OnlineOpen Article. This is a copy of an article published in Diabetic Medicine. This journal is available online at: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1464-5491Genome-wide association studies have identified >30 common variants associated with Type 2 diabetes (>5% minor allele frequency). These variants have small effects on individual risk and do not account for a large proportion of the heritable component of the disease. Monogenic forms of diabetes are caused by mutations that occur in <1:2000 individuals and follow strict patterns of inheritance. In contrast, the role of low frequency genetic variants (minor allele frequency 0.1-5%) in Type 2 diabetes is not known. The aim of this study was to assess the role of low frequency PDX1 (also called IPF1) variants in Type 2 diabetes

Edghill, EL

Ellard, Sian

Frayling, Timothy M.

Hattersley, Andrew T.

Hitman, Graham A.

Khamis, A

McCarthy, MI

Owen, KR

Walker, M

Weedon, Michael N.

Diabetic Medicine

Open Research Exeter

Short Report: GeneticsSequencing PDX1 (insulin promoter factor 1) in 1788 UKindividuals found 5% had a low frequency coding variant,but these variants are not associated with Type 2diabetesE. L. Edghill, A. Khamis, M. N. Weedon*, M. Walker†, G. A. Hitman‡, M. I. McCarthy§–**,K. R. Owen§–, S. Ellard, A. T Hattersley and T. M. Frayling*Institute of Biomedical and Clinical Science, *Genetics of Complex Traits, Peninsula College of Medicine and Dentistry, University of Exeter, †Institute of CellularMedicine, Newcastle University, ‡Department of Diabetes and Metabolism, Barts and the London NHS Trust and School of Medicine and Dentistry, London,§Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, –Oxford NIHR Biomedical Research Centre, Churchill Hospital and**Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UKAccepted 15 February 2011AbstractAim Genome-wide association studies have identified > 30 common variants associated with Type 2 diabetes (> 5% minorallele frequency). These variants have small effects on individual risk and do not account for a large proportion of the heritablecomponent of the disease.Monogenic forms of diabetes are caused bymutations that occur in < 1:2000 individuals and followstrict patterns of inheritance. In contrast, the role of low frequency genetic variants (minor allele frequency 0.1–5%) in Type 2diabetes is not known. The aim of this studywas to assess the role of low frequencyPDX1 (also called IPF1) variants in Type 2diabetes.Methods We sequenced the coding and flanking intronic regions of PDX1 in 910 patients with Type 2 diabetes and 878control subjects.Results We identified a total of 26 variants that occurred in 5.3% of individuals, 14 of which occurred once. Only D76Noccurred in > 1%.We foundnodifference in carrier frequency betweenpatients (5.7%)and control subjects (5.0%) (P = 0.46).There were also no differences between patients and control subjects when analyses were limited to subsets of variants. Thestrongest subset were those variants in the DNA binding domain where all five variants identified were only found in patients(P = 0.06).Conclusion Approximately 5% of UK individuals carry a PDX1 variant, but there is no evidence that these variants, eitherindividually or cumulatively, predispose to Type 2 diabetes. Further studies will need to consider strategies to assess the role ofmultiple variants that occur in < 1 in 1000 individuals.Diabet. Med. 28, 681–684 (2011)Keywords diabetes, genetics, polygenic, variantsAbbreviation PDX1, pancreatic and duodenal homeobox 1 geneIntroductionThe study of the genetic component to complex diseases such asType 2 diabetes has primarily focused on testing commonvariants. Genome-wide association studies focus on commonsingle nucleotide polymorphisms, where common is usuallydefined as > 5% minor allele frequency. To date, there are over30 replicated genome-wide association study case–controlassociations with Type 2 diabetes [1–3]. Despite the successfulidentification of many common variants involved in Type 2diabetes, they explain only a fraction of the estimated geneticcomponent. One possible explanation for this ‘missingheritability’ is that low frequency variants contributesubstantially to the genetic risk of Type 2 diabetes. MostCorrespondence to: Dr Emma L. Edghill, Peninsula Medical School, Institute ofBiomedical and Clinical Science, Barrack Road, Exeter EX2 5DW, UK.E-mail: emma.edghill@pms.ac.ukDIABETICMedicineDOI:10.1111/j.1464-5491.2011.03269.xª 2011 The Authors.Diabetic Medicine ª 2011 Diabetes UK 681variants in the human genome are of low frequency (< 5%) andmanymore are < 1%frequency. Such low frequency variants arepoorly captured by current genome-wide association studymicroarrays. Projects such as the 1000 Genomes Project arelikely to reveal amuch larger set of low frequency variants, but itremains a challenge to perform adequately powered associationtests of such variants with human phenotypes. Few studies havetested comprehensively the role of low frequency variants inType 2 diabetes, either genome wide or in the context ofcandidate genes. One study has tested theWFS1 gene, but foundno associations other than the well-replicated common variant(rs10010131) [4], and a second study tested part of theHNF1Agene [5] but did not identify any associated coding variants.Despite the challenges, there are several proof-of-principleexamples that suggest sequencing strategies will identify lowfrequency variants involved in common human traits. Theseinclude the identification of rarer variants in the IFIH1 gene thatprotect fromType 1 diabetes [6] and the low frequency variantsinNOD2 thathave strongpredisposingeffectsonCrohnsdisease[7]. Other approaches have shown that multiple low frequencycoding variants in one or more genes accumulate at the tails of apopulation distribution for a continuous trait such as lipid levels[8] or blood pressure [9].Mutations in the pancreatic and duodenal homeobox 1(PDX1 ⁄ IPF1) gene are a known cause of monogenic diabetes(OMIM 600733).PDX1 is a key transcription factor involved inpancreatic development, islet hormone expression and theregulation of insulin in the mature B-cell. The importance ofPDX1 in pancreatic development is highlighted by the knockoutmouse model, which has pancreatic agenesis [10]. Thisphenotype is mirrored in humans, where two different familieshave been described with pancreatic agenesis and neonataldiabetes attributable to recessive mutations [11,12].Heterozygous mutation carriers have a later age of diabetesonset. PDX1 has been previously studied as a candidate geneand in genome-wide association studies for Type 2 diabetes, butthese approaches have been limited either to sequencing insmall numbers of patients or common single nucleotidepolymorphisms (genome-wide association studies) and therehave been no robust associationswith diabetes risk. In this study,we used an extensive re-sequencing approach to test the role inType 2diabetes of a comprehensive set of low frequencyand rarePDX1 variants.Subjects and methodsCase–control cohortThe study population consisted of 910 patients with Type 2diabetes. We selected patients diagnosed under 55 years, notinsulin treated within the first year of diagnosis, with a medianage of onset of 43 years (range 17–55 years) and a median BMIof 31 kg ⁄m2 (range 18–58 kg ⁄m2). The control populationconsisted of 878 normoglycaemic individuals, who were notknown to have diabetes at time of blood collection, defined by afasting blood glucose of < 5.5mmol ⁄ l and ⁄or HbA1c < 7%(< 53 mmol ⁄ml). This population had amedian age at samplingof 35 years (range 17–86 years) and amedian BMI of 26 kg ⁄m2(range 17–49 kg ⁄m2). All participants (patients and controlsubjects) were from the South West region of the UK and ofEuropean ancestry and came from four sample collections: theExeter Family Study (control subjects), the Young Type 2diabetes Study (YTYPE 2 DIABETES) and the Diabetes inFamilies Study (Warren2).Sequencing methodsWe screened the two exons and approximately 50 bp of flankingsequence of the PDX1 gene using bidirectional sequencing usingstandard conditions and following manufacturers’ protocols(primers available on request). Sequencing reactions were runon an ABI3730 capillary machine (Applied Biosystems,Warrington, UK). Sequencing was viewed in MutationSurveyor (SoftGenetics, State College, PA, USA) (PDX1nucleotide reference NM 000209.3).We used the bioinformatic tools, SIFT, PolyPhen andMutationTaster (http://blocks.fhcrc.org/sift/SIFT_dbSNP.html,http://genetics.bwh.harvard.edu/pph/, http://www.mutationtaster.org/)to predict the effect novel variantswould have on the PDX1protein (protein reference NP 000200.1).Statistical comparisonTo compare the prevalence of individual variants andaccumulations of variants in patients with Type 2 diabetes vs.control subjects, we used Fisher’s exact test.We had 80%powerto detect variants that occurred in one control subject and sevenpatients with Type 2 diabetes at nominal levels of significance(P = 0.05)ResultsMolecular geneticsWe sequenced 1788 individuals and identified 26 low frequencyand rare variants in the PDX1 gene. The detailed distribution ofthese variants within the cases and controls is shown in Table 1.Of these 26 variants, 22 were in the coding region, of which 17altered the amino acid sequence, four were in the sequenceimmediately flanking the exons and 18were novel (Table 1). Sixof the variants were predicted likely to be deleterious in at leasttwo bioinformatic programs, with three variants (P99H, E160Vand R198C) predicted to be damaging by all three programs.Association with diabetesIndividual rare variant analysisFor each of the 26 rare variants, there was no significantdifference in frequency between patients and control subjects.Only one variant, D76N, occurred in more than 1% ofDIABETICMedicine Assessing the role of low frequency PDX1 variants in Type 2 diabetes • E. L. Edghill et al.ª 2011 The Authors.682 Diabetic Medicine ª 2011 Diabetes UKindividuals and was not associated with Type 2 diabetes (12patients vs. 11 control subjects, P = 0.83).Accumulation of low frequency variants in the PDX1 geneWe found that 5.3% of all individuals carried a variant in thePDX1 gene, with no difference between patients (5.7%) andcontrol subjects (5%) (P = 0.46). There were no differencesbetween patients and control subjects when we carried outsubgroup analysis comparing missense and frameshift variants(42 patients vs. 33 control subjects, P = 0.55), or variants thatwere unique to either patients or control subjects (13 vs. 5,P = 0.09), or unique to either patients or control subjects and inthe coding region (8 vs. 3, P = 0.22). We considered thosepredicted to be deleterious by at least two bioinformaticprograms and there was no significant difference (37 patientsvs. 32 control subjects, P = 0.70). Finally, there were nodifferences when limiting the analyses to those variants in theDNA binding domain (137–203 amino acids), although all fivevariants identified in this regionwere in thepatients (5patientsvs.0 control subjects, P = 0.06).In a secondary analysis, the 5.7%ofpatients carrying a variantwere leaner [28.3 kg ⁄m2 (18–44 kg ⁄m2)] compared withpatients not carrying a variant [31.3 kg ⁄m2 (18–58 kg ⁄m2)](P = 0.002), but not diagnosed earlier (P = 0.15).DiscussionThe identification of gene variants associated with Type 2diabetes but not captured by current genome-wise associationstudies is important for twomain reasons. First, such variants arelikely to be of low frequency and may have appreciably greatereffects on individual risk than the common variants typicallyidentified by genome-wise association studies. Second, if suchvariants alter the coding sequence of genes, they could implicatethe gene’s protein product in the aetiology of diabetes.Our study representsoneof fewattempts to sequence the entirecoding sequence of a known diabetes gene in more than athousand individuals and test the individual and cumulativevariants for association with Type 2 diabetes. Our results areanalogous with those of Fawcett et al. [4], who showed that alarge number of low frequency variants occur in the Wolframsyndrome gene, WFS1, but there is no evidence that thesevariants influence the risk of Type 2 diabetes. Eight per cent ofUK individuals carry a low frequency variant in theWFS1 geneTable 1 PDX1 rare variants identified in 910 patients with Type 2 diabetes and 878 control subjectsPosition of change AA changeCase subjects(n = 910)Control subjects(n = 878) Nucleotide changeSIFT ⁄ PolyPhen ⁄MutationTaster*c.1-25insCTCCCGG 1 1 c.-25 NA*2 N 3 3 c. 6 C > T NA*3 G > A 1 0 c. 8 G > C ) ⁄ + ⁄ )18 C > R 0 1 c. 52 T > C + ⁄ ++ ⁄ )*33 P 2 1 c. 97 T > C ⁄T > A NA33 P > T 6 5 c. 97 C > A + ⁄ ++ ⁄ +*55 G 1 3 c. 165 C > A NA76 D > N 12 11 c. 226 G > A + ⁄ ) ⁄ +*95 P > Q 1 0 c. 284 C > A ) ⁄ ) ⁄ +*96 P > S 0 1 c. 286 C > T ) ⁄ ++ ⁄ )*99 P > H 1 1 c. 296 C > A + ⁄ ++ ⁄ +*117 L > M 1 0 c. 349 C > A + ⁄ ) ⁄ +140 A > T 2 0 c. 418 G > A ) ⁄ ) ⁄ )143 P > R 1 0 c. 428 C > G + ⁄ + ⁄ +*160 E > V 1 0 c. 479 A > T + ⁄ ++ ⁄ +197 R > H 1 0 c. 590 G > A + ⁄ + ⁄ +*198 R > C 2 0 c. 592 C > T + ⁄ ++ ⁄ +239 P > Q 8 10 c. 716 C > A ) ⁄ ++ ⁄ +*242 P > L 4 3 c. 725 C > T ) ⁄ ++ ⁄ +P243insPro(GCC) 1 0 c. 726 insGCC NA*245 G > R 0 1 c. 733 G > A + ⁄ + ⁄ )*250 P 0 2 c. 750 C > A NA*264 G 1 0 c. 792 C > T NA*IVS1 + 1 (c.406 + 1G > C) 1 0 c. 406 + 1 G > C NA*IVS2 – 8 (c.407 – 8G > T) 0 1 c. 407 – 8 G > T NA*IVS2 + 4 (c.846*4G > A) 1 0 c. 846 + 4 G > A NA*Novel variant.SIFT ⁄ PolyPhen ⁄MutationTaster: ) tolerated, + not tolerated (SIFT) ⁄ ) benign, + possibly damaging, ++ probably damaging (PolyPhen) ⁄ )polymorphism, + disease causing (MutationTaster) (http://blocks.fhcrc.org/sift/SIFT_dbSNP.html, http://genetics.bwh.harvard.edu/pph/,http://www.mutationtaster.org/).AA, amino acid; NA, not applicable.DIABETICMedicineOriginal articleª 2011 The Authors.Diabetic Medicine ª 2011 Diabetes UK 683and 5% carry a low frequency variant in the PDX1 gene.Furthermore, the PDX1 variant D76N has been widely studied,but the reproducibility of associationswithType 2 diabetes havevaried [13–18]. Our data are in keeping with the recent meta-analysis of PDX1 D76N case–control studies concluding thatthere is no association with Type 2 diabetes [19].Together with theWFS1 study, our results have a number ofimplications for the study of rarer genetic variation in diabetes.First, the results suggest that a largeproportionof codingvariantswill be present at a low frequency, with all but one of the 26variants we identified in PDX1 in less than 1% of individuals.Such variants will need to confer odds ratios of 1.8 (for 1%frequency) to 4.5 (for 0.1% frequency) to be detectable atP = 5 · 10)8 in 10 000 patients with Type 2 diabetes and10 000 control subjects. Second, 14 of these variants occurredonly once in all 1788 individuals. This distribution of allelefrequencies means that testing the cumulative effects of multiplelow frequency coding variants could be used to potentiallyimprove power. The power of such cumulative tests will dependon the proportion of variants that have a functional effect.Alternatively, studies of low frequency and rare variants shouldconsider tracking variants through families and performing testsof linkage with reduced penetrance. The analysis of PDX1 infurther samples, and possibly functional studies, will strengthenthe evidence for or against the role of coding variants specific tothe DNA-binding domain of PDX1, where we found variants infive individuals with diabetes and none in control subjects.Finally, genome-wide analysis of low frequency variants in largesample populationsmay provide additional insights into the roleof low frequency variants in Type 2 diabetes.In conclusion, our study has shown that PDX1 is an excellentcandidate to capture low frequencyvariants; however, there is noevidence that these variants, either individually or cumulatively,predispose to Type 2 diabetes.Competing interestsNothing to declare.AcknowledgementsThisworkwas fundedby theEuropeanUnion (IntegratedProjectEURODIA LSHM-CT-2006-518153 in the FrameworkProgramme 6 of the European Community). ATH and SE areemployed as core members of the Peninsula NIHR ClinicalResearch Facility. 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Edghill et al.ª 2011 The Authors.684 Diabetic Medicine ª 2011 Diabetes UK

Sequencing PDX1 (insulin promoter factor 1) in 1788 UK individuals found 5% had a low frequency coding variant, but these variants are not associated with Type 2 diabetes

Sequencing PDX1 (insulin promoter factor 1) in 1788 UK individuals found 5% had a low frequency coding variant, but these variants are not associated with Type 2 diabetes

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