.9% O2/2.1% CO2 at 2.4 atms; PO2 ~238 kPa). The mRNA expression of 92 genes was then analyzed, and we identified changes in genes involved in adhesion molecule expression, angiogenesis and tissue remodeling, intracellular signaling, and cellular oxygen responses and redox signaling. We noted differences in expression between different treatment pressures, highlighting the need for further research into the use of different therapeutic protocols in the treatment of inflammatory conditions such as chronic wounds.
Abstract.
aer M, Jonathan L, Anna M, Lorna WH (2013). Expression profiling of putative type 2 diabetes susceptibility genes in human islets and in rat beta cell lines. Journal of Diabetes Mellitus, 03(01), 27-32.
Morrison F, Locke J, Arif M, Murray A, Brown JE, Harries LW (2013). Expression profiling of type 2 diabetes susceptibility genes in the pancreatic islets, adipose tissue and liver of obese mice.
Exp Clin Endocrinol Diabetes,
121(7), 413-419.
Abstract:
Expression profiling of type 2 diabetes susceptibility genes in the pancreatic islets, adipose tissue and liver of obese mice.
Type 2 diabetes (T2D) is characterized by impaired beta cell function and insulin resistance. T2D susceptibility genes identified by Genome-wide association studies (GWAS) are likely to have roles in both impaired insulin secretion from the beta cell as well as insulin resistance. The aim of this study was to use gene expression profiling to assess the effect of the diabetic milieu on the expression of genes involved in both insulin secretion and insulin resistance.We measured the expression of 43 T2D susceptibility genes in the islets, adipose and liver of leptin-deficient Ob/Ob mice compared with Ob/+ littermates. The same panel of genes were also profiled in cultured rodent adipocytes, hepatocytes and beta cells in response to high glucose conditions, to distinguish expression effects due to elevated glycemia from those on the causal pathway to diabetes or induced by other factors in the diabetic microenviroment.We found widespread deregulation of these genes in tissues from Ob/Ob mice, with differential regulation of 23 genes in adipose, 18 genes in liver and one gene (Tcf7l2) in islets of diabetic animals (Ob/Ob) compared to control (Ob/+) animals. However, these expression changes were in most cases not noted in glucose-treated adipocyte, hepatocyte or beta cell lines, indicating that they may not be an effect of hyperglycemia alone.This study indicates that expression changes are apparent with diabetes in both the insulin producing beta cells, but also in peripheral tissues involved in insulin resistance. This suggests that incidence or progression of diabetic phenotypes in a mouse model of diabetes is driven by both secretory and peripheral defects.
Abstract.
Author URL.
Melzer D, Pilling LC, Fellows AD, Holly AC, Harries LW, Ferrucci L (2013). Gene Expression Biomarkers and Longevity. Annual Review of Gerontology and Geriatrics, 33(1), 233-258.
Fellows AD, Holly AC, Pilling LC, Melzer D, Harries LW (2013). Microarray-based whole transcriptome expression profiling as a tool to understand human ageing.
, 209-220.
Abstract:
Microarray-based whole transcriptome expression profiling as a tool to understand human ageing
The human transcriptome is exceptionally complex. Over 95% of human DNA is transcribed into RNA; the transcriptome containing not only the messenger RNAs (mRNAs) but also a range of large and small non-coding regulatory RNAs and components of the translational machinery. The transcriptome is therefore at the heart of the gene-environment interaction, and this complexity, together with the diversity brought about by alternative mRNA processing gives the human transcriptome unprecedented plasticity and adaptability. Many human diseases are associated with effects at the level of the mRNA transcript, either as part of the causal pathway, or as a response to disease. Analysis of the transcriptome thus has enormous potential for the development of potential biomarkers of disease, and to increase our understanding of the disease processes themselves. In the last few decades, technological advances have made profiling of the entire transcriptome an affordable and manageable prospect. These advances have made high-throughput analysis of the output of the human genome accessible, allowing transcriptional alterations with disease phenotypes to be determined in large human populations. In this review, we will describe some of the recent advances in our understanding of fundamental human processes such as human ageing, and common chronic disorders that have been achieved by whole genome transcriptional profiling. We will outline some of the methodologies that have been employed to answer these important questions, as well as present some examples from our own laboratory where we have used these approaches to investigate mechanisms ofageing and in specific age-related disorders such as frailty and cognitive decline. Gene expression profiling has an important role to play in inferring mechanisms underlying certain diseases, and technological advances will render it an increasingly powerful tool in the future. © 2013 by Nova Science Publishers, Inc. All rights reserved.
Abstract.
Morrison FS, Locke JM, Wood AR, Tuke M, Pasko D, Murray A, Frayling T, Harries LW (2013). The splice site variant rs11078928 may be associated with a genotype-dependent alteration in expression of GSDMB transcripts.
BMC Genomics,
14Abstract:
The splice site variant rs11078928 may be associated with a genotype-dependent alteration in expression of GSDMB transcripts.
BACKGROUND: Many genetic variants have been associated with susceptibility to complex traits by genome wide association studies (GWAS), but for most, causal genes and mechanisms of action have yet to be elucidated. Using bioinformatics, we identified index and proxy variants associated with autoimmune disease susceptibility, with the potential to affect splicing of candidate genes. PCR and sequence analysis of whole blood RNA samples from population controls was then carried out for the 8 most promising variants to determine the effect of genetic variation on splicing of target genes. RESULTS: We identified 31 splice site SNPs with the potential to affect splicing, and prioritised 8 to determine the effect of genotype on candidate gene splicing. We identified that variants rs11078928 and rs2014886 were associated with altered splicing of the GSDMB and TSFM genes respectively. rs11078928, present in the asthma and autoimmune disease susceptibility locus on chromosome 17q12-21, was associated with the production of a novel Δ exon5-8 transcript of the GSDMB gene, and a separate decrease in the percentage of transcripts with inclusion of exon 6, whereas the multiple sclerosis susceptibility variant rs2014886, was associated with an alternative TFSM transcript encompassing a short cryptic exon within intron 2. CONCLUSIONS: Our findings demonstrate the utility of a bioinformatic approach in identification and prioritisation of genetic variants effecting splicing of their host genes, and suggest that rs11078928 and rs2014886 may affect the splicing of the GSDMB and TSFM genes respectively.
Abstract.
Author URL.
Holly AC, Melzer D, Pilling LC, Henley W, Hernandez DG, Singleton AB, Bandinelli S, Guralnik JM, Ferrucci L, Harries LW, et al (2013). Towards a gene expression biomarker set for human biological age.
Aging Cell,
12(2), 324-326.
Abstract:
Towards a gene expression biomarker set for human biological age
We have previously described a statistical model capable of distinguishing young (age
Abstract.
Harries LW, Fellows AD, Pilling LC, Hernandez D, Singleton A, Bandinelli S, Guralnik J, Powell J, Ferrucci L, Melzer D, et al (2012). Advancing age is associated with gene expression changes resembling mTOR inhibition: Evidence from two human populations.
Mechanisms of Ageing and Development,
133(8), 556-562.
Abstract:
Advancing age is associated with gene expression changes resembling mTOR inhibition: Evidence from two human populations
Interventions which inhibit TOR activity (including rapamycin and caloric restriction) lead to downstream gene expression changes and increased lifespan in laboratory models. However, the role of mTOR signaling in human aging is unclear.We tested the expression of mTOR-related transcripts in two independent study cohorts; the InCHIANTI population study of aging and the San Antonio Family Heart Study (SAFHS). Expression of 27/56 (InCHIANTI) and 19/44 (SAFHS) genes were associated with age after correction for multiple testing. 8 genes were robustly associated with age in both cohorts. Genes involved in insulin signaling (PTEN, PI3K, PDK1), ribosomal biogenesis (S6K), lipid metabolism (SREBF1), cellular apoptosis (SGK1), angiogenesis (VEGFB), insulin production and sensitivity (FOXO), cellular stress response (HIF1A) and cytoskeletal remodeling (PKC) were inversely correlated with age, whereas genes relating to inhibition of ribosomal components (4EBP1) and inflammatory mediators (STAT3) were positively associated with age in one or both datasets.We conclude that the expression of mTOR-related transcripts is associated with advancing age in humans. Changes seen are broadly similar to mTOR inhibition interventions associated with increased lifespan in animals. Work is needed to establish whether these changes are predictive of human longevity and whether further mTOR inhibition would be beneficial in older people. © 2012 Elsevier Ireland Ltd.
Abstract.
Harries LW, Fellows AD, Pilling LC, Hernandez D, Singleton A, Bandinelli S, Guralnik J, Powell J, Ferrucci L, Melzer D, et al (2012). Advancing age is associated with gene expression changes resembling mTOR inhibition: evidence from two human populations.
Mech Ageing Dev,
133(8), 556-562.
Abstract:
Advancing age is associated with gene expression changes resembling mTOR inhibition: evidence from two human populations.
Interventions which inhibit TOR activity (including rapamycin and caloric restriction) lead to downstream gene expression changes and increased lifespan in laboratory models. However, the role of mTOR signaling in human aging is unclear. We tested the expression of mTOR-related transcripts in two independent study cohorts; the InCHIANTI population study of aging and the San Antonio Family Heart Study (SAFHS). Expression of 27/56 (InCHIANTI) and 19/44 (SAFHS) genes were associated with age after correction for multiple testing. 8 genes were robustly associated with age in both cohorts. Genes involved in insulin signaling (PTEN, PI3K, PDK1), ribosomal biogenesis (S6K), lipid metabolism (SREBF1), cellular apoptosis (SGK1), angiogenesis (VEGFB), insulin production and sensitivity (FOXO), cellular stress response (HIF1A) and cytoskeletal remodeling (PKC) were inversely correlated with age, whereas genes relating to inhibition of ribosomal components (4EBP1) and inflammatory mediators (STAT3) were positively associated with age in one or both datasets. We conclude that the expression of mTOR-related transcripts is associated with advancing age in humans. Changes seen are broadly similar to mTOR inhibition interventions associated with increased lifespan in animals. Work is needed to establish whether these changes are predictive of human longevity and whether further mTOR inhibition would be beneficial in older people.
Abstract.
Author URL.
Fellows AD, Holly AC, Pilling LC, Melzer D, Harries LW (2012). Age related changes in mTOR-related gene expression in two primary human cell lines. Healthy Aging Research
Harries LW, Pilling LC, Hernandez LDG, Bradley-Smith R, Henley W, Singleton AB, Guralnik JM, Bandinelli S, Ferrucci L, Melzer D, et al (2012). CCAAT-enhancer-binding protein-beta expression in vivo is associated with muscle strength.
Aging Cell,
11(2), 262-268.
Abstract:
CCAAT-enhancer-binding protein-beta expression in vivo is associated with muscle strength.
Declining muscle strength is a core feature of aging. Several mechanisms have been postulated, including CCAAT/enhancer-binding protein-beta (C/EBP-β)-triggered macrophage-mediated muscle fiber regeneration after micro-injury, evidenced in a mouse model. We aimed to identify in vivo circulating leukocyte gene expression changes associated with muscle strength in the human adult population. We undertook a genome-wide expression microarray screen, using peripheral blood RNA samples from InCHIANTI study participants (aged 30 and 104). Logged expression intensities were regressed with muscle strength using models adjusted for multiple confounders. Key results were validated by real-time PCR. The Short Physical Performance Battery (SPPB) score tested walk speed, chair stand, and balance. CEBPB expression levels were associated with muscle strength (β coefficient = 0.20560, P = 1.03*10(-6), false discovery rate q = 0.014). The estimated handgrip strength in 70-year-old men in the lowest CEBPB expression tertile was 35.2 kg compared with 41.2 kg in the top tertile. CEBPB expression was also associated with hip, knee, ankle, and shoulder strength and the SPPB score (P = 0.018). Near-study-wide associations were also noted for TGF-β3 (P = 3.4*10(-5) , q = 0.12) and CEBPD expression (P = 9.7*10(-5) , q = 0.18) but not for CEBPA expression. We report here a novel finding that raised CEBPB expression in circulating leukocyte-derived RNA samples in vivo is associated with greater muscle strength and better physical performance in humans. This association may be consistent with mouse model evidence of CEBPB-triggered muscle repair: if this mechanism is confirmed, it may provide a target for intervention to protect and enhance aging muscle.
Abstract.
Author URL.
Kendall AC, Whatmore JL, Harries LW, Winyard PG, Ferguson D, Eggleton P (2012). Different oxygen treatment pressures alter oxygen responses and redox signalling gene expression in human endothelial cells.
FREE RADICAL BIOLOGY AND MEDICINE,
53, S166-S166.
Author URL.
Melzer D, Harries L, Llewellyn DJ, Bandinelli S, Guralnik JM, Ferrucci L (2012). GENE EXPRESSION ASSOCIATIONS WITH MMSE SCORES: PHAGOCYTOSIS OF CNS BETA AMYLIOD?.
GERONTOLOGIST,
52, 453-453.
Author URL.
Pilling LC, Harries LW, Powell J, Llewellyn DJ, Ferrucci L, Melzer D (2012). Genomics and successful aging: grounds for renewed optimism?.
J Gerontol a Biol Sci Med Sci,
67(5), 511-519.
Abstract:
Genomics and successful aging: grounds for renewed optimism?
BACKGROUND: Successful aging depends in part on delaying age-related disease onsets until later in life. Conditions including coronary artery disease, Alzheimer's disease, prostate cancer, and type 2 diabetes are moderately heritable. Genome-wide association studies have identified many risk associated single-nucleotide polymorphisms for these conditions, but much heritability remains unaccounted for. Nevertheless, a great deal is being learned. METHODS: Here, we review age-related disease associated single-nucleotide polymorphisms and identify key underlying pathways including lipid handling, specific immune processes, early tissue development, and cell cycle control. RESULTS: Most age-related disease associated single-nucleotide polymorphisms do not affect coding regions of genes or protein makeup but instead influence regulation of gene expression. Recent evidence indicates that evolution of gene regulatory sites is fundamental to interspecies differences. Animal models relevant to human aging may therefore need to focus more on gene regulation rather than testing major disruptions to fundamental pathway genes. Recent larger scale human studies of in vivo genome-wide expression (notably from the InCHIANTI aging study) have identified changes in splicing, the "fine tuning" of protein sequences, as a potentially important factor in decline of cellular function with age. Studies of expression with muscle strength and cognition have shown striking concordance with certain mice models of muscle repair and beta-amyloid phagocytosis respectively. CONCLUSIONS: the emerging clearer picture of the genetic architecture of age-related diseases in humans is providing new insights into the underlying pathophysiological pathways involved. Translation of genomics into new approaches to prevention, tests and treatments to extend successful aging is therefore likely in the coming decades.
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Author URL.
Harries LW, Bradley-Smith RM, Llewellyn DJ, Pilling LC, Fellows A, Henley W, Hernandez D, Guralnik JM, Bandinelli S, Singleton A, et al (2012). Leukocyte CCR2 expression is associated with mini-mental state examination score in older adults.
Rejuvenation Res,
15(4), 395-404.
Abstract:
Leukocyte CCR2 expression is associated with mini-mental state examination score in older adults.
INTRODUCTION: Circulating inflammatory markers may play an important role in cognitive impairment at older ages. Mice deficient for the chemokine (C-C motif) receptor 2 (CCR2) develop an accelerated Alzheimer-like pathology. CCR2 is also important in neurogenesis. To identify human gene transcripts most closely associated with Mini-Mental State Examination (MMSE) scores, we undertook a genome-wide and inflammation specific transcriptome screen in circulating leukocytes from a population-based sample. METHODS: We measured in vivo transcript levels by microarray analysis in 691 subjects (mean age 72.6 years) in the InCHIANTI study (Invecchiare in Chianti, aging in the Chianti area). We assessed expression associations with MMSE performance at RNA collection and prior 9-year change in MMSE score in linear regression models. RESULTS: in genome-wide analysis, raised CCR2 expression was cross-sectionally the most strongly associated transcript with lower MMSE score (beta=-0.16, p=5.1×10(-6), false discovery rate (FDR; q=0.077). Amongst inflammatory transcripts, only CCR2 expression was associated with both MMSE score and accelerated decline in score over the preceding 9 years (beta=-0.16, p=5.1×10(-6), q=0.003; and beta=-0.13, p=5.5×10(-5), q=0.03, respectively). CCR2 expression was also positively associated with apolipoprotein E (ApoE) e4 Alzheimer disease risk haplotype. CONCLUSIONS: We show for the first time that CCR2 expression is associated with lower MMSE scores in an older human population. Laboratory models of Ccr2-mediated β-amyloid removal and regulation of neurogenesis affecting cognitive function may be applicable in humans. CCR2-mediated pathways may provide a possible focus for intervention to potentiate protective reactions to Alzheimer pathology in older people, including for people with an adverse ApoE haplotype.
Abstract.
Author URL.
Harries LW (2012). Long non-coding RNAs and human disease.
Biochem Soc Trans,
40(4), 902-906.
Abstract:
Long non-coding RNAs and human disease.
The central dogma of molecular biology states that DNA is transcribed into RNA, which in turn is translated into proteins. We now know, however, that as much as 50% of the transcriptome has no protein-coding potential, but rather represents an important class of regulatory molecules responsible for the fine-tuning of gene expression. Although the role of small regulatory RNAs [microRNAs and siRNAs (small interfering RNA)] is well defined, another much less characterized category of non-coding transcripts exists, namely lncRNAs (long non-coding RNAs). Pervasively expressed by eukaryotic genomes, lncRNAs can be kilobases long and regulate their targets by influencing the epigenetic control, chromatin status, mRNA processing or translation capacity of their targets. In the present review, I outline the potential mechanisms of action of lncRNAs, the cellular processes that have been associated with them, and also explore some of the emerging evidence for their involvement in common human disease.
Abstract.
Author URL.
Locke JM, Harries LW (2012). MicroRNA expression profiling of human islets from individuals with and without type 2 diabetes: promises and pitfalls.
Biochem Soc Trans,
40(4), 800-803.
Abstract:
MicroRNA expression profiling of human islets from individuals with and without type 2 diabetes: promises and pitfalls.
Recent studies in mouse, involving the β-cell-specific deletion of Dicer1, have highlighted the crucial role of miRNAs (microRNAs) in regulating insulin secretion and consequently Type 2 diabetes. Identifying the individual miRNAs involved in human islet dysfunction may be of diagnostic and therapeutic interest. miRNA expression profiling of human islets isolated from donors with and without Type 2 diabetes may represent one of the first steps in the discovery of these specific miRNAs. The present review discusses some of the potential pitfalls and promises of such an approach.
Abstract.
Author URL.
Morrison F, Johnstone K, Murray A, Locke J, Harries LW (2012). Oxidative metabolism genes are not responsive to oxidative stress in rodent beta cell lines.
Experimental Diabetes Research,
2012Abstract:
Oxidative metabolism genes are not responsive to oxidative stress in rodent beta cell lines
Altered expression of oxidative metabolism genes has been described in the skeletal muscle of individuals with type 2 diabetes. Pancreatic beta cells contain low levels of antioxidant enzymes and are particularly susceptible to oxidative stress. In this study, we explored the effect of hyperglycemia-induced oxidative stress on a panel of oxidative metabolism genes in a rodent beta cell line. We exposed INS-1 rodent beta cells to low (5.6mmol/L), ambient (11mmol/L), and high (28mmol/L) glucose conditions for 48 hours. Increases in oxidative stress were measured using the fluorescent probe dihydrorhodamine 123. We then measured the expression levels of a panel of 90 oxidative metabolism genes by real-time PCR. Elevated reactive oxygen species (ROS) production was evident in INS-1 cells after 48 hours (P
Abstract.
Garin I, de Nanclares GP, Gastaldo E, Harries LW, Rubio-Cabezas O, Castaño L (2012). Permanent neonatal diabetes caused by creation of an ectopic splice site within the INS gene.
PLoS ONE,
7(1).
Abstract:
Permanent neonatal diabetes caused by creation of an ectopic splice site within the INS gene
Background: the aim of this study was to characterize the genetic etiology in a patient who presented with permanent neonatal diabetes at 2 months of age. Methodology/Principal Findings: Regulatory elements and coding exons 2 and 3 of the INS gene were amplified and sequenced from genomic and complementary DNA samples. A novel heterozygous INS mutation within the terminal intron of the gene was identified in the proband and her affected father. This mutation introduces an ectopic splice site leading to the insertion of 29 nucleotides from the intronic sequence into the mature mRNA, which results in a longer and abnormal transcript. Conclusions/Significance: This study highlights the importance of routinely sequencing the exon-intron boundaries and the need to carry out additional studies to confirm the pathogenicity of any identified intronic genetic variants. © 2012 Garin et al.
Abstract.
Harries LW, Watura R (2012). Septic arthritis of unilateral lumbar facet joint with contiguous abscess, without prior intervention. Case Reports, 2012(apr02 1), bcr0920114849-bcr0920114849.
Wood AR, Hernandez DG, Nalls MA, Yaghootkar H, Gibbs JR, Harries LW, Chong S, Moore M, Weedon MN, Guralnik JM, et al (2011). Allelic heterogeneity and more detailed analyses of known loci explain additional phenotypic variation and reveal complex patterns of association.
Hum Mol Genet,
20(20), 4082-4092.
Abstract:
Allelic heterogeneity and more detailed analyses of known loci explain additional phenotypic variation and reveal complex patterns of association.
The identification of multiple signals at individual loci could explain additional phenotypic variance ('missing heritability') of common traits, and help identify causal genes. We examined gene expression levels as a model trait because of the large number of strong genetic effects acting in cis. Using expression profiles from 613 individuals, we performed genome-wide single nucleotide polymorphism (SNP) analyses to identify cis-expression quantitative trait loci (eQTLs), and conditional analysis to identify second signals. We examined patterns of association when accounting for multiple SNPs at a locus and when including additional SNPs from the 1000 Genomes Project. We identified 1298 cis-eQTLs at an approximate false discovery rate 0.01, of which 118 (9%) showed evidence of a second independent signal. For this subset of 118 traits, accounting for two signals resulted in an average 31% increase in phenotypic variance explained (Wilcoxon P< 0.0001). The association of SNPs with cis gene expression could increase, stay similar or decrease in significance when accounting for linkage disequilibrium with second signals at the same locus. Pairs of SNPs increasing in significance tended to have gene expression increasing alleles on opposite haplotypes, whereas pairs of SNPs decreasing in significance tended to have gene expression increasing alleles on the same haplotypes. Adding data from the 1000 Genomes Project showed that apparently independent signals could be potentially explained by a single association signal. Our results show that accounting for multiple variants at a locus will increase the variance explained in a substantial fraction of loci, but that allelic heterogeneity will be difficult to define without resequencing loci and functional work.
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Author URL.
Melzer D, Harries L, Cipelli R, Henley W, Money C, McCormack P, Young A, Guralnik J, Ferrucci L, Bandinelli S, et al (2011). Bisphenol a exposure is associated with in vivo estrogenic gene expression in adults.
Environ Health Perspect,
119(12), 1788-1793.
Abstract:
Bisphenol a exposure is associated with in vivo estrogenic gene expression in adults.
BACKGROUND: Bisphenol a (BPA) is a synthetic estrogen commonly used in polycarbonate plastic and resin-lined food and beverage containers. Exposure of animal and cell models to doses of BPA below the recommended tolerable daily intake (TDI) of 50 μg/kg/day have been shown to alter specific estrogen-responsive gene expression, but this has not previously been shown in humans. OBJECTIVE: We investigated associations between BPA exposure and in vivo estrogenic gene expression in humans. METHODS: We studied 96 adult men from the InCHIANTI population study and examined in vivo expression of six estrogen receptor, estrogen-related receptor, and androgen receptor genes in peripheral blood leukocytes. RESULTS: the geometric mean urinary BPA concentration was 3.65 ng/mL [95% confidence interval (CI): 3.13, 4.28], giving an estimated mean excretion of 5.84 μg/day (95% CI: 5.00, 6.85), significantly below the current TDI. In age-adjusted models, there were positive associations between higher BPA concentrations and higher ESR2 [estrogen receptor 2 (ER beta)] expression (unstandardized linear regression coefficient = 0.1804; 95% CI: 0.0388, 0.3221; p = 0.013) and ESRRA (estrogen related receptor alpha) expression (coefficient = 0.1718; 95% CI: 0.0213, 0.3223; p = 0.026): These associations were little changed after adjusting for potential confounders, including obesity, serum lipid concentrations, and white cell subtype percentages. Upper-tertile BPA excretors (urinary BPA > 4.6 ng/mL) had 65% higher mean ESR2 expression than did lower-tertile BPA excretors (0-2.4 ng/mL). CONCLUSIONS: Because activation of nuclear-receptor-mediated pathways by BPA is consistently found in laboratory studies, such activation in humans provides evidence that BPA is likely to function as a xenoestrogen in this sample of adults.
Abstract.
Author URL.
Harries LW, Melzer D, Cipelli R, Henley W, Money C, McCormack P, Young A, Guralnik J, Ferrucci L, Bandinelli S, et al (2011). Bisphenol a exposure is associated with in vivo estrogenic gene expression in adults.
Environ Health Perspect,
12(119), 1788-1793.
Abstract:
Bisphenol a exposure is associated with in vivo estrogenic gene expression in adults
Background: Bisphenol a (BPA) is a synthetic estrogen commonly used in polycarbonate plastic and resin-lined food and beverage containers. Exposure of animal and cell models to doses of BPA below the recommended tolerable daily intake (TDI) of 50 μg/kg/day have been shown to alter specific estrogen-responsive gene expression, but this has not previously been shown in humans.Objective: We investigated associations between BPA exposure and in vivo estrogenic gene expression in humans.Methods: We studied 96 adult men from the InCHIANTI population study and examined in vivo expression of six estrogen receptor, estrogen-related receptor, and androgen receptor genes in peripheral blood leukocytes.Results: the geometric mean urinary BPA concentration was 3.65 ng/mL [95% confidence interval (CI): 3.13, 4.28], giving an estimated mean excretion of 5.84 μg/day (95% CI: 5.00, 6.85), significantly below the current TDI. In age-adjusted models, there were positive associations between higher BPA concentrations and higher ESR2 [estrogen receptor 2 (ER beta)] expression (unstandardized linear regression coefficient = 0.1804; 95% CI: 0.0388, 0.3221; p = 0.013) and ESRRA (estrogen related receptor alpha) expression (coefficient = 0.1718; 95% CI: 0.0213, 0.3223; p = 0.026): These associations were little changed after adjusting for potential confounders, including obesity, serum lipid concentrations, and white cell subtype percentages. Upper-tertile BPA excretors (urinary BPA > 4.6 ng/mL) had 65% higher mean ESR2 expression than did lower-tertile BPA excretors (0-2.4 ng/mL).Conclusions: Because activation of nuclear-receptor-mediated pathways by BPA is consistently found in laboratory studies, such activation in humans provides evidence that BPA is likely to function as a xenoestrogen in this sample of adults.
Abstract.
Melzer D, Harries L, Pilling L, Bandinelli S, Guralnik JM, Singleton A, Hernandez D, Ferrucci L (2011). CONFIRMATION OF MOUSE MODEL OF SARCOPENIA BY GENOME WIDE EXPRESSION STUDY IN HUMANS.
GERONTOLOGIST,
51, 506-507.
Author URL.
Kendall AC, Whatmore JL, Harries LW, Winyard PG, Smerdon GR, Eggleton P (2011). Changes in inflammatory gene expression induced by hyperbaric oxygen treatment in human endothelial cells under chronic wound conditions.
Experimental Cell ResearchAbstract:
Changes in inflammatory gene expression induced by hyperbaric oxygen treatment in human endothelial cells under chronic wound conditions
Hyperbaric oxygen (HBO) therapy involves the inhalation of 100% oxygen, whilst inside a chamber at greater than atmospheric pressure. It is an effective treatment for chronic diabetic wounds, although the molecular mechanisms involved remain unclear. We hypothesised that HBO could alter inflammatory gene expression in human endothelial cells via a reactive oxygen/nitrogen species-mediated pathway. Endothelial cells were exposed to a chronic wound model comprising hypoxia (2% O(2) at 1 atmosphere absolute (ATA); PO(2) ~2kPa) in the presence of lipopolysaccharide and TNF-α for 24h, then treated with HBO for 90min (97.5% O(2) at 2.4 ATA; PO(2) ~237kPa). 5h post-HBO, 19 genes involved in adhesion, angiogenesis, inflammation and oxidative stress were downregulated. Notably, only angiogenin gene expression, which promotes both angiogenesis and nitric oxide production (reflected by increased eNOS protein expression in this study), was upregulated. This led to a decrease in endothelial IL-8 mRNA and protein, which could help alleviate inflammatory processes during chronic wound healing. This was no longer evident 22.5h post-HBO, demonstrating the importance of daily exposures in HBO treatment protocols. These studies indicate that elevated oxygen transiently regulates inflammatory gene expression in endothelial cells, which may enhance chronic wound healing.
Abstract.
GoDARTS and UKPDS Diabetes Pharmacogenetics Study Group, Wellcome Trust Case Control Consortium 2, Zhou K, Bellenguez C, Spencer CCA, Bennett AJ, Coleman RL, Tavendale R, Hawley SA, Donnelly LA, et al (2011). Common variants near ATM are associated with glycemic response to metformin in type 2 diabetes.
Nat Genet,
43(2), 117-120.
Abstract:
Common variants near ATM are associated with glycemic response to metformin in type 2 diabetes.
Metformin is the most commonly used pharmacological therapy for type 2 diabetes. We report a genome-wide association study for glycemic response to metformin in 1,024 Scottish individuals with type 2 diabetes with replication in two cohorts including 1,783 Scottish individuals and 1,113 individuals from the UK Prospective Diabetes Study. In a combined meta-analysis, we identified a SNP, rs11212617, associated with treatment success (n = 3,920, P = 2.9 × 10(-9), odds ratio = 1.35, 95% CI 1.22-1.49) at a locus containing ATM, the ataxia telangiectasia mutated gene. In a rat hepatoma cell line, inhibition of ATM with KU-55933 attenuated the phosphorylation and activation of AMP-activated protein kinase in response to metformin. We conclude that ATM, a gene known to be involved in DNA repair and cell cycle control, plays a role in the effect of metformin upstream of AMP-activated protein kinase, and variation in this gene alters glycemic response to metformin.
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Author URL.
Johnstone KA, Diakogiannaki E, Dhayal S, Morgan NG, Harries LW (2011). Dysregulation of Hnf1b gene expression in cultured beta-cells in response to cytotoxic fatty acid.
JOP,
12(1), 6-10.
Abstract:
Dysregulation of Hnf1b gene expression in cultured beta-cells in response to cytotoxic fatty acid.
CONTEXT: Increased levels of circulating fatty acids deriving from over-nutrition are thought to contribute to the progressive beta-cell failure associated with type 2 diabetes. Pancreatic beta-cells in culture are sensitive to exposure to long-chain saturated fatty acids (e.g. palmitate) which cause cytotoxicity, whereas the monounsaturated equivalents (e.g. palmitoleate) are cytoprotective. OBJECTIVES: in this study we sought to determine whether of members of the hepatocyte nuclear factor (HNF) family of transcription factors, which are mutated in familial, young-onset, monogenic beta-cell diabetes, could play a role in fatty acid-mediated cytotoxicity in cultured beta-cells. DESIGN: We used real-time PCR to determine whether hepatocyte nuclear factor gene expression was altered in response to palmitate exposure in the BRIN-BD11 beta-cell line. RESULTS: We found that the Hnf isoforms expressed in BRIN-BD11 cells are dysregulated by palmitate exposure. The expression of Hnf1b is specifically reduced by exposure to palmitate, and this response is prevented by co-incubation with palmitoleate. CONCLUSIONS: Down-regulation of Hnf1b gene expression accompanies palmitate-mediated cytotoxicity in cultured beta-cells.
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Author URL.
Locke JM, Da Silva Xavier G, Rutter GA, Harries LW (2011). Erratum to: an alternative polyadenylation signal in TCF7L2 generates isoforms that inhibit T cell factor/lymphoid-enhancer factor (TCF/LEF)-dependent target genes. Diabetologia, 1-1.
Locke JM, Da Silva Xavier G, Rutter GA, Harries LW (2011). Erratum to: an alternative polyadenylation signal in TCF7L2 generates isoforms that inhibit T cell factor/lymphoid-enhancer factor (TCF/LEF)-dependent target genes. Diabetologia, 54(12).
Locke JM, Da Silva Xavier G, Rutter GA, Harries LW (2011). Erratum: an alternative polyadenylation signal in TCF7L2 generates isoforms that inhibit T cell factor/lymphoid-enhancer factor (TCF/LEF)-dependent target genes (Diabetologia DOI: 10.1007/s00125-011-2290-6). Diabetologia, 54(12).
Harries LW, Hernandez D, Henley W, Wood AR, Holly AC, Bradley-Smith RM, Yaghootkar H, Dutta A, Murray A, Frayling TM, et al (2011). Human aging is characterized by focused changes in gene expression and deregulation of alternative splicing.
Aging Cell,
10(5), 868-878.
Abstract:
Human aging is characterized by focused changes in gene expression and deregulation of alternative splicing.
Aging is a major risk factor for chronic disease in the human population, but there are little human data on gene expression alterations that accompany the process. We examined human peripheral blood leukocyte in-vivo RNA in a large-scale transcriptomic microarray study (subjects aged 30-104 years). We tested associations between probe expression intensity and advancing age (adjusting for confounding factors), initially in a discovery set (n= 58), following-up findings in a replication set (n=240). We confirmed expression of key results by real-time PCR. of 16,571 expressed probes, only 295 (2%) were robustly associated with age. Just six probes were required for a highly efficient model for distinguishing between young and old (area under the curve in replication set; 95%). The focused nature of age-related gene expression may therefore provide potential biomarkers of aging. Similarly, only 7 of 1065 biological or metabolic pathways were age-associated, in gene set enrichment analysis, notably including the processing of messenger RNAs (mRNAs); [P
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Author URL.
Yaghootkar H, Hernandez D, Nalls M, Wood A, Gibbs R, Harries L, Chong S, Moore M, Guralnik J, Bandinell S, et al (2011). Identifying likely causal connections between gene expression levels using a Mendelian randomization approach.
CLINICAL BIOCHEMISTRY,
44(13), S294-S294.
Author URL.
Harries LW (2011). Messenger RNA processing and its role in diabetes.
Diabet Med,
28(9), 1010-1017.
Abstract:
Messenger RNA processing and its role in diabetes.
The past few years have seen huge advances in our understanding of the genetics of diabetes. However, definition of the mechanisms that underpin these observations is less clear. It is now becoming apparent that the processes that mediate these effects are complex and interlinked, and will require consideration of other factors in addition to the DNA sequence. The information in our genes is conveyed to the cellular machinery via an intermediate molecule, RNA. However, we now understand that RNA is not merely a messenger, as RNA-based mechanisms are responsible for a large proportion of the fine-tuning of gene expression and gene regulation. The initial RNA transcript produced undergoes a series of modifications known as RNA processing to generate a mature messenger RNA (mRNA). This includes addition of the 5' cap sequences and the poly-A tail of the mRNA molecule, and removal of its intronic sequences. The exact pattern of mRNA processing may vary from cell type to cell type and differ in response to internal and external stimuli. In this review, using examples from my own work, I will outline how mRNA processing mechanisms can sometimes provide a mode of action for mutations causing monogenic diabetes, and also suggest potential explanations for phenotypic variation in this condition. The potential for mRNA processing to impact on more complex causes of diabetes as well will also be considered.
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Assam EE, Rhodes A, Ladomery M, Harries L, Sohail M (2011). Novel Alternative Splice Variants of HER2 in Invasive Breast Cancer.
CANCER RESEARCH,
71 Author URL.
Morrison FS, Johnstone KA, Harries LW (2011). Physiological effects of type 2 diabetes on mRNA processing and gene expression.
Expert Review of Endocrinology and Metabolism,
6(2), 255-267.
Abstract:
Physiological effects of type 2 diabetes on mRNA processing and gene expression
Characteristics of Type 2 diabetes include both high blood glucose (hyperglycemia) and raised cholesterol and triglycerides (hyperlipidemia). Several studies have now shown that both hyperglycemia and hyperlipidemia can alter gene expression by disrupting physiological mechanisms of gene regulation, including alternative mRNA splicing, epigenetic gene regulation and miRNA-mediated regulation of gene expression. These processes may also be influenced by intracellular oxidative stress, which is increased in diabetes and in response to hyperglycemia and hyperlipidemia. Many pathways relevant to diabetes are affected by altered gene expression, including lipid and glucose metabolism and oxidative phosphorylation. This article considers how hyperglycemia and hyperlipidemia can alter gene expression in diabetes, which could potentially contribute to the worsening of the diabetic phenotype and diabetic complications. © 2011 Expert Reviews Ltd.
Abstract.
Harries LW, Perry JRB, McCullagh P, Crundwell M (2010). Alterations in LMTK2, MSMB and HNF1B gene expression are associated with the development of prostate cancer.
BMC Cancer,
10Abstract:
Alterations in LMTK2, MSMB and HNF1B gene expression are associated with the development of prostate cancer.
BACKGROUND: Genome wide association studies (GWAS) have identified several genetic variants that are associated with prostate cancer. Most of these variants, like other GWAS association signals, are located in non-coding regions of potential candidate genes, and thus could act at the level of the mRNA transcript. METHODS: We measured the expression and isoform usage of seven prostate cancer candidate genes in benign and malignant prostate by real-time PCR, and correlated these factors with cancer status and genotype at the GWAS risk variants. RESULTS: We determined that levels of LMTK2 transcripts in prostate adenocarcinomas were only 32% of those in benign tissues (p = 3.2 x 10(-7)), and that an independent effect of genotype at variant rs6465657 on LMTK2 expression in benign (n = 39) and malignant tissues (n = 21) was also evident (P = 0.002). We also identified that whilst HNF1B(C) and MSMB2 comprised the predominant isoforms in benign tissues (90% and 98% of total HNF1B or MSMB expression), HNF1B(B) and MSMB1 were predominant in malignant tissue (95% and 96% of total HNF1B or MSMB expression; P = 1.7 x 10(-7) and 4 x 10(-4) respectively), indicating major shifts in isoform usage. CONCLUSIONS: Our results indicate that the amount or nature of mRNA transcripts expressed from the LMTK2, HNF1B and MSMB candidate genes is altered in prostate cancer, and provides further evidence for a role for these genes in this disorder. The alterations in isoform usage we detect highlights the potential importance of alternative mRNA processing and moderation of mRNA stability as potentially important disease mechanisms.
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Author URL.
Alexander HM, Young E, Harries LW, Joyner M, Newman P, Sarsfield P (2010). An unusual case of cyclin-D1-positive peripheral T cell lymphoma with a 11:14 translocation.
Journal of Hematopathology,
3(2), 77-81.
Abstract:
An unusual case of cyclin-D1-positive peripheral T cell lymphoma with a 11:14 translocation
Overexpression of cyclin D1 is commonly observed in human cancers. The best-known association is with mantle cell lymphoma in which there is a t(11:14) translocation. We describe an unusual case of high-grade T cell lymphoma, presenting as rapidly enlarging, painless lymphadenopathy in an 80-year-old female. She died 13 months after presentation, having relapsed after chemotherapy. The lymph node showed diffuse effacement by blastic lymphoid cells, positive for T cell markers and cyclin D1. Molecular genetics investigations found t(11:14) translocation. There was definite T cell clonality demonstrating clonal rearrangement of both T cell receptor gamma and beta genes but no convincing evidence of B cell clonality. These data represent the first documented case where deregulation of the cyclin D1 expression has been implicated in human T cell lymphoreticular malignancy. © 2010 Springer-Verlag.
Abstract.
Eggleton P, Harries LW, Alberigo G, Wordsworth P, Viner N, Haigh R, Donnelly S, Jones HW, O Conner TWE, Thomson AER, et al (2010). Changes in apoptotic gene expression in lymphocytes from rheumatoid arthritis and systemic lupus erythematosus patients compared with healthy lymphocytes.
Journal of Clinical Immunology,
30(5), 649-658.
Abstract:
Changes in apoptotic gene expression in lymphocytes from rheumatoid arthritis and systemic lupus erythematosus patients compared with healthy lymphocytes
Introduction Systemic lupus erythematosus (SLE) and rheumatoid arthritis have complex genetic traits, but in both autoimmune diseases, dysfunctional apoptosis appears to play a part in disease pathology. This study examined the levels of in vitro apoptosis in lymphocytes from healthy, rheumatoid arthritis (RA) and SLE individuals and related observed differences to their lymphocyte apoptosis gene profiles. Methods Materials and Methods Lymphocytes were assessed for cell death by nuclear pyknosis and DNA fragmentation. Control, SLE and RA apoptosis gene profiles were obtained by quantitative real time polymerase chain reaction (QRT-PCR) analysis.
Results and Discussion the mean levels of pyknosis in RA and SLE freshly isolated lymphocytes were significantly higher than in control lymphocytes. Ninety three apoptosis genes were analysed by QRT-PCR of mRNA from RA, SLE and healthy lymphocytes. We identified significant differences (p
Abstract.
Tarr JM, Winyard PG, Haigh R, Viner N, Eggleton P (2010). Extracellular calreticulin accumulates in the joints of rheumatoid arthritis patients and inhibits FasL (CD95L) mediated apoptosis of T cells. Arthritis and Rheumatism, 62(10), 2919-2929.
Wirsing A, Johnstone KA, Harries LW, Ellard S, Ryffel GU, Stanik J, Gasperikova D, Klimes I, Murphy R (2010). Novel monogenic diabetes mutations in the P2 promoter of the HNF4A gene are associated with impaired function in vitro. Diabetic Medicine, 27(6), 631-635.
Garin I, Edghill EL, Akerman I, Rubio-Cabezas O, Rica I, Locke JM, Maestro MA, Alshaikh A, Bundak R, Del Castillo G, et al (2010). Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis.
Proceedings of the National Academy of Sciences of the United States of America,
107(7), 3105-3110.
Abstract:
Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis
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.
Abstract.
Hanlon K, Harries LW, Ellard S, Rudin CE (2009). Evaluation of 13q14 status in multiple myeloma by digital single nucleotide polymorphism technology.
J Mol Diagn,
11(5), 450-457.
Abstract:
Evaluation of 13q14 status in multiple myeloma by digital single nucleotide polymorphism technology.
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.
Abstract.
Author URL.
Hanlon K, Ellard S, Rudin CE, Thorne S, Davies T, Harries LW (2009). Evaluation of 13q14 status in patients with chronic lymphocytic leukemia using single nucleotide polymorphism-based techniques.
J Mol Diagn,
11(4), 298-305.
Abstract:
Evaluation of 13q14 status in patients with chronic lymphocytic leukemia using single nucleotide polymorphism-based techniques.
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.
Abstract.
Author URL.
Adalat S, Woolf AS, Johnstone KA, Wirsing A, Harries LW, Long DA, Hennekam RC, Ledermann SE, Rees L, van't Hoff W, et al (2009). HNF1B mutations associate with hypomagnesemia and renal magnesium wasting.
J Am Soc Nephrol,
20(5), 1123-1131.
Abstract:
HNF1B mutations associate with hypomagnesemia and renal magnesium wasting.
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 (
Abstract.
Author URL.
Bockenhauer D, Adalat S, Johnstone KA, Wirsing A, Harries LW, Long DA, Hennekam RC, Ledermann SE, Rees L, van't Hoff W, et al (2009). Hepatocyte Nuclear Factor 1B mutations are associated with hypomagnesemia and renal magnesium wasting.
PEDIATRIC NEPHROLOGY,
24(4), 885-885.
Author URL.
Hanlon K, Rudin CE, Harries LW (2009). Investigating the targets of MIR-15a and MIR-16-1 in patients with chronic lymphocytic leukemia (CLL).
PLoS One,
4(9).
Abstract:
Investigating the targets of MIR-15a and MIR-16-1 in patients with chronic lymphocytic leukemia (CLL).
BACKGROUND: MicroRNAs (miRNAs) are short, noncoding RNAs that regulate the expression of multiple target genes. Deregulation of miRNAs is common in human tumorigenesis. The miRNAs, MIR-15a/16-1, at chromosome band 13q14 are down-regulated in the majority of patients with chronic lymphocytic leukaemia (CLL). METHODOLOGY/PRINCIPAL FINDINGS: We have measured the expression of MIR-15a/16-1, and 92 computationally-predicted MIR-15a/16-1 target genes in CLL patients and in normal controls. We identified 35 genes that are deregulated in CLL patients, 5 of which appear to be specific targets of the MIR-15a/16-1 cluster. These targets included 2 genes (BAZ2A and RNF41) that were significantly up-regulated (p
Abstract.
Author URL.
Harries LW, Brown JE, Gloyn AL (2009). Species-specific differences in the expression of the HNF1A, HNF1B and HNF4A genes.
PLoS One,
4(11).
Abstract:
Species-specific differences in the expression of the HNF1A, HNF1B and HNF4A genes.
BACKGROUND: the HNF1A, HNF1B and HNF4A genes are part of an autoregulatory network in mammalian pancreas, liver, kidney and gut. The layout of this network appears to be similar in rodents and humans, but inactivation of HNF1A, HNF1B or HNF4A genes in animal models cause divergent phenotypes to those seen in man. We hypothesised that some differences may arise from variation in the expression profile of alternatively processed isoforms between species. METHODOLOGY/PRINCIPAL FINDINGS: We measured the expression of the major isoforms of the HNF1A, HNF1B and HNF4A genes in human and rodent pancreas, islet, liver and kidney by isoform-specific quantitative real-time PCR and compared their expression by the comparative Ct (DeltaDeltaCt) method. We found major changes in the expression profiles of the HNF genes between humans and rodents. The principal difference lies in the expression of the HNF1A gene, which exists as three isoforms in man, but as a single isoform only in rodents. More subtle changes were to the balance of HNF1B and HNF4A isoforms between species; the repressor isoform HNF1B(C) comprised only 6% in human islets compared with 24-26% in rodents (p = 0.006) whereas HNF4A9 comprised 22% of HNF4A expression in human pancreas but only 11% in rodents (p = 0.001). CONCLUSIONS/SIGNIFICANCE: the differences we note in the isoform-specific expression of the human and rodent HNF1A, HNF1B and HNF4A genes may impact on the absolute activity of these genes, and therefore on the activity of the pancreatic transcription factor network as a whole. We conclude that alterations to expression of HNF isoforms may underlie some of the phenotypic variation caused by mutations in these genes.
Abstract.
Author URL.
Flanagan SE, Clauin S, Bellanné-Chantelot C, de Lonlay P, Harries LW, Gloyn AL, Ellard S (2009). Update of mutations in the genes encoding the pancreatic beta-cell K(ATP) channel subunits Kir6.2 (KCNJ11) and sulfonylurea receptor 1 (ABCC8) in diabetes mellitus and hyperinsulinism.
Hum Mutat,
30(2), 170-180.
Abstract:
Update of mutations in the genes encoding the pancreatic beta-cell K(ATP) channel subunits Kir6.2 (KCNJ11) and sulfonylurea receptor 1 (ABCC8) in diabetes mellitus and hyperinsulinism.
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.
Abstract.
Author URL.
Locke JM, Ellard S, Norwood VF, Harries LW (2009). Variants in the isoform-specific coding regions of the <i>HNF1A, HNF4A</i> and <i>HNF1B</i> genes are not a common cause of familial, young-onset diabetes or renal cysts and diabetes (RCAD).
DIABETIC MEDICINE,
26(5), 570-570.
Author URL.
Harries LW, Sloman MJ, Sellers EA, Hattersley AT, Ellard S (2008). DIABETES SUSCEPTIBILITY IN THE CANADIAN OJI-CREE POPULATION IS MODERATED BY ABNORMAL mRNA PROCESSING OF HNF1A G319S TRANSCRIPTS.
DiabetesAbstract:
DIABETES SUSCEPTIBILITY IN THE CANADIAN OJI-CREE POPULATION IS MODERATED BY ABNORMAL mRNA PROCESSING OF HNF1A G319S TRANSCRIPTS.
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.
Abstract.
Harries LW, Sloman MJ, Sellers EAC, Hattersley AT, Ellard S (2008). Diabetes susceptibility in the Canadian Oji-Cree population is moderated by abnormal mRNA processing of <i>HNF1A</i> G319S transcripts.
DIABETES,
57(7), 1978-1982.
Author URL.
Edghill EL, Oram RA, Owens M, Stals KL, Harries LW, Hattersley AT, Ellard S, Bingham C (2008). Hepatocyte nuclear factor-1beta gene deletions--a common cause of renal disease.
Nephrol Dial Transplant,
23(2), 627-635.
Abstract:
Hepatocyte nuclear factor-1beta gene deletions--a common cause of renal disease.
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.
Abstract.
Author URL.
Shield JPH, Flanagan SE, Mackay DJ, Harries LW, Proks P, Girard C, Ashcroft FM, Temple IK, Ellard S (2008). Mosaic paternal uniparental isodisomy and an ABCC8 gene mutation in a patient with permanent neonatal diabetes and hemihypertrophy.
Diabetes,
57(1), 255-258.
Abstract:
Mosaic paternal uniparental isodisomy and an ABCC8 gene mutation in a patient with permanent neonatal diabetes and hemihypertrophy.
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.
Abstract.
Author URL.
Locke JM, Harries LW (2008). RNA processing and mRNA surveillance in monogenic diabetes.
Gene Regul Syst Bio,
2, 203-212.
Abstract:
RNA processing and mRNA surveillance in monogenic diabetes.
In the eukaryotic cell a number of molecular mechanisms exist to regulate the nature and quantity of transcripts intended for translation. For monogenic diabetes an understanding of these processes is aiding scientists and clinicians in studying and managing this disease. Knowledge of RNA processing and mRNA surveillance pathways is helping to explain disease mechanisms, form genotype-phenotype relationships, and identifying new regions within genes to screen for mutations. Furthermore, recent insights into the regulatory role of micro RNAs (miRNAs) and RNA editing in the pancreas suggests that these mechanisms may also be important in the progression to the diabetic state.
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Author URL.
Murphy R, Baptista J, Holly J, Umpleby AM, Ellard S, Harries LW, Crolla J, Cundy T, Hattersley AT (2008). Severe intrauterine growth retardation and atypical diabetes associated with a translocation breakpoint disrupting regulation of the insulin-like growth factor 2 gene.
J Clin Endocrinol Metab,
93(11), 4373-4380.
Abstract:
Severe intrauterine growth retardation and atypical diabetes associated with a translocation breakpoint disrupting regulation of the insulin-like growth factor 2 gene.
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.
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Author URL.
Harries LW, Locke JM, Shields B, Hanley NA, Hanley KP, Steele A, Njølstad PR, Ellard S, Hattersley AT (2008). The diabetic phenotype in HNF4A mutation carriers is moderated by the expression of HNF4A isoforms from the P1 promoter during fetal development.
Diabetes,
57(6), 1745-1752.
Abstract:
The diabetic phenotype in HNF4A mutation carriers is moderated by the expression of HNF4A isoforms from the P1 promoter during fetal development.
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.
Abstract.
Author URL.
Frayling TM, Timpson NJ, Weedon MN, Zeggini E, Freathy RM, Lindgren CM, Perry JRB, Elliott KS, Lango H, Rayner NW, et al (2007). A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity.
Science,
316(5826), 889-894.
Abstract:
A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity.
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.
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Author URL.
Melzer D, Frayling TM, Murray A, Hurst AJ, Harries LW, Song H, Khaw K, Luben R, Surtees PG, Bandinelli SS, et al (2007). A common variant of the p16(INK4a) genetic region is associated with physical function in older people.
Mech Ageing Dev,
128(5-6), 370-377.
Abstract:
A common variant of the p16(INK4a) genetic region is associated with physical function in older people.
p16(INK4a) is active in cell senescence, ageing and tumor suppression. Deletion of the small p16(INK4a)/ARF/p15(INK4b) region occurs in many cancers. We screened 25 common polymorphisms across the region and three related genes for associations with physical functioning in older people. In an initial sample of 938 (aged 65-80 years) from the EPIC study (Norfolk, UK), the rs2811712 SNP minor allele (located between the shared p16(INK4a)/ARF locus and p15(INK4b)) was associated with reduced physical impairment. This association remained after testing an additional 1319 EPIC-Norfolk samples (p-value=0.013, total n=2257), and on independent replication in the InCHIANTI study (n=709, p=0.015), and at one-sided significance in Iowa-EPESE (n=419, p=0.079). Overall (n=3372), the prevalence of severely limited physical function was 15.0% in common homozygotes and 7.0% in rare homozygotes (per minor allele odds ratio=1.48, 95% CI: 1.17-1.88, p=0.001, adjusted for age, sex and study). This estimate was similar excluding screening set 1 (OR=1.45, 95% CI: 1.09-1.92, p=0.010, n=2434). These findings require further replication, but provide the first direct evidence that the p16(INK4a)/ARF/p15(INK4b) genetic region and the senescence machinery are active in physical ageing in heterogeneous human populations. The mechanism involved may be via greater cellular restorative activity and reduced stem cell senescence.
Abstract.
Author URL.
Wolstencroft EC, Hanlon K, Harries LW, Standen GR, Sternberg A, Ellard S (2007). Development of a quantitative real-time polymerase chain reaction assay for the detection of the JAK2 V617F mutation.
J Mol Diagn,
9(1), 42-46.
Abstract:
Development of a quantitative real-time polymerase chain reaction assay for the detection of the JAK2 V617F mutation.
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.
Abstract.
Author URL.
Locke JM, Harries LW, Sloman MJ, Sellers EA, Hattersley AT, Ellard S (2007). Diabetes susceptibility in the Canadian Oji-Cree population may be moderated by abnormal mRNA processing of <i>HNFIA</i> G319S transcripts.
DIABETIC MEDICINE,
24, 36-36.
Author URL.
Singh R, Edghill E, Bingham C, Ellard S, Hattersley AT, Harries LW (2007). Low prevalence of mitochondrial DNA 3243A>G point mutation in Caucasians with unexplained renal disease.
Diabet Med,
24(7), 804-806.
Author URL.
Edghill EL, Gloyn AL, Goriely A, Harries LW, Flanagan SE, Rankin J, Hattersley AT, Ellard S (2007). Origin of de novo KCNJ11 mutations and risk of neonatal diabetes for subsequent siblings.
J Clin Endocrinol Metab,
92(5), 1773-1777.
Abstract:
Origin of de novo KCNJ11 mutations and risk of neonatal diabetes for subsequent siblings.
CONTEXT: Activating mutations in the KCNJ11 gene, which encodes the Kir6.2 subunit of the pancreatic beta-cell K(ATP) channel, result in permanent and transient neonatal diabetes. The majority of KCNJ11 mutations are spontaneous, but the parental origin of these mutations is not known. OBJECTIVE: Our objective was to determine the parental origin of de novo KCNJ11 mutations and investigate the possibility of mosaicism in transmitting parents. DESIGN: We identified 68 index cases with a KCNJ11 mutation where neither parent was known to be affected. DNA was available from both parents of 41 probands. The parental origin of the mutation was determined in 18 families by examination of pedigrees, microsatellite analysis, or allele-specific PCR. RESULTS: a nonsignificant excess of paternally derived mutations was found with 13 of 18 (72%) shown to have arisen on the paternal allele. There was no evidence to suggest an association with increased age at conception. In two families, there were half-siblings with permanent neonatal diabetes born to an unaffected father, suggesting germline mosaicism that was confirmed by the presence of the R201C mutation in one father's semen. Somatic mosaicism was detected in one unaffected mother, and this mutation will also be present in her germ cells. CONCLUSION: De novo KCNJ11 mutations can arise either during gametogenesis or embryogenesis. The possibility of germline mosaicism means that future siblings are at increased risk of neonatal diabetes, and we recommend that molecular genetic testing is routinely offered at birth for subsequent siblings of children with de novo KCNJ11 mutations.
Abstract.
Author URL.
Ellard S, Thomas K, Edghill EL, Owens M, Ambye L, Cropper J, Little J, Strachan M, Stride A, Ersoy B, et al (2007). Partial and whole gene deletion mutations of the GCK and HNF1A genes in maturity-onset diabetes of the young.
Diabetologia,
50(11), 2313-2317.
Abstract:
Partial and whole gene deletion mutations of the GCK and HNF1A genes in maturity-onset diabetes of the young.
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.
Abstract.
Author URL.
Ellard S, Flanagan SE, Girard CA, Patch A-M, Harries LW, Parrish A, Edghill EL, Mackay DJG, Proks P, Shimomura K, et al (2007). Permanent neonatal diabetes caused by dominant, recessive, or compound heterozygous SUR1 mutations with opposite functional effects.
Am J Hum Genet,
81(2), 375-382.
Abstract:
Permanent neonatal diabetes caused by dominant, recessive, or compound heterozygous SUR1 mutations with opposite functional effects.
Heterozygous activating mutations in the KCNJ11 gene encoding the pore-forming Kir6.2 subunit of the pancreatic beta cell K(ATP) channel are the most common cause of permanent neonatal diabetes (PNDM). Patients with PNDM due to a heterozygous activating mutation in the ABCC8 gene encoding the SUR1 regulatory subunit of the K(ATP) channel have recently been reported. We studied a cohort of 59 patients with permanent diabetes who received a diagnosis before 6 mo of age and who did not have a KCNJ11 mutation. ABCC8 gene mutations were identified in 16 of 59 patients and included 8 patients with heterozygous de novo mutations. A recessive mode of inheritance was observed in eight patients with homozygous, mosaic, or compound heterozygous mutations. Functional studies of selected mutations showed a reduced response to ATP consistent with an activating mutation that results in reduced insulin secretion. A novel mutational mechanism was observed in which a heterozygous activating mutation resulted in PNDM only when a second, loss-of-function mutation was also present.
Abstract.
Author URL.
Singh R, Hattersley AT, Harries LW (2007). Reduced peripheral blood mitochondrial DNA content is not a risk factor for Type 2 diabetes.
Diabet Med,
24(7), 784-787.
Abstract:
Reduced peripheral blood mitochondrial DNA content is not a risk factor for Type 2 diabetes.
AIMS: Mitochondrial depletion in pancreatic beta cells is known to reduce glucose stimulated insulin secretion. We aimed to determine whether the offspring of patients with early onset Type 2 diabetes had reduced peripheral blood mitochondrial content relative to control subjects and whether this could lead to a predisposition to type 2 diabetes in later life. METHODS: We measured the levels of mitochondria relative to a single copy genomic target by real time polymerase chain reaction in a series of peripheral blood samples taken from the offspring of Caucasian patients with Type 2 diabetes and matched controls. Measures of insulin sensitivity and beta cell function were also taken. RESULTS: in contrast with previous studies, mitochondrial DNA content was not decreased in the offspring of patients with Type 2 diabetes relative to matched controls in our cohort. Conversely, we noted a small proliferation in mitochondrial numbers in our case subjects. In agreement with these findings, no correlations with either insulin sensitivity or beta cell function were noted. CONCLUSIONS: Our results indicate that reduced mitochondrial DNA content in peripheral blood is not a risk factor for the development of Type 2 diabetes in the offspring of patients with early onset Type 2 diabetes.
Abstract.
Author URL.
Zeggini E, Weedon MN, Lindgren CM, Frayling TM, Elliott KS, Lango H, Timpson NJ, Perry JRB, Rayner NW, Freathy RM, et al (2007). Replication of genome-wide association signals in UK samples reveals risk loci for type 2 diabetes.
Science,
316(5829), 1336-1341.
Abstract:
Replication of genome-wide association signals in UK samples reveals risk loci for type 2 diabetes.
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.
Abstract.
Author URL.
Harries LW (2006). Alternate mRNA processing of the hepatocyte nuclear factor genes and its role in monogenic diabetes.
Expert Rev Endocrinol Metab,
1(6), 715-726.
Abstract:
Alternate mRNA processing of the hepatocyte nuclear factor genes and its role in monogenic diabetes.
Variation in mRNA processing has the capacity to exert fine control over gene expression in most cell types. The hepatic nuclear factor genes, like approximately 74% of the genome, produce multiple transcripts. Hepatic nuclear factor isoforms exhibit both spatial and temporal variation in expression. In this review, the known isoforms of the hepatocyte nuclear factor-1α, hepatocyte nuclear factor-1β and hepatocyte nuclear factor-4α genes are described and their properties are compared. Finally, data are discussed regarding the influence of hepatocyte nuclear factor-1α alternate mRNA processing on the clinical phenotype of maturity-onset diabetes of the young.
Abstract.
Author URL.
Welters HJ, Senkel S, Klein-Hitpass L, Erdmann S, Thomas H, Harries LW, Pearson ER, Bingham C, Hattersley AT, Ryffel GU, et al (2006). Conditional expression of hepatocyte nuclear factor-1beta, the maturity-onset diabetes of the young-5 gene product, influences the viability and functional competence of pancreatic beta-cells.
J Endocrinol,
190(1), 171-181.
Abstract:
Conditional expression of hepatocyte nuclear factor-1beta, the maturity-onset diabetes of the young-5 gene product, influences the viability and functional competence of pancreatic beta-cells.
Mutations in the gene encoding hepatocyte nuclear factor (HNF)1beta result in maturity-onset diabetes of the young-(MODY)5, by impairing insulin secretory responses and, possibly, by reducing beta-cell mass. The functional role of HNF1beta in normal beta-cells is poorly understood; therefore, in the present study, wild-type (WT) HNF1beta, or one of two naturally occurring MODY5 mutations (an activating mutation, P328L329del, or a dominant-negative form, A263insGG) were conditionally expressed in the pancreatic beta-cell line, insulin-1 (INS-1), and the functional consequences examined. Surprisingly, overexpression of the dominant-negative mutant did not modify any of the functional properties of the cells studied (including insulin secretion, cell growth and viability). By contrast, expression of WT HNF1beta was associated with a time- and dose-dependent inhibition of INS-1 cell proliferation and a marked increase in apoptosis. Induction of WT HNF1beta also inhibited the insulin secretory response to nutrient stimuli, membrane depolarisation or activation of protein kinases a and C and this correlated with a significant decrease in pancrease-duodenum homeobox-1 protein levels. The attenuation of insulin secretion was, however, dissociated from the inhibition of proliferation and loss of viability, since expression of the P328L329del mutant led to a reduced rate of cell proliferation, but failed to induce apoptosis or to alter insulin secretion. Taken together, the present results suggest that mature rodent beta-cells are sensitive to increased expression of WT HNF1beta and they imply that the levels of this protein are tightly regulated to maintain secretory competence and cell viability.
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Harries LW, Ellard S, Stride A, Morgan NG, Hattersley AT (2006). Isomers of the TCF1 gene encoding hepatocyte nuclear factor-1 alpha show differential expression in the pancreas and define the relationship between mutation position and clinical phenotype in monogenic diabetes.
Hum Mol Genet,
15(14), 2216-2224.
Abstract:
Isomers of the TCF1 gene encoding hepatocyte nuclear factor-1 alpha show differential expression in the pancreas and define the relationship between mutation position and clinical phenotype in monogenic diabetes.
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.
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Wickham CL, Harries LW, Sarsfield P, Joyner MV, Ellard S (2006). Large variation in t(11;14)(q13;q32) and t(14;18)(q32;q21) translocation product size is confirmed by sequence analysis of PCR products.
Clin Lab Haematol,
28(4), 248-253.
Abstract:
Large variation in t(11;14)(q13;q32) and t(14;18)(q32;q21) translocation product size is confirmed by sequence analysis of PCR products.
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.
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Singh R, Ellard S, Hattersley A, Harries LW (2006). Rapid and sensitive real-time polymerase chain reaction method for detection and quantification of 3243A>G mitochondrial point mutation.
J Mol Diagn,
8(2), 225-230.
Abstract:
Rapid and sensitive real-time polymerase chain reaction method for detection and quantification of 3243A>G mitochondrial point mutation.
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.
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Harries LW, Wickham CL, Evans JC, Rule SA, Joyner MV, Ellard S (2005). Analysis of haematopoietic chimaerism by quantitative real-time polymerase chain reaction.
Bone Marrow Transplant,
35(3), 283-290.
Abstract:
Analysis of haematopoietic chimaerism by quantitative real-time polymerase chain reaction.
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.
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Harries LW, Bingham C, Bellanne-Chantelot C, Hattersley AT, Ellard S (2005). The position of premature termination codons in the hepatocyte nuclear factor -1 beta gene determines susceptibility to nonsense-mediated decay.
Hum Genet,
118(2), 214-224.
Abstract:
The position of premature termination codons in the hepatocyte nuclear factor -1 beta gene determines susceptibility to nonsense-mediated decay.
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, Q243fsdelC, P328L329fsdelCCTCT and A373fsdel29) showed evidence of NMD with levels of mutant transcript at 71% (p=0.009), 24% (p=0.008), 22% (p=0.008) and 3% (p=0.016) of the wild-type allele respectively. Comparable results were derived from lymphoblastoid cells and renal tubule cells isolated from a patient's overnight urine confirming that cell lines provide a good model for mRNA analysis. Two mutations (H69fsdelAC and P159fsdelT) produced transcripts unexpectedly immune to NMD. We conclude that truncating mutant transcripts of the HNF-1beta gene do not conform to the known rules governing NMD susceptibility, but instead demonstrate a previously unreported 5' to 3' polarity. We hypothesise that this may be due to reinitiation of translation downstream of the premature termination codon. Our study suggests that reinitiation of translation may be an important mechanism in the evasion of NMD, but that other factors such as the distance from the native initiation codon may also play a part.
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Theron T, Fousteri MI, Volker M, Harries LW, Botta E, Stefanini M, Fujimoto M, Andressoo J-O, Mitchell J, Jaspers NGJ, et al (2005). Transcription-associated breaks in xeroderma pigmentosum group D cells from patients with combined features of xeroderma pigmentosum and Cockayne syndrome.
Mol Cell Biol,
25(18), 8368-8378.
Abstract:
Transcription-associated breaks in xeroderma pigmentosum group D cells from patients with combined features of xeroderma pigmentosum and Cockayne syndrome.
Defects in the XPD gene can result in several clinical phenotypes, including xeroderma pigmentosum (XP), trichothiodystrophy, and, less frequently, the combined phenotype of XP and Cockayne syndrome (XP-D/CS). We previously showed that in cells from two XP-D/CS patients, breaks were introduced into cellular DNA on exposure to UV damage, but these breaks were not at the sites of the damage. In the present work, we show that three further XP-D/CS patients show the same peculiar breakage phenomenon. We show that these breaks can be visualized inside the cells by immunofluorescence using antibodies to either gamma-H2AX or poly-ADP-ribose and that they can be generated by the introduction of plasmids harboring methylation or oxidative damage as well as by UV photoproducts. Inhibition of RNA polymerase II transcription by four different inhibitors dramatically reduced the number of UV-induced breaks. Furthermore, the breaks were dependent on the nucleotide excision repair (NER) machinery. These data are consistent with our hypothesis that the NER machinery introduces the breaks at sites of transcription initiation. During transcription in UV-irradiated XP-D/CS cells, phosphorylation of the carboxy-terminal domain of RNA polymerase II occurred normally, but the elongating form of the polymerase remained blocked at lesions and was eventually degraded.
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Harries LW, Ellard S, Jones RWA, Hattersley AT, Bingham C (2004). Abnormal splicing of hepatocyte nuclear factor-1 beta in the renal cysts and diabetes syndrome.
Diabetologia,
47(5), 937-942.
Abstract:
Abnormal splicing of hepatocyte nuclear factor-1 beta in the renal cysts and diabetes syndrome.
AIMS/HYPOTHESIS: Mutations in the hepatocyte nuclear factor-1 beta ( HNF-1 beta) gene result in disorders of renal development, typically involving renal cysts and early-onset diabetes (the RCAD syndrome/ MODY5). Sixteen mutations have been reported, including three splicing mutations of the intron 2 splice donor site. Because tissues showing abundant expression (kidney, liver, pancreas, gut, lung and gonads) are not easily accessible for analysis in living subjects, it has previously proven difficult to determine the effect of HNF-1 beta mutations at the mRNA level. This is the aim of the present study. METHODS: We have developed a nested RT-PCR assay that exploits the presence of ectopic HNF-1 beta transcripts in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines derived from subjects carrying HNF-1 beta splice site mutations. RESULTS: We report a fourth mutation of the intron 2 splice donor site, IVS2nt+2insT. Sequence analysis of ectopic HNF-1 beta transcripts showed that both IVS2nt+2insT and IVS2nt+1G>T result in the deletion of exon 2 and are predicted to result in premature termination of the HNF-1 beta protein. Mutant transcripts were less abundant than the normal transcripts but there was no evidence of nonsense-mediated decay. CONCLUSIONS/INTERPRETATION: This is the first study to define the pathogenic consequences of mutations within the HNF-1 beta gene by mRNA analysis. This type of approach is a useful and important tool to define mutational mechanisms and determine pathogenicity.
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Harries LW, Hattersley AT, Ellard S (2004). Messenger RNA transcripts of the hepatocyte nuclear factor-1alpha gene containing premature termination codons are subject to nonsense-mediated decay.
Diabetes,
53(2), 500-504.
Abstract:
Messenger RNA transcripts of the hepatocyte nuclear factor-1alpha gene containing premature termination codons are subject to nonsense-mediated decay.
Mutations in the hepatocyte nuclear factor-1alpha (HNF-1a) gene cause maturity-onset diabetes of the young (MODY). Approximately 30% of these mutations generate mRNA transcripts harboring premature termination codons (PTCs). Degradation of such transcripts by the nonsense-mediated decay (NMD) pathway has been reported for many genes. To determine whether PTC mutant transcripts of the HNF-1alpha gene elicit NMD, we have developed a novel quantitative RT-PCR assay. We performed quantification of ectopically expressed mutant transcripts relative to normal transcripts in lymphoblastoid cell lines using a coding single nucleotide polymorphism (cSNP) as a marker. The nonsense mutations R171X, I414G415ATCG-->CCA, and P291fsinsC showed reduced mutant mRNA expression to 40% (P = 0.009),
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Gloyn AL, Cummings EA, Edghill EL, Harries LW, Scott R, Costa T, Temple IK, Hattersley AT, Ellard S (2004). Permanent neonatal diabetes due to paternal germline mosaicism for an activating mutation of the KCNJ11 Gene encoding the Kir6.2 subunit of the beta-cell potassium adenosine triphosphate channel.
J Clin Endocrinol Metab,
89(8), 3932-3935.
Abstract:
Permanent neonatal diabetes due to paternal germline mosaicism for an activating mutation of the KCNJ11 Gene encoding the Kir6.2 subunit of the beta-cell potassium adenosine triphosphate channel.
Activating mutations in the KCNJ11 gene encoding for the Kir6.2 subunit of the beta-cell ATP-sensitive potassium channel have recently been shown to be a common cause of permanent neonatal diabetes. In 80% of probands, these are isolated cases resulting from de novo mutations. We describe a family in which two affected paternal half-siblings were found to be heterozygous for the previously reported R201C mutation. Direct sequencing of leukocyte DNA showed that their clinically unaffected mothers and father were genotypically normal. Quantitative real-time PCR analysis of the father's leukocyte DNA detected no trace of mutant DNA. These results are consistent with the father being a mosaic for the mutation, which is restricted to his germline. This is the first report of germline mosaicism in any form of monogenic diabetes. The high percentage of permanent neonatal diabetes cases due to de novo KCNJ11 mutations suggests that germline mosaicism may be common. The possibility of germline mosaicism should be considered when counseling recurrence risks for the parents of a child with an apparently de novo KCNJ11 activating mutation.
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Bulman MP, Harries LW, Hansen T, Shepherd M, Kelly WF, Hattersley AT, Ellard S (2002). Abnormal splicing of hepatocyte nuclear factor 1 alpha in maturity-onset diabetes of the young.
Diabetologia,
45(10), 1463-1467.
Abstract:
Abnormal splicing of hepatocyte nuclear factor 1 alpha in maturity-onset diabetes of the young.
AIMS/HYPOTHESIS: Mutations in the HNF-1 alpha gene result in maturity-onset diabetes of the young (MODY); an early-onset, dominantly inherited form of diabetes caused by pancreatic beta-cell dysfunction. Splice site mutations represent approximately 10% of reported HNF-1 alpha mutations. No studies to date have investigated the effect of splice site mutations on mRNA processing because the tissues with abundant HNF-1alpha expression (liver, pancreas, kidney and gut) are not easily accessible for analysis. This study aimed to define the pathogenic mechanism in three novel splice site mutations by analysing illegitimate transcripts. METHODS: to assess the consequence of potential HNF-1 alpha splice site mutations we developed a nested reverse transcriptase PCR (RT-PCR) assay for the amplification of illegitimate HNF-1 alpha transcripts in Epstein Barr virus transformed lymphoblastoid cell lines. RESULTS: Sequencing the illegitimate HNF-1 alpha transcripts showed that the splice donor site mutation IVS8nt+1G>A leads to complete skipping of exon 8, the splice acceptor site mutation IVS4nt-2A>G causes skipping of exon 5 with the recruitment of a cryptic splice acceptor site within intron 5 and the cryptic splice acceptor site mutation (IVS7nt-6G>A) resulted in the skipping of exon 7. All three changes are predicted to result in premature termination of the HNF-1alpha protein, providing further evidence for their role as pathogenic mutations. CONCLUSION/INTERPRETATION: We conclude that the sequencing of illegitimate transcripts from lymphoblastoid cell lines is helpful in the assessment of intronic variation in HNF-1 alpha that could alter splicing. This analysis of the mRNA is required to define mutational mechanisms and confirm pathogenic status.
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Smith G, Stubbins MJ, Harries LW, Wolf CR (1998). Molecular genetics of the human cytochrome P450 monooxygenase superfamily.
Xenobiotica,
28(12), 1129-1165.
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Matthias C, Bockmühl U, Jahnke V, Harries LW, Wolf CR, Jones PW, Alldersea J, Worrall SF, Hand P, Fryer AA, et al (1998). The glutathione S-transferase GSTP1 polymorphism: effects on susceptibility to oral/pharyngeal and laryngeal carcinomas.
Pharmacogenetics,
8(1), 1-6.
Abstract:
The glutathione S-transferase GSTP1 polymorphism: effects on susceptibility to oral/pharyngeal and laryngeal carcinomas.
We have examined the hypothesis that the polymorphic, glutathione S-transferase GSTP1 gene is a susceptibility candidate for squamous cell cancer of the oral/pharynx and larynx. We describe GSTP1 genotype frequencies in 380 cases and 180 controls. We found a lower frequency of GSTP1 AA in the oral/pharyngeal cases compared with controls (p = 0.003, odds ratio = 0.47) after correction for age and gender. We used an immunohistochemical approach to show widespread expression of the GSTP1 subunit throughout the pharynx and larynx. In uninfiltrated tissue, strong positivity was found throughout the squamous cell epithelium with the exception of the basal cell layer. The cilia of the respiratory epithelium of the larynx also showed positivity for GSTP1. In tumour tissue, expression of GSTP1 was similar in pharyngeal and laryngeal samples. These data are the first to show that polymorphism at GSTP1 mediates susceptibility to squamous cell cancer of the upper aerodigestive tract. No significant interactions were identified between GSTP1 and GSTM1, GSTM3, GSTT1 and the cytochrome P450 CYP1A1, CYP2D6 and CYP1A1 genotypes.
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Ryberg D, Skaug V, Hewer A, Phillips DH, Harries LW, Wolf CR, Ogreid D, Ulvik A, Vu P, Haugen A, et al (1997). Genotypes of glutathione transferase M1 and P1 and their significance for lung DNA adduct levels and cancer risk.
Carcinogenesis,
18(7), 1285-1289.
Abstract:
Genotypes of glutathione transferase M1 and P1 and their significance for lung DNA adduct levels and cancer risk.
The A-G polymorphism at codon 104 in the glutathione S-transferase P1 (GSTP1) gene was examined in 138 male lung cancer patients and 297 healthy controls. The patients had significantly higher frequency of the GG genotype (15.9%) and a lower frequency of AA (38.4%) than the controls (9.1% and 51.5%, respectively). The level of hydrophobic DNA-adducts were determined in lung tissue from 70 current smokers. Patients with the GG genotype had a significantly higher adduct level than patients with AA (15.5 +/- 10.2 vs 7.9 +/- 5.1 per 10(8) nucleotides, P = 0.006). We also analyzed the deletion polymorphism in the GSTM1 gene in 135 male patients and 342 controls. The patients were stratified according to histology, smoking dose, age, adduct level and mutational types found in the tumors (Ki-ras and p53 genes). The results consistently indicated that the GSTM1 null genotype was associated with a slightly increased lung cancer risk. When the combined GST M1 and P1 genotypes were examined, patients with the combination null and AG or GG had significantly higher adduct levels than all other genotype combinations (P = 0.011). The distribution of combined genotypes was also significantly different in cases and controls, mainly due to increased frequency of the combination GSTM1 null and GSTP1 AG or GG among patients.
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Harries LW, Stubbins MJ, Forman D, Howard GC, Wolf CR (1997). Identification of genetic polymorphisms at the glutathione S-transferase Pi locus and association with susceptibility to bladder, testicular and prostate cancer.
Carcinogenesis,
18(4), 641-644.
Abstract:
Identification of genetic polymorphisms at the glutathione S-transferase Pi locus and association with susceptibility to bladder, testicular and prostate cancer.
Two variant glutathione S-transferase cDNAs have been described at the GSTP1 locus, which differ by a single base pair (A-G) substitution at nucleotide 313 of the GSTP1 cDNA. This results in an amino acid substitution which alters the function of the enzyme. In this study, a novel PCR assay has been developed which demonstrates that these two variant cDNAs represent distinct GSTP1 alleles (GSTP1a and GSTP1b). In a study of individuals with different forms of cancer, the GSTP1b allele is found to be strongly associated with bladder cancer and testicular cancer. In controls 6.5% of individuals were homozygous for the GSTP1b allele. In bladder cancer cases, this rose to 19.7% [n = 71, odds ratio 3.6 (1.4-9.2), P = 0.006] and in testicular cancer to 18.7% [n = 155, odds ratio 3.3 (1.5-7.7), P = 0.002]. In addition, in prostate cancer a highly significant decrease in the frequency of the GSTP1a homozygotes was observed [control 51.0% versus 27.8% cancer cases, n = 36, odds ratio 0.4 (0.02-3.3), P = 0.008]. Increases in the frequency of GSTP1b homozygotes was also observed in lung cancer and chronic obstructive pulmonary disease. However, these were not statistically significant. No change in breast or colon cancer allele frequencies was observed.
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Stubbins MJ, Harries LW, Smith G, Tarbit MH, Wolf CR (1996). Genetic analysis of the human cytochrome P450 CYP2C9 locus.
Pharmacogenetics,
6(5), 429-439.
Abstract:
Genetic analysis of the human cytochrome P450 CYP2C9 locus.
Cytochrome P450 CYP2C9 metabolizes a wide variety of clinically important drugs, including phenytoin, tolbutamide, warfarin and a large number of non-steroidal anti-inflammatory drugs. Previous studies have shown that even relatively conservative changes in the amino acid composition of this enzyme can affect both its activity and substrate specificity. To date six different human CYP2C9 cDNA sequences, as well as the highly homologous CYP2C10 sequence have been reported suggesting that the CYP2C9 gene is polymorphic. Only nine single base substitutions in the coding region of CYP2C9 account for the differences seen between the CYP2C9 proteins. In this report we have developed polymerase chain reaction (PCR)-based assays to distinguish all seven sequences, and have determined their allele frequencies in the Caucasian population. of the seven sequences studied in one hundred individuals only three appeared to be CYP2C9 alleles. These alleles termed CYP2C9*1, CYP2C9*2 and CYP2C9*3 had allele frequencies of 0.79, 0.125 and 0.085 respectively. The CYP2C10 gene could not be found in any of the samples studied. The assays developed here will allow the prediction of CYP2C9 phenotype, thus identifying those individuals who may exhibit different drug pharmacokinetics for CYP2C9 substrates.
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Abbott C, Malas S, Pilz A, Pate L, Ali R, Peters J (1994). Linkage mapping around the ragged (Ra) and wasted (wst) loci on distal mouse chromosome 2.
Genomics,
20(1), 94-98.
Abstract:
Linkage mapping around the ragged (Ra) and wasted (wst) loci on distal mouse chromosome 2.
Mice that are heterozygous for the ragged (Ra) mutation, which is semidominant, have ragged coats caused by an absence of certain hair types. Ra/Ra homozygous mice usually die soon after birth, are naked, and have edema. Mice that are homozygous for the recessive mutation wasted (wst) appear normal until soon after weaning, but then develop tremors and ataxia, undergo atrophy of the thymus and spleen, and die by around 28 days of age. The Ra and wst loci map to distal mouse chromosome 2, but have never been positioned with respect to molecular markers. We have now mapped each of these genes in interspecific backcrosses that were also typed for available molecular markers. The results show that Ra maps very close to D2Mit74 and Acra-4, with no recombinants in 165 mice, whereas wst maps 3 cM distal to the most telomeric molecular marker on mouse chromosome 2, Acra-4.
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