Analysis of the Type 2 Diabetes Associated Single Nucleotide Polymorphisms in the Genes IRS1 and KCNJ11and PPARG2 in Type 1 Diabetes.pdf

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Brief Genetics Report

Analysis of the Type 2 DiabetesAssociated SingleNucleotide Polymorphisms in the Genes IRS1, KCNJ11,

and PPARG2in Type 1 DiabetesChristina Eftychi,1 Joanna M.M. Howson,1 Bryan J. Barratt,1 Adrian Vella,1 Felicity Payne,1

Deborah J. Smyth,1 Rebecca C.J. Twells,1 Neil M. Walker,1 Helen E. Rance,1 Eva Tuomilehto-Wolf,2

Jaakko Tuomilehto,2,3 Dag E. Undlien,4 Kjersti S. Rnningen,5 Cristian Guja,6

Constantin Ionescu-Trgoviste,6 David A. Savage,7 and John A. Todd1

It has been proposed that type 1 and 2 diabetes might

share common pathophysiological pathways and, tosome extent, genetic background. However, to datethere has been no convincing data to establish a molec-ular genetic link between them. We have genotypedthree single nucleotide polymorphisms associated withtype 2 diabetes in a large type 1 diabetic family collec-tion of European descent: Gly972Arg in the insulinreceptor substrate 1 (IRS1) gene, Glu23Lys in the po-tassium inwardly-rectifying channel gene (KCNJ11),and Pro12Ala in the peroxisome proliferativeactivatedreceptor 2 gene (PPARG2). We were unable to confirma recently published association of the IRS1 Gly972Arg

variant with type 1 diabetes. Moreover, KCNJ11Glu23Lys showed no association with type 1 diabetes

(P > 0.05). However, the PPARG2 Pro12Ala variantshowed evidence of association (RR 1.15, 95% CI 1.041.28, P 0.008). Additional studies need to be con-ducted to confirm this result. Diabetes 53:870873,2004

Type 1 and 2 diabetes have been considered genet-ically and pathophysiologically distinct diseases,although clinically, it can sometimes be difficultto distinguish between them in individuals with

deficient or absent endogenous insulin secretion, a featurecommon to both categories. Evidence does exist suggest-ing that there may be shared etiological features (13).Occasionally, individuals who develop diabetes late in lifeinitially present in a similar manner to type 2 diabetes, butdevelop progressive -cell failure together with evidenceof autoimmunity to islet cells, which is characteristic oftype 1 diabetes. This so-called latent autoimmune diabetesin adults could suggest an overlap in the pathogenesis oftype 1 and 2 diabetes in some cases. Furthermore, a recentstudy (4) has reported that early in the course of type 1diabetes, individuals with evidence of islet autoimmunitymay have normal fasting glucose but abnormal postpran-dial glucose tolerance. This pattern of defective postpran-dial secretion of insulin in response to nutrient intake isoften observed in type 2 diabetes as well (4).

There has been some evidence of familial clustering oftype 1 and 2 diabetes (5,6). However, the HLA region, themajor locus in type 1 diabetes, has only infrequently beenassociated with type 2 diabetes (7). Moreover, both dis-eases have been associated with variation in the insulingene but with predisposition determined by opposite

alleles of the 5 variable number tandem repeat locus (8).Recently, genetic studies in type 2 diabetes have begun toreveal disease-associated polymorphisms that have estab-lished some level of general acceptance because they havebeen replicated by independent studies. Two polymor-

phisms that are well established as associated with type 2diabetes are the single nucleotide polymorphisms (SNPs)Pro12Ala in the peroxisome proliferatoractivated recep-tor gene isoform 2 (PPARG2), resulting from a CGtransversion, and Glu23Lys in the potassium inwardlyrectifying channel gene (KCNJ11), resulting from an AGtransition (9). The reported effect sizes are modest (oddsratio [OR] 1.21.3) in meta-analyses (9,10). A large collab-

orative collection of type 1 diabetic families is now avail-

From the 1Juvenile Diabetes Research Foundation/Wellcome Trust Diabetesand Inflammation Laboratory, Cambridge Institute for Medical Research,University of Cambridge, Cambridge, U.K.; the 2Diabetes and Genetic Epide-miology Unit, National Public Health Institute, University of Helsinki, Hel-

sinki, Finland; the 3

Department of Public Health, University of Helsinki,Helsinki, Finland; the 4Institute of Medical Genetics, Ulleval UniversityHospital, University of Oslo, Oslo, Norway; the 5Laboratory of MolecularEpidemiology, Division of Epidemiology, Norwegian Institute of PublicHealth, Oslo, Norway; the 6Clinic of Diabetes, Institute of Diabetes, Nutritionand Metabolic Diseases, Bucharest, Romania; and the 7Department of MedicalGenetics, Queens University Belfast, Belfast City Hospital, Belfast, NorthernIreland.

Address correspondence and reprint requests to John A. Todd, JDRF/WTDiabetes and Inflammation Laboratory, Cambridge Institute for MedicalResearch, Wellcome Trust/MRC Building, Hills Rd., Cambridge CB2 2XY, U.K.E-mail: [email protected].

Recieved for publication 3 October 2003 and accepted in revised form 31October 2003.

ICAM1, intercellular adhesion molecule 1; IRS1, insulin receptor substrate1; KCNJ11, potassium inwardly rectifying channel gene; PPAR, peroxisome

proliferatoractivated receptor; PPARG2, peroxisome proliferatoractivatedreceptor2 gene; SNP, single nucleotide polymorphism; TDT, transmission/disequilibrium test.

2004 by the American Diabetes Association.

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able for genetic analysis (11) with sufficient power todetect effects in the OR range of 1.21.3. Hence, in the

present report we investigated the possibility that the twotype 2 diabetesassociated SNPs were also associatedwith type 1 diabetes in an effort to begin to establish ifoverlaps exist in the two disease pathways. We have alsotested the insulin receptor substrate 1 (IRS1) Gly972Arg

polymorphism because it has been reported to be associ-ated with type 1 diabetes (12), although it is not as wellestablished in type 2 diabetes as thePPARG2andKCNJ11SNPs.

The PPARG gene encodes peroxisome proliferatoractivated receptor (PPAR), a member of the nuclearreceptor superfamily that has regulatory functions inadipocyte differentiation and glucose homeostasis (13).PPAR is a target for a large family of antidiabetic drugs,the thiazolidinediones, that act as PPAR agonists andincrease hepatic and peripheral insulin action in type 2diabetes (14). In a meta-analysis of published studiesthrough February 2003 (9), an OR of 1.27 was estimatedfor the Pro allele of isoform 2 (Pvalue 2 108) in type

2 diabetes. This association is supported by functionaldata, as in vitro studies have shown that the Ala alleledecreases the DNA-binding affinity of the PPAR2, thusreducing its transcriptional activity (15).

The KCNJ11 Glu23Lys variant has also been consis-tently reported (10,16,17) to be associated with type 2diabetes. KCNJ11 encodes for KIR6.2, which comprisesone of the two subunits of the -cell ATP-sensitive potas-sium channel that regulates insulin secretion. A meta-analysis combining published case-control studies up toSeptember 2002 (10) gave an OR of 1.23 (95% CI 1.121.36,

P 1.5 105) for the Lys allele, suggesting that thisallele is associated with type 2 diabetes. In addition,

functional data shows that the Lys variant may alter type 2diabetes susceptibility by increasing the threshold ATPconcentration necessary for insulin release, thus inducingspontaneous overactivity of pancreatic -cells (18). In-creasing the metabolic activity of -cells might confersusceptibility to type 1 diabetes as well (19).

The insulin receptor substrate proteins (IRS1 and IRS2)are expressed in a variety of insulin responsive cells andtissues, mediating metabolic and growth-promoting ac-tions of insulin and IGF-1. Observations from knockoutmice suggest a role for these proteins in regulation of-cell function (20), and the human IRS1 Gly972Arg vari-ant has been reported to be associated with type 2diabetes (9). Meta-analysis, based on 3,408 case subjects

and 5,419 control subjects from studies before January

2002 (21), resulted in an OR of 1.25 (95% CI 1.05 1.48). Ofdirect relevance to our study is a recent study (12)reporting an association of this variant with type 1 diabe-tes, with an OR of 2.5 (P 0.0008) in a case-control studyand a transmission/disequilibrium test (TDT) in simplexfamilies giving aPvalue 0.02.

Therefore, we genotyped these three polymorphisms in

2,434 type 1 diabetic families of European descent. Paren-tal genotype frequencies were found to be consistent withHardy-Weinberg equilibrium. TDT and conditional logisticregression analyses were performed, and the results aresummarized in Table 1. In this large dataset of type 1diabetic families, we were unable to confirm the reportedassociation ofIRS1 (12), although we had 95% power todetect the reported effect (OR 2.5,P 0.0008, minor allelefrequency 8%). Similarly, the KCNJ11 Glu23Lys variantwas not associated with type 1 diabetes, as a nonsignifi-cant deviation from 50:50 transmission from heterozygous

parents was observed.The PPARG2 Pro12Ala SNP, however, showed some

evidence of association with the more common Pro alleleconferring risk to type 1 diabetes (relative risk [RR] 1.15,95% CI 1.04 1.28, P 0.008), as is the case for type 2diabetes. TDT results indicated a transmission to affectedindividuals of 54% (769 of 1,437 informative transmissions,

P 0.008). To confirm that this result was not due togenotyping error, we regenotyped the PPARG2 Pro12AlaSNP on the reverse strand and obtained 99.77% concor-dance. In agreement with the reports in type 2 diabetes,the minor allele (G) is protective in type 1 diabetes.Genotype RRs were calculated by conditional logisticregression analysis for the Pro12Ala SNP, giving values of0.85 (0.76 0.95, P 0.005) and 0.87 (0.611.24, P 0.44)for the C/G and G/G genotypes, respectively, relative to theC/C genotype. Conversely, relative to the G/G genotype,the C/G and C/C genotypes have RRs of 0.98 (0.69 1.39)and 1.15 (0.811.64), respectively. The nonsignificant re-sult for the G/G genotype (and the risks relative to the G/Ggenotype) is likely a result of its low frequency.

Our failure to replicate the Italian study (12), whichreported an association betweenIRS1Gly972Arg and type1 diabetes, is perhaps not surprising given the modestsample size previously studied. Nevertheless, we geno-typed this SNP with two technologies, as family studies ofrare variants may be compromised because apparentundertransmission of alleles can be observed due togenotyping errors. A concordance rate of 99.74% was

observed between genotypes generated by the two meth-

TABLE 1Type 1 diabetes SNP association analysis

SNP

Reported riskallele for type

2 diabetesFrequency

(%)

TDT CLR

T U P P RR 95% CI

*IRSIGly972Arg Arg 5.9 312 327 0.54 0.77 0.95 0.821.11KCNJ11 Glu23Lys Lys 42 1268 1300 0.54 0.72 0.97 0.851.10

PPARG2 Pro12Ala Pro 86 769 668 0.0084 0.018 1.15 1.041.28Pvalues were calculated based on the null hypothesis of no association of the polymorphism. Conditional logistic regression (CLR) Pvaluesare for2 on 2 degrees of freedom; RRs are for the reported risk allele. Frequency given is the frequency in unaffected parents of the reportedrisk allele for type 2 diabetes. *Successfully typed in 2,161 families (2,887 affected offspring). Successfully typed in 2,090 families (2,734affected offspring). Successfully typed in 2,355 families (3,157 affected offspring). T, transmitted; U, untransmitted.

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ods. It is unlikely that this common SNP could be geneti-cally associated with type 1 diabetes in central Italy andnot in other European countries. In these data we foundno evidence of population heterogeneity (2 [4 df] 1.93,

P 0.75, in five populations). Hence, our study highlightsthe importance of large datasets to investigate the multi-genic basis of type 1 diabetes, type 2 diabetes, and othercommon multifactorial diseases.

If the PPARG2 Pro12Ala association is confirmed inother studies, then a molecular link would be establishedbetween type 1 and 2 diabetes involving a protective effectof the minor allele (Ala). Other studies have reported thatthiazolidinediones possess anti-inflammatory propertiesand decrease diabetes incidence in nonobese diabetic(NOD) mice (22,23). Prevention of autoimmune diabetesin NOD mice by thiazolidinediones has been associatedwith suppression of intercellular adhesion molecule 1(ICAM1) expression and alteration of Th1/Th2 cytokinebalance (24). Interestingly, we have recently reported (25)an association of the Gly241Arg polymorphism ofICAM1with type 1 diabetes. It is possible that PPAR agonists

may be worth testing in prevention of immune rejection oftransplanted islets.

RESEARCH DESIGN AND METHODS

All families were Caucasian of European descent and were composed of two

parents and at least one affected child. The families comprised up to 458

Diabetes U.K. Warren 1 multiplex from the U.K. (26), 328 U.S. multiplex from

the Human Biological Data Interchange (27), 80 Yorkshire simplex from the

U.K., 250 Belfast multiplex/simplex (28) from the U.K., 159 Norwegian

simplex, 233 Romanian simplex, and 926 Finnish multiplex/simplex (29). All

DNA samples were collected with appropriate ethical approval and informed

consent.

Genotyping.SNPs were genotyped by the TaqMan 5 nuclease assay accord-

ing to the manufacturers instructions (Applied Biosystems, Warrington, U.K.).

TaqMan primers and probes were designed by Applied Biosystems. The

PPARG2 Pro12Ala SNP was independently typed by the TaqMan 5 nuclease

assay on both strands. IRS1 Gly972 Arg was also genotyped by restriction

enzyme digest with XmaI, incorrectly reported as MvaI in Federici et al. (12).

All genotyping data were double scored to minimize error.

Statistical analysis. All statistical analyses were performed in Stata (http://

www.stata.com) using the Genassoc package (http://www-gene.cimr.cam.

ac.uk/clayton/software/stata). A modified test was used to evaluate Hardy-

Weinberg equilibrium that allows for allelic frequencies to differ between

known population groups. Allelic association was tested using the TDT.

Pseudo-control subjects, generated by conditioning on parental genotype,

allowed us to test genotypic association with conditional logistic regression

(30). This also allowed us to test for population heterogeneity by adding

appropriate interaction terms into the regression equation. Robust variance

estimates were used for the calculation ofPvalues and 95% CIs in order to

correct for nonindependence of transmissions within families with more than

one affected offspring. As a quality control measure, the transmission of

PPARG2 Pro12Ala alleles to unaffected offspring was evaluated by TDT andfound to be nonsignificant (data not shown), ruling out genotyping error as a

possible explanation for the association observed with type 1 diabetes.

ACKNOWLEDGMENTS

We thank the Juvenile Diabetes Research Foundation andthe Wellcome Trust forfinancial support.

We also thank the Human Biological Data Interchangeand Diabetes U.K. for U.S. and U.K. multiplex families,respectively, the Norwegian Study Group for ChildhoodDiabetes for sample collection of Norwegian samples,Sarah Field and Tasneem Hassanali for DNA preparation,and David Clayton, Jason Cooper, and Chris Lowe for

discussions.

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Documents

Analysis of the Type 2 Diabetes Associated Single Nucleotide Polymorphisms in the Genes IRS1 and KCNJ11and PPARG2 in Type 1 Diabetes.pdf