Brief Genetics Report Analysis of the Type 2 DiabetesAssociated Single Nucleotide Polymorphisms in the Genes IRS1, KCNJ11, and

PPARG2 in Type 1 Diabetes

Christina 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-T rgovis te,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, to some extent, genetic background. However, to date there has been no convincing data to establish a molecular genetic link between them. We have genotyped three single nucleotide polymorphisms associated with type 2 diabetes in a large type 1 diabetic family collection of European descent: Gly972Arg in the insulin receptor substrate 1 (IRS1) gene, Glu23Lys in the potassium inwardly-rectifying channel gene (KCNJ11), and Pro12Ala in the peroxisome proliferativeactivated receptor 2 gene (PPARG2). We were unable to conrm a recently published association of the IRS1 Gly972Arg variant with type 1 diabetes. Moreover, KCNJ11 Glu23Lys showed no association with type 1 diabetes (P > 0.05). However, the PPARG2 Pro12Ala variant showed evidence of association (RR 1.15, 95% CI 1.04 1.28, P 0.008). Additional studies need to be conducted to conrm this result. Diabetes 53:870 873, 2004

From the 1Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inammation Laboratory, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, U.K.; the 2Diabetes and Genetic Epidemiology Unit, National Public Health Institute, University of Helsinki, Helsinki, Finland; the 3Department of Public Health, University of Helsinki, Helsinki, Finland; the 4Institute of Medical Genetics, Ulleval University Hospital, University of Oslo, Oslo, Norway; the 5Laboratory of Molecular Epidemiology, Division of Epidemiology, Norwegian Institute of Public Health, Oslo, Norway; the 6Clinic of Diabetes, Institute of Diabetes, Nutrition and Metabolic Diseases, Bucharest, Romania; and the 7Department of Medical Genetics, Queens University Belfast, Belfast City Hospital, Belfast, Northern Ireland. Address correspondence and reprint requests to John A. Todd, JDRF/WT Diabetes and Inammation Laboratory, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Rd., Cambridge CB2 2XY, U.K. E-mail: john.todd@cimr.cam.ac.uk. Recieved for publication 3 October 2003 and accepted in revised form 31 October 2003. ICAM1, intercellular adhesion molecule 1; IRS1, insulin receptor substrate 1; KCNJ11, potassium inwardly rectifying channel gene; PPAR, peroxisome proliferatoractivated receptor ; PPARG2, peroxisome proliferatoractivated receptor 2 gene; SNP, single nucleotide polymorphism; TDT, transmission/ disequilibrium test. 2004 by the American Diabetes Association. 870

ype 1 and 2 diabetes have been considered genetically and pathophysiologically distinct diseases, although clinically, it can sometimes be difcult to distinguish between them in individuals with decient or absent endogenous insulin secretion, a feature common to both categories. Evidence does exist suggesting that there may be shared etiological features (13). Occasionally, individuals who develop diabetes late in life initially present in a similar manner to type 2 diabetes, but develop progressive -cell failure together with evidence of autoimmunity to islet cells, which is characteristic of type 1 diabetes. This so-called latent autoimmune diabetes in adults could suggest an overlap in the pathogenesis of type 1 and 2 diabetes in some cases. Furthermore, a recent study (4) has reported that early in the course of type 1 diabetes, individuals with evidence of islet autoimmunity may have normal fasting glucose but abnormal postprandial glucose tolerance. This pattern of defective postprandial secretion of insulin in response to nutrient intake is often observed in type 2 diabetes as well (4). There has been some evidence of familial clustering of type 1 and 2 diabetes (5,6). However, the HLA region, the major locus in type 1 diabetes, has only infrequently been associated with type 2 diabetes (7). Moreover, both diseases have been associated with variation in the insulin gene 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 to reveal disease-associated polymorphisms that have established some level of general acceptance because they have been replicated by independent studies. Two polymorphisms that are well established as associated with type 2 diabetes are the single nucleotide polymorphisms (SNPs) Pro12Ala in the peroxisome proliferatoractivated receptor gene isoform 2 (PPARG2), resulting from a CG transversion, and Glu23Lys in the potassium inwardly rectifying channel gene (KCNJ11), resulting from an AG transition (9). The reported effect sizes are modest (odds ratio [OR] 1.21.3) in meta-analyses (9,10). A large collaborative collection of type 1 diabetic families is now availDIABETES, VOL. 53, MARCH 2004

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TABLE 1 Type 1 diabetes SNP association analysis Reported risk allele for type 2 diabetes Arg Lys Pro Frequency (%) 5.9 42 86 TDT T 312 1268 769 U 327 1300 668 P 0.54 0.54 0.0084 P 0.77 0.72 0.018 CLR RR 0.95 0.97 1.15 95% CI 0.821.11 0.851.10 1.041.28

SNP *IRSI Gly972Arg KCNJ11 Glu23Lys PPARG2 Pro12Ala

P values were calculated based on the null hypothesis of no association of the polymorphism. Conditional logistic regression (CLR) P values are for 2 on 2 degrees of freedom; RRs are for the reported risk allele. Frequency given is the frequency in unaffected parents of the reported risk allele for type 2 diabetes. *Successfully typed in 2,161 families (2,887 affected offspring). Successfully typed in 2,090 families (2,734 affected offspring). Successfully typed in 2,355 families (3,157 affected offspring). T, transmitted; U, untransmitted.

able for genetic analysis (11) with sufcient power to detect effects in the OR range of 1.21.3. Hence, in the present report we investigated the possibility that the two type 2 diabetesassociated SNPs were also associated with type 1 diabetes in an effort to begin to establish if overlaps exist in the two disease pathways. We have also tested the insulin receptor substrate 1 (IRS1) Gly972Arg polymorphism because it has been reported to be associated with type 1 diabetes (12), although it is not as well established in type 2 diabetes as the PPARG2 and KCNJ11 SNPs. The PPARG gene encodes peroxisome proliferator activated receptor (PPAR), a member of the nuclear receptor superfamily that has regulatory functions in adipocyte differentiation and glucose homeostasis (13). PPAR is a target for a large family of antidiabetic drugs, the thiazolidinediones, that act as PPAR agonists and increase hepatic and peripheral insulin action in type 2 diabetes (14). In a meta-analysis of published studies through February 2003 (9), an OR of 1.27 was estimated for the Pro allele of isoform 2 (P value 2 108) in type 2 diabetes. This association is supported by functional data, as in vitro studies have shown that the Ala allele decreases the DNA-binding afnity of the PPAR2, thus reducing its transcriptional activity (15). The KCNJ11 Glu23Lys variant has also been consistently reported (10,16,17) to be associated with type 2 diabetes. KCNJ11 encodes for KIR6.2, which comprises one of the two subunits of the -cell ATP-sensitive potassium channel that regulates insulin secretion. A metaanalysis combining published case-control studies up to September 2002 (10) gave an OR of 1.23 (95% CI 1.121.36, P 1.5 105) for the Lys allele, suggesting that this allele is associated with type 2 diabetes. In addition, functional data shows that the Lys variant may alter type 2 diabetes susceptibility by increasing the threshold ATP concentration necessary for insulin release, thus inducing spontaneous overactivity of pancreatic -cells (18). Increasing the metabolic activity of -cells might confer susceptibility to type 1 diabetes as well (19). The insulin receptor substrate proteins (IRS1 and IRS2) are expressed in a variety of insulin responsive cells and tissues, mediating metabolic and growth-promoting actions of insulin and IGF-1. Observations from knockout mice suggest a role for these proteins in regulation of -cell function (20), and the human IRS1 Gly972Arg variant has been reported to be associated with type 2 diabetes (9). Meta-analysis, based on 3,408 case subjects and 5,419 control subjects from studies before January
DIABETES, VOL. 53, MARCH 2004

2002 (21), resulted in an OR of 1.25 (95% CI 1.051.48). Of direct relevance to our study is a recent study (12) reporting an association of this variant with type 1 diabetes, with an OR of 2.5 (P 0.0008) in a case-control study and a transmission/disequilibrium test (TDT) in simplex families giving a P value 0.02. Therefore, we genotyped these three polymorphisms in 2,434 type 1 diabetic families of European descent. Parental genotype frequencies were found to be consistent with Hardy-Weinberg equilibrium. TDT and conditional logistic regression analyses were performed, and the results are summarized in Table 1. In this large dataset of type 1 diabetic families, we were unable to conrm the reported association of IRS1 (12), although we had 95% power to detect the reported effect (OR 2.5, P 0.0008, minor allele frequency 8%). Similarly, the KCNJ11 Glu23Lys variant was not associated with type 1 diabetes, as a nonsignicant 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 allele conferring 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 2 diabetes. TDT results indicated a transmission to affected individuals of 54% (769 of 1,437 informative transmissions, P 0.008). To conrm that this result was not due to genotyping error, we regenotyped the PPARG2 Pro12Ala SNP on the reverse strand and obtained 99.77% concordance. 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 logistic regression analysis for the Pro12Ala SNP, giving values of 0.85 (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 the C/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 nonsignicant result for the G/G genotype (and the risks relative to the G/G genotype) is likely a result of its low frequency. Our failure to replicate the Italian study (12), which reported an association between IRS1 Gly972Arg and type 1 diabetes, is perhaps not surprising given the modest sample size previously studied. Nevertheless, we genotyped this SNP with two technologies, as family studies of rare variants may be compromised because apparent undertransmission of alleles can be observed due to genotyping errors. A concordance rate of 99.74% was observed between genotypes generated by the two meth871

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ods. It is unlikely that this common SNP could be genetically associated with type 1 diabetes in central Italy and not in other European countries. In these data we found no evidence of population heterogeneity (2 [4 df] 1.93, P 0.75, in ve populations). Hence, our study highlights the importance of large datasets to investigate the multigenic basis of type 1 diabetes, type 2 diabetes, and other common multifactorial diseases. If the PPARG2 Pro12Ala association is conrmed in other studies, then a molecular link would be established between type 1 and 2 diabetes involving a protective effect of the minor allele (Ala). Other studies have reported that thiazolidinediones possess anti-inammatory properties and decrease diabetes incidence in nonobese diabetic (NOD) mice (22,23). Prevention of autoimmune diabetes in NOD mice by thiazolidinediones has been associated with suppression of intercellular adhesion molecule 1 (ICAM1) expression and alteration of Th1/Th2 cytokine balance (24). Interestingly, we have recently reported (25) an association of the Gly241Arg polymorphism of ICAM1 with type 1 diabetes. It is possible that PPAR agonists may be worth testing in prevention of immune rejection of transplanted 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 according 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 modied test was used to evaluate HardyWeinberg 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 of P values 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 and found to be nonsignicant (data not shown), ruling out genotyping error as a possible explanation for the association observed with type 1 diabetes.

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ACKNOWLEDGMENTS

We thank the Juvenile Diabetes Research Foundation and the Wellcome Trust for nancial support. We also thank the Human Biological Data Interchange and Diabetes U.K. for U.S. and U.K. multiplex families, respectively, the Norwegian Study Group for Childhood Diabetes 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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