Prof Anna L Gloyn

Research Area: Genetics and Genomics
Technology Exchange: Biobanking, Human genetics, SNP typing and Transcript profiling
Scientific Themes: Diabetes, Endocrinology & Metabolism and Genes, Genetics, Epigenetics & Genomics
Keywords: genetics, beta-cell dysfunction, diabetes, glucokinase, insulin resistance and cytogenetics
Web Links:

Anna Gloyn is currently a Wellcome Trust Senior Fellow in Basic Biomedical Science & Professor of Molecular Genetics & Metabolism based jointly at the Oxford Centre for Diabetes Endocrinology and Metabolism (OCDEM) and the Wellcome Trust Centre for Human Genetics (WTCHG) at the University of Oxford.

Anna completed her DPhil at the University of Oxford under the supervision of the late Professor Robert Turner. Her post-doctoral training was carried out at the University of Exeter under the mentorship of Professors Andrew Hattersley & Sian Ellard and at the University of Pennsylvania in Philadelphia under the mentorship of Professor Franz Matschinsky.

Anna’s research is focused on using naturally occurring mutations in humans as tools to identity critical regulatory pathways and insights into normal physiology. Anna’s early post-doctoral research led to the identification a new genetic aetiology for permanent and transient neonatal diabetes due to KCNJ11 mutations and resulted in one of the first examples of the determination of the molecular genetic aetiology leading to improved treatment options for patients.  Whilst in Oxford Anna's team discovered a novel genetic cause of constitutive insulin sensitivity in humans due to mutations in the PTEN gene highlighting the complex interplay between pathways involved in cell-growth and metabolism.

Anna's current research projects are focused on the translation of genetic association signals for type 2 diabetes and glycaemic traits mechanisms for beta-cell dysfunction and diabetes. Her group uses a variety of complementary approaches, including human genetics, genomics, physiology and islet-biology to dissect out the molecular mechanisms driving disease pathogenesis.

Anna is an active member of multiple internal genetic discovery efforts including:  NIH/Pharma funded Accelerated Medicines PartnershipDIAGRAM (Diabetes Genetics Replication and Meta-analysis), MAGIC (Meta-analysis of Glucose and Insulin traits Consortium), Type 2 Diabetes Genetic Exploration by Next-generation sequencing in multi-Ethnic Samples (T2D-GENES) and the Genetics of Type 2 Diabetes (GoT2D). She is also involved in the IMI funded STEMBANCC project which is working to deliver human IPS cell derived beta-cell models for drug discovery efforts. 

Anna’s work has been recognized both nationally and internationally as she is a recipient of  a European Association for the Study of Diabetes (EASD) Rising Star Award (2005), the RD Lawrence Named Lecturer (Diabetes UK Annual Professional Conference 2009), the GB Morgagni Silver Medal (2014) and the EASD Minkowski Prize (2014).

The Gloyn group currently consists of a multidisciplinary team of clinical and basic scientists.

Current members of the team

  • Dr Nicola Beer - Naomi Berry Research Fellow
  • Dr Martijn van de Bunt - Novo Nordisk Research Fellow
  • Dr Benoit Hastoy
  • Dr Vibe Nylander
  • Dr Jason Torres
  • Dr Carla Burrows
  • Ms Benoite Champion
  • Dr Fernando Abaitua

Current DPhil Students

  • Natasha Ng (2012-)
  • Soren Thomsen (2013-)
  • Sara Althari (2014-)
  • Mahesh Umapathysivam (2014-)
  • Katia Mattis (2015-)
  • Shahana Sengupta (2015-)
  • Claire Duff (2016-)
  • Anjte Grotz (2016-)

Past DPhil Students

  • Matthias Thurner (2016)
  • Dr Neelam Hassanali (2015)
  • Dr Martijn van de Bunt (2014)
  • Dr Aparna Pal (2014)
  • Dr Bahram Jafar-Mohammadi (2012)
  • Dr Matthew Rees (2012)
  • Dr Mary Travers (2012)
  • Dr Sara Suliman (2011)
  • Dr Laura McCulloch (2011)
  • Dr Nicola Beer (2011)

Alumni

  • Dr Anne Raimondo
  • Mrs Amy Barrett
  • Dr Jana Rundle
  • Dr Nicholas Tribble
  • Dr Tom Waterfield
  • Dr Juveria Siddiqui
  • Dr Kara Osbak

Name Department Institution Country
Prof Mark McCarthy OCDEM Oxford University, Oxford Centre for Diabetes, Endocrinology & Metabolism United Kingdom
Prof Patrik Rorsman FRS FMedSci OCDEM Oxford University, Oxford Centre for Diabetes, Endocrinology & Metabolism United Kingdom
Prof Fredrik Karpe OCDEM Oxford University, Oxford Centre for Diabetes, Endocrinology & Metabolism United Kingdom
Prof Katharine Owen OCDEM Oxford University, Oxford Centre for Diabetes, Endocrinology & Metabolism United Kingdom
Prof Paul Johnson OCDEM Oxford University, Oxford Centre for Diabetes, Endocrinology & Metabolism United Kingdom
Professor Cecilia Lindgren (NDM) Big Data Institute Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Professor Andrew P Morris (NDM) Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Prof Tatjana Sauka-Spengler Nuffield Division of Clinical Laboratory Sciences Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof Jim R Hughes Nuffield Division of Clinical Laboratory Sciences Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Dr Francis S Collins NIH United States
Prof Sian Ellard Peninsula Medical School United Kingdom
Prof Andrew Hattersley Peninsula Medical School United Kingdom
Thomsen SK, Gloyn AL. 2017. Human genetics as a model for target validation: finding new therapies for diabetes. Diabetologia, 60 (6), pp. 960-970. | Show Abstract | Read more

Type 2 diabetes is a global epidemic with major effects on healthcare expenditure and quality of life. Currently available treatments are inadequate for the prevention of comorbidities, yet progress towards new therapies remains slow. A major barrier is the insufficiency of traditional preclinical models for predicting drug efficacy and safety. Human genetics offers a complementary model to assess causal mechanisms for target validation. Genetic perturbations are 'experiments of nature' that provide a uniquely relevant window into the long-term effects of modulating specific targets. Here, we show that genetic discoveries over the past decades have accurately predicted (now known) therapeutic mechanisms for type 2 diabetes. These findings highlight the potential for use of human genetic variation for prospective target validation, and establish a framework for future applications. Studies into rare, monogenic forms of diabetes have also provided proof-of-principle for precision medicine, and the applicability of this paradigm to complex disease is discussed. Finally, we highlight some of the limitations that are relevant to the use of genome-wide association studies (GWAS) in the search for new therapies for diabetes. A key outstanding challenge is the translation of GWAS signals into disease biology and we outline possible solutions for tackling this experimental bottleneck.

Carrat GR, Hu M, Nguyen-Tu MS, Chabosseau P, Gaulton KJ, van de Bunt M, Siddiq A, Falchi M, Thurner M, Canouil M et al. 2017. Decreased STARD10 Expression Is Associated with Defective Insulin Secretion in Humans and Mice. Am J Hum Genet, 100 (2), pp. 238-256. | Show Abstract | Read more

Genetic variants near ARAP1 (CENTD2) and STARD10 influence type 2 diabetes (T2D) risk. The risk alleles impair glucose-induced insulin secretion and, paradoxically but characteristically, are associated with decreased proinsulin:insulin ratios, indicating improved proinsulin conversion. Neither the identity of the causal variants nor the gene(s) through which risk is conferred have been firmly established. Whereas ARAP1 encodes a GTPase activating protein, STARD10 is a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer protein family. By integrating genetic fine-mapping and epigenomic annotation data and performing promoter-reporter and chromatin conformational capture (3C) studies in β cell lines, we localize the causal variant(s) at this locus to a 5 kb region that overlaps a stretch-enhancer active in islets. This region contains several highly correlated T2D-risk variants, including the rs140130268 indel. Expression QTL analysis of islet transcriptomes from three independent subject groups demonstrated that T2D-risk allele carriers displayed reduced levels of STARD10 mRNA, with no concomitant change in ARAP1 mRNA levels. Correspondingly, β-cell-selective deletion of StarD10 in mice led to impaired glucose-stimulated Ca(2+) dynamics and insulin secretion and recapitulated the pattern of improved proinsulin processing observed at the human GWAS signal. Conversely, overexpression of StarD10 in the adult β cell improved glucose tolerance in high fat-fed animals. In contrast, manipulation of Arap1 in β cells had no impact on insulin secretion or proinsulin conversion in mice. This convergence of human and murine data provides compelling evidence that the T2D risk associated with variation at this locus is mediated through reduction in STARD10 expression in the β cell.

Thomsen SK, Ceroni A, van de Bunt M, Burrows C, Barrett A, Scharfmann R, Ebner D, McCarthy MI, Gloyn AL. 2016. Systematic Functional Characterization of Candidate Causal Genes for Type 2 Diabetes Risk Variants. Diabetes, 65 (12), pp. 3805-3811. | Show Abstract | Read more

Most genetic association signals for type 2 diabetes risk are located in noncoding regions of the genome, hindering translation into molecular mechanisms. Physiological studies have shown a majority of disease-associated variants to exert their effects through pancreatic islet dysfunction. Systematically characterizing the role of regional transcripts in β-cell function could identify the underlying disease-causing genes, but large-scale studies in human cellular models have previously been impractical. We developed a robust and scalable strategy based on arrayed gene silencing in the human β-cell line EndoC-βH1. In a screen of 300 positional candidates selected from 75 type 2 diabetes regions, each gene was assayed for effects on multiple disease-relevant phenotypes, including insulin secretion and cellular proliferation. We identified a total of 45 genes involved in β-cell function, pointing to possible causal mechanisms at 37 disease-associated loci. The results showed a strong enrichment for genes implicated in monogenic diabetes. Selected effects were validated in a follow-up study, including several genes (ARL15, ZMIZ1, and THADA) with previously unknown or poorly described roles in β-cell biology. We have demonstrated the feasibility of systematic functional screening in a human β-cell model and successfully prioritized plausible disease-causing genes at more than half of the regions investigated.

Fuchsberger C, Flannick J, Teslovich TM, Mahajan A, Agarwala V, Gaulton KJ, Ma C, Fontanillas P, Moutsianas L, McCarthy DJ et al. 2016. The genetic architecture of type 2 diabetes. Nature, 536 (7614), pp. 41-47. | Show Abstract | Read more

The genetic architecture of common traits, including the number, frequency, and effect sizes of inherited variants that contribute to individual risk, has been long debated. Genome-wide association studies have identified scores of common variants associated with type 2 diabetes, but in aggregate, these explain only a fraction of the heritability of this disease. Here, to test the hypothesis that lower-frequency variants explain much of the remainder, the GoT2D and T2D-GENES consortia performed whole-genome sequencing in 2,657 European individuals with and without diabetes, and exome sequencing in 12,940 individuals from five ancestry groups. To increase statistical power, we expanded the sample size via genotyping and imputation in a further 111,548 subjects. Variants associated with type 2 diabetes after sequencing were overwhelmingly common and most fell within regions previously identified by genome-wide association studies. Comprehensive enumeration of sequence variation is necessary to identify functional alleles that provide important clues to disease pathophysiology, but large-scale sequencing does not support the idea that lower-frequency variants have a major role in predisposition to type 2 diabetes.

Zeggini E, Gloyn AL, Hansen T. 2016. Insights into metabolic disease from studying genetics in isolated populations: stories from Greece to Greenland. Diabetologia, 59 (5), pp. 938-941. | Show Abstract | Read more

Over the last 10 years substantial progress has been made in our understanding of the genetic basis for type 2 diabetes and related traits. These developments have been facilitated by technological advancements that have allowed comprehensive genome-wide assessments of the impact of common genetic variation on disease risk. Current efforts are now focused on extending this to genetic variants in the rare and low-frequency spectrum by capitalising on next-generation sequencing technologies. This review discusses the important contributions that studies in isolated populations are making to this effort for diabetes and metabolic disease, drawing on specific examples from populations in Greece and Greenland. This review summarises a presentation given at the 'Exciting news in genetics of diabetes' symposium at the 2015 annual meeting of the EASD, with topics presented by Eleftheria Zeggini and Torben Hansen, and an overview by the Session Chair, Anna Gloyn.

Beer NL, Gloyn AL. 2016. Genome-edited human stem cell-derived beta cells: a powerful tool for drilling down on type 2 diabetes GWAS biology F1000Research, 5 pp. 1711-1711. | Show Abstract | Read more

© 2016 Beer NL and Gloyn AL. Type 2 diabetes (T2D) is a disease of pandemic proportions, one defined by acomplex aetiological mix of genetic, epigenetic, environmental, and lifestyle riskfactors. Whilst the last decade of T2D genetic research has identified morethan 100 loci showing strong statistical association with disease susceptibility,our inability to capitalise upon these signals reflects, in part, a lack ofappropriate human cell models for study. This review discusses the impact oftwo complementary, state-of-the-art technologies on T2D genetic research: thegeneration of stem cell-derived, endocrine pancreas-lineage cells and theediting of their genomes. Such models facilitate investigation ofdiabetes-associated genomic perturbations in a physiologically representativecell context and allow the role of both developmental and adult islet dysfunctionin T2D pathogenesis to be investigated. Accordingly, we interrogate the rolethat patient-derived induced pluripotent stem cell models are playing inunderstanding cellular dysfunction in monogenic diabetes, and howsite-specific nucleases such as the clustered regularly interspaced shortpalindromic repeats (CRISPR)-Cas9 system are helping to confirm genescrucial to human endocrine pancreas development. We also highlight the novelbiology gleaned in the absence of patient lines, including an ability to model thewhole phenotypic spectrum of diabetes phenotypes occurring both in utero andin adult cells, interrogating the non-coding 'islet regulome' for disease-causingperturbations, and understanding the role of other islet cell types in aberrantglycaemia. This article aims to reinforce the importance of investigating T2Dsignals in cell models reflecting appropriate species, genomic context,developmental time point, and tissue type.

Kim YJ, Lee J, Kim BJ, T2D-Genes Consortium, Park T. 2015. A new strategy for enhancing imputation quality of rare variants from next-generation sequencing data via combining SNP and exome chip data. BMC Genomics, 16 (1), pp. 1109. | Show Abstract | Read more

BACKGROUND: Rare variants have gathered increasing attention as a possible alternative source of missing heritability. Since next generation sequencing technology is not yet cost-effective for large-scale genomic studies, a widely used alternative approach is imputation. However, the imputation approach may be limited by the low accuracy of the imputed rare variants. To improve imputation accuracy of rare variants, various approaches have been suggested, including increasing the sample size of the reference panel, using sequencing data from study-specific samples (i.e., specific populations), and using local reference panels by genotyping or sequencing a subset of study samples. While these approaches mainly utilize reference panels, imputation accuracy of rare variants can also be increased by using exome chips containing rare variants. The exome chip contains 250 K rare variants selected from the discovered variants of about 12,000 sequenced samples. If exome chip data are available for previously genotyped samples, the combined approach using a genotype panel of merged data, including exome chips and SNP chips, should increase the imputation accuracy of rare variants. RESULTS: In this study, we describe a combined imputation which uses both exome chip and SNP chip data simultaneously as a genotype panel. The effectiveness and performance of the combined approach was demonstrated using a reference panel of 848 samples constructed using exome sequencing data from the T2D-GENES consortium and 5,349 sample genotype panels consisting of an exome chip and SNP chip. As a result, the combined approach increased imputation quality up to 11 %, and genomic coverage for rare variants up to 117.7 % (MAF < 1 %), compared to imputation using the SNP chip alone. Also, we investigated the systematic effect of reference panels on imputation quality using five reference panels and three genotype panels. The best performing approach was the combination of the study specific reference panel and the genotype panel of combined data. CONCLUSIONS: Our study demonstrates that combined datasets, including SNP chips and exome chips, enhances both the imputation quality and genomic coverage of rare variants.

van de Bunt M, Manning Fox JE, Dai X, Barrett A, Grey C, Li L, Bennett AJ, Johnson PR, Rajotte RV, Gaulton KJ et al. 2015. Transcript Expression Data from Human Islets Links Regulatory Signals from Genome-Wide Association Studies for Type 2 Diabetes and Glycemic Traits to Their Downstream Effectors. PLoS Genet, 11 (12), pp. e1005694. | Show Abstract | Read more

The intersection of genome-wide association analyses with physiological and functional data indicates that variants regulating islet gene transcription influence type 2 diabetes (T2D) predisposition and glucose homeostasis. However, the specific genes through which these regulatory variants act remain poorly characterized. We generated expression quantitative trait locus (eQTL) data in 118 human islet samples using RNA-sequencing and high-density genotyping. We identified fourteen loci at which cis-exon-eQTL signals overlapped active islet chromatin signatures and were coincident with established T2D and/or glycemic trait associations. ‎At some, these data provide an experimental link between GWAS signals and biological candidates, such as DGKB and ADCY5. At others, the cis-signals implicate genes with no prior connection to islet biology, including WARS and ZMIZ1. At the ZMIZ1 locus, we show that perturbation of ZMIZ1 expression in human islets and beta-cells influences exocytosis and insulin secretion, highlighting a novel role for ZMIZ1 in the maintenance of glucose homeostasis. Together, these findings provide a significant advance in the mechanistic insights of T2D and glycemic trait association loci.

Gaulton KJ, Ferreira T, Lee Y, Raimondo A, Mägi R, Reschen ME, Mahajan A, Locke A, Rayner NW, Robertson N et al. 2015. Genetic fine mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci. Nat Genet, 47 (12), pp. 1415-1425. | Show Abstract | Read more

We performed fine mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in or near KCNQ1. 'Credible sets' of the variants most likely to drive each distinct signal mapped predominantly to noncoding sequence, implying that association with T2D is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine mapping implicated rs10830963 as driving T2D association. We confirmed that the T2D risk allele for this SNP increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease.

Ferdaoussi M, Dai X, Jensen MV, Wang R, Peterson BS, Huang C, Ilkayeva O, Smith N, Miller N, Hajmrle C et al. 2015. Isocitrate-to-SENP1 signaling amplifies insulin secretion and rescues dysfunctional β cells. J Clin Invest, 125 (10), pp. 3847-3860. | Show Abstract | Read more

Insulin secretion from β cells of the pancreatic islets of Langerhans controls metabolic homeostasis and is impaired in individuals with type 2 diabetes (T2D). Increases in blood glucose trigger insulin release by closing ATP-sensitive K+ channels, depolarizing β cells, and opening voltage-dependent Ca2+ channels to elicit insulin exocytosis. However, one or more additional pathway(s) amplify the secretory response, likely at the distal exocytotic site. The mitochondrial export of isocitrate and engagement with cytosolic isocitrate dehydrogenase (ICDc) may be one key pathway, but the mechanism linking this to insulin secretion and its role in T2D have not been defined. Here, we show that the ICDc-dependent generation of NADPH and subsequent glutathione (GSH) reduction contribute to the amplification of insulin exocytosis via sentrin/SUMO-specific protease-1 (SENP1). In human T2D and an in vitro model of human islet dysfunction, the glucose-dependent amplification of exocytosis was impaired and could be rescued by introduction of signaling intermediates from this pathway. Moreover, islet-specific Senp1 deletion in mice caused impaired glucose tolerance by reducing the amplification of insulin exocytosis. Together, our results identify a pathway that links glucose metabolism to the amplification of insulin secretion and demonstrate that restoration of this axis rescues β cell function in T2D.

Chakera AJ, Steele AM, Gloyn AL, Shepherd MH, Shields B, Ellard S, Hattersley AT. 2015. Recognition and Management of Individuals With Hyperglycemia Because of a Heterozygous Glucokinase Mutation. Diabetes Care, 38 (7), pp. 1383-1392. | Show Abstract | Read more

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

Manning Fox JE, Lyon J, Dai XQ, Wright RC, Hayward J, van de Bunt M, Kin T, Shapiro AM, McCarthy MI, Gloyn AL et al. 2015. Human islet function following 20 years of cryogenic biobanking. Diabetologia, 58 (7), pp. 1503-1512. | Show Abstract | Read more

AIMS/HYPOTHESIS: There are potential advantages to the low-temperature (-196 °C) banking of isolated islets, including the maintenance of viable islets for future research. We therefore assessed the in vitro and in vivo function of islets cryopreserved for nearly 20 years. METHODS: Human islets were cryopreserved from 1991 to 2001 and thawed between 2012 and 2014. These were characterised by immunostaining, patch-clamp electrophysiology, insulin secretion, transcriptome analysis and transplantation into a streptozotocin (STZ)-induced mouse model of diabetes. RESULTS: The cryopreservation time was 17.6 ± 0.4 years (n = 43). The thawed islets stained positive with dithizone, contained insulin-positive and glucagon-positive cells, and displayed levels of apoptosis and transcriptome profiles similar to those of freshly isolated islets, although their insulin content was lower. The cryopreserved beta cells possessed ion channels and exocytotic responses identical to those of freshly isolated beta cells. Cells from a subset of five donors demonstrated similar perifusion insulin secretion profiles pre- and post-cryopreservation. The transplantation of cryopreserved islets into the diabetic mice improved their glucose tolerance but did not completely normalise their blood glucose levels. Circulating human insulin and insulin-positive grafts were detectable at 10 weeks post-transplantation. CONCLUSIONS/INTERPRETATION: We have demonstrated the potential for long-term banking of human islets for research, which could enable the use of tissue from a large number of donors with future technologies to gain new insight into diabetes.

Raimondo A, Rees MG, Gloyn AL. 2015. Glucokinase regulatory protein: complexity at the crossroads of triglyceride and glucose metabolism. Curr Opin Lipidol, 26 (2), pp. 88-95. | Show Abstract | Read more

PURPOSE OF REVIEW: Glucokinase regulator (GCKR) encodes glucokinase regulatory protein (GKRP), a hepatocyte-specific inhibitor of the glucose-metabolizing enzyme glucokinase (GCK). Genome-wide association studies have identified a common coding variant within GCKR associated with multiple metabolic traits. This review focuses on recent insights into the critical role of GKRP in hepatic glucose metabolism that have stemmed from the study of human genetics. This knowledge has improved our understanding of glucose and lipid physiology and informed the development of targeted molecular therapeutics for diabetes. RECENT FINDINGS: Rare GCKR variants have effects on GKRP expression, localization, and activity. These variants are collectively associated with hypertriglyceridaemia but are not causal. Crystal structures of GKRP and the GCK-GKRP complex have been solved, providing greater insight into the molecular interactions between these proteins. Finally, small molecules have been identified that directly bind GKRP and reduce blood glucose levels in rodent models of diabetes. SUMMARY: GCKR variants across the allelic spectrum have effects on glucose and lipid homeostasis. Functional analysis has highlighted numerous molecular mechanisms for GKRP dysfunction. Hepatocyte-specific GCK activation via small molecule GKRP inhibition may be a new avenue for type 2 diabetes treatment, particularly considering evidence indicating GKRP loss-of-function alone does not cause hypertriglyceridaemia.

Mahajan A, Sim X, Ng HJ, Manning A, Rivas MA, Highland HM, Locke AE, Grarup N, Im HK, Cingolani P et al. 2015. Identification and functional characterization of G6PC2 coding variants influencing glycemic traits define an effector transcript at the G6PC2-ABCB11 locus. PLoS Genet, 11 (1), pp. e1004876. | Show Abstract | Read more

Genome wide association studies (GWAS) for fasting glucose (FG) and insulin (FI) have identified common variant signals which explain 4.8% and 1.2% of trait variance, respectively. It is hypothesized that low-frequency and rare variants could contribute substantially to unexplained genetic variance. To test this, we analyzed exome-array data from up to 33,231 non-diabetic individuals of European ancestry. We found exome-wide significant (P<5×10-7) evidence for two loci not previously highlighted by common variant GWAS: GLP1R (p.Ala316Thr, minor allele frequency (MAF)=1.5%) influencing FG levels, and URB2 (p.Glu594Val, MAF = 0.1%) influencing FI levels. Coding variant associations can highlight potential effector genes at (non-coding) GWAS signals. At the G6PC2/ABCB11 locus, we identified multiple coding variants in G6PC2 (p.Val219Leu, p.His177Tyr, and p.Tyr207Ser) influencing FG levels, conditionally independent of each other and the non-coding GWAS signal. In vitro assays demonstrate that these associated coding alleles result in reduced protein abundance via proteasomal degradation, establishing G6PC2 as an effector gene at this locus. Reconciliation of single-variant associations and functional effects was only possible when haplotype phase was considered. In contrast to earlier reports suggesting that, paradoxically, glucose-raising alleles at this locus are protective against type 2 diabetes (T2D), the p.Val219Leu G6PC2 variant displayed a modest but directionally consistent association with T2D risk. Coding variant associations for glycemic traits in GWAS signals highlight PCSK1, RREB1, and ZHX3 as likely effector transcripts. These coding variant association signals do not have a major impact on the trait variance explained, but they do provide valuable biological insights.

Majithia AR, Flannick J, Shahinian P, Guo M, Bray MA, Fontanillas P, Gabriel SB, GoT2D Consortium, NHGRI JHS/FHS Allelic Spectrum Project, SIGMA T2D Consortium et al. 2014. Rare variants in PPARG with decreased activity in adipocyte differentiation are associated with increased risk of type 2 diabetes. Proc Natl Acad Sci U S A, 111 (36), pp. 13127-13132. | Show Abstract | Read more

Peroxisome proliferator-activated receptor gamma (PPARG) is a master transcriptional regulator of adipocyte differentiation and a canonical target of antidiabetic thiazolidinedione medications. In rare families, loss-of-function (LOF) mutations in PPARG are known to cosegregate with lipodystrophy and insulin resistance; in the general population, the common P12A variant is associated with a decreased risk of type 2 diabetes (T2D). Whether and how rare variants in PPARG and defects in adipocyte differentiation influence risk of T2D in the general population remains undetermined. By sequencing PPARG in 19,752 T2D cases and controls drawn from multiple studies and ethnic groups, we identified 49 previously unidentified, nonsynonymous PPARG variants (MAF < 0.5%). Considered in aggregate (with or without computational prediction of functional consequence), these rare variants showed no association with T2D (OR = 1.35; P = 0.17). The function of the 49 variants was experimentally tested in a novel high-throughput human adipocyte differentiation assay, and nine were found to have reduced activity in the assay. Carrying any of these nine LOF variants was associated with a substantial increase in risk of T2D (OR = 7.22; P = 0.005). The combination of large-scale DNA sequencing and functional testing in the laboratory reveals that approximately 1 in 1,000 individuals carries a variant in PPARG that reduces function in a human adipocyte differentiation assay and is associated with a substantial risk of T2D.

Chakera AJ, Raimondo A, Homsen S, Colclough K, Barrett A, De-Franco E, Chatelas A, van de Bunt M, Flanagan SE, Hattersley AT et al. 2014. The phenotypic severity of homozygous GCK mutations causing neonatal or adolescent-onset diabetes is mediated through thermostability in addition to enzyme activity DIABETOLOGIA, 57 pp. S155-S155.

Cited:

43

Scopus

Ng MCY, Shriner D, Chen BH, Li J, Chen W-M, Guo X, Liu J, Bielinski SJ, Yanek LR, Nalls MA et al. 2014. Meta-Analysis of Genome-Wide Association Studies in African Americans Provides Insights into the Genetic Architecture of Type 2 Diabetes PLoS Genetics, 10 (8), pp. e1004517-e1004517. | Read more

Raimondo A, Chakera AJ, Thomsen SK, Colclough K, Barrett A, De Franco E, Chatelas A, Demirbilek H, Akcay T, Alawneh H et al. 2014. Phenotypic severity of homozygous GCK mutations causing neonatal or childhood-onset diabetes is primarily mediated through effects on protein stability. Hum Mol Genet, 23 (24), pp. 6432-6440. | Show Abstract | Read more

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

Rees MG, Raimondo A, Wang J, Ban MR, Davis MI, Barrett A, Ranft J, Jagdhuhn D, Waterstradt R, Baltrusch S et al. 2014. Inheritance of rare functional GCKR variants and their contribution to triglyceride levels in families. Hum Mol Genet, 23 (20), pp. 5570-5578. | Show Abstract | Read more

Significant resources have been invested in sequencing studies to investigate the role of rare variants in complex disease etiology. However, the diagnostic interpretation of individual rare variants remains a major challenge, and may require accurate variant functional classification and the collection of large numbers of variant carriers. Utilizing sequence data from 458 individuals with hypertriglyceridemia and 333 controls with normal plasma triglyceride levels, we investigated these issues using GCKR, encoding glucokinase regulatory protein. Eighteen rare non-synonymous GCKR variants identified in these 791 individuals were comprehensively characterized by a range of biochemical and cell biological assays, including a novel high-throughput-screening-based approach capable of measuring all variant proteins simultaneously. Functionally deleterious variants were collectively associated with hypertriglyceridemia, but a range of in silico prediction algorithms showed little consistency between algorithms and poor agreement with functional data. We extended our study by obtaining sequence data on family members; however, functional variants did not co-segregate with triglyceride levels. Therefore, despite evidence for their collective functional and clinical relevance, our results emphasize the low predictive value of rare GCKR variants in individuals and the complex heritability of lipid traits.

Kavvoura FK, Raimondo A, Thanabalasingham G, Barrett A, Webster AL, Shears D, Mann NP, Ellard S, Gloyn AL, Owen KR. 2014. Reclassification of diabetes etiology in a family with multiple diabetes phenotypes. J Clin Endocrinol Metab, 99 (6), pp. E1067-E1071. | Show Abstract | Read more

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

Zelent B, Raimondo A, Barrett A, Buettger CW, Chen P, Gloyn AL, Matschinsky FM. 2014. Analysis of the co-operative interaction between the allosterically regulated proteins GK and GKRP using tryptophan fluorescence. Biochem J, 459 (3), pp. 551-564. | Show Abstract | Read more

Hepatic glucose phosphorylation by GK (glucokinase) is regulated by GKRP (GK regulatory protein). GKRP forms a cytosolic complex with GK followed by nuclear import and storage, leading to inhibition of GK activity. This process is initiated by low glucose, but reversed nutritionally by high glucose and fructose or pharmacologically by GKAs (GK activators) and GKRPIs (GKRP inhibitors). To study the regulation of this process by glucose, fructose-phosphate esters and a GKA, we measured the TF (tryptophan fluorescence) of human WT (wild-type) and GKRP-P446L (a mutation associated with high serum triacylglycerol) in the presence of non-fluorescent GK with its tryptophan residues mutated. Titration of GKRP-WT by GK resulted in a sigmoidal increase in TF, suggesting co-operative PPIs (protein-protein interactions) perhaps due to the hysteretic nature of GK. The affinity of GK for GKRP was decreased and binding co-operativity increased by glucose, fructose 1-phosphate and GKA, reflecting disruption of the GK-GKRP complex. Similar studies with GKRP-P446L showed significantly different results compared with GKRP-WT, suggesting impairment of complex formation and nuclear storage. The results of the present TF-based biophysical analysis of PPIs between GK and GKRP suggest that hepatic glucose metabolism is regulated by a metabolite-sensitive drug-responsive co-operative molecular switch, involving complex formation between these two allosterically regulated proteins.

Tattikota SG, Rathjen T, McAnulty SJ, Wessels HH, Akerman I, van de Bunt M, Hausser J, Esguerra JL, Musahl A, Pandey AK et al. 2014. Argonaute2 mediates compensatory expansion of the pancreatic β cell. Cell Metab, 19 (1), pp. 122-134. | Show Abstract | Read more

Pancreatic β cells adapt to compensate for increased metabolic demand during insulin resistance. Although the microRNA pathway has an essential role in β cell proliferation, the extent of its contribution is unclear. Here, we report that miR-184 is silenced in the pancreatic islets of insulin-resistant mouse models and type 2 diabetic human subjects. Reduction of miR-184 promotes the expression of its target Argonaute2 (Ago2), a component of the microRNA-induced silencing complex. Moreover, restoration of miR-184 in leptin-deficient ob/ob mice decreased Ago2 and prevented compensatory β cell expansion. Loss of Ago2 during insulin resistance blocked β cell growth and relieved the regulation of miR-375-targeted genes, including the growth suppressor Cadm1. Lastly, administration of a ketogenic diet to ob/ob mice rescued insulin sensitivity and miR-184 expression and restored Ago2 and β cell mass. This study identifies the targeting of Ago2 by miR-184 as an essential component of the compensatory response to regulate proliferation according to insulin sensitivity.

Rees MG, Davis MI, Shen M, Titus S, Raimondo A, Barrett A, Gloyn AL, Collins FS, Simeonov A. 2014. A panel of diverse assays to interrogate the interaction between glucokinase and glucokinase regulatory protein, two vital proteins in human disease. PLoS One, 9 (2), pp. e89335. | Show Abstract | Read more

Recent genetic and clinical evidence has implicated glucokinase regulatory protein (GKRP) in the pathogenesis of type 2 diabetes and related traits. The primary role of GKRP is to bind and inhibit hepatic glucokinase (GCK), a critically important protein in human health and disease that exerts a significant degree of control over glucose metabolism. As activation of GCK has been associated with improved glucose tolerance, perturbation of the GCK-GKRP interaction represents a potential therapeutic target for pharmacological modulation. Recent structural and kinetic advances are beginning to provide insight into the interaction of these two proteins. However, tools to comprehensively assess the GCK-GKRP interaction, particularly in the context of small molecules, would be a valuable resource. We therefore developed three robust and miniaturized assays for assessing the interaction between recombinant human GCK and GKRP: an HTRF assay, a diaphorase-coupled assay, and a luciferase-coupled assay. The assays are complementary, featuring distinct mechanisms of detection (luminescence, fluorescence, FRET). Two assays rely on GCK enzyme activity modulation by GKRP while the FRET-based assay measures the GCK-GKRP protein-protein interaction independent of GCK enzymatic substrates and activity. All three assays are scalable to low volumes in 1536-well plate format, with robust Z' factors (>0.7). Finally, as GKRP sequesters GCK in the hepatocyte nucleus at low glucose concentrations, we explored cellular models of GCK localization and translocation. Previous findings from freshly isolated rat hepatocytes were confirmed in cryopreserved rat hepatocytes, and we further extended this study to cryopreserved human hepatocytes. Consistent with previous reports, there were several key differences between the rat and human systems, with our results suggesting that human hepatocytes can be used to interrogate GCK translocation in response to small molecules. The assay panel developed here should help direct future investigation of the GCK-GKRP interaction in these or other physiologically relevant human systems.

Pasquali L, Gaulton KJ, Rodríguez-Seguí SA, Mularoni L, Miguel-Escalada I, Akerman İ, Tena JJ, Morán I, Gómez-Marín C, van de Bunt M et al. 2014. Pancreatic islet enhancer clusters enriched in type 2 diabetes risk-associated variants. Nat Genet, 46 (2), pp. 136-143. | Show Abstract | Read more

Type 2 diabetes affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. Pancreatic islet dysfunction is central in type 2 diabetes pathogenesis, and understanding islet genome regulation could therefore provide valuable mechanistic insights. We have now mapped and examined the function of human islet cis-regulatory networks. We identify genomic sequences that are targeted by islet transcription factors to drive islet-specific gene activity and show that most such sequences reside in clusters of enhancers that form physical three-dimensional chromatin domains. We find that sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers and identify trait-associated variants that disrupt DNA binding and islet enhancer activity. Our studies illustrate how islet transcription factors interact functionally with the epigenome and provide systematic evidence that the dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes.

Garg SK, Shah VN. 2014. Newer Therapies for Diabetes Management Diabetes Technology & Therapeutics, 16 (S1), pp. S-119-S-127. | Read more

Thomsen SK, Gloyn AL. 2014. The pancreatic β cell: Recent insights from human genetics Trends in Endocrinology and Metabolism, 25 (8), pp. 425-434. | Show Abstract | Read more

Diabetes mellitus is a metabolic disease characterised by relative or absolute pancreatic β cell dysfunction. Genetic variants implicated in disease risk can be identified by studying affected individuals. To understand the mechanisms driving genetic associations, variants must be translated through causative transcripts to biological insights. Studies into the genetic basis of Mendelian forms of diabetes have successfully identified genes involved in both β cell function and pancreatic development. For type 2 diabetes (T2D), genome-wide association studies (GWASs) are uncovering an ever-increasing number of susceptibility variants that exert their effect through β cell dysfunction, but translation to mechanistic understanding has in most cases been slow. Improved annotations of the islet genome and advances in whole-genome and -exome sequencing (WHS and WES) have facilitated recent progress. © 2014 Elsevier Ltd.

Thomsen SK, Gloyn AL. 2014. The pancreatic β cell: recent insights from human genetics. Trends Endocrinol Metab, 25 (8), pp. 425-434. | Show Abstract | Read more

Diabetes mellitus is a metabolic disease characterised by relative or absolute pancreatic β cell dysfunction. Genetic variants implicated in disease risk can be identified by studying affected individuals. To understand the mechanisms driving genetic associations, variants must be translated through causative transcripts to biological insights. Studies into the genetic basis of Mendelian forms of diabetes have successfully identified genes involved in both β cell function and pancreatic development. For type 2 diabetes (T2D), genome-wide association studies (GWASs) are uncovering an ever-increasing number of susceptibility variants that exert their effect through β cell dysfunction, but translation to mechanistic understanding has in most cases been slow. Improved annotations of the islet genome and advances in whole-genome and -exome sequencing (WHS and WES) have facilitated recent progress.

Gardner DS, Owen KR, Gloyn AL. 2014. Translating advances in our understanding of the genetics of diabetes into the clinic Frontiers in Diabetes, 23 pp. 173-186. | Read more

Rees MG, Gloyn AL. 2014. Translating genetic association signals for diabetes and metabolic traits into molecular mechanisms for disease Frontiers in Diabetes, 23 pp. 133-145. | Read more

Zhang Q, Ramracheya R, Lahmann C, Tarasov A, Bengtsson M, Braha O, Braun M, Brereton M, Collins S, Galvanovskis J et al. 2013. Role of KATP channels in glucose-regulated glucagon secretion and impaired counterregulation in type 2 diabetes. Cell Metab, 18 (6), pp. 871-882. | Show Abstract | Read more

Glucagon, secreted by pancreatic islet α cells, is the principal hyperglycemic hormone. In diabetes, glucagon secretion is not suppressed at high glucose, exacerbating the consequences of insufficient insulin secretion, and is inadequate at low glucose, potentially leading to fatal hypoglycemia. The causal mechanisms remain unknown. Here we show that α cell KATP-channel activity is very low under hypoglycemic conditions and that hyperglycemia, via elevated intracellular ATP/ADP, leads to complete inhibition. This produces membrane depolarization and voltage-dependent inactivation of the Na(+) channels involved in action potential firing that, via reduced action potential height and Ca(2+) entry, suppresses glucagon secretion. Maneuvers that increase KATP channel activity, such as metabolic inhibition, mimic the glucagon secretory defects associated with diabetes. Low concentrations of the KATP channel blocker tolbutamide partially restore glucose-regulated glucagon secretion in islets from type 2 diabetic organ donors. These data suggest that impaired metabolic control of the KATP channels underlies the defective glucose regulation of glucagon secretion in type 2 diabetes.

Ng HJ, Gloyn AL. 2013. Bridging the gap between genetic associations and molecular mechanisms for type 2 diabetes. Curr Diab Rep, 13 (6), pp. 778-785. | Show Abstract | Read more

Type 2 diabetes is a global pandemic for which there is currently no disease-modifying treatment. New and targeted therapeutics are greatly needed, but progress in identifying novel targets for therapeutic intervention is severely hampered by poor understanding of disease pathogenesis. Over the past 6 years, the success of genome-wide association studies has led to an unprecedented increase in the number of loci robustly associating with type 2 diabetes risk. Each of these signals offers the opportunity to uncover biological insights into disease pathogenesis, which, if harnessed effectively, hold the promise to deliver new pathways for therapeutic intervention, strategies for patient stratification, and potentially, biomarkers for identifying those at greatest risk of developing diabetes. We review the progress that has been made and the approaches being adopted and discuss the inherent challenges in moving from association signals, which largely map to poorly annotated sequence, to transcripts, mechanisms, and disease biology.

Hassanali N, Rundle JK, Thanabalasingham G, Owen KR, Wilson JF, McCarthy MI, Gloyn AL. 2013. Assessment of the pathogenicity of the previously reported D526N Hepatocyte Nuclear factor-1 alpha (HNF1A) variant using a combined genetic, in silico and functional approach DIABETIC MEDICINE, 30 pp. 61-61.

Kavvoura FK, Thanabalasingham G, Webster AL, Mann N, Ellard S, Gloyn AL, Owen KR. 2013. One is never enough: a case report of three different diabetes phenotypes in a single family DIABETIC MEDICINE, 30 pp. 1-1.

McCarthy MI, Rorsman P, Gloyn AL. 2013. TCF7L2 and diabetes: a tale of two tissues, and of two species. Cell Metab, 17 (2), pp. 157-159. | Show Abstract | Read more

Human genetics is revealing ever more variants that influence propensity to common diseases, but progress in translating these discoveries into the biological mechanisms responsible for predisposition continues to lag behind. A recent paper in Cell (Boj et al., 2012) using rodent models to examine how diabetes-associated variants near TCF7L2 perturb metabolic regulation provides surprising results.

van de Bunt M, Gaulton KJ, Parts L, Moran I, Johnson PR, Lindgren CM, Ferrer J, Gloyn AL, McCarthy MI. 2013. The miRNA profile of human pancreatic islets and beta-cells and relationship to type 2 diabetes pathogenesis. PLoS One, 8 (1), pp. e55272. | Show Abstract | Read more

Recent advances in the understanding of the genetics of type 2 diabetes (T2D) susceptibility have focused attention on the regulation of transcriptional activity within the pancreatic beta-cell. MicroRNAs (miRNAs) represent an important component of regulatory control, and have proven roles in the development of human disease and control of glucose homeostasis. We set out to establish the miRNA profile of human pancreatic islets and of enriched beta-cell populations, and to explore their potential involvement in T2D susceptibility. We used Illumina small RNA sequencing to profile the miRNA fraction in three preparations each of primary human islets and of enriched beta-cells generated by fluorescence-activated cell sorting. In total, 366 miRNAs were found to be expressed (i.e. >100 cumulative reads) in islets and 346 in beta-cells; of the total of 384 unique miRNAs, 328 were shared. A comparison of the islet-cell miRNA profile with those of 15 other human tissues identified 40 miRNAs predominantly expressed (i.e. >50% of all reads seen across the tissues) in islets. Several highly-expressed islet miRNAs, such as miR-375, have established roles in the regulation of islet function, but others (e.g. miR-27b-3p, miR-192-5p) have not previously been described in the context of islet biology. As a first step towards exploring the role of islet-expressed miRNAs and their predicted mRNA targets in T2D pathogenesis, we looked at published T2D association signals across these sites. We found evidence that predicted mRNA targets of islet-expressed miRNAs were globally enriched for signals of T2D association (p-values <0.01, q-values <0.1). At six loci with genome-wide evidence for T2D association (AP3S2, KCNK16, NOTCH2, SCL30A8, VPS26A, and WFS1) predicted mRNA target sites for islet-expressed miRNAs overlapped potentially causal variants. In conclusion, we have described the miRNA profile of human islets and beta-cells and provide evidence linking islet miRNAs to T2D pathogenesis.

Thanabalasingham G, Huffman JE, Kattla JJ, Novokmet M, Rudan I, Gloyn AL, Hayward C, Adamczyk B, Reynolds RM, Muzinic A et al. 2013. Mutations in HNF1A result in marked alterations of plasma glycan profile. Diabetes, 62 (4), pp. 1329-1337. | Show Abstract | Read more

A recent genome-wide association study identified hepatocyte nuclear factor 1-α (HNF1A) as a key regulator of fucosylation. We hypothesized that loss-of-function HNF1A mutations causal for maturity-onset diabetes of the young (MODY) would display altered fucosylation of N-linked glycans on plasma proteins and that glycan biomarkers could improve the efficiency of a diagnosis of HNF1A-MODY. In a pilot comparison of 33 subjects with HNF1A-MODY and 41 subjects with type 2 diabetes, 15 of 29 glycan measurements differed between the two groups. The DG9-glycan index, which is the ratio of fucosylated to nonfucosylated triantennary glycans, provided optimum discrimination in the pilot study and was examined further among additional subjects with HNF1A-MODY (n = 188), glucokinase (GCK)-MODY (n = 118), hepatocyte nuclear factor 4-α (HNF4A)-MODY (n = 40), type 1 diabetes (n = 98), type 2 diabetes (n = 167), and nondiabetic controls (n = 98). The DG9-glycan index was markedly lower in HNF1A-MODY than in controls or other diabetes subtypes, offered good discrimination between HNF1A-MODY and both type 1 and type 2 diabetes (C statistic ≥ 0.90), and enabled us to detect three previously undetected HNF1A mutations in patients with diabetes. In conclusion, glycan profiles are altered substantially in HNF1A-MODY, and the DG9-glycan index has potential clinical value as a diagnostic biomarker of HNF1A dysfunction.

Mughal SA, Park R, Nowak N, Gloyn AL, Karpe F, Matile H, Malecki MT, McCarthy MI, Stoffel M, Owen KR. 2013. Apolipoprotein M can discriminate HNF1A-MODY from Type 1 diabetes. Diabet Med, 30 (2), pp. 246-250. | Show Abstract | Read more

AIMS: Missed diagnosis of maturity-onset diabetes of the young (MODY) has led to an interest in biomarkers that enable efficient prioritization of patients for definitive molecular testing. Apolipoprotein M (apoM) was suggested as a biomarker for hepatocyte nuclear factor 1 alpha (HNF1A)-MODY because of its reduced expression in Hnf1a(-/-) mice. However, subsequent human studies examining apoM as a biomarker have yielded conflicting results. We aimed to evaluate apoM as a biomarker for HNF1A-MODY using a highly specific and sensitive ELISA. METHODS: ApoM concentration was measured in subjects with HNF1A-MODY (n = 69), Type 1 diabetes (n = 50), Type 2 diabetes (n = 120) and healthy control subjects (n = 100). The discriminative accuracy of apoM and of the apoM/HDL ratio for diabetes aetiology was evaluated. RESULTS: Mean (standard deviation) serum apoM concentration (μmol/l) was significantly lower for subjects with HNF1A-MODY [0.86 (0.29)], than for those with Type 1 diabetes [1.37 (0.26), P = 3.1 × 10(-18) ) and control subjects [1.34 (0.22), P = 7.2 × 10(-19) ). There was no significant difference in apoM concentration between subjects with HNF1A-MODY and Type 2 diabetes [0.89 (0.28), P = 0.13]. The C-statistic measure of discriminative accuracy for apoM was 0.91 for HNF1A-MODY vs. Type 1 diabetes, indicating high discriminative accuracy. The apoM/HDL ratio was significantly lower in HNF1A-MODY than other study groups. However, this ratio did not perform well in discriminating HNF1A-MODY from either Type 1 diabetes (C-statistic = 0.79) or Type 2 diabetes (C-statistic = 0.68). CONCLUSIONS: We confirm an earlier report that serum apoM levels are lower in HNF1A-MODY than in controls. Serum apoM provides good discrimination between HNF1A-MODY and Type 1 diabetes and warrants further investigation for clinical utility in diabetes diagnostics.

Travers ME, Mackay DJ, Dekker Nitert M, Morris AP, Lindgren CM, Berry A, Johnson PR, Hanley N, Groop LC, McCarthy MI, Gloyn AL. 2013. Insights into the molecular mechanism for type 2 diabetes susceptibility at the KCNQ1 locus from temporal changes in imprinting status in human islets. Diabetes, 62 (3), pp. 987-992. | Show Abstract | Read more

The molecular basis of type 2 diabetes predisposition at most established susceptibility loci remains poorly understood. KCNQ1 maps within the 11p15.5 imprinted domain, a region with an established role in congenital growth phenotypes. Variants intronic to KCNQ1 influence diabetes susceptibility when maternally inherited. By use of quantitative PCR and pyrosequencing of human adult islet and fetal pancreas samples, we investigated the imprinting status of regional transcripts and aimed to determine whether type 2 diabetes risk alleles influence regional DNA methylation and gene expression. The results demonstrate that gene expression patterns differ by developmental stage. CDKN1C showed monoallelic expression in both adult and fetal tissue, whereas PHLDA2, SLC22A18, and SLC22A18AS were biallelically expressed in both tissues. Temporal changes in imprinting were observed for KCNQ1 and KCNQ1OT1, with monoallelic expression in fetal tissues and biallelic expression in adult samples. Genotype at the type 2 diabetes risk variant rs2237895 influenced methylation levels of regulatory sequence in fetal pancreas but without demonstrable effects on gene expression. We demonstrate that CDKN1C, KCNQ1, and KCNQ1OT1 are most likely to mediate diabetes susceptibility at the KCNQ1 locus and identify temporal differences in imprinting status and methylation effects, suggesting that diabetes risk effects may be mediated in early development.

Morán I, Akerman I, van de Bunt M, Xie R, Benazra M, Nammo T, Arnes L, Nakić N, García-Hurtado J, Rodríguez-Seguí S et al. 2012. Human β cell transcriptome analysis uncovers lncRNAs that are tissue-specific, dynamically regulated, and abnormally expressed in type 2 diabetes. Cell Metab, 16 (4), pp. 435-448. | Show Abstract | Read more

A significant portion of the genome is transcribed as long noncoding RNAs (lncRNAs), several of which are known to control gene expression. The repertoire and regulation of lncRNAs in disease-relevant tissues, however, has not been systematically explored. We report a comprehensive strand-specific transcriptome map of human pancreatic islets and β cells, and uncover >1100 intergenic and antisense islet-cell lncRNA genes. We find islet lncRNAs that are dynamically regulated and show that they are an integral component of the β cell differentiation and maturation program. We sequenced the mouse islet transcriptome and identify lncRNA orthologs that are regulated like their human counterparts. Depletion of HI-LNC25, a β cell-specific lncRNA, downregulated GLIS3 mRNA, thus exemplifying a gene regulatory function of islet lncRNAs. Finally, selected islet lncRNAs were dysregulated in type 2 diabetes or mapped to genetic loci underlying diabetes susceptibility. These findings reveal a new class of islet-cell genes relevant to β cell programming and diabetes pathophysiology.

Raimondo A, Rees MG, Wang J, Barrett A, Collins FS, Hegele RA, Gloyn AL. 2012. Functional analysis of GCKR mutations identified in subjects with hypertriglyceridaemia demonstrates the inherent challenges in clinical interpretation of rare variants DIABETOLOGIA, 55 pp. S78-S78.

Pal A, Barber TM, Van de Bunt M, Rudge SA, Zhang Q, Lachlan KL, Cooper NS, Linden H, Levy JC, Wakelam MJ et al. 2012. PTEN mutations as a cause of constitutive insulin sensitivity and obesity. N Engl J Med, 367 (11), pp. 1002-1011. | Show Abstract | Read more

BACKGROUND: Epidemiologic and genetic evidence links type 2 diabetes, obesity, and cancer. The tumor-suppressor phosphatase and tensin homologue (PTEN) has roles in both cellular growth and metabolic signaling. Germline PTEN mutations cause a cancer-predisposition syndrome, providing an opportunity to study the effect of PTEN haploinsufficiency in humans. METHODS: We measured insulin sensitivity and beta-cell function in 15 PTEN mutation carriers and 15 matched controls. Insulin signaling was measured in muscle and adipose-tissue biopsy specimens from 5 mutation carriers and 5 well-matched controls. We also assessed the effect of PTEN haploinsufficiency on obesity by comparing anthropometric indexes between the 15 patients and 2097 controls from a population-based study of healthy adults. Body composition was evaluated by means of dual-emission x-ray absorptiometry and skinfold thickness. RESULTS: Measures of insulin resistance were lower in the patients with a PTEN mutation than in controls (e.g., mean fasting plasma insulin level, 29 pmol per liter [range, 9 to 99] vs. 74 pmol per liter [range, 22 to 185]; P=0.001). This finding was confirmed with the use of hyperinsulinemic euglycemic clamping, showing a glucose infusion rate among carriers 2 times that among controls (P=0.009). The patients' insulin sensitivity could be explained by the presence of enhanced insulin signaling through the PI3K-AKT pathway, as evidenced by increased AKT phosphorylation. The PTEN mutation carriers were obese as compared with population-based controls (mean body-mass index [the weight in kilograms divided by the square of the height in meters], 32 [range, 23 to 42] vs. 26 [range, 15 to 48]; P<0.001). This increased body mass in the patients was due to augmented adiposity without corresponding changes in fat distribution. CONCLUSIONS: PTEN haploinsufficiency is a monogenic cause of profound constitutive insulin sensitization that is apparently obesogenic. We demonstrate an apparently divergent effect of PTEN mutations: increased risks of obesity and cancer but a decreased risk of type 2 diabetes owing to enhanced insulin sensitivity. (Funded by the Wellcome Trust and others.).

Rees MG, Gloyn AL. 2013. Small molecular glucokinase activators: has another new anti-diabetic therapeutic lost favour? Br J Pharmacol, 168 (2), pp. 335-338. | Show Abstract | Read more

UNLABELLED: Glucokinase activators (GKAs) represent one of the leading hopes for the next generation of type 2 diabetes (T2D) therapeutics, showing efficacy in reducing blood glucose and HbA1c levels in animal models of T2D and short-term human trials. While the hypoglycaemic risks of GCK activation in pancreatic beta-cells have long been appreciated, the hepatic effects of GKAs have generally been perceived to be without significant side effect. In this issue of the British Journal of Pharmacology, De Ceuninck et al. report that acute and chronic GKA treatment of normoglycaemic and hyperglycaemic rodent models results in significant accumulation of triglycerides in the liver. This suggests GKA-mediated activation of hepatic glucose uptake and suppression of endogenous glucose production may come at a significant cost; namely, the development of hepatic steatosis. This raises important questions regarding the safety of GKAs and emphasizes that both plasma and hepatic lipid profiles should be carefully monitored in on-going and future studies of these molecules. LINKED ARTICLE: This article is a commentary on De Ceuninck et al., pp. 339-353 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2012.02184.x.

Kailey B, van de Bunt M, Cheley S, Johnson PR, MacDonald PE, Gloyn AL, Rorsman P, Braun M. 2012. SSTR2 is the functionally dominant somatostatin receptor in human pancreatic β- and α-cells. Am J Physiol Endocrinol Metab, 303 (9), pp. E1107-E1116. | Show Abstract | Read more

Somatostatin-14 (SST) inhibits insulin and glucagon secretion by activating G protein-coupled somatostatin receptors (SSTRs), of which five isoforms exist (SSTR1-5). In mice, the effects on pancreatic β-cells are mediated by SSTR5, whereas α-cells express SSTR2. In both cell types, SSTR activation results in membrane hyperpolarization and suppression of exocytosis. Here, we examined the mechanisms by which SST inhibits secretion from human β- and α-cells and the SSTR isoforms mediating these effects. Quantitative PCR revealed high expression of SSTR2, with lower levels of SSTR1, SSTR3, and SSTR5, in human islets. Immunohistochemistry showed expression of SSTR2 in both β- and α-cells. SST application hyperpolarized human β-cells and inhibited action potential firing. The membrane hyperpolarization was unaffected by tolbutamide but antagonized by tertiapin-Q, a blocker of G protein-gated inwardly rectifying K⁺ channels (GIRK). The effect of SST was mimicked by an SSTR2-selective agonist, whereas a SSTR5 agonist was marginally effective. SST strongly (>70%) reduced depolarization-evoked exocytosis in both β- and α-cells. A slightly weaker inhibition was observed in both cell types after SSTR2 activation. SSTR3- and SSTR1-selective agonists moderately reduced the exocytotic responses in β- and α-cells, respectively, whereas SSTR4- and SSTR5-specific agonists were ineffective. SST also reduced voltage-gated P/Q-type Ca²⁺ currents in β-cells, but normalization of Ca²⁺ influx to control levels by prolonged depolarizations only partially restored exocytosis. We conclude that SST inhibits secretion from both human β- and α-cells by activating GIRK and suppressing electrical activity, reducing P/Q-type Ca²⁺ currents, and directly inhibiting exocytosis. These effects are mediated predominantly by SSTR2 in both cell types.

Beer NL, Osbak KK, van de Bunt M, Tribble ND, Steele AM, Wensley KJ, Edghill EL, Colcough K, Barrett A, Valentínová L et al. 2012. Insights into the pathogenicity of rare missense GCK variants from the identification and functional characterization of compound heterozygous and double mutations inherited in cis. Diabetes Care, 35 (7), pp. 1482-1484. | Show Abstract | Read more

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

Rosengren AH, Braun M, Mahdi T, Andersson SA, Travers ME, Shigeto M, Zhang E, Almgren P, Ladenvall C, Axelsson AS et al. 2012. Reduced insulin exocytosis in human pancreatic β-cells with gene variants linked to type 2 diabetes. Diabetes, 61 (7), pp. 1726-1733. | Show Abstract | Read more

The majority of genetic risk variants for type 2 diabetes (T2D) affect insulin secretion, but the mechanisms through which they influence pancreatic islet function remain largely unknown. We functionally characterized human islets to determine secretory, biophysical, and ultrastructural features in relation to genetic risk profiles in diabetic and nondiabetic donors. Islets from donors with T2D exhibited impaired insulin secretion, which was more pronounced in lean than obese diabetic donors. We assessed the impact of 14 disease susceptibility variants on measures of glucose sensing, exocytosis, and structure. Variants near TCF7L2 and ADRA2A were associated with reduced glucose-induced insulin secretion, whereas susceptibility variants near ADRA2A, KCNJ11, KCNQ1, and TCF7L2 were associated with reduced depolarization-evoked insulin exocytosis. KCNQ1, ADRA2A, KCNJ11, HHEX/IDE, and SLC2A2 variants affected granule docking. We combined our results to create a novel genetic risk score for β-cell dysfunction that includes aberrant granule docking, decreased Ca(2+) sensitivity of exocytosis, and reduced insulin release. Individuals with a high risk score displayed an impaired response to intravenous glucose and deteriorating insulin secretion over time. Our results underscore the importance of defects in β-cell exocytosis in T2D and demonstrate the potential of cellular phenotypic characterization in the elucidation of complex genetic disorders.

Palmer ND, McDonough CW, Hicks PJ, Roh BH, Wing MR, An SS, Hester JM, Cooke JN, Bostrom MA, Rudock ME et al. 2012. A genome-wide association search for type 2 diabetes genes in African Americans. PLoS One, 7 (1), pp. e29202. | Show Abstract | Read more

African Americans are disproportionately affected by type 2 diabetes (T2DM) yet few studies have examined T2DM using genome-wide association approaches in this ethnicity. The aim of this study was to identify genes associated with T2DM in the African American population. We performed a Genome Wide Association Study (GWAS) using the Affymetrix 6.0 array in 965 African-American cases with T2DM and end-stage renal disease (T2DM-ESRD) and 1029 population-based controls. The most significant SNPs (n = 550 independent loci) were genotyped in a replication cohort and 122 SNPs (n = 98 independent loci) were further tested through genotyping three additional validation cohorts followed by meta-analysis in all five cohorts totaling 3,132 cases and 3,317 controls. Twelve SNPs had evidence of association in the GWAS (P<0.0071), were directionally consistent in the Replication cohort and were associated with T2DM in subjects without nephropathy (P<0.05). Meta-analysis in all cases and controls revealed a single SNP reaching genome-wide significance (P<2.5×10(-8)). SNP rs7560163 (P = 7.0×10(-9), OR (95% CI) = 0.75 (0.67-0.84)) is located intergenically between RND3 and RBM43. Four additional loci (rs7542900, rs4659485, rs2722769 and rs7107217) were associated with T2DM (P<0.05) and reached more nominal levels of significance (P<2.5×10(-5)) in the overall analysis and may represent novel loci that contribute to T2DM. We have identified novel T2DM-susceptibility variants in the African-American population. Notably, T2DM risk was associated with the major allele and implies an interesting genetic architecture in this population. These results suggest that multiple loci underlie T2DM susceptibility in the African-American population and that these loci are distinct from those identified in other ethnic populations.

van de Bunt M, Gloyn AL. 2012. A tale of two glucose transporters: how GLUT2 re-emerged as a contender for glucose transport into the human beta cell. Diabetologia, 55 (9), pp. 2312-2315. | Show Abstract | Read more

Finding novel causes for monogenic forms of diabetes is important as, alongside the clinical implications of such a discovery, it can identify critical proteins and pathways required for normal beta cell function in humans. It is increasingly apparent that there are significant differences between rodent and human islets. One example that has generated interest is the relative importance of the glucose transporter GLUT2 in rodent and human beta cells. The central role of GLUT2 in rodent beta cells is well established, but a number of studies have suggested that other glucose transporters, namely GLUT1 and GLUT3, may play an important role in facilitating glucose transport into human beta cells. In this issue of Diabetologia Sansbury et al (DOI: 10.1007/s00125-012-2595-0 ) report homozygous loss of function mutations in SLC2A2, which encodes GLUT2, as a rare cause of neonatal diabetes. Evidence for a beta cell defect in these subjects comes from very low birthweights, lack of endogenous insulin secretion and a requirement for insulin therapy. Neonatal diabetes is not a consistent feature of SLC2A2 mutations. It is only found in a small percentage of cases (~4%) and the diabetes largely resolves before 18 months of age. This discovery is significant as it suggests that GLUT2 plays an important role in human beta cells, but the interplay and relative roles of other transporters differ from those in rodents. This finding should encourage efforts to delineate the precise role of GLUT2 in the human beta cell at different developmental time points and is a further reminder of critical differences between human and rodent islets.

Valentínová L, Beer NL, Staník J, Tribble ND, van de Bunt M, Hučková M, Barrett A, Klimeš I, Gašperíková D, Gloyn AL. 2012. Identification and functional characterisation of novel glucokinase mutations causing maturity-onset diabetes of the young in Slovakia. PLoS One, 7 (4), pp. e34541. | Show Abstract | Read more

Heterozygous glucokinase (GCK) mutations cause a subtype of maturity-onset diabetes of the young (GCK-MODY). Over 600 GCK mutations have been reported of which ∼65% are missense. In many cases co-segregation has not been established and despite the importance of functional studies in ascribing pathogenicity for missense variants these have only been performed for <10% of mutations. The aim of this study was to determine the minimum prevalence of GCK-MODY amongst diabetic subjects in Slovakia by sequencing GCK in 100 Slovakian probands with a phenotype consistent with GCK-MODY and to explore the pathogenicity of identified variants through family and functional studies. Twenty-two mutations were identified in 36 families (17 missense) of which 7 (I110N, V200A, N204D, G258R, F419S, c.580-2A>C, c.1113-1114delGC) were novel. Parental DNA was available for 22 probands (covering 14/22 mutations) and co-segregation established in all cases. Bioinformatic analysis predicted all missense mutations to be damaging. Nine (I110N, V200A, N204D, G223S, G258R, F419S, V244G, L315H, I436N) mutations were functionally evaluated. Basic kinetic analysis explained pathogenicity for 7 mutants which showed reduced glucokinase activity with relative activity indices (RAI) between 0.6 to <0.001 compared to wild-type GCK (1.0). For the remaining 2 mutants additional molecular mechanisms were investigated. Differences in glucokinase regulatory protein (GKRP) -mediated-inhibition of GCK were observed for both L315H & I436N when compared to wild type (IC(50) 14.6±0.1 mM & 20.3±1.6 mM vs.13.3±0.1 mM respectively [p<0.03]). Protein instability as assessed by thermal lability studies demonstrated that both L315H and I436N show marked thermal instability compared to wild-type GCK (RAI at 55°C 8.8±0.8% & 3.1±0.4% vs. 42.5±3.9% respectively [p<0.001]). The minimum prevalence of GCK-MODY amongst Slovakian patients with diabetes was 0.03%. In conclusion, we have identified 22 GCK mutations in 36 Slovakian probands and demonstrate that combining family, bioinformatic and functional studies can aid the interpretation of variants identified by molecular diagnostic screening.

Gloyn AL, Faber JH, Malmodin D, Thanabalasingham G, Lam F, Ueland PM, McCarthy MI, Owen KR, Baunsgaard D. 2012. Metabolic profiling in Maturity-onset diabetes of the young (MODY) and young onset type 2 diabetes fails to detect robust urinary biomarkers. PLoS One, 7 (7), pp. e40962. | Show Abstract | Read more

It is important to identify patients with Maturity-onset diabetes of the young (MODY) as a molecular diagnosis determines both treatment and prognosis. Genetic testing is currently expensive and many patients are therefore not assessed and are misclassified as having either type 1 or type 2 diabetes. Biomarkers could facilitate the prioritisation of patients for genetic testing. We hypothesised that patients with different underlying genetic aetiologies for their diabetes could have distinct metabolic profiles which may uncover novel biomarkers. The aim of this study was to perform metabolic profiling in urine from patients with MODY due to mutations in the genes encoding glucokinase (GCK) or hepatocyte nuclear factor 1 alpha (HNF1A), type 2 diabetes (T2D) and normoglycaemic control subjects. Urinary metabolic profiling by Nuclear Magnetic Resonance (NMR) and ultra performance liquid chromatography hyphenated to Q-TOF mass spectrometry (UPLC-MS) was performed in a Discovery set of subjects with HNF1A-MODY (n = 14), GCK-MODY (n = 17), T2D (n = 14) and normoglycaemic controls (n = 34). Data were used to build a valid partial least squares discriminate analysis (PLS-DA) model where HNF1A-MODY subjects could be separated from the other diabetes subtypes. No single metabolite contributed significantly to the separation of the patient groups. However, betaine, valine, glycine and glucose were elevated in the urine of HNF1A-MODY subjects compared to the other subgroups. Direct measurements of urinary amino acids and betaine in an extended dataset did not support differences between patients groups. Elevated urinary glucose in HNF1A-MODY is consistent with the previously reported low renal threshold for glucose in this genetic subtype. In conclusion, we report the first metabolic profiling study in monogenic diabetes and show that, despite the distinct biochemical pathways affected, there are unlikely to be robust urinary biomarkers which distinguish monogenic subtypes from T2D. Our results have implications for studies investigating metabolic profiles in complex traits including T2D.

Dastani Z, Hivert MF, Timpson N, Perry JR, Yuan X, Scott RA, Henneman P, Heid IM, Kizer JR, Lyytikäinen LP et al. 2012. Novel loci for adiponectin levels and their influence on type 2 diabetes and metabolic traits: a multi-ethnic meta-analysis of 45,891 individuals. PLoS Genet, 8 (3), pp. e1002607. | Show Abstract | Read more

Circulating levels of adiponectin, a hormone produced predominantly by adipocytes, are highly heritable and are inversely associated with type 2 diabetes mellitus (T2D) and other metabolic traits. We conducted a meta-analysis of genome-wide association studies in 39,883 individuals of European ancestry to identify genes associated with metabolic disease. We identified 8 novel loci associated with adiponectin levels and confirmed 2 previously reported loci (P = 4.5×10(-8)-1.2×10(-43)). Using a novel method to combine data across ethnicities (N = 4,232 African Americans, N = 1,776 Asians, and N = 29,347 Europeans), we identified two additional novel loci. Expression analyses of 436 human adipocyte samples revealed that mRNA levels of 18 genes at candidate regions were associated with adiponectin concentrations after accounting for multiple testing (p<3×10(-4)). We next developed a multi-SNP genotypic risk score to test the association of adiponectin decreasing risk alleles on metabolic traits and diseases using consortia-level meta-analytic data. This risk score was associated with increased risk of T2D (p = 4.3×10(-3), n = 22,044), increased triglycerides (p = 2.6×10(-14), n = 93,440), increased waist-to-hip ratio (p = 1.8×10(-5), n = 77,167), increased glucose two hours post oral glucose tolerance testing (p = 4.4×10(-3), n = 15,234), increased fasting insulin (p = 0.015, n = 48,238), but with lower in HDL-cholesterol concentrations (p = 4.5×10(-13), n = 96,748) and decreased BMI (p = 1.4×10(-4), n = 121,335). These findings identify novel genetic determinants of adiponectin levels, which, taken together, influence risk of T2D and markers of insulin resistance.

Gloyn AL, Pal A, Karpe F. 2012. PTEN Haploinsufficiency, Obesity, and Insulin Sensitivity New England Journal of Medicine, 367 (25), pp. 2450-2451. | Read more

Rees MG, Ng D, Ruppert S, Turner C, Beer NL, Swift AJ, Morken MA, Below JE, Blech I, NISC Comparative Sequencing Program et al. 2012. Correlation of rare coding variants in the gene encoding human glucokinase regulatory protein with phenotypic, cellular, and kinetic outcomes. J Clin Invest, 122 (1), pp. 205-217. | Show Abstract | Read more

Defining the genetic contribution of rare variants to common diseases is a major basic and clinical science challenge that could offer new insights into disease etiology and provide potential for directed gene- and pathway-based prevention and treatment. Common and rare nonsynonymous variants in the GCKR gene are associated with alterations in metabolic traits, most notably serum triglyceride levels. GCKR encodes glucokinase regulatory protein (GKRP), a predominantly nuclear protein that inhibits hepatic glucokinase (GCK) and plays a critical role in glucose homeostasis. The mode of action of rare GCKR variants remains unexplored. We identified 19 nonsynonymous GCKR variants among 800 individuals from the ClinSeq medical sequencing project. Excluding the previously described common missense variant p.Pro446Leu, all variants were rare in the cohort. Accordingly, we functionally characterized all variants to evaluate their potential phenotypic effects. Defects were observed for the majority of the rare variants after assessment of cellular localization, ability to interact with GCK, and kinetic activity of the encoded proteins. Comparing the individuals with functional rare variants to those without such variants showed associations with lipid phenotypes. Our findings suggest that, while nonsynonymous GCKR variants, excluding p.Pro446Leu, are rare in individuals of mixed European descent, the majority do affect protein function. In sum, this study utilizes computational, cell biological, and biochemical methods to present a model for interpreting the clinical significance of rare genetic variants in common disease.

McDonald TJ, Owen KR, Gloyn AL, Hattersley AT. 2011. Response to Comment on: McDonald et al. High-Sensitivity CRP Discriminates HNF1A-MODY From Other Subtypes of Diabetes. Diabetes Care 2011;34:1860-1862. Diabetes Care, 34 (12), pp. e187. | Read more

Bown MJ, Jones GT, Harrison SC, Wright BJ, Bumpstead S, Baas AF, Gretarsdottir S, Badger SA, Bradley DT, Burnand K et al. 2011. Abdominal aortic aneurysm is associated with a variant in low-density lipoprotein receptor-related protein 1. Am J Hum Genet, 89 (5), pp. 619-627. | Show Abstract | Read more

Abdominal aortic aneurysm (AAA) is a common cause of morbidity and mortality and has a significant heritability. We carried out a genome-wide association discovery study of 1866 patients with AAA and 5435 controls and replication of promising signals (lead SNP with a p value < 1 × 10(-5)) in 2871 additional cases and 32,687 controls and performed further follow-up in 1491 AAA and 11,060 controls. In the discovery study, nine loci demonstrated association with AAA (p < 1 × 10(-5)). In the replication sample, the lead SNP at one of these loci, rs1466535, located within intron 1 of low-density-lipoprotein receptor-related protein 1 (LRP1) demonstrated significant association (p = 0.0042). We confirmed the association of rs1466535 and AAA in our follow-up study (p = 0.035). In a combined analysis (6228 AAA and 49182 controls), rs1466535 had a consistent effect size and direction in all sample sets (combined p = 4.52 × 10(-10), odds ratio 1.15 [1.10-1.21]). No associations were seen for either rs1466535 or the 12q13.3 locus in independent association studies of coronary artery disease, blood pressure, diabetes, or hyperlipidaemia, suggesting that this locus is specific to AAA. Gene-expression studies demonstrated a trend toward increased LRP1 expression for the rs1466535 CC genotype in arterial tissues; there was a significant (p = 0.029) 1.19-fold (1.04-1.36) increase in LRP1 expression in CC homozygotes compared to TT homozygotes in aortic adventitia. Functional studies demonstrated that rs1466535 might alter a SREBP-1 binding site and influence enhancer activity at the locus. In conclusion, this study has identified a biologically plausible genetic variant associated specifically with AAA, and we suggest that this variant has a possible functional role in LRP1 expression.

Hollingsworth MT, Hart GW, Paulson JC, Stansell E, Canis K, Huang IC, Panico M, Morris H, Haslam S, Farzan M et al. 2011. Program and abstracts for the 2011 Meeting of the Society for Glycobiology. Glycobiology, 21 (11), pp. 1454-1531. | Show Abstract | Read more

Cell surface mucins configure the cell surface by presenting extended protein backbones that are heavily O-glycosylated. The glycopeptide structures establish physicochemical properties at the cell surface that enable and block the formation of biologically important molecular complexes. Some mucins, such as MUC1, associate with receptor tyrosine kinases and other cell surface receptors, and engage in signal transduction in order to communicate information regarding conditions at the cell surface to the nucleus. In that context, the MUC1 cytoplasmic tail (MUC1CT) receives phosphorylation signals from receptor tyrosine kinases and serine/threonine kinases, which enables its association with different signaling complexes that conduct these signals to the nucleus and perhaps other subcellular organelles. We have detected the MUC1CT at promoters of over 500 genes, in association with several different transcription factors, and have shown that promoter occupancy can vary under different growth factor conditions. However, the full biochemical nature of the nuclear forms of MUC1 and its function at these promoter regions remain undefined. I will present evidence that nuclear forms of the MUC1CT include extracellular and cytoplasmic tail domains. In addition, I will discuss evidence for a hypothesis that the MUC1CT possesses a novel catalytic function that enables remodeling of the transcription factor occupancy of promoters, and thereby engages in regulation of gene expression.

Rees MG, Wincovitch S, Schultz J, Waterstradt R, Beer NL, Baltrusch S, Collins FS, Gloyn AL. 2012. Cellular characterisation of the GCKR P446L variant associated with type 2 diabetes risk. Diabetologia, 55 (1), pp. 114-122. | Show Abstract | Read more

AIMS/HYPOTHESIS: Translation of genetic association signals into molecular mechanisms for diabetes has been slow. The glucokinase regulatory protein (GKRP; gene symbol GCKR) P446L variant, associated with inverse modulation of glucose- and lipid-related traits, has been shown to alter the kinetics of glucokinase (GCK) inhibition. As GCK inhibition is associated with nuclear sequestration, we aimed to determine whether this variant also alters the direct interaction between GKRP and GCK and their intracellular localisation. METHODS: Fluorescently tagged rat and human wild-type (WT)- or P446L-GCKR and GCK were transiently transfected into HeLa cells and mouse primary hepatocytes. Whole-cell and nuclear fluorescence was quantified in individual cells exposed to low- or high-glucose conditions (5.5 or 25 mmol/l glucose, respectively). Interaction between GCK and GKRP was measured by sensitised emission-based fluorescence resonance energy transfer (FRET) efficiency. RESULTS: P446L-GKRP had a decreased degree of nuclear localisation, ability to sequester GCK and direct interaction with GCK as measured by FRET compared with WT-GKRP. Decreased interaction was observed between WT-GKRP and GCK at high compared with low glucose, but not between P446L-GKRP and GCK. Rat WT-GKRP and P446L-GKRP behaved quite differently: both variants responded to high glucose by diminished sequestration of GCK but showed no effect of the P446L variant on nuclear localisation or GCK sequestration. CONCLUSIONS/INTERPRETATION: Our study suggests the common human P446L-GKRP variant protein results in elevated hepatic glucose uptake and disposal by increasing active cytosolic GCK. This would increase hepatic lipid biosynthesis but decrease fasting plasma glucose concentrations and provides a potential mechanism for the protective effect of this allele on type 2 diabetes risk.

McCulloch LJ, van de Bunt M, Braun M, Frayn KN, Clark A, Gloyn AL. 2011. GLUT2 (SLC2A2) is not the principal glucose transporter in human pancreatic beta cells: implications for understanding genetic association signals at this locus. Mol Genet Metab, 104 (4), pp. 648-653. | Show Abstract | Read more

SLC2A2 encoding glucose transporter -2 (GLUT2) acts as the primary glucose transporter and sensor in rodent pancreatic islets and is widely assumed to play a similar role in humans. In healthy adults SLC2A2 variants are associated with elevated fasting plasma glucose (fpg) concentrations but physiological characterisation does not support a defect in pancreatic beta-cell function. Interspecies differences can create barriers for the follow up of disease association signals. We hypothesised that GLUT2 is not the principal glucose transporter in human beta-cells and that SLC2A2 variants exert their effect on fpg levels through defects in other tissues. SLC2A1-4 (GLUT 1-4) mRNA expression levels were determined in human and mouse islets, beta-cells, liver, muscle and adipose tissue by qRT-PCR whilst GLUT1-3 protein levels were examined by immunohistochemistry. The presence of all three glucose transporters was demonstrated in human and mouse islets and purified beta-cells. Quantitative expression profiling demonstrated that Slc2a2 is the predominant glucose transporter (expression >10 fold higher that Slc2a1) in mouse islets whilst SLC2A1 and SLC2A3 predominate in both human islets and beta-cells (expression 2.8 and 2.7 fold higher than SLC2A2 respectively). Our data therefore suggest that GLUT2 is unlikely to be the principal glucose transporter in human beta-cells and that SLC2A2 defects in other metabolic tissues drive the observed differences in glucose levels between carriers of SLC2A2 variants. Direct extrapolation from rodent to human islet glucose transporter activity is unlikely to be appropriate.

Thanabalasingham G, Shah N, Vaxillaire M, Hansen T, Tuomi T, Gašperíková D, Szopa M, Tjora E, James TJ, Kokko P et al. 2011. A large multi-centre European study validates high-sensitivity C-reactive protein (hsCRP) as a clinical biomarker for the diagnosis of diabetes subtypes. Diabetologia, 54 (11), pp. 2801-2810. | Show Abstract | Read more

AIMS/HYPOTHESIS: An accurate molecular diagnosis of diabetes subtype confers clinical benefits; however, many individuals with monogenic diabetes remain undiagnosed. Biomarkers could help to prioritise patients for genetic investigation. We recently demonstrated that high-sensitivity C-reactive protein (hsCRP) levels are lower in UK patients with hepatocyte nuclear factor 1 alpha (HNF1A)-MODY than in other diabetes subtypes. In this large multi-centre study we aimed to assess the clinical validity of hsCRP as a diagnostic biomarker, examine the genotype-phenotype relationship and compare different hsCRP assays. METHODS: High-sensitivity CRP levels were analysed in individuals with HNF1A-MODY (n = 457), glucokinase (GCK)-MODY (n = 404), hepatocyte nuclear factor 4 alpha (HNF4A)-MODY (n = 54) and type 2 diabetes (n = 582) from seven European centres. Three common assays for hsCRP analysis were evaluated. We excluded 121 participants (8.1%) with hsCRP values >10 mg/l. The discriminative power of hsCRP with respect to diabetes aetiology was assessed by receiver operating characteristic curve-derived C-statistic. RESULTS: In all centres and irrespective of the assay method, meta-analysis confirmed significantly lower hsCRP levels in those with HNF1A-MODY than in those with other aetiologies (z score -21.8, p < 5 × 10(-105)). HNF1A-MODY cases with missense mutations had lower hsCRP levels than those with truncating mutations (0.03 vs 0.08 mg/l, p < 5 × 10(-5)). High-sensitivity CRP values between assays were strongly correlated (r (2) ≥ 0.91, p ≤ 1 × 10(-5)). Across the seven centres, the C-statistic for distinguishing HNF1A-MODY from young adult-onset type 2 diabetes ranged from 0.79 to 0.97, indicating high discriminative accuracy. CONCLUSIONS/INTERPRETATION: In the largest study to date, we have established that hsCRP is a clinically valid biomarker for HNF1A-MODY in European populations. Given the modest costs and wide availability, hsCRP could translate rapidly into clinical practice, considerably improving diagnosis rates in monogenic diabetes.

Pal A, Godsland I, Owen KR, Whitaker L, Bishop T, Newton-Bishop J, McCarthy MI, Gloyn AL. 2011. Investigating the role of the recently described Type 2 diabetes associated gene CDKN2A (p16) on pancreatic islet function using a human model of CDKN2A haploinsufficiency (vol 28, pg 53, 2011) DIABETIC MEDICINE, 28 (7), pp. 881-881. | Read more

McDonald TJ, Shields BM, Lawry J, Owen KR, Gloyn AL, Ellard S, Hattersley AT. 2011. High-sensitivity CRP discriminates HNF1A-MODY from other subtypes of diabetes. Diabetes Care, 34 (8), pp. 1860-1862. | Show Abstract | Read more

OBJECTIVE: Maturity-onset diabetes of the young (MODY) as a result of mutations in hepatocyte nuclear factor 1-α (HNF1A) is often misdiagnosed as type 1 diabetes or type 2 diabetes. Recent work has shown that high-sensitivity C-reactive protein (hs-CRP) levels are lower in HNF1A-MODY than type 1 diabetes, type 2 diabetes, or glucokinase (GCK)-MODY. We aim to replicate these findings in larger numbers and other MODY subtypes. RESEARCH DESIGN AND METHODS: hs-CRP levels were assessed in 750 patients (220 HNF1A, 245 GCK, 54 HNF4-α [HNF4A], 21 HNF1-β (HNF1B), 53 type 1 diabetes, and 157 type 2 diabetes). RESULTS: hs-CRP was lower in HNF1A-MODY (median [IQR] 0.3 [0.1-0.6] mg/L) than type 2 diabetes (1.40 [0.60-3.45] mg/L; P < 0.001) and type 1 diabetes (1.10 [0.50-1.85] mg/L; P < 0.001), HNF4A-MODY (1.45 [0.46-2.88] mg/L; P < 0.001), GCK-MODY (0.60 [0.30-1.80] mg/L; P < 0.001), and HNF1B-MODY (0.60 [0.10-2.8] mg/L; P = 0.07). hs-CRP discriminated HNF1A-MODY from type 2 diabetes with hs-CRP <0.75 mg/L showing 79% sensitivity and 70% specificity (receiver operating characteristic area under the curve = 0.84). CONCLUSIONS: hs-CRP levels are lower in HNF1A-MODY than other forms of diabetes and may be used as a biomarker to select patients for diagnostic HNF1A genetic testing.

Steele AM, Tribble ND, Caswell R, Wensley KJ, Hattersley AT, Gloyn AL, Ellard S. 2011. The previously reported T342P GCK missense variant is not a pathogenic mutation causing MODY. Diabetologia, 54 (8), pp. 2202-2205. | Read more

Beer NL, van de Bunt M, Colclough K, Lukacs C, Arundel P, Chik CL, Grimsby J, Ellard S, Gloyn AL. 2011. Discovery of a novel site regulating glucokinase activity following characterization of a new mutation causing hyperinsulinemic hypoglycemia in humans. J Biol Chem, 286 (21), pp. 19118-19126. | Show Abstract | Read more

Type 2 diabetes is a global problem, and current ineffective therapeutic strategies pave the way for novel treatments like small molecular activators targeting glucokinase (GCK). GCK activity is fundamental to beta cell and hepatocyte glucose metabolism, and heterozygous activating and inactivating GCK mutations cause hyperinsulinemic hypoglycemia (HH) and maturity onset diabetes of the young (MODY) respectively. Over 600 naturally occurring inactivating mutations have been reported, whereas only 13 activating mutations are documented to date. We report two novel GCK HH mutations (V389L and T103S) at residues where MODY mutations also occur (V389D and T103I). Using recombinant proteins with in vitro assays, we demonstrated that both HH mutants had a greater relative activity index than wild type (6.0 for V389L, 8.4 for T103S, and 1.0 for wild type). This was driven by an increased affinity for glucose (S(0.5), 3.3 ± 0.1 and 3.5 ± 0.1 mm, respectively) versus wild type (7.5 ± 0.1 mm). Correspondingly, the V389D and T103I MODY mutants had markedly reduced relative activity indexes (<0.1). T103I had an altered affinity for glucose (S(0.5), 24.9 ± 0.6 mm), whereas V389D also exhibited a reduced affinity for ATP and decreased catalysis rate (S(0.5), 78.6 ± 4.5 mm; ATP(K(m)), 1.5 ± 0.1 mm; K(cat), 10.3 ± 1.1s(-1)) compared with wild type (ATP(K(m)), 0.4 ± <0.1; K(cat), 62.9 ± 1.2). Both Thr-103 mutants showed reduced inhibition by the endogenous hepatic inhibitor glucokinase regulatory protein. Molecular modeling demonstrated that Thr-103 maps to the allosteric activator site, whereas Val-389 is located remotely to this position and all other previously reported activating mutations, highlighting α-helix 11 as a novel region regulating GCK activity. Our data suggest that pharmacological manipulation of GCK activity at locations distal from the allosteric activator site is possible.

Cited:

48

Scopus

Thanabalasingham G, Shah N, Vaxillaire M, Hansen T, Tuomi T, Gašperíková D, Szopa M, Tjora E, James TJ, Kokko P et al. 2011. A large multi-centre European study validates high-sensitivity C-reactive protein (hsCRP) as a clinical biomarker for the diagnosis of diabetes subtypes Diabetologia, 54 (11), pp. 2801-2810. | Show Abstract | Read more

Aims/hypothesis: An accurate molecular diagnosis of diabetes subtype confers clinical benefits; however, many individuals with monogenic diabetes remain undiagnosed. Biomarkers could help to prioritise patients for genetic investigation. We recently demonstrated that high-sensitivity C-reactive protein (hsCRP) levels are lower in UK patients with hepatocyte nuclear factor 1 alpha (HNF1A)-MODY than in other diabetes subtypes. In this large multi-centre study we aimed to assess the clinical validity of hsCRP as a diagnostic biomarker, examine the genotype-phenotype relationship and compare different hsCRP assays. Methods: High-sensitivity CRP levels were analysed in individuals with HNF1A-MODY (n = 457), glucokinase (GCK)-MODY (n = 404), hepatocyte nuclear factor 4 alpha (HNF4A)-MODY (n = 54) and type 2 diabetes (n = 582) from seven European centres. Three common assays for hsCRP analysis were evaluated. We excluded 121 participants (8.1%) with hsCRP values > 10 mg/l. The discriminative power of hsCRP with respect to diabetes aetiology was assessed by receiver operating characteristic curve-derived C-statistic. Results: In all centres and irrespective of the assay method, meta-analysis confirmed significantly lower hsCRP levels in those with HNF1A-MODY than in those with other aetiologies (z score -21.8, p < 5 × 10 -105 ). HNF1A-MODY cases with missense mutations had lower hsCRP levels than those with truncating mutations (0.03 vs 0.08 mg/l, p < 5 × 10 -5 ). High-sensitivity CRP values between assays were strongly correlated (r 2 ≥ 0.91, p ≤ 1 × 10 -5 ). Across the seven centres, the C-statistic for distinguishing HNF1A-MODY from young adult-onset type 2 diabetes ranged from 0.79 to 0.97, indicating high discriminative accuracy. Conclusions/interpretation: In the largest study to date, we have established that hsCRP is a clinically valid biomarker for HNF1A-MODY in European populations. Given the modest costs and wide availability, hsCRP could translate rapidly into clinical practice, considerably improving diagnosis rates in monogenic diabetes. © 2011 Springer-Verlag.

Jafar-Mohammadi B, Groves CJ, Gjesing AP, Herrera BM, Winckler W, Stringham HM, Morris AP, Lauritzen T, Doney ASF, Morris AD et al. 2011. A role for coding functional variants in HNF4A in type 2 diabetes susceptibility Diabetologia, 54 (1), pp. 111-119. | Show Abstract | Read more

Aims/hypothesis: Rare mutations in the gene HNF4A, encoding the transcription factor hepatocyte nuclear factor 4α (HNF-4A), account for ∼5% of cases of MODY and more frequent variants in this gene may be involved in multifactorial forms of diabetes. Two low-frequency, non-synonymous variants in HNF4A (V255M, minor allele frequency [MAF] ∼0.1%; T130I, MAF ∼3.0%) - known to influence downstream HNF-4A target gene expression - are of interest, but previous type 2 diabetes association reports were inconclusive. We aimed to evaluate the contribution of these variants to type 2 diabetes susceptibility through large-scale association analysis. Methods: We genotyped both variants in at least 5,745 cases and 14,756 population controls from the UK and Denmark. We also undertook an expanded association analysis that included previously reported and novel genotype data obtained in Danish, Finnish, Canadian and Swedish samples. A meta-analysis incorporating all published association studies of the T130I variant was subsequently carried out in a maximum sample size of 14,279 cases and 26,835 controls. Results: We found no association between V255M and type 2 diabetes in either the initial (p=0.28) or the expanded analysis (p=0.44). However, T130I demonstrated a modest association with type 2 diabetes in the UK and Danish samples (additive per allele OR 1.17 [95% CI 1.08-1.28] ; p=1.5×10 -4 ), which was strengthened in the metaanalysis (OR 1.20 [95% CI 1.10-1.30]; p=2.1×10 -5 ). Conclusions/ interpretation: Our data are consistent with T130I as a low-frequency variant influencing type 2 diabetes risk, but are not conclusive when judged against stringent standards for genome-wide significance. This study exemplifies the difficulties encountered in association testing of low-frequency variants. © Springer-Verlag 2010.

Rees MG, Wincovitch S, Schultz J, Waterstradt R, Beer NL, Baltrusch S, Collins FS, Gloyn AL. 2011. Cellular characterisation of the GCKR P446L variant associated with type 2 diabetes risk Diabetologia, pp. 1-9.

Cited:

56

Scopus

Neville MJ, Collins JM, Gloyn AL, McCarthy MI, Karpe F. 2011. Comprehensive human adipose tissue mRNA and MicroRNA endogenous control selection for quantitative real-time-PCR normalization Obesity, 19 (4), pp. 888-892. | Show Abstract | Read more

The accurate quantification of cellular and tissue mRNA and microRNA content is reliant upon the selection of stable endogenous control transcripts for normalizing quantitative real-time-PCR (qRT-PCR) data. Using the combination of unbiased and informed approaches and a wide range of human adipose tissues and cells, we sought to identify invariant control transcripts for mRNA and microRNA. A total of 26 mRNA transcript candidates were selected from the literature. MicroRNA candidates were selected from a microRNA-microarray (Agilent, n = 22 tissues), and together with candidates from the literature resulted in 14 different microRNAs. The variability of these mRNA and microRNA transcripts were then tested in a large (n = 180) collection of a variety of human adipose tissues and cell samples. Phosphoglycerate kinase-1 (PGK1) and peptidylprolyl isomerase A (PPIA) were identified as the most stable mRNAs across all tissues and panels. MiR-103 was overall the most stable microRNA transcript across all biological backgrounds. Several proposed and commonly used normalization transcripts were found to be highly variable. We then tested the effect on expression of two established adipocyte-related transcripts (fatty acid binding protein 4 (FABP4) and microRNA-145 (miR-145)), either normalized to the optimal or a commonly used controls transcript. This test clearly indicated that spurious results could arise from using less stable control transcripts for mRNA and microRNA qRT-PCR. © 2010 The Obesity Society.

Voight BF, Scott LJ, Steinthorsdottir V, Morris AP, Dina C, Welch RP, Zeggini E, Huth C, Aulchenko YS, Thorleifsson G et al. 2011. Corrigendum: Twelve type 2 diabetes susceptibility loci identified through large-scale association analysis. Nat Genet, 43 (4), pp. 388. | Read more

Strawbridge RJ, Dupuis J, Prokopenko I, Barker A, Ahlqvist E, Rybin D, Petrie JR, Travers ME, Bouatia-Naji N, Dimas AS et al. 2011. Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes. Diabetes, 60 (10), pp. 2624-2634. | Show Abstract | Read more

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis.

McCulloch LJ, van de Bunt M, Braun M, Frayn KN, Clark A, Gloyn AL. 2011. GLUT2 (SLC2A2) is not the principal glucose transporter in human pancreatic beta cells: Implications for understanding genetic association signals at this locus Molecular Genetics and Metabolism,

Steele AM, Tribble ND, Caswell R, Wensley KJ, Hattersley AT, Gloyn AL, Ellard S. 2011. The previously reported T342P GCK missense variant is not a pathogenic mutation causing MODY Diabetologia, 54 (8), pp. 2202-2205. | Read more

Jafar-Mohammadi B, Groves CJ, Owen KR, Frayling TM, Hattersley AT, McCarthy MI, Gloyn AL. 2010. Role of low frequency variants in HNF1A exons 8-10 encoding hepatocyte nuclear factor-1 alpha isoform A to susceptibility to Type 2 diabetes IRISH JOURNAL OF MEDICAL SCIENCE, 179 pp. 538-538.

Neville MJ, Collins JM, Gloyn AL, McCarthy MI, Karpe F. 2011. Comprehensive human adipose tissue mRNA and microRNA endogenous control selection for quantitative real-time-PCR normalization. Obesity (Silver Spring), 19 (4), pp. 888-892. | Show Abstract | Read more

The accurate quantification of cellular and tissue mRNA and microRNA content is reliant upon the selection of stable endogenous control transcripts for normalizing quantitative real-time-PCR (qRT-PCR) data. Using the combination of unbiased and informed approaches and a wide range of human adipose tissues and cells, we sought to identify invariant control transcripts for mRNA and microRNA. A total of 26 mRNA transcript candidates were selected from the literature. MicroRNA candidates were selected from a microRNA-microarray (Agilent, n = 22 tissues), and together with candidates from the literature resulted in 14 different microRNAs. The variability of these mRNA and microRNA transcripts were then tested in a large (n = 180) collection of a variety of human adipose tissues and cell samples. Phosphoglycerate kinase-1 (PGK1) and peptidylprolyl isomerase A (PPIA) were identified as the most stable mRNAs across all tissues and panels. MiR-103 was overall the most stable microRNA transcript across all biological backgrounds. Several proposed and commonly used normalization transcripts were found to be highly variable. We then tested the effect on expression of two established adipocyte-related transcripts (fatty acid binding protein 4 (FABP4) and microRNA-145 (miR-145)), either normalized to the optimal or a commonly used controls transcript. This test clearly indicated that spurious results could arise from using less stable control transcripts for mRNA and microRNA qRT-PCR.

Jafar-Mohammadi B, Groves CJ, Gjesing AP, Herrera BM, Winckler W, Stringham HM, Morris AP, Lauritzen T, Doney AS, Morris AD et al. 2011. A role for coding functional variants in HNF4A in type 2 diabetes susceptibility. Diabetologia, 54 (1), pp. 111-119. | Show Abstract | Read more

AIMS/HYPOTHESIS: Rare mutations in the gene HNF4A, encoding the transcription factor hepatocyte nuclear factor 4α (HNF-4A), account for ~5% of cases of MODY and more frequent variants in this gene may be involved in multifactorial forms of diabetes. Two low-frequency, non-synonymous variants in HNF4A (V255M, minor allele frequency [MAF] ~0.1%; T130I, MAF ~3.0%)-known to influence downstream HNF-4A target gene expression-are of interest, but previous type 2 diabetes association reports were inconclusive. We aimed to evaluate the contribution of these variants to type 2 diabetes susceptibility through large-scale association analysis. METHODS: We genotyped both variants in at least 5,745 cases and 14,756 population controls from the UK and Denmark. We also undertook an expanded association analysis that included previously reported and novel genotype data obtained in Danish, Finnish, Canadian and Swedish samples. A meta-analysis incorporating all published association studies of the T130I variant was subsequently carried out in a maximum sample size of 14,279 cases and 26,835 controls. RESULTS: We found no association between V255M and type 2 diabetes in either the initial (p = 0.28) or the expanded analysis (p = 0.44). However, T130I demonstrated a modest association with type 2 diabetes in the UK and Danish samples (additive per allele OR 1.17 [95% CI 1.08-1.28]; p = 1.5 × 10⁻⁴), which was strengthened in the meta-analysis (OR 1.20 [95% CI 1.10-1.30]; p = 2.1 × 10⁻⁵). CONCLUSIONS/INTERPRETATION: Our data are consistent with T130I as a low-frequency variant influencing type 2 diabetes risk, but are not conclusive when judged against stringent standards for genome-wide significance. This study exemplifies the difficulties encountered in association testing of low-frequency variants.

van de Bunt M, Gloyn AL. 2010. From genetic association to molecular mechanism. Curr Diab Rep, 10 (6), pp. 452-466. | Show Abstract | Read more

Over the past 3 years, there has been a dramatic increase in the number of confirmed type 2 diabetes (T2D) susceptibility loci, most arising through the implementation of genome-wide association studies (GWAS). However, progress toward the understanding of disease mechanisms has been slowed by modest effect sizes and the fact that most GWAS signals map away from coding sequence: the presumption is that their effects are mediated through regulation of nearby transcripts, but the identities of the genes concerned are often far from clear. In this review we describe the progress that has been made to date in translating association signals into molecular mechanisms with a focus on the most tractable signals (eg, KCNJ11/ABCC8, SLC30A8, GCKR) and those in which human, animal, and cellular models (FTO, TCF7L2, G6PC2) have provided insights into the role in T2D pathogenesis. Finally, the challenges for the field with the advent of genome-scale next-generation resequencing efforts are discussed.

Owen KR, Thanabalasingham G, James TJ, Karpe F, Farmer AJ, McCarthy MI, Gloyn AL. 2010. Assessment of high-sensitivity C-reactive protein levels as diagnostic discriminator of maturity-onset diabetes of the young due to HNF1A mutations. Diabetes Care, 33 (9), pp. 1919-1924. | Show Abstract | Read more

OBJECTIVE: Despite the clinical importance of an accurate diagnosis in individuals with monogenic forms of diabetes, restricted access to genetic testing leaves many patients with undiagnosed diabetes. Recently, common variation near the HNF1 homeobox A (HNF1A) gene was shown to influence C-reactive protein levels in healthy adults. We hypothesized that serum levels of high-sensitivity C-reactive protein (hs-CRP) could represent a clinically useful biomarker for the identification of HNF1A mutations causing maturity-onset diabetes of the young (MODY). RESEARCH DESIGN AND METHODS: Serum hs-CRP was measured in subjects with HNF1A-MODY (n = 31), autoimmune diabetes (n = 316), type 2 diabetes (n = 240), and glucokinase (GCK) MODY (n = 24) and in nondiabetic individuals (n = 198). The discriminative accuracy of hs-CRP was evaluated through receiver operating characteristic (ROC) curve analysis, and performance was compared with standard diagnostic criteria. Our primary analyses excluded approximately 11% of subjects in whom the single available hs-CRP measurement was >10 mg/l. RESULTS: Geometric mean (SD range) hs-CRP levels were significantly lower (P <or= 0.009) for HNF1A-MODY individuals, 0.20 (0.03-1.14) mg/l, than for any other group: autoimmune diabetes 0.58 (0.10-2.75) mg/l, type 2 diabetes 1.33 (0.28-6.14) mg/l, GCK-MODY 1.01 (0.19-5.33) mg/l, and nondiabetic 0.48 (0.10-2.42) mg/l. The ROC-derived C-statistic for discriminating HNF1A-MODY and type 2 diabetes was 0.8. Measurement of hs-CRP, either alone or in combination with current diagnostic criteria, was superior to current diagnostic criteria alone. Sensitivity and specificity for the combined criteria approached 80%. CONCLUSIONS: Serum hs-CRP levels are markedly lower in HNF1A-MODY than in other forms of diabetes. hs-CRP has potential as a widely available, cost-effective screening test to support more precise targeting of MODY diagnostic testing.

Gloyn AL, McCarthy MI. 2010. Variation across the allele frequency spectrum. Nat Genet, 42 (8), pp. 648-650. | Show Abstract | Read more

A new study finds that individuals with high plasma triglyceride levels carry approximately twice as many rare, coding genetic variants within four candidate genes identified through genome-wide association studies than individuals without these high levels. This study demonstrates the overlap of rare and common variant signals at loci associated with lipid levels and shows the value of efforts to extend susceptibility variant discovery to embrace the full allele-frequency spectrum. © 2010 Nature America, Inc. All rights reserved.

Cited:

1027

Scopus

Voight BF, Scott LJ, Steinthorsdottir V, Morris AP, Dina C, Welch RP, Zeggini E, Huth C, Aulchenko YS, Thorleifsson G et al. 2010. Twelve type 2 diabetes susceptibility loci identified through large-scale association analysis NATURE GENETICS, 42 (7), pp. 579-U155. | Show Abstract | Read more

By combining genome-wide association data from 8,130 individuals with type 2 diabetes (T2D) and 38,987 controls of European descent and following up previously unidentified meta-analysis signals in a further 34,412 cases and 59,925 controls, we identified 12 new T2D association signals with combined P 5 × 10 8. These include a second independent signal at the KCNQ1 locus; the first report, to our knowledge, of an X-chromosomal association (near DUSP9); and a further instance of overlap between loci implicated in monogenic and multifactorial forms of diabetes (at HNF1A). The identified loci affect both beta-cell function and insulin action, and, overall, T2D association signals show evidence of enrichment for genes involved in cell cycle regulation. We also show that a high proportion of T2D susceptibility loci harbor independent association signals influencing apparently unrelated complex traits. © 2010 Nature America, Inc. All rights reserved.

Voight BF, Scott LJ, Steinthorsdottir V, Morris AP, Dina C, Welch RP, Zeggini E, Huth C, Aulchenko YS, Thorleifsson G et al. 2010. Twelve type 2 diabetes susceptibility loci identified through large-scale association analysis. Nat Genet, 42 (7), pp. 579-589. | Show Abstract | Read more

By combining genome-wide association data from 8,130 individuals with type 2 diabetes (T2D) and 38,987 controls of European descent and following up previously unidentified meta-analysis signals in a further 34,412 cases and 59,925 controls, we identified 12 new T2D association signals with combined P<5x10(-8). These include a second independent signal at the KCNQ1 locus; the first report, to our knowledge, of an X-chromosomal association (near DUSP9); and a further instance of overlap between loci implicated in monogenic and multifactorial forms of diabetes (at HNF1A). The identified loci affect both beta-cell function and insulin action, and, overall, T2D association signals show evidence of enrichment for genes involved in cell cycle regulation. We also show that a high proportion of T2D susceptibility loci harbor independent association signals influencing apparently unrelated complex traits.

Herrera BM, Lockstone HE, Taylor JM, Ria M, Barrett A, Collins S, Kaisaki P, Argoud K, Fernandez C, Travers ME et al. 2010. Global microRNA expression profiles in insulin target tissues in a spontaneous rat model of type 2 diabetes. Diabetologia, 53 (6), pp. 1099-1109. | Show Abstract | Read more

AIMS/HYPOTHESIS: MicroRNAs regulate a broad range of biological mechanisms. To investigate the relationship between microRNA expression and type 2 diabetes, we compared global microRNA expression in insulin target tissues from three inbred rat strains that differ in diabetes susceptibility. METHODS: Using microarrays, we measured the expression of 283 microRNAs in adipose, liver and muscle tissue from hyperglycaemic (Goto-Kakizaki), intermediate glycaemic (Wistar Kyoto) and normoglycaemic (Brown Norway) rats (n = 5 for each strain). Expression was compared across strains and validated using quantitative RT-PCR. Furthermore, microRNA expression variation in adipose tissue was investigated in 3T3-L1 adipocytes exposed to hyperglycaemic conditions. RESULTS: We found 29 significantly differentiated microRNAs (p(adjusted) < 0.05): nine in adipose tissue, 18 in liver and two in muscle. Of these, five microRNAs had expression patterns that correlated with the strain-specific glycaemic phenotype. MiR-222 (p(adjusted) = 0.0005) and miR-27a (p(adjusted) = 0.006) were upregulated in adipose tissue; miR-195 (p(adjusted) = 0.006) and miR-103 (p(adjusted) = 0.04) were upregulated in liver; and miR-10b (p(adjusted) = 0.004) was downregulated in muscle. Exposure of 3T3-L1 adipocytes to increased glucose concentration upregulated the expression of miR-222 (p = 0.008), miR-27a (p = 0.02) and the previously reported miR-29a (p = 0.02). Predicted target genes of these differentially expressed microRNAs are involved in pathways relevant to type 2 diabetes. CONCLUSION: The expression patterns of miR-222, miR-27a, miR-195, miR-103 and miR-10b varied with hyperglycaemia, suggesting a role for these microRNAs in the pathophysiology of type 2 diabetes, as modelled by the Gyoto-Kakizaki rat. We observed similar patterns of expression of miR-222, miR-27a and miR-29a in adipocytes as a response to increased glucose levels, which supports our hypothesis that altered expression of microRNAs accompanies primary events related to the pathogenesis of type 2 diabetes.

Dupuis J, Langenberg C, Prokopenko I, Saxena R, Soranzo N, Jackson AU, Wheeler E, Glazer NL, Bouatia-Naji N, Gloyn AL et al. 2010. New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk. Nat Genet, 42 (2), pp. 105-116. | Show Abstract | Read more

Levels of circulating glucose are tightly regulated. To identify new loci influencing glycemic traits, we performed meta-analyses of 21 genome-wide association studies informative for fasting glucose, fasting insulin and indices of beta-cell function (HOMA-B) and insulin resistance (HOMA-IR) in up to 46,186 nondiabetic participants. Follow-up of 25 loci in up to 76,558 additional subjects identified 16 loci associated with fasting glucose and HOMA-B and two loci associated with fasting insulin and HOMA-IR. These include nine loci newly associated with fasting glucose (in or near ADCY5, MADD, ADRA2A, CRY2, FADS1, GLIS3, SLC2A2, PROX1 and C2CD4B) and one influencing fasting insulin and HOMA-IR (near IGF1). We also demonstrated association of ADCY5, PROX1, GCK, GCKR and DGKB-TMEM195 with type 2 diabetes. Within these loci, likely biological candidate genes influence signal transduction, cell proliferation, development, glucose-sensing and circadian regulation. Our results demonstrate that genetic studies of glycemic traits can identify type 2 diabetes risk loci, as well as loci containing gene variants that are associated with a modest elevation in glucose levels but are not associated with overt diabetes.

Dupuis J, Langenberg C, Prokopenko I, Saxena R, Soranzo N, Jackson AU, Wheeler E, Glazer NL, Bouatia-Naji N, Gloyn AL et al. 2010. Erratum: New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk Nature Genetics, 42 (5), pp. 464-464. | Read more

Ding SY, Tribble ND, Kraft CA, Markwardt M, Gloyn AL, Rizzo MA. 2010. Naturally occurring glucokinase mutations are associated with defects in posttranslational S-nitrosylation. Mol Endocrinol, 24 (1), pp. 171-177. | Show Abstract | Read more

Posttranslational activation of glucokinase (GCK) through S-nitrosylation has been recently observed in the insulin-secreting pancreatic beta-cell; however, the function of this molecular mechanism in regulating the physiology of insulin secretion is not well understood. To more fully understand the function of posttranslational regulation of GCK, we examined two naturally occurring GCK mutations that map to residues proximal to the S-nitrosylated cysteine and cause mild fasting hyperglycemia (maturity-onset diabetes of the young; subtype glucokinase). The kinetics of recombinantly generated GCK-R369P and GCK-V367M were assessed in vitro. The GCK-R369P protein has greatly reduced catalytic activity (relative activity index 0.05 vs. 1.00 for wild type), whereas the GCK-V367M has near normal kinetics (relative activity index 1.26 vs. 1.00 for wild type). Quantitative imaging and biochemical assays were used to assess the effect of these mutants on the metabolic response to glucose, GCK activation, and S-nitrosylation of GCK in betaTC3 insulinoma cells. Expression of either mutant in betaTC3 cells did not affect the metabolic response to 5 mM glucose. However, expression of either mutant blocked the effects of insulin on glucose-stimulated nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate reduction, suggesting defects in posttranslational regulation of GCK. Each of these mutations blocked GCK activation, and prevented posttranslational cysteine S-nitrosylation. Our findings link defects in hormone-regulated GCK S-nitrosylation to hyperglycemia and support a role for posttranslational regulation of GCK S-nitrosylation as a vital regulatory mechanism for glucose-stimulated insulin secretion.

Kong A, Steinthorsdottir V, Masson G, Thorleifsson G, Sulem P, Besenbacher S, Jonasdottir A, Sigurdsson A, Kristinsson KT, Jonasdottir A et al. 2009. Parental origin of sequence variants associated with complex diseases. Nature, 462 (7275), pp. 868-874. | Show Abstract | Read more

Effects of susceptibility variants may depend on from which parent they are inherited. Although many associations between sequence variants and human traits have been discovered through genome-wide associations, the impact of parental origin has largely been ignored. Here we show that for 38,167 Icelanders genotyped using single nucleotide polymorphism (SNP) chips, the parental origin of most alleles can be determined. For this we used a combination of genealogy and long-range phasing. We then focused on SNPs that associate with diseases and are within 500 kilobases of known imprinted genes. Seven independent SNP associations were examined. Five-one with breast cancer, one with basal-cell carcinoma and three with type 2 diabetes-have parental-origin-specific associations. These variants are located in two genomic regions, 11p15 and 7q32, each harbouring a cluster of imprinted genes. Furthermore, we observed a novel association between the SNP rs2334499 at 11p15 and type 2 diabetes. Here the allele that confers risk when paternally inherited is protective when maternally transmitted. We identified a differentially methylated CTCF-binding site at 11p15 and demonstrated correlation of rs2334499 with decreased methylation of that site.

Suliman SG, Stanik J, McCulloch LJ, Wilson N, Edghill EL, Misovicova N, Gasperikova D, Sandrikova V, Elliott KS, Barak L et al. 2009. Severe insulin resistance and intrauterine growth deficiency associated with haploinsufficiency for INSR and CHN2: new insights into synergistic pathways involved in growth and metabolism. Diabetes, 58 (12), pp. 2954-2961. | Show Abstract | Read more

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

Pal A, Farmer AJ, Dudley C, Selwood MP, Barrow BA, Klyne R, Grew JP, McCarthy MI, Gloyn AL, Owen KR. 2010. Evaluation of serum 1,5 anhydroglucitol levels as a clinical test to differentiate subtypes of diabetes. Diabetes Care, 33 (2), pp. 252-257. | Show Abstract | Read more

OBJECTIVE: Assignment of the correct molecular diagnosis in diabetes is necessary for informed decisions regarding treatment and prognosis. Better clinical markers would facilitate discrimination and prioritization for genetic testing between diabetes subtypes. Serum 1,5 anhydroglucitol (1,5AG) levels were reported to differentiate maturity-onset diabetes of the young due to HNF1A mutations (HNF1A-MODY) from type 2 diabetes, but this requires further validation. We evaluated serum 1,5AG in a range of diabetes subtypes as an adjunct for defining diabetes etiology. RESEARCH DESIGN AND METHODS: 1,5AG was measured in U.K. subjects with: HNF1A-MODY (n = 23), MODY due to glucokinase mutations (GCK-MODY, n = 23), type 1 diabetes (n = 29), latent autoimmune diabetes in adults (LADA, n = 42), and type 2 diabetes (n = 206). Receiver operating characteristic curve analysis was performed to assess discriminative accuracy of 1,5AG for diabetes etiology. RESULTS: Mean (SD range) 1,5AG levels were: GCK-MODY 13.06 microg/ml (5.74-29.74), HNF1A-MODY 4.23 microg/ml (2.12-8.44), type 1 diabetes 3.09 microg/ml (1.45-6.57), LADA 3.46 microg/ml (1.42-8.45), and type 2 diabetes 5.43 (2.12-13.23). Levels in GCK-MODY were higher than in other groups (P < 10(-4) vs. each group). HNF1A-MODY subjects showed no difference in unadjusted 1,5AG levels from type 2 diabetes, type 1 diabetes, and LADA. Adjusting for A1C revealed a difference between HNF1A-MODY and type 2 diabetes (P = 0.001). The discriminative accuracy of unadjusted 1,5AG levels was 0.79 for GCK-MODY versus type 2 diabetes and 0.86 for GCK-MODY versus HNF1A-MODY but was only 0.60 for HNF1A-MODY versus type 2 diabetes. CONCLUSIONS: In our dataset, serum 1,5AG performed well in discriminating GCK-MODY from other diabetes subtypes, particularly HNF1A-MODY. Measurement of 1,5AG levels could inform decisions regarding MODY diagnostic testing.

Gloyn AL. 2009. RD Lawrence Lecture 2009. Old genes, new tricks: learning about blood glucose regulation from naturally occurring genetic variation in humans. Diabet Med, 26 (11), pp. 1083-1089. | Show Abstract | Read more

The study of rare monogenic forms of diabetes and pancreatic B-cell dysfunction provides an unrivalled opportunity to link a specific change in gene function with precise cellular consequences and clinical phenotype in humans. Over the past 20 years there has been considerable success in determining the genetic aetiology of a number of rare monogenic forms of diabetes, which has had a significant impact on both our understanding of normal physiology and on translational medicine. The impact of these discoveries has been substantial, with insights into both developmental biology and normal physiology. There are clear examples where determining the genetic aetiology for individuals with rare monogenic subtypes of diabetes has led to improved treatment. Although formerly in the shadow of the monogenic diabetes field, over the past 3 years there has been staggering progress in our understanding of the genetic basis of Type 2 diabetes. This has been largely as a result of genome-wide association studies and has seen the list of 'diabetes susceptibility genes' increase from three to close to 20. There is now encouraging evidence to support a potential role for genetics in determining the response of individuals with Type 2 diabetes to different therapeutic options. One of the challenges that lies ahead is determining how the non-coding genetic variants exert their pathogenicity. It is possible that parallels can be drawn from functional work on rare regulatory mutations causing monogenic forms of diabetes. However, it is more likely that comprehensive approaches will be necessary.

Beer NL, Tribble ND, McCulloch LJ, Roos C, Johnson PR, Orho-Melander M, Gloyn AL. 2009. The P446L variant in GCKR associated with fasting plasma glucose and triglyceride levels exerts its effect through increased glucokinase activity in liver. Hum Mol Genet, 18 (21), pp. 4081-4088. | Show Abstract | Read more

Genome-wide association studies have identified a number of signals for both Type 2 Diabetes and related quantitative traits. For the majority of loci, the transition from association signal to mutational mechanism has been difficult to establish. Glucokinase (GCK) regulates glucose storage and disposal in the liver where its activity is regulated by glucokinase regulatory protein (GKRP; gene name GCKR). Fructose-6 and fructose-1 phosphate (F6P and F1P) enhance or reduce GKRP-mediated inhibition, respectively. A common GCKR variant (P446L) is reproducibly associated with triglyceride and fasting plasma glucose levels in the general population. The aim of this study was to determine the mutational mechanism responsible for this genetic association. Recombinant human GCK and both human wild-type (WT) and P446L-GKRP proteins were generated. GCK kinetic activity was observed spectrophotometrically using an NADP(+)-coupled assay. WT and P446L-GKRP-mediated inhibition of GCK activity and subsequent regulation by phosphate esters were determined. Assays matched for GKRP activity demonstrated no difference in dose-dependent inhibition of GCK activity or F1P-mediated regulation. However, the response to physiologically relevant F6P levels was significantly attenuated with P446L-GKRP (n = 18; P <or= 0.03). Experiments using equimolar concentrations of both regulatory proteins confirmed these findings (n = 9; P < 0.001). In conclusion, P446L-GKRP has reduced regulation by physiological concentrations of F6P, resulting indirectly in increased GCK activity. Altered GCK regulation in liver is predicted to enhance glycolytic flux, promoting hepatic glucose metabolism and elevating concentrations of malonyl-CoA, a substrate for de novo lipogenesis, providing a mutational mechanism for the reported association of this variant with raised triglycerides and lower glucose levels.

Osbak KK, Colclough K, Saint-Martin C, Beer NL, Bellanné-Chantelot C, Ellard S, Gloyn AL. 2009. Update on mutations in glucokinase (GCK), which cause maturity-onset diabetes of the young, permanent neonatal diabetes, and hyperinsulinemic hypoglycemia. Hum Mutat, 30 (11), pp. 1512-1526. | Show Abstract | Read more

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

Hamming KS, Soliman D, Matemisz LC, Niazi O, Lang Y, Gloyn AL, Light PE. 2009. Coexpression of the type 2 diabetes susceptibility gene variants KCNJ11 E23K and ABCC8 S1369A alter the ATP and sulfonylurea sensitivities of the ATP-sensitive K(+) channel. Diabetes, 58 (10), pp. 2419-2424. | Show Abstract | Read more

OBJECTIVE: In the pancreatic beta-cell, ATP-sensitive K(+) (K(ATP)) channels couple metabolism with excitability and consist of Kir6.2 and SUR1 subunits encoded by KCNJ11 and ABCC8, respectively. Sulfonylureas, which inhibit the K(ATP) channel, are used to treat type 2 diabetes. Rare activating mutations cause neonatal diabetes, whereas the common variants, E23K in KCNJ11 and S1369A in ABCC8, are in strong linkage disequilibrium, constituting a haplotype that predisposes to type 2 diabetes. To date it has not been possible to establish which of these represents the etiological variant, and functional studies are inconsistent. Furthermore, there have been no studies of the S1369A variant or the combined effect of the two on K(ATP) channel function. RESEARCH DESIGN AND METHODS: The patch-clamp technique was used to study the nucleotide sensitivity and sulfonylurea inhibition of recombinant human K(ATP) channels containing either the K23/A1369 or E23/S1369 variants. RESULTS: ATP sensitivity of the K(ATP) channel was decreased in the K23/A1369 variant (half-maximal inhibitory concentration [IC(50)] = 8.0 vs. 2.5 mumol/l for the E23/S1369 variant), although there was no difference in ADP sensitivity. The K23/A1369 variant also displayed increased inhibition by gliclazide, an A-site sulfonylurea drug (IC(50) = 52.7 vs. 188.7 nmol/l for the E23/S1369 variant), but not by glibenclamide (AB site) or repaglinide (B site). CONCLUSIONS: Our findings indicate that the common K23/A1369 variant K(ATP) channel displays decreased ATP inhibition that may contribute to the observed increased risk for type 2 diabetes. Moreover, the increased sensitivity of the K23/A1369 variant to the A-site sulfonylurea drug gliclazide may provide a pharmacogenomic therapeutic approach for patients with type 2 diabetes who are homozygous for both risk alleles.

Beer NL, Tribble ND, Roos C, Orho-Melander M, Gloyn AL. 2009. Functional characterisation of the glucokinase regulatory protein gene variant P446L shows diminished regulation by fructose-6 phosphate resulting in increased glucokinase activity DIABETOLOGIA, 52 pp. S103-S104.

Tribble ND, Beer N, Grimsby J, Baltrusch S, Gloyn AL. 2009. Investigating novel mutational mechanisms for glucokinase mutations with near normal or paradoxical kinetics DIABETOLOGIA, 52 pp. S104-S104.

Gasperíková D, Tribble ND, Staník J, Hucková M, Misovicová N, van de Bunt M, Valentínová L, Barrow BA, Barák L, Dobránsky R et al. 2009. Identification of a novel beta-cell glucokinase (GCK) promoter mutation (-71G>C) that modulates GCK gene expression through loss of allele-specific Sp1 binding causing mild fasting hyperglycemia in humans. Diabetes, 58 (8), pp. 1929-1935. | Show Abstract | Read more

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

Gloyn AL, Braun M, Rorsman P. 2009. Type 2 diabetes susceptibility gene TCF7L2 and its role in beta-cell function. Diabetes, 58 (4), pp. 800-802. | Read more

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), pp. 170-180. | Show Abstract | Read more

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.

Cited:

315

Scopus

Kong A, Steinthorsdottir V, Masson G, Thorleifsson G, Sulem P, Besenbacher S, Jonasdottir A, Sigurdsson A, Kristinsson KT, Jonasdottir A et al. 2009. Parental origin of sequence variants associated with complex diseases Nature, 462 (7275), pp. 868-874. | Read more

Gloyn AL, van de Bunt M, Stratton IM, Lonie L, Tucker L, Ellard S, Holman RR. 2009. Prevalence of GCK mutations in individuals screened for fasting hyperglycaemia. Diabetologia, 52 (1), pp. 172-174. | Read more

Harries LW, Brown JE, Gloyn AL. 2009. Species-specific differences in the expression of the HNF1A, HNF1B and HNF4A genes. PLoS One, 4 (11), pp. e7855. | Show Abstract | Read more

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.

Jafar-Mohammadi B, Groves CJ, Owen KR, Frayling TM, Hattersley AT, McCarthy MI, Gloyn AL. 2009. Low frequency variants in the exons only encoding isoform A of HNF1A do not contribute to susceptibility to type 2 diabetes. PLoS One, 4 (8), pp. e6615. | Show Abstract | Read more

BACKGROUND: There is considerable interest in the hypothesis that low frequency, intermediate penetrance variants contribute to the proportion of Type 2 Diabetes (T2D) susceptibility not attributable to the common variants uncovered through genome-wide association approaches. Genes previously implicated in monogenic and multifactorial forms of diabetes are obvious candidates in this respect. In this study, we focussed on exons 8-10 of the HNF1A gene since rare, penetrant mutations in these exons (which are only transcribed in selected HNF1A isoforms) are associated with a later age of diagnosis of Maturity onset diabetes of the young (MODY) than mutations in exons 1-7. The age of diagnosis in the subgroup of HNF1A-MODY individuals with exon 8-10 mutations overlaps with that of early multifactorial T2D, and we set out to test the hypothesis that these exons might also harbour low-frequency coding variants of intermediate penetrance that contribute to risk of multifactorial T2D. METHODOLOGY AND PRINCIPAL FINDINGS: We performed targeted capillary resequencing of HNF1A exons 8-10 in 591 European T2D subjects enriched for genetic aetiology on the basis of an early age of diagnosis (< or =45 years) and/or family history of T2D (> or =1 affected sibling). PCR products were sequenced and compared to the published HNF1A sequence. We identified several variants (rs735396 [IVS9-24T>C], rs1169304 [IVS8+29T>C], c.1768+44C>T [IVS9+44C>T] and rs61953349 [c.1545G>A, p.T515T] but no novel non-synonymous coding variants were detected. CONCLUSIONS AND SIGNIFICANCE: We conclude that low frequency, nonsynonymous coding variants in the terminal exons of HNF1A are unlikely to contribute to T2D-susceptibility in European samples. Nevertheless, the rationale for seeking low-frequency causal variants in genes known to contain rare, penetrant mutations remains strong and should motivate efforts to screen other genes in a similar fashion.

Edghill EL, McCulloch L, Fulton P, Beer N, Hattersley AT, Gloyn AL. 2009. Mutations in the third gene shown to alter fasting glucose levels in the population (G6PC2) are not a common cause of monogenic forms of pancreatic B-cell dysfunction. Diabet Med, 26 (1), pp. 113-114. | Read more

Pal A, Barber TM, McCulloch L, Smith R, Barrow BA, Walker L, Gloyn AL. 2008. Enhanced insulin sensitivity in obese patients with a rare cancer predisposition syndrome (Cowden syndrome) due to PTEN mutations: further evidence for the Yin-Yang hypothesis DIABETOLOGIA, 51 pp. S138-S138.

Gloyn AL, McCarthy MI. 2008. Genetics: how the UKPDS contributed to determining the genetic landscape of Type 2 diabetes. Diabet Med, 25 Suppl 2 (SUPPL. 2), pp. 35-40. | Show Abstract | Read more

The identification and functional characterisation of genetic variants that either cause or predispose to diabetes is a major focus of biomedical research. The molecular basis is now known for the majority of monogenic forms of diabetes arising from pancreatic beta-cell dysfunction; however finding the genetic variants underlying susceptibility to Type 2 diabetes (T2DM) has been a greater technical, statistical and biological challenge. The advent of biology-agnostic approaches made possible by the improved arsenal of research platforms and genetic tools available has increased the number of known T2DM genes dramatically and provided important insights into the pathophysiology of T2DM. Over the past 18 months, the list of T2DM susceptibility genes has grown from three to close to 20, illustrating the substantial progress which has been made. These recent milestones have not only illustrated the limited knowledge we have of the pancreatic beta-cell, but have also reinforced our belief in the involvement of common genetic variants in the genes involved in monogenic forms of diabetes in the susceptibility to T2DM and have clearly shown a primary role for pancreatic beta-cell dysfunction in T2DM. Both of these concepts were explored in the early work of the UK Prospective Diabetes Study (UKPDS) genetics research groups.

Christesen HB, Tribble ND, Molven A, Siddiqui J, Sandal T, Brusgaard K, Ellard S, Njølstad PR, Alm J, Brock Jacobsen B et al. 2008. Activating glucokinase (GCK) mutations as a cause of medically responsive congenital hyperinsulinism: prevalence in children and characterisation of a novel GCK mutation. Eur J Endocrinol, 159 (1), pp. 27-34. | Show Abstract | Read more

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

Turkkahraman D, Bircan I, Tribble ND, Akçurin S, Ellard S, Gloyn AL. 2008. Permanent neonatal diabetes mellitus caused by a novel homozygous (T168A) glucokinase (GCK) mutation: initial response to oral sulphonylurea therapy. J Pediatr, 153 (1), pp. 122-126. | Show Abstract | Read more

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

van de Bunt M, Edghill EL, Hussain K, Ellard S, Gloyn AL. 2008. Gene duplications resulting in over expression of glucokinase are not a common cause of hypoglycaemia of infancy in humans. Mol Genet Metab, 94 (2), pp. 268-269. | Read more

Gloyn AL, Tribble ND, van de Bunt M, Barrett A, Johnson PR. 2008. Glucokinase (GCK) and other susceptibility genes for beta-cell dysfunction: the candidate approach. Biochem Soc Trans, 36 (Pt 3), pp. 306-311. | Show Abstract | Read more

There are well-documented examples in the literature of where determining the genetic aetiology of a disorder has provided insights into important regulatory pathways and protein interactions, and, more recently, has led to improved treatment options for patients. The studies of monogenic forms of beta-cell dysfunction are no exception. Naturally occurring mutations in the gene for the beta-cell enzyme glucokinase (GCK) result in both hyper- and hypo-glycaemia. Over 200 mutations have been described, and careful study of the mutational mechanisms for a number of these has provided important insights into glucokinase regulation. Increased understanding of post-translational regulatory mechanisms holds the promise of novel pharmacotherapeutic options for the treatment of T2DM (Type 2 diabetes mellitus). It is well established that common genetic variation in genes involved in monogenic forms of beta-cell dysfunction contributes to susceptibility to T2DM. Recent genome-wide scans for association have identified a number of novel T2DM susceptibility genes which probably influence beta-cell mass and/or function. Their identification allows the investigation of the role of rare mutations in monogenic beta-cell dysfunction. Current results indicate the importance of these genes in pancreatic development and suggest that mutations which result in a severe functional defect could be lethal.

Katulanda P, Groves CJ, Barrett A, Sheriff R, Matthews DR, McCarthy MI, Gloyn AL. 2008. Prevalence and clinical characteristics of maternally inherited diabetes and deafness caused by the mt3243A > G mutation in young adult diabetic subjects in Sri Lanka. Diabet Med, 25 (3), pp. 370-374. | Show Abstract | Read more

AIMS: The maternally inherited mt3243A > G mutation is associated with a variable clinical phenotype including diabetes and deafness (MIDD). We aimed to determine the prevalence and clinical characteristics of MIDD in a large South Asian cohort of young adult-onset diabetic patients from Sri Lanka. METHODS: DNA was available from 994 subjects (age of diagnosis 16-40 years, age at recruitment < or = 45 years). Mutation screening was performed using a QRT-PCR method on an ABI 7900HT system using sequence-specific probes. Samples with heteroplasm > or = 5.0% were considered positive. RESULTS: Nine (four males) mutation-positive subjects were identified (prevalence 0.9%). They were diagnosed at a younger age (25.9 +/- 4.8 years vs. 31.9 +/- 5.6 years, P = 0.002) and were lean (body mass index [BMI] 18.7 +/- 2.7 kg/m(2) vs. 24.7 +/- 4.0 kg/m(2), P < 0.001) compared to NMCs. One mutation-positive subject (11.1%) had metabolic syndrome, compared to 633 (64.3%) of NMCs. Insulin therapy within 6 months of diagnosis was used in four (44.0%) carriers compared to 6.9% of NMCs (P = 0.002). Combined screening criteria of any two of maternal history of diabetes, personal history of hearing impairment and family history of hearing impairment only identified five (55%) of the carriers, with a positive predictive value of 7.4%. CONCLUSIONS: The prevalence of mt3243A > G mutation among young adult-onset diabetic subjects from Sri Lanka was 0.9%. Our study demonstrates that a maternal family history of diabetes and either a personal and/or family history of deafness only distinguish half of patients with MIDD from Sri Lankan subjects with young-onset diabetes.

Flanagan SE, Patch A-M, Mackay DJG, Edghill EL, Gloyn A-L, Robinson D, Shield JPH, Temple K, Ellard S, Hattersley AT. 2008. Mutations in ATP-sensitive K+ channel genes cause transient neonatal diabetes and permanent diabetes in childhood or adulthood (vol 56, pg 1930, 2007) DIABETES, 57 (2), pp. 523-523.

Flanagan SE, Patch AM, Mackay DJG, Edghill EL, Gloyn AL, Robinson D, Shield JPH, Temple K, Ellard S, Hattersley AT. 2008. Erratum Diabetes, 57 (2), pp. 523-523. | Read more

Waterfield T, Gloyn AL. 2008. Monogenic β-cell dysfunction in children: clinical phenotypes, genetic etiology and mutational pathways Pediatric Health, 2 (4), pp. 517-532. | Show Abstract | Read more

Monogenic diabetes accounts for 1-2% of all cases of diabetes mellitus and presentation is often in childhood. Recognizing the clinical features of monogenic β-cell dysfunction prevents misdiagnosis and allows for more effective management and genetic counseling. Monogenic β-cell dysfunction is a diverse collection of clinical phenotypes underpinned by common mutational pathways. Mutations affecting the glycolytic glucokinase enzyme, the mitochondria, the K ATP channels and transcription factors have been known for some time. Until recently, the role of endoplasmic reticulum stress was underestimated in the pathogenesis of diabetes. It is becoming increasingly clear that endoplasmic reticulum stress is an important etiological factor in the development of monogenic and polygenic diabetes. In this article, we aim to define the etiology of pediatric monogenic β-cell dysfunction and provide guidance on the investigation and management of children presenting with monogenic β-cell dysfunction. © 2008 Future Medicine Ltd.

Wabitsch M, Lahr G, Van de Bunt M, Marchant C, Lindner M, von Puttkamer J, Fenneberg A, Debatin KM, Klein R, Ellard S et al. 2007. Heterogeneity in disease severity in a family with a novel G68V GCK activating mutation causing persistent hyperinsulinaemic hypoglycaemia of infancy. Diabet Med, 24 (12), pp. 1393-1399. | Show Abstract | Read more

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

Minton JA, van de Bunt M, Boustred C, Hussain K, Hattersley AT, Ellard S, Gloyn AL. 2007. Mutations in HHEX are not a common cause of monogenic forms of beta cell dysfunction. Diabetologia, 50 (9), pp. 2019-2022. | Read more

Arden C, Trainer A, de la Iglesia N, Scougall KT, Gloyn AL, Lange AJ, Shaw JA, Matschinsky FM, Agius L. 2007. Cell biology assessment of glucokinase mutations V62M and G72R in pancreatic beta-cells: evidence for cellular instability of catalytic activity. Diabetes, 56 (7), pp. 1773-1782. | Show Abstract | Read more

Mutations in the glucokinase (GK) gene cause defects in blood glucose homeostasis. In some cases (V62M and G72R), the phenotype cannot be explained by altered enzyme kinetics or protein instability. We used transient and stable expression of green fluorescent protein (GFP) GK chimaeras in MIN6 beta-cells to study the phenotype defect of V62M and G72R. GK activity in lysates of MIN6 cell lines stably expressing wild-type or mutant GFP GK showed the expected affinity for glucose and response to pharmacological activators, indicating the expression of catalytically active enzymes. MIN6 cells stably expressing GFP V62M or GFP G72R had a lower GK activity-to-GK immunoreactivity ratio and GK activity-to-GK mRNA ratio but not GK immunoreactivity-to-GK mRNA ratio than wild-type GFP GK. Heterologous expression of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2/FDP2) in cell lines increased GK activity for wild-type GK and V62M but not for G72R, whereas expression of liver GK regulatory protein (GKRP) increased GK activity for wild type but not V62M or G72R. Lack of interaction of these mutants with GKRP was also evident in hepatocyte transfections from the lack of nuclear accumulation. These results suggest that cellular loss of GK catalytic activity rather than impaired translation or enhanced protein degradation may account for the hyperglycemia in subjects with V62M and G72R mutations.

Flanagan SE, Patch AM, Mackay DJ, Edghill EL, Gloyn AL, Robinson D, Shield JP, Temple K, Ellard S, Hattersley AT. 2007. Mutations in ATP-sensitive K+ channel genes cause transient neonatal diabetes and permanent diabetes in childhood or adulthood. Diabetes, 56 (7), pp. 1930-1937. | Show Abstract | Read more

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

Barber TM, Bennett AJ, Gloyn AL, Groves CJ, Sovio U, Ruokonen A, Martikainen H, Pouta A, Taponen S, Weedon MN et al. 2007. Relationship between E23K (an established type II diabetes-susceptibility variant within KCNJ11), polycystic ovary syndrome and androgen levels. Eur J Hum Genet, 15 (6), pp. 679-684. | Show Abstract | Read more

Polycystic ovary syndrome (PCOS) is strongly associated with hyperinsulinaemia and type II diabetes (T2D). Sequence variation within KCNJ11 (encoding Kir6.2, the beta-cell inwardly rectifying potassium channel) is implicated in the pathogenesis of neonatal diabetes, hyperinsulinaemia of infancy and multifactorial T2D. Comprehensive tagging studies have demonstrated that the KCNJ11 E23K variant (or ABCC8 A1369S in LD>0.9) is responsible for the known association between KCNJ11 and T2D. Given the phenotypic overlap between PCOS and T2D, we investigated whether E23K is involved in susceptibility to PCOS and related traits. Case-control analyses for the KCNJ11 E23K variant were performed in (a) 374 PCOS cases and 2574 controls of UK British/Irish origin, and (b) 550 women with PCOS symptoms and 1114 controls from a Finnish birth cohort. The relationship between E23K genotype and androgen levels (a key intermediate phenotype relevant to PCOS) in 1380 samples was studied. The UK case-control analysis revealed no association between E23K genotypes and PCOS status (P=0.49; Cochran-Armitage test), and no significant relationship between E23K genotype and androgen measures in the samples for which these phenotypes were available (P=0.19). Similarly, the Finnish case-control analysis showed no association between E23K genotypes and PCOS status (P=0.75; Cochran-Armitage test), and no significant relationship between E23K genotype and androgen measures in the samples for which these phenotypes were available (Finnish controls, P=0.25; Finnish cases, P=0.08). In conclusion, these data (involving >4600 subjects) provide no evidence that common variants of the KCNJ11 E23K polymorphism have a major influence on PCOS susceptibility, though modest effect sizes (OR<1.25) cannot be excluded.

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), pp. 1773-1777. | Show Abstract | Read more

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.

Suliman SGI, Gasperikova D, Stanik J, Sandrikova V, Misovicova N, Wilson N, Elliot K, Barak L, Volpi E, Klimes I, Gloyn AL. 2007. Insulin resistance and intrauterine growth retardation due to a novel balanced translocation [46,t (7;19) (p15.2;p13.2)1 which disrupts the insulin receptor gene DIABETIC MEDICINE, 24 pp. 26-26.

Gloyn AL, Marchant C, Linder M, von Puttkamer J, Wasser A, Clark A, Ellard S, Lahr G, Wabitsch M. 2007. Phenotypic heterogeneity in a family with a novel activating glucokinase (GCK) mutation (G68V) causing familial persistent hyperinsulinaemic hypoglycaemia of infancy DIABETIC MEDICINE, 24 pp. 36-36.

Lahr G, Gloyn AL, Debatin KM, Wabitsch M. 2007. Persistent hyperinsulinemic hypoglycemia in a 7 yr old girl due to a novel activating glucokinase mutation EXPERIMENTAL AND CLINICAL ENDOCRINOLOGY & DIABETES, 115 pp. S11-S11.

van de Bunt M, Gloyn AL. 2007. Monogenic disorders of the pancreatic β-cell: personalizing treatment for rare forms of diabetes and hypoglycemia Personalized Medicine, 4 (3), pp. 247-259. | Show Abstract | Read more

Over the past 10-20 years, our understanding of the genetic etiology of monogenic disorders of the pancreatic β-cell has greatly improved. This has enabled clinicians to provide patients with more accurate information regarding prognosis and inheritance and has influenced treatment. Maturity-onset diabetes of the young and neonatal diabe tes are two such examples. Patients with maturity-onset diabetes of the young due to glucokinase mutations can usually be managed by diet alone, while those affected by HNF-1α and HNF-4α mutations respond well to low doses of sulfonylureas. The identification of mutations in the ATP-dependent potassium channel genes KCNJ11 and ABCC8 as the most common cause of permanent neonatal diabetes has improved treatment regimes for affected children. In addition to enabling patients to stop insulin injections, their glycemic control has also improved. These advances show the importance of unravelling the genetics of a disease to achieve the best individualized treatment for the patients affected. © 2007 Future Medicine Ltd.

Porter JR, Rangasami JJ, Ellard S, Gloyn AL, Shields BM, Edwards J, Anderson JM, Shaw NJ, Hattersley AT, Frayling TM et al. 2006. Asian MODY: are we missing an important diagnosis? Diabet Med, 23 (11), pp. 1257-1260. | Show Abstract | Read more

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

Gloyn AL, Ellard S. 2006. Defining the genetic aetiology of monogenic diabetes can improve treatment. Expert Opin Pharmacother, 7 (13), pp. 1759-1767. | Show Abstract | Read more

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

Gloyn AL, Mackay DJ, Weedon MN, McCarthy MI, Walker M, Hitman G, Knight BA, Owen KR, Hattersley AT, Frayling TM. 2006. Assessment of the role of common genetic variation in the transient neonatal diabetes mellitus (TNDM) region in type 2 diabetes: a comparative genomic and tagging single nucleotide polymorphism approach. Diabetes, 55 (8), pp. 2272-2276. | Show Abstract | Read more

Recent evidence supports the strong overlap between genes implicated in monogenic diabetes and susceptibility to type 2 diabetes. Transient neonatal diabetes mellitus (TNDM) is a rare disorder associated with overexpression of genes at a paternally expressed imprinted locus on chromosome 6q24. There are two overlapping genes in this region: the transcription factor zinc finger protein associated with cell cycle control and apoptosis (ZAC also known as PLAGL1) and HYMA1, which encodes an untranslated mRNA. Several type 2 diabetes linkage studies have reported linkage to chromosome 6q22-25. We hypothesized that common genetic variation at this TNDM region influences type 2 diabetes susceptibility. In addition to the coding regions, we used comparative genomic analysis to identify conserved noncoding regions, which were resequenced for single nucleotide polymorphism (SNP) discovery in 47 individuals. Twenty-six SNPs were identified. Fifteen tag SNPs (tSNPs) were successfully genotyped in a large case-control (n = 3,594) and family-based (n = 1,654) study. We did not find any evidence of association or overtransmission of any tSNP to affected offspring or of a parent-of-origin effect. Using a study sufficiently powered to detect odds ratios of <1.2, we conclude that common variation in the TNDM region does not play an important role in the genetic susceptibility to type 2 diabetes.

Gloyn AL, Diatloff-Zito C, Edghill EL, Bellanné-Chantelot C, Nivot S, Coutant R, Ellard S, Hattersley AT, Robert JJ. 2006. KCNJ11 activating mutations are associated with developmental delay, epilepsy and neonatal diabetes syndrome and other neurological features. Eur J Hum Genet, 14 (7), pp. 824-830. | Show Abstract | Read more

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

Flanagan SE, Edghill EL, Gloyn AL, Ellard S, Hattersley AT. 2006. Mutations in KCNJ11, which encodes Kir6.2, are a common cause of diabetes diagnosed in the first 6 months of life, with the phenotype determined by genotype. Diabetologia, 49 (6), pp. 1190-1197. | Show Abstract | Read more

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

Gloyn AL, Siddiqui J, Ellard S. 2006. Mutations in the genes encoding the pancreatic beta-cell KATP channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) in diabetes mellitus and hyperinsulinism. Hum Mutat, 27 (3), pp. 220-231. | Show Abstract | Read more

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

Proks P, Girard C, Haider S, Gloyn AL, Hattersley AT, Sansom MS, Ashcroft FM. 2005. A gating mutation at the internal mouth of the Kir6.2 pore is associated with DEND syndrome. EMBO Rep, 6 (5), pp. 470-475. | Show Abstract | Read more

Inwardly rectifying potassium (Kir) channels control cell membrane K+ fluxes and electrical signalling in diverse cell types. Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the pore-forming subunit of the ATP-sensitive (K(ATP)) channel, cause permanent neonatal diabetes mellitus. However, the I296L mutation also results in developmental delay, muscle weakness and epilepsy. We investigated the functional effects of the I296L mutation by expressing wild-type or mutant Kir6.2/SUR1 channels in Xenopus oocytes. The mutation caused a marked increase in resting whole-cell K(ATP) currents by reducing channel inhibition by ATP, in both homomeric and simulated heterozygous states. Kinetic analysis showed that the mutation impaired ATP sensitivity indirectly, by stabilizing the open state of the channel and possibly also by means of an allosteric effect on ATP binding and/or transduction. The results implicate a new region in Kir-channel gating and suggest that disease severity is correlated with the extent of reduction in ATP sensitivity.

Gloyn AL, Odili S, Zelent D, Buettger C, Castleden HA, Steele AM, Stride A, Shiota C, Magnuson MA, Lorini R et al. 2005. Insights into the structure and regulation of glucokinase from a novel mutation (V62M), which causes maturity-onset diabetes of the young. J Biol Chem, 280 (14), pp. 14105-14113. | Show Abstract | Read more

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

Gloyn AL, Reimann F, Girard C, Edghill EL, Proks P, Pearson ER, Temple IK, Mackay DJ, Shield JP, Freedenberg D et al. 2005. Relapsing diabetes can result from moderately activating mutations in KCNJ11. Hum Mol Genet, 14 (7), pp. 925-934. | Show Abstract | Read more

Neonatal diabetes can either remit and hence be transient or else may be permanent. These two phenotypes were considered to be genetically distinct. Abnormalities of 6q24 are the commonest cause of transient neonatal diabetes (TNDM). Mutations in KCNJ11, which encodes Kir6.2, the pore-forming subunit of the ATP-sensitive potassium channel (K(ATP)), are the commonest cause of permanent neonatal diabetes (PNDM). In addition to diabetes, some KCNJ11 mutations also result in marked developmental delay and epilepsy. These mutations are more severe on functional characterization. We investigated whether mutations in KCNJ11 could also give rise to TNDM. We identified the three novel heterozygous mutations (G53S, G53R, I182V) in three of 11 probands with clinically defined TNDM, who did not have chromosome 6q24 abnormalities. The mutations co-segregated with diabetes within families and were not found in 100 controls. All probands had insulin-treated diabetes diagnosed in the first 4 months and went into remission by 7-14 months. Functional characterization of the TNDM associated mutations was performed by expressing the mutated Kir6.2 with SUR1 in Xenopus laevis oocytes. All three heterozygous mutations resulted in a reduction in the sensitivity to ATP when compared with wild-type (IC(50) approximately 30 versus approximately 7 microM, P-value for is all <0.01); however, this was less profoundly reduced than with the PNDM associated mutations. In conclusion, mutations in KCNJ11 are the first genetic cause for remitting as well as permanent diabetes. This suggests that a fixed ion channel abnormality can result in a fluctuating glycaemic phenotype. The multiple phenotypes associated with activating KCNJ11 mutations may reflect their severity in vitro.

Massa O, Iafusco D, D'Amato E, Gloyn AL, Hattersley AT, Pasquino B, Tonini G, Dammacco F, Zanette G, Meschi F et al. 2005. KCNJ11 activating mutations in Italian patients with permanent neonatal diabetes. Hum Mutat, 25 (1), pp. 22-27. | Show Abstract | Read more

Permanent neonatal diabetes mellitus (PNDM) is a rare condition characterized by severe hyperglycemia constantly requiring insulin treatment from its onset. Complete deficiency of glucokinase (GCK) can cause PNDM; however, the genetic etiology is unknown in most PNDM patients. Recently, heterozygous activating mutations of KCNJ11, encoding Kir6.2, the pore forming subunit of the ATP-dependent potassium (K(ATP)) channel of the pancreatic beta-cell, were found in patients with PNDM. Closure of the K(ATP) channel exerts a pivotal role in insulin secretion by modifying the resting membrane potential that leads to insulin exocytosis. We screened the KCNJ11 gene in 12 Italian patients with PNDM (onset within 3 months from birth) and in six patients with non-autoimmune, insulin-requiring diabetes diagnosed during the first year of life. Five different heterozygous mutations were identified: c.149G>C (p.R50P), c.175G>A (p.V59M), c.509A>G (p.K170R), c.510G>C (p.K170N), and c.601C>T (p.R201C) in eight patients with diabetes diagnosed between day 3 and 182. Mutations at Arg50 and Lys170 residues are novel. Four patients also presented with motor and/or developmental delay as previously reported. We conclude that KCNJ11 mutations are a common cause of PNDM either in isolation or associated with developmental delay. Permanent diabetes of non autoimmune origin can present up to 6 months from birth in individuals with KCNJ11 and EIF2AK3 mutations. Therefore, we suggest that the acronym PNDM be replaced with the more comprehensive permanent diabetes mellitus of infancy (PDMI), linking it to the gene product (e.g., GCK-PDMI, KCNJ11-PDMI) to avoid confusion between patients with early-onset, autoimmune type 1 diabetes.

Porter JR, Shaw NJ, Barrett TG, Hattersley AT, Ellard S, Gloyn AL. 2005. Permanent neonatal diabetes in an Asian infant. J Pediatr, 146 (1), pp. 131-133. | Show Abstract | Read more

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

Proks P, Antcliff JF, Lippiat J, Gloyn AL, Hattersley AT, Ashcroft FM. 2004. Molecular basis of Kir6.2 mutations associated with neonatal diabetes or neonatal diabetes plus neurological features. Proc Natl Acad Sci U S A, 101 (50), pp. 17539-17544. | Show Abstract | Read more

Inwardly rectifying potassium channels (Kir channels) control cell membrane K(+) fluxes and electrical signaling in diverse cell types. Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the pore-forming subunit of the ATP-sensitive (K(ATP)) channel, cause permanent neonatal diabetes mellitus (PNDM). For some mutations, PNDM is accompanied by marked developmental delay, muscle weakness, and epilepsy (severe disease). To determine the molecular basis of these different phenotypes, we expressed wild-type or mutant (R201C, Q52R, or V59G) Kir6.2/sulfonylurea receptor 1 channels in Xenopus oocytes. All mutations increased resting whole-cell K(ATP) currents by reducing channel inhibition by ATP, but, in the simulated heterozygous state, mutations causing PNDM alone (R201C) produced smaller K(ATP) currents and less change in ATP sensitivity than mutations associated with severe disease (Q52R and V59G). This finding suggests that increased K(ATP) currents hyperpolarize pancreatic beta cells and impair insulin secretion, whereas larger K(ATP) currents are required to influence extrapancreatic cell function. We found that mutations causing PNDM alone impair ATP sensitivity directly (at the binding site), whereas those associated with severe disease act indirectly by biasing the channel conformation toward the open state. The effect of the mutation on ATP sensitivity in the heterozygous state reflects the different contributions of a single subunit in the Kir6.2 tetramer to ATP inhibition and to the energy of the open state. Our results also show that mutations in the slide helix of Kir6.2 (V59G) influence the channel kinetics, providing evidence that this domain is involved in Kir channel gating, and suggest that the efficacy of sulfonylurea therapy in PNDM may vary with genotype.

Sellick GS, Barker KT, Stolte-Dijkstra I, Fleischmann C, Coleman RJ, Garrett C, Gloyn AL, Edghill EL, Hattersley AT, Wellauer PK et al. 2004. Mutations in PTF1A cause pancreatic and cerebellar agenesis. Nat Genet, 36 (12), pp. 1301-1305. | Show Abstract | Read more

Individuals with permanent neonatal diabetes mellitus usually present within the first three months of life and require insulin treatment. We recently identified a locus on chromosome 10p13-p12.1 involved in permanent neonatal diabetes mellitus associated with pancreatic and cerebellar agenesis in a genome-wide linkage search of a consanguineous Pakistani family. Here we report the further linkage analysis of this family and a second family of Northern European descent segregating an identical phenotype. Positional cloning identified the mutations 705insG and C886T in the gene PTF1A, encoding pancreas transcription factor 1alpha, as disease-causing sequence changes. Both mutations cause truncation of the expressed PTF1A protein C-terminal to the basic-helix-loop-helix domain. Reporter-gene studies using a minimal PTF1A deletion mutant indicate that the deleted region defines a new domain that is crucial for the function of this protein. PTF1A is known to have a role in mammalian pancreatic development, and the clinical phenotype of the affected individuals implicated the protein as a key regulator of cerebellar neurogenesis. The essential role of PTF1A in normal cerebellar development was confirmed by detailed neuropathological analysis of Ptf1a(-/-) mice.

Edghill EL, Gloyn AL, Gillespie KM, Lambert AP, Raymond NT, Swift PG, Ellard S, Gale EA, Hattersley AT. 2004. Activating mutations in the KCNJ11 gene encoding the ATP-sensitive K+ channel subunit Kir6.2 are rare in clinically defined type 1 diabetes diagnosed before 2 years. Diabetes, 53 (11), pp. 2998-3001. | Show Abstract | Read more

We have recently shown that permanent neonatal diabetes can be caused by activating mutations in KCNJ11 that encode the Kir6.2 subunit of the beta-cell ATP-sensitive K(+) channel. Some of these patients were diagnosed after 3 months of age and presented with ketoacidosis and marked hyperglycemia, which could have been diagnosed as type 1 diabetes. We hypothesized that KCNJ11 mutations could present clinically as type 1 diabetes. We screened the KCNJ11 gene for mutations in 77 U.K. type 1 diabetic subjects diagnosed under the age of 2 years. One patient was found to be heterozygous for the missense mutation R201C. She had low birth weight, was diagnosed at 5 weeks, and did not have a high risk predisposing HLA genotype. A novel variant, R176C, was identified in one diabetic subject but did not cosegregate with diabetes within the family. In conclusion, we have shown that heterozygous activating mutations in the KCNJ11 gene are a rare cause of clinically defined type 1 diabetes diagnosed before 2 years. Although activating KCNJ11 mutations are rare in patients diagnosed with type 1 diabetes, the identification of a KCNJ11 mutation may have important treatment implications.

Vaxillaire M, Populaire C, Busiah K, Cavé H, Gloyn AL, Hattersley AT, Czernichow P, Froguel P, Polak M. 2004. Kir6.2 mutations are a common cause of permanent neonatal diabetes in a large cohort of French patients. Diabetes, 53 (10), pp. 2719-2722. | Show Abstract | Read more

Permanent neonatal diabetes (PND), requiring insulin within the first months of life, is unexplained at the molecular level in most cases. It has very recently been shown that heterozygous activating mutations in the KCNJ11 gene, encoding the Kir6.2 subunit of the pancreatic ATP-sensitive K(+) channel involved in the regulation of insulin secretion, cause PND. In the present study, we screened the KCNJ11 gene for mutations in French patients with PND. Patients were recruited through the French network for the study of neonatal diabetes. Seventeen at-term babies with a median age at diagnosis of diabetes of 64 days (range 1-260) were included. We identified in nine patients seven heterozygous nonsynonymous mutations: three of them (V59M, R201C, and R201H) were already described, and the four novel mutations resulted in an amino acid change of Kir6.2 at positions F35L, G53N, E322K, and Y330C. More patients with a Kir6.2 mutation (six of nine) were reported to have a smaller birth weight than those without mutation (two of eight). Although Kir6.2 mutation carriers do not represent a phenotypically specific form of PND, an impaired function of Kir6.2 is associated with in utero insulin secretory insufficiency and growth retardation. In conclusion, we confirmed that Kir6.2 mutations are a common cause (53%) of PND in Caucasians.

Sagen JV, Raeder H, Hathout E, Shehadeh N, Gudmundsson K, Baevre H, Abuelo D, Phornphutkul C, Molnes J, Bell GI et al. 2004. Permanent neonatal diabetes due to mutations in KCNJ11 encoding Kir6.2: patient characteristics and initial response to sulfonylurea therapy. Diabetes, 53 (10), pp. 2713-2718. | Show Abstract | Read more

Permanent neonatal diabetes (PND) can be caused by mutations in the transcription factors insulin promoter factor (IPF)-1, eukaryotic translation initiation factor-2alpha kinase 3 (EIF2AK3), and forkhead box-P3 and in key components of insulin secretion: glucokinase (GCK) and the ATP-sensitive K(+) channel subunit Kir6.2. We sequenced the gene encoding Kir6.2 (KCNJ11) in 11 probands with GCK-negative PND. Heterozygous mutations were identified in seven probands, causing three novel (F35V, Y330C, and F333I) and two known (V59M and R201H) Kir6.2 amino acid substitutions. Only two probands had a family history of diabetes. Subjects with the V59M mutation had neurological features including motor delay. Three mutation carriers tested had an insulin secretory response to tolbutamide, but not to glucose or glucagon. Glibenclamide was introduced in increasing doses to investigate whether sulfonylurea could replace insulin. At a glibenclamide dose of 0.3-0.4 mg. kg(-1). day(-1), insulin was discontinued. Blood glucose did not deteriorate, and HbA(1c) was stable or fell during 2-6 months of follow-up. An oral glucose tolerance test performed in one subject revealed that glucose-stimulated insulin release was restored. Mutations in Kir6.2 were the most frequent cause of PND in our cohort. Apparently insulin-dependent patients with mutations in Kir6.2 may be managed on an oral sulfonylurea with sustained metabolic control rather than insulin injections, illustrating the principle of pharmacogenetics applied in diabetes treatment.

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), pp. 3932-3935. | Show Abstract | Read more

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.

Gloyn AL, Pearson ER, Antcliff JF, Proks P, Bruining GJ, Slingerland AS, Howard N, Srinivasan S, Silva JM, Molnes J et al. 2004. Activating mutations in the gene encoding the ATP-sensitive potassium-channel subunit Kir6.2 and permanent neonatal diabetes. N Engl J Med, 350 (18), pp. 1838-1849. | Show Abstract | Read more

BACKGROUND: Patients with permanent neonatal diabetes usually present within the first three months of life and require insulin treatment. In most, the cause is unknown. Because ATP-sensitive potassium (K(ATP)) channels mediate glucose-stimulated insulin secretion from the pancreatic beta cells, we hypothesized that activating mutations in the gene encoding the Kir6.2 subunit of this channel (KCNJ11) cause neonatal diabetes. METHODS: We sequenced the KCNJ11 gene in 29 patients with permanent neonatal diabetes. The insulin secretory response to intravenous glucagon, glucose, and the sulfonylurea tolbutamide was assessed in patients who had mutations in the gene. RESULTS: Six novel, heterozygous missense mutations were identified in 10 of the 29 patients. In two patients the diabetes was familial, and in eight it arose from a spontaneous mutation. Their neonatal diabetes was characterized by ketoacidosis or marked hyperglycemia and was treated with insulin. Patients did not secrete insulin in response to glucose or glucagon but did secrete insulin in response to tolbutamide. Four of the patients also had severe developmental delay and muscle weakness; three of them also had epilepsy and mild dysmorphic features. When the most common mutation in Kir6.2 was coexpressed with sulfonylurea receptor 1 in Xenopus laevis oocytes, the ability of ATP to block mutant K(ATP) channels was greatly reduced. CONCLUSIONS: Heterozygous activating mutations in the gene encoding Kir6.2 cause permanent neonatal diabetes and may also be associated with developmental delay, muscle weakness, and epilepsy. Identification of the genetic cause of permanent neonatal diabetes may facilitate the treatment of this disease with sulfonylureas.

McCarthy MI, Gloyn AL. 2003. Genetics for Endocrinologists: The Molecular Genetic Basis of Endocrine Disorders. Clin Endocrinol (Oxf), 59 (6), pp. 826. | Read more

Gloyn AL. 2003. Glucokinase (GCK) mutations in hyper- and hypoglycemia: maturity-onset diabetes of the young, permanent neonatal diabetes, and hyperinsulinemia of infancy. Hum Mutat, 22 (5), pp. 353-362. | Show Abstract | Read more

Glucokinase is a key regulatory enzyme in the pancreatic beta-cell. It plays a crucial role in the regulation of insulin secretion and has been termed the pancreatic beta-cell sensor. Given its central role in the regulation of insulin release, it is understandable that mutations in the gene encoding glucokinase (GCK) can cause both hyperglycemia and hypoglycemia. Heterozygous inactivating mutations in GCK cause maturity-onset diabetes of the young (MODY), characterized by mild hyperglycemia, which is present at birth, but is often only detected later in life during screening for other purposes. Homozygous inactivating GCK mutations result in a more severe phenotype, presenting at birth as permanent neonatal diabetes mellitus (PNDM). Several heterozygous activating GCK mutations that cause hypoglycemia have also been reported. A total of 195 mutations in the GCK gene have been described, in a total of 285 families. There are no common mutations and the mutations are distributed throughout the gene. Mutations that cause hypoglycemia are located in various exons in a discrete region of the protein termed the heterotropic allosteric activator site. The identification of a GCK mutation in hyper- and hypoglycemia has implications for the clinical course and clinical management of the disorder.

Thomson KL, Gloyn AL, Colclough K, Batten M, Allen LI, Beards F, Hattersley AT, Ellard S. 2003. Identification of 21 novel glucokinase (GCK) mutations in UK and European Caucasians with maturity-onset diabetes of the young (MODY). Hum Mutat, 22 (5), pp. 417. | Show Abstract | Read more

Maturity-onset diabetes of the young (MODY) resulting from mutations in the glucokinase (GCK) gene accounts for approximately 20% of MODY in the UK. We have performed fluorescent single stranded conformation polymorphism (F-SSCP) analysis or direct sequencing of the GCK gene in 212 patients referred as part of a research cohort or for diagnostic molecular genetic testing. Mutation screening has identified 43 different mutations in 61 individuals, of which 21 are novel. This report details the mutations identified and their associated clinical features.

Gloyn AL, Noordam K, Willemsen MA, Ellard S, Lam WW, Campbell IW, Midgley P, Shiota C, Buettger C, Magnuson MA et al. 2003. Insights into the biochemical and genetic basis of glucokinase activation from naturally occurring hypoglycemia mutations. Diabetes, 52 (9), pp. 2433-2440. | Show Abstract | Read more

Glucokinase (GCK) is a key regulatory enzyme in the pancreatic beta-cell and catalyzes the rate-limiting step for beta-cell glucose metabolism. We report two novel GCK mutations (T65I and W99R) that have arisen de novo in two families with familial hypoglycemia. Insulin levels, although inappropriately high for the degree of hypoglycemia, remain regulated by fluctuations in glycemia, and pancreatic histology was normal. These mutations are within the recently identified heterotropic allosteric activator site in the theoretical model of human beta-cell glucokinase. Functional analysis of the purified recombinant glutathionyl S-transferase fusion proteins of T65I and W99R GCK revealed that the kinetic changes result in a relative increased activity index (a measure of the enzyme's phosphorylating potential) of 9.81 and 6.36, respectively, compared with wild-type. The predicted thresholds for glucose-stimulated insulin release using mathematical modeling were 3.1 (T65I) and 2.8 (W99R) mmol/l, which were in line with the patients' fasting glucose. In conclusion, we have identified two novel spontaneous GCK-activating mutations whose clinical phenotype clearly differs from mutations in ATP-sensitive K(+) channel genes. In vitro studies confirm the validity of structural and functional models of GCK and the putative allosteric activator site, which is a potential drug target for the treatment of type 2 diabetes.

Weedon MN, Gloyn AL, Frayling TM, Hattersley AT, Davey Smith G, Ben-Shlomo Y. 2003. Quantitative traits associated with the Type 2 diabetes susceptibility allele in Kir6.2. Diabetologia, 46 (7), pp. 1021-1023. | Read more

Gloyn AL. 2003. The search for type 2 diabetes genes. Ageing Res Rev, 2 (2), pp. 111-127. | Show Abstract | Read more

Type 2 diabetes (T2DM) is a serious disease with severe complications. Around one in 10 people alive today suffer from type 2 diabetes or are destined to develop it before they die. Inheritance plays an important role in the cause of type 2 diabetes. A considerable amount of research is devoted to defining the genes involved in the aetiology of this widespread disease. This information is crucial if we are to improve our methods of preventing and treating diabetes. Over the last 25 years there have been considerable advances in our understanding of the genetics of diabetes. Important discoveries have been made in dissecting the genes involved in rare monogenic forms of type 2 diabetes which has become a paradigm for genetic studies of type 2 diabetes. This review focuses on the main approaches currently adopted and our current understanding of the genes involved in susceptibility to type 2 diabetes.

Gloyn AL, Weedon MN, Owen KR, Turner MJ, Knight BA, Hitman G, Walker M, Levy JC, Sampson M, Halford S et al. 2003. Large-scale association studies of variants in genes encoding the pancreatic beta-cell KATP channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) confirm that the KCNJ11 E23K variant is associated with type 2 diabetes. Diabetes, 52 (2), pp. 568-572. | Show Abstract | Read more

The genes ABCC8 and KCNJ11, which encode the subunits sulfonylurea receptor 1 (SUR1) and inwardly rectifying potassium channel (Kir6.2) of the beta-cell ATP-sensitive potassium (K(ATP)) channel, control insulin secretion. Common polymorphisms in these genes (ABCC8 exon 16-3t/c, exon 18 T/C, KCNJ11 E23K) have been variably associated with type 2 diabetes, but no large ( approximately 2,000 subjects) case-control studies have been performed. We evaluated the role of these three variants by studying 2,486 U.K. subjects: 854 with type 2 diabetes, 1,182 population control subjects, and 150 parent-offspring type 2 diabetic trios. The E23K allele was associated with diabetes in the case-control study (odds ratio [OR] 1.18 [95% CI 1.04-1.34], P = 0.01) but did not show familial association with diabetes. Neither the exon 16 nor the exon 18 ABCC8 variants were associated with diabetes (1.04 [0.91-1.18], P = 0.57; 0.93 [0.71-1.23], P = 0.63, respectively). Meta-analysis of all case-control data showed that the E23K allele was associated with type 2 diabetes (K allele OR 1.23 [1.12-1.36], P = 0.000015; KK genotype 1.65 [1.34-2.02], P = 0.000002); but the ABCC8 variants were not associated. Our results confirm that E23K increases risk of type 2 diabetes and show that large-scale association studies are important for the identification of diabetes susceptibility alleles.

Cited:

528

Scopus

Gloyn AL, Weedon MN, Owen KR, Turner MJ, Knight BA, Hitman G, Walker M, Levy JC, Sampson M, Halford S et al. 2003. Large-scale association studies of variants in genes encoding the pancreatic beta-cell K-ATP channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) confirm that the KCNJ11 E23K variant is associated with type 2 diabetes DIABETES, 52 (2), pp. 568-572. | Show Abstract | Read more

The genes ABCC8 and KCNJ11, which encode the subunits sulfonylurea receptor 1 (SUR1) and inwardly rectifying potassium channel (Kir6.2) of the β-cell ATP-sensitive potassium (K ATP ) channel, control insulin secretion. Common polymorphisms in these genes (ABCC8 exon 16-3t/c, exon 18 T/C, KCNJ11 E23K) have been variably associated with type 2 diabetes, but no large (∼2,000 subjects) case-control studies have been performed. We evaluated the role of these three variants by studying 2,486 U.K. subjects: 854 with type 2 diabetes, 1,182 population control subjects, and 150 parent-offspring type 2 diabetic trios. The E23K allele was associated with diabetes in the case-control study (odds ratio [OR] 1.18 [95% CI 1.04-1.34] , P = 0.01) but did not show familial association with diabetes. Neither the exon 16 nor the exon 18 ABCC8 variants were associated with diabetes (1.04 [0.91-1.18], P = 0.57; 0.93 [0.71-1.23] , P = 0.63, respectively). Meta-analysis of all case-control data showed that the E23K allele was associated with type 2 diabetes (K allele OR 1.23 [1.12-1.36], P = 0.000015; KK genotype 1.65 [1.34-2.02] , P = 0.000002); but the ABCC8 variants were not associated. Our results confirm that E23K increases risk of type 2 diabetes and show that large-scale association studies are important for the identification of diabetes susceptibility alleles.

Gloyn AL, Hashim Y, Ashcroft SJH, Ashfield R, Wiltshire S, Turner RC. 2003. Erratum Diabetic Medicine, 20 (3), pp. 252-252. | Read more

Frayling TM, Hattersley AT, McCarthy A, Holly J, Mitchell SM, Gloyn AL, Owen K, Davies D, Smith GD, Ben-Shlomo Y. 2002. A putative functional polymorphism in the IGF-I gene: association studies with type 2 diabetes, adult height, glucose tolerance, and fetal growth in U.K. populations. Diabetes, 51 (7), pp. 2313-2316. | Show Abstract | Read more

IGF-I has a critical role in growth and metabolism. A microsatellite polymorphism 1 kb upstream to the IGF-I gene has recently been associated with several adult phenotypes. In a large Dutch cohort, the absence of the commonest allele (Z) was associated with reduced serum IGF-I levels, reduced height, and an increased risk of type 2 diabetes and myocardial infarction. This result has not been replicated, and the role of this polymorphism in these traits in U.K. subjects is not known. We sought further evidence for the involvement of this variant in type 2 diabetes using a case-control study and IGF-I and diabetes-related traits in a population cohort of 640 U.K. individuals aged 25 years. Absence of the common allele was not associated with type 2 diabetes (odds ratio 0.70, 95% CI 0.47-1.04 for X/X versus Z/Z genotype, chi(2) test for trend across genotypes, P = 0.018). In the population cohort, the common allele (Z) was associated with decreased IGF-I levels (P = 0.01), contrary to the Dutch study, but not with adult height (P = 0.23), glucose tolerance (P = 0.84), oral glucose tolerance test-derived values of beta-cell function (P = 0.90), or insulin resistance (P = 0.66). There was no association with measures of fetal growth, including birth weight (P = 0.17). Our results do not support the previous associations and suggest that the promoter microsatellite is unlikely to be functionally important.

Gloyn AL, Ellard S, Shepherd M, Howell RT, Parry EM, Jefferson A, Levy ER, Hattersley AT. 2002. Maturity-onset diabetes of the young caused by a balanced translocation where the 20q12 break point results in disruption upstream of the coding region of hepatocyte nuclear factor-4alpha (HNF4A) gene. Diabetes, 51 (7), pp. 2329-2333. | Show Abstract | Read more

Monogenic human disorders have been used as paradigms for complex genetic disease and as tools for establishing important insights into mechanisms of gene regulation and transcriptional control. Maturity-onset diabetes of the young (MODY) is a monogenic dominantly inherited form of diabetes that is characterized by defective insulin secretion from the pancreatic beta-cells. A wide variety of mutation types in five different genes have been identified that result in this condition. There have been no reports of a chromosome deletion or translocation resulting in MODY. We report a pedigree where MODY cosegregates with a balanced translocation [karyotype 46, XX t(3;20) (p21.2;q12)]. The chromosome 20 break point, 20q12, is within the region of one of the known MODY genes, hepatocyte nuclear factor-4alpha (HNF4A). Fluorescence in situ hybridization analysis demonstrated that the break point does not disrupt the coding region of this gene, but it lies at least 6 kb upstream of the conventional promoter (P1). We propose that this mutation disrupts the spatial relationship between the recently described alternate distal pancreatic promoter (P2) and HNF4A. This is the first case of MODY due to a balanced translocation, and it provides evidence to confirm the crucial role of an upstream regulator of HNF4A gene expression in the beta-cell.

Mitchell SM, Gloyn AL, Owen KR, Hattersley AT, Frayling TM. 2002. The role of the HNF4alpha enhancer in type 2 diabetes. Mol Genet Metab, 76 (2), pp. 148-151. | Show Abstract | Read more

The genetic causes of type 2 diabetes are not well understood. The disease has been linked to chromosome 20q12-q13.1 a region which harbors the transcription factor HNF4alpha. Mutations in the coding region of HNF4alpha cause maturity onset diabetes of the young, an autosomal dominant form of diabetes, but do not account for the linkage to this region. An enhancer element has recently been characterized 6 kb 5' of the HNF4alpha P1 promoter containing binding sites for the transcription factors HNF1, HNF4, HNF3, and C/EBP, which are overlapped by glucocorticoid consensus sites. We hypothesized that variation in the enhancer element disrupts HNF4alpha expression in the liver and increases susceptibility to type 2 diabetes. We screened for variants of the enhancer element in 39 white UK young onset diabetic subjects, giving >95% power to identify variants with minor allele frequencies of >5%. No variants of the enhancer element were found in this population. We conclude that variation in the HNF4alpha enhancer element is not a common cause of susceptibility to type 2 diabetes.

Gloyn AL, Desai M, Clark A, Levy JC, Holman RR, Frayling TM, Hattersley AT, Ashcroft SJ. 2002. Human calcium/calmodulin-dependent protein kinase II gamma gene (CAMK2G): cloning, genomic structure and detection of variants in subjects with type II diabetes. Diabetologia, 45 (4), pp. 580-583. | Show Abstract | Read more

AIMS/HYPOTHESIS: Ca(2+)/calmodulin-dependent protein kinase II, is expressed in the pancreatic beta cells and is activated by glucose and other secretagogues in a manner correlating with insulin secretion. The activation of Ca(2+)/calmodulin-dependent protein kinase II mediates some of the actions of Ca(2+) on the exocytosis of insulin. We therefore investigated the gene encoding the gamma isoform ( CAMK2G) which has been shown to be expressed in human beta cells as a candidate gene for Type II (non-insulin-dependent) diabetes mellitus. METHODS: Human CAMK2G was cloned from a total human P1 artificial chromosome library using a partial Ca(2+)/calmodulin-dependent protein kinase gamma(E) cDNA probe. Positive PAC clones were localised to chromosome 10q22 by fluorescence in situ hybridisation. To obtain structural information and the sequences of the exon-intron boundaries, the published genomic structures of the rat and mouse genes allowed the putative exon-intron boundaries of human CAMK2G to be amplified by vectorette polymerase chain reaction and sequenced. Sequence variants in each exon were identified using single stranded conformational polymorphism analysis. RESULTS: The human CAMK2G gene comprises 22 exons which range in size between 43 to 230 bp. Screening of the exons and exon-intron boundaries identified two single nucleotide polymorphisms. These did not show association with diabetes in 122 patients and 144 control subjects. CONCLUSIONS/INTERPRETATION: We have identified the genomic structure of CAMK2G to enable further study of this potential candidate gene. Variation in this gene is not strongly associated with diabetes in Caucasians in the United Kingdom. We have identified two single nucleotide polymorphisms which, with appropriately large case control studies, can be used to assess the role of CAMK2G in the susceptibility to Type II diabetes.

Gloyn AL, Ellard S, Shield JP, Temple IK, Mackay DJ, Polak M, Barrett T, Hattersley AT. 2002. Complete glucokinase deficiency is not a common cause of permanent neonatal diabetes. Diabetologia, 45 (2), pp. 290. | Read more

Mitchell SMS, Gloyn AL, Owen KR, Hattersley AT, Frayling TM. 2002. The role of the HNF4α enhancer in type 2 diabetes variants of the HNF4α enhancer are not a common cause of susceptibility to type 2 diabetes Molecular Genetics and Metabolism, 76 (2), pp. 148-151. | Show Abstract | Read more

The genetic causes of type 2 diabetes are not well understood. The disease has been linked to chromosome 20q12-q13.1 a region which harbors the transcription factor HNF4α. Mutations in the coding region of HNF4α cause maturity onset diabetes of the young, an autosomal dominant form of diabetes, but do not account for the linkage to this region. An enhancer element has recently been characterized 6 kb 5′ of the HNF4α P1 promoter containing binding sites for the transcription factors HNF1, HNF4, HNF3, and C/EBP, which are overlapped by glucocorticoid consensus sites. We hypothesized that variation in the enhancer element disrupts HNF4α expression in the liver and increases susceptibility to type 2 diabetes. We screened for variants of the enhancer element in 39 white UK young onset diabetic subjects, giving > 95% power to identify variants with minor allele frequencies of > 5%. No variants of the enhancer element were found in this population. We conclude that variation in the HNF4α enhancer element is not a common cause of susceptibility to type 2 diabetes. © 2002 Elsevier Science (USA). All rights reserved.

Gloyn AL, McCarthy MI. 2001. The genetics of type 2 diabetes. Best Pract Res Clin Endocrinol Metab, 15 (3), pp. 293-308. | Show Abstract | Read more

Type 2 diabetes mellitus is not a single disease but a genetically heterogeneous group of metabolic disorders sharing glucose intolerance. The precise underlying biochemical defects are unknown and almost certainly include impairments of both insulin secretion and action. The rapidly increasing prevalence of T2D world wide makes it a major cause of morbidity and mortality. Understanding the genetic aetiology of T2D will facilitate its diagnosis, treatment and prevention. The results of linkage and association studies to date demonstrate that, as with other common diseases, multiple genes are involved in the susceptibility to T2D, each making a modest contribution to the overall risk. The completion of the draft human genome sequence and a brace of novel tools for genomic analysis promise to accelerate progress towards a more complete molecular description of T2D.

Gloyn AL, Ashcroft SJ. 2001. The beta-cell Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) beta3 isoform containing a proline-rich tandem repeat in the association domain can be found in the human genome. Diabetologia, 44 (6), pp. 787. | Read more

Gloyn AL, Hashim Y, Ashcroft SJ, Ashfield R, Wiltshire S, Turner RC, UK Prospective Diabetes Study (UKPDS 53). 2001. Association studies of variants in promoter and coding regions of beta-cell ATP-sensitive K-channel genes SUR1 and Kir6.2 with Type 2 diabetes mellitus (UKPDS 53). Diabet Med, 18 (3), pp. 206-212. | Show Abstract | Read more

AIMS: The beta-cell ATP-sensitive potassium channel consists of two subunits, SUR1 and Kir6.2. Population association studies have shown that three variants in SUR1 and one in Kir6.2 are associated with Type 2 diabetes. These polymorphisms do not result in a functional change or affect splicing, suggesting that they could be in linkage disequilibrium with a pathogenic mutation. The present study aimed firstly to screen the promoter regions of SUR1 and Kir6.2 to determine whether mutations in linkage disequilibrium with the silent variants lie in regulatory regions, which might lead to changes in gene expression. Secondly, novel and previously described variants associated with Type 2 diabetes (SUR1 exon 16-3t, exon 18 T, and Kir6.2 E23K) were investigated in the UKPDS cohort. METHODS: The promoter sequences of both genes were screened by single-stranded conformational polymorphism analysis for variants associated with Type 2 diabetes. The previously reported variants were evaluated in 364 Type 2 diabetic and 328 normoglycaemic control subjects. RESULTS: Two variants were detected in the SUR1 promoter, a three base insertion (caa) at -522 bp and a single base substitution at - 679 bp (c-->g). Neither of the variants were associated with diabetes, nor were they in a sequence consensus region for transcription factors. No association with diabetes was observed for either SUR1 variant. However, in contrast, analysis of the Kir6.2 E23K variant showed that the KK homozygosity was more frequent in Type 2 diabetic than control subjects. Variants were not associated with clinical characteristics nor did they affect response to sulphonylurea therapy CONCLUSION: There is no support at present for mutations in either Kir6.2 or SUR1 promoter sequences contributing to Type 2 diabetes. However, the minimal promoter region of SUR1 has yet to be investigated. The E23K variant of Kir6.2 is associated with Type 2 diabetes mellitus in the UKPDS cohort.

Gloyn AL. 2001. The genetics of diabetes: A progress report Practical Diabetes International, 18 (7), pp. 246-250. | Show Abstract | Read more

Inheritance plays an important role in the cause of diabetes. A considerable amount of research is devoted to defining the genes involved in the aetiology of this widespread disease. This information is crucial if we are to improve our methods of preventing and treating diabetes. Over the last 25 years there have been considerable advances in our understanding of the genetics of both type 1 (insulin dependent) and type 2 (non-insulin dependent) diabetes. Important discoveries have been made in dissecting the genes involved in a rare monogenic form of type 2 diabetes, which has become a paradigm for genetic studies of type 2 diabetes. Copyright © 2001 John Wiley & Sons, Ltd.

Hashim Y, Gloyn A, Turner RC. 1997. Screening of sulphonylurea receptor 1 (SUR1) for mutations in different types of NIDDM DIABETOLOGIA, 40 pp. 643-643.

Thomsen SK, McCarthy MI, Gloyn AL. 2016. The Importance of Context: Uncovering Species- and Tissue-Specific Effects of Genetic Risk Variants for Type 2 Diabetes. Front Endocrinol (Lausanne), 7 (AUG), pp. 112. | Read more

van de Bunt M, Lako M, Barrett A, Gloyn AL, Hansson M, McCarthy MI, Beer NL, Honoré C. 2016. Insights into islet development and biology through characterization of a human iPSC-derived endocrine pancreas model. Islets, 8 (3), pp. 83-95. | Show Abstract | Read more

Directed differentiation of stem cells offers a scalable solution to the need for human cell models recapitulating islet biology and T2D pathogenesis. We profiled mRNA expression at 6 stages of an induced pluripotent stem cell (iPSC) model of endocrine pancreas development from 2 donors, and characterized the distinct transcriptomic profiles associated with each stage. Established regulators of endodermal lineage commitment, such as SOX17 (log2 fold change [FC] compared to iPSCs = 14.2, p-value = 4.9 × 10(-5)) and the pancreatic agenesis gene GATA6 (log2 FC = 12.1, p-value = 8.6 × 10(-5)), showed transcriptional variation consistent with their known developmental roles. However, these analyses highlighted many other genes with stage-specific expression patterns, some of which may be novel drivers or markers of islet development. For example, the leptin receptor gene, LEPR, was most highly expressed in published data from in vivo-matured cells compared to our endocrine pancreas-like cells (log2 FC = 5.5, p-value = 2.0 × 10(-12)), suggesting a role for the leptin pathway in the maturation process. Endocrine pancreas-like cells showed significant stage-selective expression of adult islet genes, including INS, ABCC8, and GLP1R, and enrichment of relevant GO-terms (e.g. "insulin secretion"; odds ratio = 4.2, p-value = 1.9 × 10(-3)): however, principal component analysis indicated that in vitro-differentiated cells were more immature than adult islets. Integration of the stage-specific expression information with genetic data from T2D genome-wide association studies revealed that 46 of 82 T2D-associated loci harbor genes present in at least one developmental stage, facilitating refinement of potential effector transcripts. Together, these data show that expression profiling in an iPSC islet development model can further understanding of islet biology and T2D pathogenesis.

Althari S, Gloyn AL. 2015. When is it MODY? Challenges in the Interpretation of Sequence Variants in MODY Genes The Review of Diabetic Studies, 12 (3-4), pp. 330-348. | Read more

Pal A, Potjer TP, Thomsen SK, Ng HJ, Barrett A, Scharfmann R, James TJ, Bishop DT, Karpe F, Godsland IF et al. 2016. Loss-of-Function Mutations in the Cell-Cycle Control Gene CDKN2A Impact on Glucose Homeostasis in Humans. Diabetes, 65 (2), pp. 527-533. | Show Abstract | Read more

At the CDKN2A/B locus, three independent signals for type 2 diabetes risk are located in a noncoding region near CDKN2A. The disease-associated alleles have been implicated in reduced β-cell function, but the underlying mechanism remains elusive. In mice, β-cell-specific loss of Cdkn2a causes hyperplasia, while overexpression leads to diabetes, highlighting CDKN2A as a candidate effector transcript. Rare CDKN2A loss-of-function mutations are a cause of familial melanoma and offer the opportunity to determine the impact of CDKN2A haploinsufficiency on glucose homeostasis in humans. To test the hypothesis that such individuals have improved β-cell function, we performed oral and intravenous glucose tolerance tests on mutation carriers and matched control subjects. Compared with control subjects, carriers displayed increased insulin secretion, impaired insulin sensitivity, and reduced hepatic insulin clearance. These results are consistent with a model whereby CDKN2A loss affects a range of different tissues, including pancreatic β-cells and liver. To test for direct effects of CDKN2A-loss on β-cell function, we performed knockdown in a human β-cell line, EndoC-bH1. This revealed increased insulin secretion independent of proliferation. Overall, we demonstrated that CDKN2A is an important regulator of glucose homeostasis in humans, thus supporting its candidacy as an effector transcript for type 2 diabetes-associated alleles in the region.

van de Bunt M, Manning Fox JE, Dai X, Barrett A, Grey C, Li L, Bennett AJ, Johnson PR, Rajotte RV, Gaulton KJ et al. 2015. Transcript Expression Data from Human Islets Links Regulatory Signals from Genome-Wide Association Studies for Type 2 Diabetes and Glycemic Traits to Their Downstream Effectors. PLoS Genet, 11 (12), pp. e1005694. | Show Abstract | Read more

The intersection of genome-wide association analyses with physiological and functional data indicates that variants regulating islet gene transcription influence type 2 diabetes (T2D) predisposition and glucose homeostasis. However, the specific genes through which these regulatory variants act remain poorly characterized. We generated expression quantitative trait locus (eQTL) data in 118 human islet samples using RNA-sequencing and high-density genotyping. We identified fourteen loci at which cis-exon-eQTL signals overlapped active islet chromatin signatures and were coincident with established T2D and/or glycemic trait associations. ‎At some, these data provide an experimental link between GWAS signals and biological candidates, such as DGKB and ADCY5. At others, the cis-signals implicate genes with no prior connection to islet biology, including WARS and ZMIZ1. At the ZMIZ1 locus, we show that perturbation of ZMIZ1 expression in human islets and beta-cells influences exocytosis and insulin secretion, highlighting a novel role for ZMIZ1 in the maintenance of glucose homeostasis. Together, these findings provide a significant advance in the mechanistic insights of T2D and glycemic trait association loci.

Gaulton KJ, Ferreira T, Lee Y, Raimondo A, Mägi R, Reschen ME, Mahajan A, Locke A, Rayner NW, Robertson N et al. 2015. Genetic fine mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci. Nat Genet, 47 (12), pp. 1415-1425. | Show Abstract | Read more

We performed fine mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in or near KCNQ1. 'Credible sets' of the variants most likely to drive each distinct signal mapped predominantly to noncoding sequence, implying that association with T2D is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine mapping implicated rs10830963 as driving T2D association. We confirmed that the T2D risk allele for this SNP increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease.

Mahajan A, Sim X, Ng HJ, Manning A, Rivas MA, Highland HM, Locke AE, Grarup N, Im HK, Cingolani P et al. 2015. Identification and functional characterization of G6PC2 coding variants influencing glycemic traits define an effector transcript at the G6PC2-ABCB11 locus. PLoS Genet, 11 (1), pp. e1004876. | Show Abstract | Read more

Genome wide association studies (GWAS) for fasting glucose (FG) and insulin (FI) have identified common variant signals which explain 4.8% and 1.2% of trait variance, respectively. It is hypothesized that low-frequency and rare variants could contribute substantially to unexplained genetic variance. To test this, we analyzed exome-array data from up to 33,231 non-diabetic individuals of European ancestry. We found exome-wide significant (P<5×10-7) evidence for two loci not previously highlighted by common variant GWAS: GLP1R (p.Ala316Thr, minor allele frequency (MAF)=1.5%) influencing FG levels, and URB2 (p.Glu594Val, MAF = 0.1%) influencing FI levels. Coding variant associations can highlight potential effector genes at (non-coding) GWAS signals. At the G6PC2/ABCB11 locus, we identified multiple coding variants in G6PC2 (p.Val219Leu, p.His177Tyr, and p.Tyr207Ser) influencing FG levels, conditionally independent of each other and the non-coding GWAS signal. In vitro assays demonstrate that these associated coding alleles result in reduced protein abundance via proteasomal degradation, establishing G6PC2 as an effector gene at this locus. Reconciliation of single-variant associations and functional effects was only possible when haplotype phase was considered. In contrast to earlier reports suggesting that, paradoxically, glucose-raising alleles at this locus are protective against type 2 diabetes (T2D), the p.Val219Leu G6PC2 variant displayed a modest but directionally consistent association with T2D risk. Coding variant associations for glycemic traits in GWAS signals highlight PCSK1, RREB1, and ZHX3 as likely effector transcripts. These coding variant association signals do not have a major impact on the trait variance explained, but they do provide valuable biological insights.

Raimondo A, Chakera AJ, Thomsen SK, Colclough K, Barrett A, De Franco E, Chatelas A, Demirbilek H, Akcay T, Alawneh H et al. 2014. Phenotypic severity of homozygous GCK mutations causing neonatal or childhood-onset diabetes is primarily mediated through effects on protein stability. Hum Mol Genet, 23 (24), pp. 6432-6440. | Show Abstract | Read more

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

Rees MG, Raimondo A, Wang J, Ban MR, Davis MI, Barrett A, Ranft J, Jagdhuhn D, Waterstradt R, Baltrusch S et al. 2014. Inheritance of rare functional GCKR variants and their contribution to triglyceride levels in families. Hum Mol Genet, 23 (20), pp. 5570-5578. | Show Abstract | Read more

Significant resources have been invested in sequencing studies to investigate the role of rare variants in complex disease etiology. However, the diagnostic interpretation of individual rare variants remains a major challenge, and may require accurate variant functional classification and the collection of large numbers of variant carriers. Utilizing sequence data from 458 individuals with hypertriglyceridemia and 333 controls with normal plasma triglyceride levels, we investigated these issues using GCKR, encoding glucokinase regulatory protein. Eighteen rare non-synonymous GCKR variants identified in these 791 individuals were comprehensively characterized by a range of biochemical and cell biological assays, including a novel high-throughput-screening-based approach capable of measuring all variant proteins simultaneously. Functionally deleterious variants were collectively associated with hypertriglyceridemia, but a range of in silico prediction algorithms showed little consistency between algorithms and poor agreement with functional data. We extended our study by obtaining sequence data on family members; however, functional variants did not co-segregate with triglyceride levels. Therefore, despite evidence for their collective functional and clinical relevance, our results emphasize the low predictive value of rare GCKR variants in individuals and the complex heritability of lipid traits.

Pasquali L, Gaulton KJ, Rodríguez-Seguí SA, Mularoni L, Miguel-Escalada I, Akerman İ, Tena JJ, Morán I, Gómez-Marín C, van de Bunt M et al. 2014. Pancreatic islet enhancer clusters enriched in type 2 diabetes risk-associated variants. Nat Genet, 46 (2), pp. 136-143. | Show Abstract | Read more

Type 2 diabetes affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. Pancreatic islet dysfunction is central in type 2 diabetes pathogenesis, and understanding islet genome regulation could therefore provide valuable mechanistic insights. We have now mapped and examined the function of human islet cis-regulatory networks. We identify genomic sequences that are targeted by islet transcription factors to drive islet-specific gene activity and show that most such sequences reside in clusters of enhancers that form physical three-dimensional chromatin domains. We find that sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers and identify trait-associated variants that disrupt DNA binding and islet enhancer activity. Our studies illustrate how islet transcription factors interact functionally with the epigenome and provide systematic evidence that the dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes.

Thomsen SK, Gloyn AL. 2014. The pancreatic β cell: Recent insights from human genetics Trends in Endocrinology and Metabolism, 25 (8), pp. 425-434. | Show Abstract | Read more

Diabetes mellitus is a metabolic disease characterised by relative or absolute pancreatic β cell dysfunction. Genetic variants implicated in disease risk can be identified by studying affected individuals. To understand the mechanisms driving genetic associations, variants must be translated through causative transcripts to biological insights. Studies into the genetic basis of Mendelian forms of diabetes have successfully identified genes involved in both β cell function and pancreatic development. For type 2 diabetes (T2D), genome-wide association studies (GWASs) are uncovering an ever-increasing number of susceptibility variants that exert their effect through β cell dysfunction, but translation to mechanistic understanding has in most cases been slow. Improved annotations of the islet genome and advances in whole-genome and -exome sequencing (WHS and WES) have facilitated recent progress. © 2014 Elsevier Ltd.

Travers ME, Mackay DJ, Dekker Nitert M, Morris AP, Lindgren CM, Berry A, Johnson PR, Hanley N, Groop LC, McCarthy MI, Gloyn AL. 2013. Insights into the molecular mechanism for type 2 diabetes susceptibility at the KCNQ1 locus from temporal changes in imprinting status in human islets. Diabetes, 62 (3), pp. 987-992. | Show Abstract | Read more

The molecular basis of type 2 diabetes predisposition at most established susceptibility loci remains poorly understood. KCNQ1 maps within the 11p15.5 imprinted domain, a region with an established role in congenital growth phenotypes. Variants intronic to KCNQ1 influence diabetes susceptibility when maternally inherited. By use of quantitative PCR and pyrosequencing of human adult islet and fetal pancreas samples, we investigated the imprinting status of regional transcripts and aimed to determine whether type 2 diabetes risk alleles influence regional DNA methylation and gene expression. The results demonstrate that gene expression patterns differ by developmental stage. CDKN1C showed monoallelic expression in both adult and fetal tissue, whereas PHLDA2, SLC22A18, and SLC22A18AS were biallelically expressed in both tissues. Temporal changes in imprinting were observed for KCNQ1 and KCNQ1OT1, with monoallelic expression in fetal tissues and biallelic expression in adult samples. Genotype at the type 2 diabetes risk variant rs2237895 influenced methylation levels of regulatory sequence in fetal pancreas but without demonstrable effects on gene expression. We demonstrate that CDKN1C, KCNQ1, and KCNQ1OT1 are most likely to mediate diabetes susceptibility at the KCNQ1 locus and identify temporal differences in imprinting status and methylation effects, suggesting that diabetes risk effects may be mediated in early development.

Pal A, Barber TM, Van de Bunt M, Rudge SA, Zhang Q, Lachlan KL, Cooper NS, Linden H, Levy JC, Wakelam MJ et al. 2012. PTEN mutations as a cause of constitutive insulin sensitivity and obesity. N Engl J Med, 367 (11), pp. 1002-1011. | Show Abstract | Read more

BACKGROUND: Epidemiologic and genetic evidence links type 2 diabetes, obesity, and cancer. The tumor-suppressor phosphatase and tensin homologue (PTEN) has roles in both cellular growth and metabolic signaling. Germline PTEN mutations cause a cancer-predisposition syndrome, providing an opportunity to study the effect of PTEN haploinsufficiency in humans. METHODS: We measured insulin sensitivity and beta-cell function in 15 PTEN mutation carriers and 15 matched controls. Insulin signaling was measured in muscle and adipose-tissue biopsy specimens from 5 mutation carriers and 5 well-matched controls. We also assessed the effect of PTEN haploinsufficiency on obesity by comparing anthropometric indexes between the 15 patients and 2097 controls from a population-based study of healthy adults. Body composition was evaluated by means of dual-emission x-ray absorptiometry and skinfold thickness. RESULTS: Measures of insulin resistance were lower in the patients with a PTEN mutation than in controls (e.g., mean fasting plasma insulin level, 29 pmol per liter [range, 9 to 99] vs. 74 pmol per liter [range, 22 to 185]; P=0.001). This finding was confirmed with the use of hyperinsulinemic euglycemic clamping, showing a glucose infusion rate among carriers 2 times that among controls (P=0.009). The patients' insulin sensitivity could be explained by the presence of enhanced insulin signaling through the PI3K-AKT pathway, as evidenced by increased AKT phosphorylation. The PTEN mutation carriers were obese as compared with population-based controls (mean body-mass index [the weight in kilograms divided by the square of the height in meters], 32 [range, 23 to 42] vs. 26 [range, 15 to 48]; P<0.001). This increased body mass in the patients was due to augmented adiposity without corresponding changes in fat distribution. CONCLUSIONS: PTEN haploinsufficiency is a monogenic cause of profound constitutive insulin sensitization that is apparently obesogenic. We demonstrate an apparently divergent effect of PTEN mutations: increased risks of obesity and cancer but a decreased risk of type 2 diabetes owing to enhanced insulin sensitivity. (Funded by the Wellcome Trust and others.).

Rees MG, Ng D, Ruppert S, Turner C, Beer NL, Swift AJ, Morken MA, Below JE, Blech I, NISC Comparative Sequencing Program et al. 2012. Correlation of rare coding variants in the gene encoding human glucokinase regulatory protein with phenotypic, cellular, and kinetic outcomes. J Clin Invest, 122 (1), pp. 205-217. | Show Abstract | Read more

Defining the genetic contribution of rare variants to common diseases is a major basic and clinical science challenge that could offer new insights into disease etiology and provide potential for directed gene- and pathway-based prevention and treatment. Common and rare nonsynonymous variants in the GCKR gene are associated with alterations in metabolic traits, most notably serum triglyceride levels. GCKR encodes glucokinase regulatory protein (GKRP), a predominantly nuclear protein that inhibits hepatic glucokinase (GCK) and plays a critical role in glucose homeostasis. The mode of action of rare GCKR variants remains unexplored. We identified 19 nonsynonymous GCKR variants among 800 individuals from the ClinSeq medical sequencing project. Excluding the previously described common missense variant p.Pro446Leu, all variants were rare in the cohort. Accordingly, we functionally characterized all variants to evaluate their potential phenotypic effects. Defects were observed for the majority of the rare variants after assessment of cellular localization, ability to interact with GCK, and kinetic activity of the encoded proteins. Comparing the individuals with functional rare variants to those without such variants showed associations with lipid phenotypes. Our findings suggest that, while nonsynonymous GCKR variants, excluding p.Pro446Leu, are rare in individuals of mixed European descent, the majority do affect protein function. In sum, this study utilizes computational, cell biological, and biochemical methods to present a model for interpreting the clinical significance of rare genetic variants in common disease.

Rees MG, Wincovitch S, Schultz J, Waterstradt R, Beer NL, Baltrusch S, Collins FS, Gloyn AL. 2012. Cellular characterisation of the GCKR P446L variant associated with type 2 diabetes risk. Diabetologia, 55 (1), pp. 114-122. | Show Abstract | Read more

AIMS/HYPOTHESIS: Translation of genetic association signals into molecular mechanisms for diabetes has been slow. The glucokinase regulatory protein (GKRP; gene symbol GCKR) P446L variant, associated with inverse modulation of glucose- and lipid-related traits, has been shown to alter the kinetics of glucokinase (GCK) inhibition. As GCK inhibition is associated with nuclear sequestration, we aimed to determine whether this variant also alters the direct interaction between GKRP and GCK and their intracellular localisation. METHODS: Fluorescently tagged rat and human wild-type (WT)- or P446L-GCKR and GCK were transiently transfected into HeLa cells and mouse primary hepatocytes. Whole-cell and nuclear fluorescence was quantified in individual cells exposed to low- or high-glucose conditions (5.5 or 25 mmol/l glucose, respectively). Interaction between GCK and GKRP was measured by sensitised emission-based fluorescence resonance energy transfer (FRET) efficiency. RESULTS: P446L-GKRP had a decreased degree of nuclear localisation, ability to sequester GCK and direct interaction with GCK as measured by FRET compared with WT-GKRP. Decreased interaction was observed between WT-GKRP and GCK at high compared with low glucose, but not between P446L-GKRP and GCK. Rat WT-GKRP and P446L-GKRP behaved quite differently: both variants responded to high glucose by diminished sequestration of GCK but showed no effect of the P446L variant on nuclear localisation or GCK sequestration. CONCLUSIONS/INTERPRETATION: Our study suggests the common human P446L-GKRP variant protein results in elevated hepatic glucose uptake and disposal by increasing active cytosolic GCK. This would increase hepatic lipid biosynthesis but decrease fasting plasma glucose concentrations and provides a potential mechanism for the protective effect of this allele on type 2 diabetes risk.

Owen KR, Thanabalasingham G, James TJ, Karpe F, Farmer AJ, McCarthy MI, Gloyn AL. 2010. Assessment of high-sensitivity C-reactive protein levels as diagnostic discriminator of maturity-onset diabetes of the young due to HNF1A mutations. Diabetes Care, 33 (9), pp. 1919-1924. | Show Abstract | Read more

OBJECTIVE: Despite the clinical importance of an accurate diagnosis in individuals with monogenic forms of diabetes, restricted access to genetic testing leaves many patients with undiagnosed diabetes. Recently, common variation near the HNF1 homeobox A (HNF1A) gene was shown to influence C-reactive protein levels in healthy adults. We hypothesized that serum levels of high-sensitivity C-reactive protein (hs-CRP) could represent a clinically useful biomarker for the identification of HNF1A mutations causing maturity-onset diabetes of the young (MODY). RESEARCH DESIGN AND METHODS: Serum hs-CRP was measured in subjects with HNF1A-MODY (n = 31), autoimmune diabetes (n = 316), type 2 diabetes (n = 240), and glucokinase (GCK) MODY (n = 24) and in nondiabetic individuals (n = 198). The discriminative accuracy of hs-CRP was evaluated through receiver operating characteristic (ROC) curve analysis, and performance was compared with standard diagnostic criteria. Our primary analyses excluded approximately 11% of subjects in whom the single available hs-CRP measurement was >10 mg/l. RESULTS: Geometric mean (SD range) hs-CRP levels were significantly lower (P <or= 0.009) for HNF1A-MODY individuals, 0.20 (0.03-1.14) mg/l, than for any other group: autoimmune diabetes 0.58 (0.10-2.75) mg/l, type 2 diabetes 1.33 (0.28-6.14) mg/l, GCK-MODY 1.01 (0.19-5.33) mg/l, and nondiabetic 0.48 (0.10-2.42) mg/l. The ROC-derived C-statistic for discriminating HNF1A-MODY and type 2 diabetes was 0.8. Measurement of hs-CRP, either alone or in combination with current diagnostic criteria, was superior to current diagnostic criteria alone. Sensitivity and specificity for the combined criteria approached 80%. CONCLUSIONS: Serum hs-CRP levels are markedly lower in HNF1A-MODY than in other forms of diabetes. hs-CRP has potential as a widely available, cost-effective screening test to support more precise targeting of MODY diagnostic testing.

Unravelling molecular mechanisms for type 2 diabetes using genetic and genomic approaches

There are now over 100 regions of the human genome which robustly influence the risk of developing type 2 diabetes (T2D).  Each of these association signals provides an opportunity to understand the causal mechanisms driving T2D pathogenesis.  Broad physiological characterisation of T2D-associated variants in humans has elucidated their role in regulation of glucose levels, insulin secretion and/or action, identifying pathways involved in T2D pathogenesis, and demonstrating that most impact ...

View project

1447