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author:("glyn, Anna L.")
1.  Insights Into the Molecular Mechanism for Type 2 Diabetes Susceptibility at the KCNQ1 Locus From Temporal Changes in Imprinting Status in Human Islets 
Diabetes  2013;62(3):987-992.
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.
PMCID: PMC3581222  PMID: 23139357
2.  Identification and Functional Characterization of G6PC2 Coding Variants Influencing Glycemic Traits Define an Effector Transcript at the G6PC2-ABCB11 Locus 
Mahajan, Anubha | Sim, Xueling | Ng, Hui Jin | Manning, Alisa | Rivas, Manuel A. | Highland, Heather M. | Locke, Adam E. | Grarup, Niels | Im, Hae Kyung | Cingolani, Pablo | Flannick, Jason | Fontanillas, Pierre | Fuchsberger, Christian | Gaulton, Kyle J. | Teslovich, Tanya M. | Rayner, N. William | Robertson, Neil R. | Beer, Nicola L. | Rundle, Jana K. | Bork-Jensen, Jette | Ladenvall, Claes | Blancher, Christine | Buck, David | Buck, Gemma | Burtt, Noël P. | Gabriel, Stacey | Gjesing, Anette P. | Groves, Christopher J. | Hollensted, Mette | Huyghe, Jeroen R. | Jackson, Anne U. | Jun, Goo | Justesen, Johanne Marie | Mangino, Massimo | Murphy, Jacquelyn | Neville, Matt | Onofrio, Robert | Small, Kerrin S. | Stringham, Heather M. | Syvänen, Ann-Christine | Trakalo, Joseph | Abecasis, Goncalo | Bell, Graeme I. | Blangero, John | Cox, Nancy J. | Duggirala, Ravindranath | Hanis, Craig L. | Seielstad, Mark | Wilson, James G. | Christensen, Cramer | Brandslund, Ivan | Rauramaa, Rainer | Surdulescu, Gabriela L. | Doney, Alex S. F. | Lannfelt, Lars | Linneberg, Allan | Isomaa, Bo | Tuomi, Tiinamaija | Jørgensen, Marit E. | Jørgensen, Torben | Kuusisto, Johanna | Uusitupa, Matti | Salomaa, Veikko | Spector, Timothy D. | Morris, Andrew D. | Palmer, Colin N. A. | Collins, Francis S. | Mohlke, Karen L. | Bergman, Richard N. | Ingelsson, Erik | Lind, Lars | Tuomilehto, Jaakko | Hansen, Torben | Watanabe, Richard M. | Prokopenko, Inga | Dupuis, Josee | Karpe, Fredrik | Groop, Leif | Laakso, Markku | Pedersen, Oluf | Florez, Jose C. | Morris, Andrew P. | Altshuler, David | Meigs, James B. | Boehnke, Michael | McCarthy, Mark I. | Lindgren, Cecilia M. | Gloyn, Anna L.
PLoS Genetics  2015;11(1):e1004876.
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.
Author Summary
Understanding how FI and FG levels are regulated is important because their derangement is a feature of T2D. Despite recent success from GWAS in identifying regions of the genome influencing glycemic traits, collectively these loci explain only a small proportion of trait variance. Unlocking the biological mechanisms driving these associations has been challenging because the vast majority of variants map to non-coding sequence, and the genes through which they exert their impact are largely unknown. In the current study, we sought to increase our understanding of the physiological pathways influencing both traits using exome-array genotyping in up to 33,231 non-diabetic individuals to identify coding variants and consequently genes associated with either FG or FI levels. We identified novel association signals for both traits including the receptor for GLP-1 agonists which are a widely used therapy for T2D. Furthermore, we identified coding variants at several GWAS loci which point to the genes underlying these association signals. Importantly, we found that multiple coding variants in G6PC2 result in a loss of protein function and lower fasting glucose levels.
PMCID: PMC4307976  PMID: 25625282
3.  Phenotypic severity of homozygous GCK mutations causing neonatal or childhood-onset diabetes is primarily mediated through effects on protein stability 
Human molecular genetics  2014;23(24):6432-6440.
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 (r2 = 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 (r2 = 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.
PMCID: PMC4240195  PMID: 25015100
4.  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  2012;35(7):1482-1484.
To demonstrate the importance of using a combined genetic and functional approach to correctly interpret a genetic test for monogenic diabetes.
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.
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.
These data highlight the need for family/functional studies, even for previously reported pathogenic mutations.
PMCID: PMC3379612  PMID: 22611063
5.  Pancreatic islet enhancer clusters enriched in type 2 diabetes risk–associated variants 
Nature genetics  2014;46(2):136-143.
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 for type 2 diabetes pathogenesis, and therefore understanding islet genome regulation could 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 3D 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 dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes.
PMCID: PMC3935450  PMID: 24413736
6.  The pancreatic β cell: recent insights from human genetics 
•Genes involved in pancreatic development are implicated in Mendelian diabetes.•Studying the genetic basis of diabetes has delivered insights into β cell biology.•Most genetic associations influence diabetes risk through defects in β cell function.•Sequencing-based methods and improved genomic annotations have facilitated advances.
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.
PMCID: PMC4229643  PMID: 24986330
pancreatic β cell; human genetics; diabetes; GWAS
7.  Phenotypic severity of homozygous GCK mutations causing neonatal or childhood-onset diabetes is primarily mediated through effects on protein stability 
Human Molecular Genetics  2014;23(24):6432-6440.
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 (r2 = 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 (r2 = 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.
PMCID: PMC4240195  PMID: 25015100
8.  PTEN Mutations as a Cause of Constitutive Insulin Sensitivity and Obesity 
The New England journal of medicine  2012;367(11):1002-1011.
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.
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.
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.
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.)
PMCID: PMC4072504  PMID: 22970944
9.  Inheritance of rare functional GCKR variants and their contribution to triglyceride levels in families 
Human Molecular Genetics  2014;23(20):5570-5578.
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.
PMCID: PMC4168830  PMID: 24879641
10.  Severe Insulin Resistance and Intrauterine Growth Deficiency Associated With Haploinsufficiency for INSR and CHN2 
Diabetes  2009;58(12):2954-2961.
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.
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.
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 β-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.
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.
PMCID: PMC2780873  PMID: 19720790
11.  Identification of a Novel β-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  2009;58(8):1929-1935.
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 β-cell promoter region, which currently is not routinely screened, could contain pathogenic mutations; therefore, we sequenced this region in 60 such probands.
The β-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.
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 (≥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.
A novel β-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 β-cell GCK promoter should be included.
PMCID: PMC2712784  PMID: 19411616
12.  Analysis of the co-operative interaction between the allosterically regulated proteins GK and GKRP using tryptophan fluorescence 
Biochemical Journal  2014;459(Pt 3):551-564.
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.
Using tryptophan fluorescence we explore in a unique way the co-operative interaction between two allosterically regulated proteins (GK and GKRP), which operate as a critical drug-sensitive molecular switch in hepatic carbohydrate metabolism.
PMCID: PMC4109836  PMID: 24568320
allosteric regulation; co-operativity; diabetes; fructose phosphate ester; glucokinase; glucokinase regulatory protein; metabolic regulation; F1P, fructose 1-phosphate; F6P, fructose 6-phosphate; GCK/GK, glucokinase; GCKR/GKRP, GK regulatory protein; GKA, GK activator; GKRPI, GKRP inhibitor; hGK, human GK; hGKRP, human GKRP; MH, D-mannoheptulose; NATA, N-acetyl-L-tryptophanamide; NPC, nuclear pore complex; PPI, protein–protein interaction; rGKRP, rat GKRP; SIS, sugar isomerase; T2D, Type 2 diabetes; TF, tryptophan fluorescence; WT, wild-type; xGK, Xenopus GK; xGKRP, Xenopus GKRP
13.  Mutations in HNF1A Result in Marked Alterations of Plasma Glycan Profile 
Diabetes  2013;62(4):1329-1337.
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.
PMCID: PMC3609552  PMID: 23274891
14.  Reclassification of Diabetes Etiology in a Family With Multiple Diabetes Phenotypes 
Maturity-onset diabetes of the young (MODY) is uncommon; however, accurate diagnosis facilitates personalized management and informs prognosis in probands and relatives.
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).
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.
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.
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.
PMCID: PMC4186945  PMID: 24606082
15.  A Panel of Diverse Assays to Interrogate the Interaction between Glucokinase and Glucokinase Regulatory Protein, Two Vital Proteins in Human Disease 
PLoS ONE  2014;9(2):e89335.
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.
PMCID: PMC3929664  PMID: 24586696
16.  Argonaute2 Mediates Compensatory Expansion of the Pancreatic β Cell 
Cell Metabolism  2014;19(1):122-134.
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.
Graphical Abstract
•Silencing of miR-184 during insulin resistance promotes its target Ago2•Loss of Ago2 during insulin resistance blocks pancreatic β cell proliferation•Ago2 mediates the suppression of Cadm1 by miR-375 in the β cell•Administration of the ketogenic diet to ob/ob mice rescues miR-184 in islets
Tattikota et al. find that as β cells adapt to increased metabolic demand during insulin resistance in obesity, miR-184 is silenced to alleviate repression of its target Argonaute2, a component of the microRNA-induced silencing complex. Argonaute2 promotes compensatory β cell proliferation via miR-375 and its target genes, including the growth suppressor Cadm1.
PMCID: PMC3945818  PMID: 24361012
17.  Role of KATP Channels in Glucose-Regulated Glucagon Secretion and Impaired Counterregulation in Type 2 Diabetes 
Cell Metabolism  2013;18(6):871-882.
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 Ca2+ 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.
Graphical Abstract
•KATP channel closure stimulates insulin secretion but inhibits glucagon release•α cell depolarization reduces voltage-gated Ca2+ entry and glucagon release•An activating KATP channel mutation impairs glucagon release in mice•KATP channel closure corrects glucagon secretion defect in type 2 diabetic islets
PMCID: PMC3851686  PMID: 24315372
18.  Human β Cell Transcriptome Analysis Uncovers lncRNAs That Are Tissue-Specific, Dynamically Regulated, and Abnormally Expressed in Type 2 Diabetes 
Cell metabolism  2012;16(4):435-448.
A significant portion of the genome is transcribed as long non-coding 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.
PMCID: PMC3475176  PMID: 23040067
19.  Reduced Insulin Exocytosis in Human Pancreatic β-Cells With Gene Variants Linked to Type 2 Diabetes 
Diabetes  2012;61(7):1726-1733.
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 Ca2+ 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.
PMCID: PMC3379663  PMID: 22492527
20.  The miRNA Profile of Human Pancreatic Islets and Beta-Cells and Relationship to Type 2 Diabetes Pathogenesis 
PLoS ONE  2013;8(1):e55272.
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.
PMCID: PMC3555946  PMID: 23372846
22.  Genome-Wide Association Identifies Nine Common Variants Associated With Fasting Proinsulin Levels and Provides New Insights Into the Pathophysiology of Type 2 Diabetes 
Strawbridge, Rona J. | Dupuis, Josée | Prokopenko, Inga | Barker, Adam | Ahlqvist, Emma | Rybin, Denis | Petrie, John R. | Travers, Mary E. | Bouatia-Naji, Nabila | Dimas, Antigone S. | Nica, Alexandra | Wheeler, Eleanor | Chen, Han | Voight, Benjamin F. | Taneera, Jalal | Kanoni, Stavroula | Peden, John F. | Turrini, Fabiola | Gustafsson, Stefan | Zabena, Carina | Almgren, Peter | Barker, David J.P. | Barnes, Daniel | Dennison, Elaine M. | Eriksson, Johan G. | Eriksson, Per | Eury, Elodie | Folkersen, Lasse | Fox, Caroline S. | Frayling, Timothy M. | Goel, Anuj | Gu, Harvest F. | Horikoshi, Momoko | Isomaa, Bo | Jackson, Anne U. | Jameson, Karen A. | Kajantie, Eero | Kerr-Conte, Julie | Kuulasmaa, Teemu | Kuusisto, Johanna | Loos, Ruth J.F. | Luan, Jian'an | Makrilakis, Konstantinos | Manning, Alisa K. | Martínez-Larrad, María Teresa | Narisu, Narisu | Nastase Mannila, Maria | Öhrvik, John | Osmond, Clive | Pascoe, Laura | Payne, Felicity | Sayer, Avan A. | Sennblad, Bengt | Silveira, Angela | Stančáková, Alena | Stirrups, Kathy | Swift, Amy J. | Syvänen, Ann-Christine | Tuomi, Tiinamaija | van 't Hooft, Ferdinand M. | Walker, Mark | Weedon, Michael N. | Xie, Weijia | Zethelius, Björn | Ongen, Halit | Mälarstig, Anders | Hopewell, Jemma C. | Saleheen, Danish | Chambers, John | Parish, Sarah | Danesh, John | Kooner, Jaspal | Östenson, Claes-Göran | Lind, Lars | Cooper, Cyrus C. | Serrano-Ríos, Manuel | Ferrannini, Ele | Forsen, Tom J. | Clarke, Robert | Franzosi, Maria Grazia | Seedorf, Udo | Watkins, Hugh | Froguel, Philippe | Johnson, Paul | Deloukas, Panos | Collins, Francis S. | Laakso, Markku | Dermitzakis, Emmanouil T. | Boehnke, Michael | McCarthy, Mark I. | Wareham, Nicholas J. | Groop, Leif | Pattou, François | Gloyn, Anna L. | Dedoussis, George V. | Lyssenko, Valeriya | Meigs, James B. | Barroso, Inês | Watanabe, Richard M. | Ingelsson, Erik | Langenberg, Claudia | Hamsten, Anders | Florez, Jose C.
Diabetes  2011;60(10):2624-2634.
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.
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.
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.
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.
PMCID: PMC3178302  PMID: 21873549
23.  High-Sensitivity CRP Discriminates HNF1A-MODY From Other Subtypes of Diabetes 
Diabetes Care  2011;34(8):1860-1862.
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.
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).
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).
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.
PMCID: PMC3142017  PMID: 21700917
24.  Metabolic Profiling in Maturity-Onset Diabetes of the Young (MODY) and Young Onset Type 2 Diabetes Fails to Detect Robust Urinary Biomarkers 
PLoS ONE  2012;7(7):e40962.
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.
PMCID: PMC3408469  PMID: 22859960
25.  Identification and Functional Characterisation of Novel Glucokinase Mutations Causing Maturity-Onset Diabetes of the Young in Slovakia 
PLoS ONE  2012;7(4):e34541.
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 (IC50 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.
PMCID: PMC3321013  PMID: 22493702

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