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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1942
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 6 3629-3637
Copyright © 2005 by The Endocrine Society

Analysis of Separate and Combined Effects of Common Variation in KCNJ11 and PPARG on Risk of Type 2 Diabetes

Sara K. Hansen, Eva-Maria D. Nielsen, Jakob Ek, Gitte Andersen, Charlotte Glümer, Bendix Carstensen, Peter Mouritzen, Thomas Drivsholm, Knut Borch-Johnsen, Torben Jørgensen, Torben Hansen and Oluf Pedersen

Steno Diabetes Center and Hagedorn Research Institute (S.K.H., E.-M.D.N., J.E., G.A., C.G., B.C., K.B.-J., T.H., O.P.), Gentofte, Copenhagen, Denmark; Research Center for Prevention and Health, Copenhagen County, Glostrup University Hospital (C.G., T.D., K.B.-J., T.J.), Glostrup, Denmark; Exiqon A/S (P.M.), Vedbæk, Denmark; and Faculty of Health Science, University of Aarhus (K.B.-J., O.P.), Aarhus, Denmark

Address all correspondence and requests for reprints to: Professor Oluf Pedersen, M.D., D.M.Sc., Steno Diabetes Center and Hagedorn Research Institute, Niels Steensens Vej 2, DK-2820 Gentofte, Denmark. E-mail: oluf{at}steno.dk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The separate and combined effects of the PPARG Pro12Ala polymorphism and the KCNJ11 Glu23Lys polymorphisms on risk of type 2 diabetes were investigated in relatively large-scale, case-control studies. Separate effects of the variants were examined among 1187/1461 type 2 diabetic patients and 4791/4986 middle-aged, glucose-tolerant subjects. The combined analysis involved 1164 type 2 diabetic patients and 4733 middle-aged, glucose-tolerant subjects. In the separate analyses, the K allele of the KCNJ11 Glu23Lys associated with type 2 diabetes (odds ratio, 1.19; P = 0.0002), whereas the PPARG Pro12Ala showed no significant association with type 2 diabetes. The combined analysis indicated that the two polymorphisms acted in an additive manner to increase the risk of type 2 diabetes, and we found no evidence for a synergistic interaction between them. Analysis of a model with equal additive effects of the two variants showed that the odds ratio for type 2 diabetes increased with 1.14/risk allele (P = 0.003). Together, the two polymorphisms conferred a population-attributable risk for type 2 diabetes of 28%. In conclusion, our results showed no evidence of a synergistic interaction between the KCNJ11 Glu23Lys and PPARG Pro12Ala polymorphisms, but indicated that they may act in an additive manner to increase the risk of type 2 diabetes.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE COMMON FORM of type 2 diabetes is generally assumed to be a polygenic disorder in which the development of the disease is triggered by both genetic and environmental risk factors and potentially aggravated by additive or synergistic interplays between these susceptibility factors. Some rare subtypes of type 2 diabetes with a Mendelian form of inheritance have been described (1). However, despite a massive effort from several investigators to identify genetic risk factors, the inherited background for the common, late-onset form of type 2 diabetes still remains largely unknown.

However, few genetic variants have consistently been reported to increase the risk of type 2 diabetes (1, 2). Among these, the most convincing candidates are the Pro12Ala (P12A) polymorphism of the gene encoding the peroxisome proliferator-activated receptor-{gamma}2 (PPAR-{gamma}2; PPARG) (3) and the Glu23Lys (E23K) polymorphism of the gene encoding the ATP-sensitive potassium channel subunit KIR6.2 (KCNJ11) (4).

The PPAR-{gamma}2 is a member of the nuclear hormone receptor family of transcription factors and is involved in adipocyte differentiation (5). Rare mutations in PPARG cause monogenic syndromes of severe insulin resistance and obesity (6, 7), whereas common variation in the PPARG is implicated in susceptibility of the polygenic form of type 2 diabetes. Initially, the common allele of the P12A polymorphism was reported to associate with an increased risk of type 2 diabetes (3), which has subsequently been replicated in several studies, including two meta-analyses showing that the common allele increases risk of type 2 diabetes with an odds ratio (OR) of approximately 1.25 and a population-attributable risk of about 25% (2, 8, 9, 10, 11, 12, 13). Furthermore, the P12A polymorphism has been shown to associate with altered insulin sensitivity (14, 15, 16, 17). Functional studies have demonstrated an increased activity of the PPAR-{gamma}2 protein containing a proline compared with the protein containing an alanine at codon 12 (3, 18), which is predicted to induce an increase in adipose tissue mass and a decrease in insulin sensitivity (3). These results strengthen the idea that the common allele of P12A is likely to be diabetogenic.

The KIR6.2 protein together with the SUR1 protein constitute the pancreatic ß-cell ATP-sensitive potassium (KATP) channel, which plays a crucial role in the glucose-induced insulin secretion (19, 20). The human genes encoding both the KIR6.2 (KCNJ11) and the SUR1 (ABCC8) have thus for several years been considered obvious candidate genes for type 2 diabetes. Mice lacking the KIR6.2 gene are characterized by impaired glucose- and tolbutamide-induced insulin secretion (21). However, the glucose tolerance in these mice is only mildly impaired, presumably due to an increased glucose-lowering effect by insulin (21). Rare loss of function mutations in KCNJ11 cause decreased function of the KATP channels, leading to persistent hyperinsulinemic hypoglycemia of infancy in humans (22, 23). Activating mutations in KCNJ11 cause permanent neonatal diabetes due to overactive KATP channels, resulting in reduced insulin secretion (24). As a candidate gene for type 2 diabetes, the frequent KCNJ11 E23K polymorphism has been analyzed in several studies, most of which have reported a positive association between the minor K allele and type 2 diabetes (4, 25, 26, 27, 28, 29, 30). A recent meta analysis of 11 published association studies adding up to a total of 5083 type 2 diabetic patients and 4747 control subjects showed a significant association between the K allele of the KCNJ11 E23K polymorphism and type 2 diabetes [odds ratio (OR), 1.15; P < 10–5] (30). Furthermore, a functional effect of this polymorphism, leading to an overactive KIR6.2 channel with a decreased sensitivity toward ATP, has been reported (31). Conceivably, the association between the KCNJ11 E23K polymorphism and type 2 diabetes may be explained in part by a decreased insulin release caused by the polymorphism.

In the present report we have investigated the separate and combined effects of the KCNJ11 E23K and the PPARG P12A polymorphisms on risk of type 2 diabetes in relatively large-scale, case-control studies. Furthermore, in genotype-quantitative trait analyses of middle-aged, glucose-tolerant individuals from a relatively large population-based study, we analyzed the separate effect of the KCNJ11 E23K polymorphism on insulin secretion, the separate effect of the PPARG P12A on insulin resistance and lipid metabolism, and the combined effect of both polymorphisms on the relevant quantitative traits.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

From the population-based Inter99 study cohort that was established at the Research Center for Prevention and Health during 1999–2001 (32), both glucose-tolerant subjects and type 2 diabetic patients were recruited for the present case-control and genotype-quantitative trait analyses (Table 1Go). Glucose-tolerant subjects for the case-control studies were also recruited from two smaller, population-based study samples from the Research Center for Prevention and Health (33) and the Steno Diabetes Center, respectively (Table 1Go). All individuals from the three population-based study samples were randomly recruited through the Danish Central Population Register from the same geographical area as the type 2 diabetic patients. The remaining type 2 diabetic patients were recruited from the out-patient clinics in the greater Copenhagen area during 1992–2001 (Table 1Go).


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  		TABLE 1. Characteristics of the glucose-tolerant control subjects and the type 2 diabetic patients enrolled in the case-control studies for the KCNJ11 E23K and the PPARG P12A polymorphisms, respectively

 
The case-control studies of the KCNJ11 E23K and the PPARG P12A polymorphisms comprised 1187 and 1461 type 2 diabetic patients, respectively (834/1094 diabetic patients from the out-patient clinics and 353/367 diabetic patients from the population-based Inter99 study; Table 1Go). Of these patients, 20% were treated with diet alone, 60% were treated with oral hypoglycemic agent, 15% were treated with insulin, and 5% received a combination of insulin and oral hypoglycemic agent. Diabetes was diagnosed in accordance with the World Health Organization 1999 criteria. The glucose-tolerant control subjects in the case-control studies of the KCNJ11 E23K polymorphism and the PPARG P12A polymorphism comprised 4273/4470 individuals who were recruited from the population-based Inter99 study and 518/516 subjects from the two smaller population-based study samples, respectively (Table 1Go). The age- and sex-matched glucose-tolerant subjects were selected by defining a group with similar mean age and distribution of sex as the total group of type 2 diabetic patients (Table 1Go). All control subjects were given a standard 75-g oral glucose tolerance test (OGTT), and only glucose-tolerant subjects were included as control subjects in the studies.

The case-control studies of the KCNJ11 E23K and the PPARG P12A polymorphisms were extensions of previously published case-control studies (15, 28). The total 803 type 2 diabetic patients and 862 glucose-tolerant subjects from the previous case-control study of the KCNJ11 E23K polymorphism (28) are also included in the present case-control study, which thereby also comprises the 115 subjects from another reported case-control study (34). For the PPARG P12A polymorphism, the total of 654 type 2 diabetic patients and 742 glucose-tolerant subjects from the previous case-control study (15) are included in the present case-control study. Genotype-quantitative-trait analyses of the KCNJ11 E23K and PPARG P12A polymorphisms were performed among the 4273 and 4470 middle-aged, glucose-tolerant individuals from the Inter99 study, respectively. All participants were Danish Caucasians by self-report. Informed consent was obtained from all study participants before participation. The studies were approved by the ethical committee of Copenhagen and were performed in accordance with the principles of the Declaration of Helsinki II.

Biochemical and physiological assays

The standard 75-g OGTTs were performed in the morning after a 12-h overnight fast. Blood samples for measurements of plasma glucose, serum insulin, and serum C peptide were drawn in the fasting state and at 30 and 120 min during the OGTT. Blood samples were also analyzed for fasting levels of serum lipids. Plasma glucose, serum insulin, serum C peptide, and serum lipids were analyzed using routine methods at Steno Diabetes Center.

The insulinogenic index for serum insulin and serum C peptide was calculated as: (insulin/C peptide at 30 min – fasting insulin/C peptide)/glucose at 30 min. The incremental area under the curve for insulin and C peptide during 0–120 min were calculated using the trapezoidal method. The homeostasis model assessment of insulin resistance (HOMA IR) was calculated as (fasting plasma glucose x fasting serum insulin)/22.5.

Body mass index (BMI) was calculated as [weight (kilograms)/(height (meters))2]. Weight and height were measured in the standing position in indoor light clothes without shoes.

Genotyping of the PPARG P12A polymorphism

All samples were genotyped for the PPARG P12A polymorphism, applying a chip-based, matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis of PCR-generated primer extension products as previously described (35). The genotyping success rate was 99%. To elucidate the genotyping error rate, we examined 90 replicate samples and found one mismatched sample. The genotype was designated wild type in one sample and heterozygous in the replicate sample. This indicates a genotyping error rate of approximately 1%. The genotype for the mismatched sample was not used in the subsequent analyses. The genotype distribution of the PPARG P12A polymorphism was in Hardy-Weinberg equilibrium.

Of the total 6447 samples with genotypes of the PPARG P12A polymorphism, 1396 samples (654 type 2 diabetic patients and 742 glucose-tolerant subjects) had previously been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP) (15). When comparing the genotypes from the two different genotyping methods, we observed only one mismatch, which indicates that the concordance rate is almost 100%.

Genotyping of the KCNJ11 E23K polymorphism

The 1665 DNA samples from the initial case-control study were genotyped using a PCR-RFLP-based method as previously described (28). The remaining samples were genotyped using the GenoView locked nucleic acid (LNA) assay (Exiqon A/S, Vedbæk, Denmark). The GenoView LNA assay was performed by competitive hybridization to PCR amplicons with two allele-specific LNA probes labeled with Cy3 and Cy5; PCR amplification was carried out in a 15-µl volume containing 50–100 ng genomic DNA, 1x PCR buffer, 0.3 µmol/liter of each primer, 0.4 mmol/liter deoxy-NTP, 0.6 U AmpliTaq Gold polymerase (Applied Biosystems, Inc., Foster City, CA), and 3 mmol/liter MgCl2 using a GeneAmp 9600 or 9700 thermal cycler (Applied Biosystems, Inc.). The cycling program was as follows: denaturation at 95 C for 10 min; 32 cycles of 95 C for 45 sec, annealing at 62 C for 45 sec, and elongation at 72 C for 1 min; and final elongation at 72 C for 10 min. The sequences of the PCR primers were: forward primer, 5'-cga gga ata cgt gct gac ac-3'; and reverse primer, 5'-acg ttg cag ttg cct ttc tt-3'. The sequences of the LNA probes were: 5'-ctT Ggc agG g-3', 5'-ctC ggc Agg g-3' for Cy3 and Cy5, respectively (uppercase letters, LNA; lowercase letters: DNA, variable nucleotides are underlined).

The PCR amplicons were then immobilized and denatured in separate wells of a streptavidin immobilizer microtiter plate (Exiqon); prewashing of the plate was made with three washes with 100 µl/well 5x SSCT buffer [1x SSCT = 150 mM NaCl, 15 mM sodium citrate (pH 7.0), and 0.05% (vol/vol) Tween 20]. Twenty microliters of a solution of nonpurified biotin-labeled PCR amplicons in 5x SSCT (10 µl PCR product and 10 µl 10x SSCT) were added to the wells and incubated with gentle agitation for 1 h at room temperature. The wells were aspirated, and 30 µl/well denaturation solution (400 mM NaOH and 0.25% Tween 20) were added and incubated for 5 min at room temperature, followed by aspiration and washing three times in 100 µl/well with 2x SSCT.

Hybridization was performed with gentle agitation for 2 h at 42 C in 20 µl/well of the Cy3 and Cy5 LNA probe pairs, each diluted to 0.01 µM in 1x SSCT. After incubation, the wells were aspirated and washed three times in 100 µl/well with 0.15x SSCT, then 100 µl/well 1x SSCT were added. The plates were kept in dark at 4 C until scanning on a Typhoon 8600 (Amersham Biosciences, Little Chalfont, UK) using normal sensitivity settings, photomultiplier voltage at 850 V, and 200 µm image resolution. For excitation, green laser (532 nm) and red laser (633 nm) were used for Cy3 and Cy5, respectively. The emission filters used were 580 BP 30 nm (Cy3) and 670 BP 30 nm (Cy5). Fluorescence was quantified using ImageQuant version 5.1 image analysis software (Amersham Biosciences). The signals were imported into Exiqon single nucleotide polymorphism (SNP) analysis software (www.exiqon.com), in which the SNP genotypes were scored.

The genotyping success rates were 97% and 92% for the RFLP-based and LNA-based assays, respectively. To elucidate the genotyping error rate, we examined 70 replicate samples, and among these we observed no mismatches. The genotype distribution of the KCNJ11 E23K polymorphism was in Hardy-Weinberg equilibrium.

Statistical analyses

Differences in minor allele frequencies and in genotype distribution among type 2 diabetic patients and control subjects were analyzed, and the corresponding ORs were estimated using logistic regression or likelihood ratio tests. All genotype distributions were tested for Hardy-Weinberg equilibrium using likelihood ratio tests.

Differences in continuous variables for both separate and combined effects of the KCNJ11 E23K and PPARG P12A polymorphisms were tested using a general linear model for ANOVA with adjustments for age, sex, and body mass index. All residuals were tested for normal distribution, and transformation of the variables (ln or cube root transformation) was made if necessary. In the genotype-quantitative trait analyses of both KCNJ11 E23K and PPARG P12A polymorphisms, several prediabetic traits were analyzed. The problem of multiple testing is therefore relevant. However, based on results from other studies, a potential association of the KCNJ11 E23K polymorphism with altered insulin secretion was part of our primary working hypothesis, which also included a potential effect of the PPARG P12A polymorphism on insulin sensitivity and/or serum lipids. Therefore, we have chosen not to correct the P values of the quantitative trait analyses for multiple testing.

To avoid assumptions regarding mode of inheritance, all case-control analyses and analyses of differences in continuous variables of the separate effect of the KCNJ11 E23K and the PPARG P12A polymorphisms were performed using additive, recessive and dominant models.

The analyses of the combined effects of the KCNJ11 E23K and PPARG P12A polymorphisms on risk of type 2 diabetes were performed on subjects with data relating to age, sex, genotypes of the KCNJ11 E23K and PPARG P12A polymorphisms, and glucose tolerance status. To assess the effects of the polymorphisms, we used a logistic regression model, with adjustment for age and sex. The combined effects of the KCNJ11 E23K polymorphism and the PPARG P12A polymorphism were modeled with both a general interaction model and a model for the independent additive effects. The trend test for the combined equal additive effects was made using logistic regression with adjustment for age and sex.

The population attributable risks were estimated as the fraction of the total number of subjects from the population-based Inter99 cohort with an OR greater than 1.

The Statistical Package for Social Science (SPSS) for Windows (version 11.5, SPSS, Inc., Chicago, IL), the SAS System for Windows 8.02, the R program version 1.9.0, and the Web-AssoTest program (available at www.ekstroem.com) were used for the statistical analyses. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Analysis of the separate effect of the KCNJ11 E23K polymorphism on risk of diabetes among the total 1187 type 2 diabetic patients and the total 4791 glucose-tolerant subjects showed that the minor K allele associated with type 2 diabetes [Table 2Go; OR for the minor K allele, 1.19 (95% confidence interval [CI], 1.09–1.30; P = 0.0002; OR for the EK and KK genotypes compared with EE, 1.20 (95% CI, 1.04–1.38) and 1.41 (95% CI, 1.17–1.71), respectively; P = 0.001]. An additional case-control study comparing the same 1187 type 2 diabetic patients with a selected age- and sex-matched subgroup of 1454 glucose-tolerant subjects from the total group of 4791 glucose-tolerant subjects also showed a significant association with type 2 diabetes [Table 2Go; OR for the minor K allele, 1.15 (95% CI, 1.03–1.28); P = 0.02; OR for the EK and KK genotypes compared with EE, 1.17 (95% CI, 0.99–1.38) and 1.30 (95% CI, 1.03–1.65), respectively; P = 0.05]. The estimated population-attributable risk for type 2 diabetes for the separate effect of the KCNJ11 E23K polymorphism was 13%.


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  		TABLE 2. Case-control study of the KCNJ11 E23K polymorphism comprising the total 1187 type 2 diabetic patients compared with a total of 4791 glucose-tolerant subjects or with 1454 age- and sex-matched glucose-tolerant subjects selected from the total group of 4791 glucose-tolerant subjects, respectively

 
The analyses of the separate impact of the PPARG P12A polymorphism showed no significant difference in allele frequency of the common P allele or in genotype distribution among the total 1461 type 2 diabetic patients compared with the total 4986 glucose-tolerant subjects [Table 3Go; OR for the P allele: 1.07 (95% CI, 0.95–1.21); P = 0.25, OR for the PA and PP genotypes compared with AA, 1.05 (95% CI, 0.69–1.62) and 1.13 (95% CI, 0.75–1.71), respectively; P = 0.52]. When comparing the same 1461 type 2 diabetic patients with a selected age- and sex-matched subgroup of 1497 glucose-tolerant subjects from the total group of 4986 glucose-tolerant subjects, the P allele was borderline significantly more frequent among the type 2 diabetic patients (87.0%) compared with the glucose-tolerant subjects [85.3%; Table 3Go; OR for the P allele, 1.15 (95% CI, 0.99–1.33); P = 0.07; OR for the AP and PP genotypes compared with AA, 1.27 (95% CI, 0.77–2.09) and 1.43 (95% CI, 0.88–2.31), respectively; P = 0.17].


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  		TABLE 3. Case-control study of the PPARG P12A polymorphism comprising a total of 1461 type 2 diabetic patients compared with a total of 4986 glucose-tolerant subjects or with 1497 age- and sex-matched glucose-tolerant subjects selected from the total 4986 glucose-tolerant subjects, respectively

 
Our results showed that the KCNJ11 E23K polymorphism was significantly associated with type 2 diabetes, whereas the PPARG P12A P-allele was nonsignificantly more frequent among the type 2 diabetic patients. Nevertheless, based on results from other pertinent studies in the literature showing evidence for increased risks of type 2 diabetes and relevant functional effects conferred by each of the two polymorphisms, the KCNJ11 E23K and the PPARG P12A variants were subjected to analyses of their combined impact on risk of type 2 diabetes.

The combined effect of the KCNJ11 E23K and PPARG P12A polymorphisms on risk of type 2 diabetes was studied among 1164 type 2 diabetic patients from both the out-patient clinics and the population-based Inter99 cohort and from 4733 glucose-tolerant subjects from the Inter99 cohort and the two smaller population-based study samples, adding up to a total of 5897 subjects, who were successfully genotyped for both the KCNJ11 E23K and PPARG P12A variants. The distribution of the total 5897 type 2 diabetic and glucose-tolerant subjects in the nine possible genotype combinations of the KCNJ11 E23K and the PPARG P12A polymorphisms is shown in Table 4Go. ORs for the nine genotype combinations were estimated by two different models: one model with independent additive effects of both polymorphisms, and a second model including both the independent additive effects of both polymorphisms and a general interaction between the two polymorphisms. In the model with independent additive effects, we found an OR of 1.17 (95% CI, 1.05–1.30; P = 0.003) for the minor allele of the KCNJ11 E23K polymorphism and an OR of 1.08 (95% CI, 0.93–1.25; P = 0.33) for the P allele of the PPARG P12A polymorphism, respectively. The model including the general interaction between the two variants was not significantly different from the model without the interaction (P = 0.50). The results of these analyses suggest that jointly the KCNJ11 E23K and PPARG P12A polymorphisms increase the risk of type 2 diabetes in an additive manner without any significant synergistic interaction (epistasis) between the two variants. The independent effects of the KCNJ11 E23K and PPARG P12A polymorphisms on risk of type 2 diabetes were not statistical significantly different (P = 0.4). Thus, based on the assumption of equal additive effects of the two variants, we reduced the nine groups to five groups according to the number of risk alleles (n = 0–4), where the K allele of the KCNJ11 E23K polymorphism and the P allele of the PPARG P12A polymorphism were considered as the risk alleles. Estimation of ORs for the five groups in the model with equal additive effects showed an increased risk of type 2 diabetes with increasing number of risk alleles, with an increase in OR per allele of 1.14 (95% CI, 1.05–1.24; test for trend, P = 0.003; Fig. 1Go). In this model, the estimated population-attributable risk for type 2 diabetes of the combined impact of the KCNJ11 E23K and PPARG P12A polymorphisms was 28%.


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  		TABLE 4. The distribution of the nine genotype combinations of the KCNJ11 E23K and the PPARG P12A polymorphism among 1164 type 2 diabetic patients and 4733 glucose-tolerant subjects (shown both as the total number and the percentage of subjects in each group)

 


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  		FIG. 1. Graphic presentation of ORs and corresponding 95% CI for type 2 diabetes estimated from 1164 type 2 diabetic patients and 4733 glucose-tolerant control subjects categorized into five groups (n = 0–4) according to the number of risk alleles, where the K allele of the KCNJ11 E23K polymorphism and the P allele of the PPARG P12A polymorphism were considered as type 2 diabetes risk alleles. The group with no risk alleles comprised 42 subjects (six type 2 diabetic patients/36 glucose-tolerant subjects, respectively); the group with one risk allele comprised 623 subjects (117/506); the group with two risk alleles comprised 2396 subjects (420/1976); the group with three risk alleles comprised 2196 subjects (478/1718); the group with four risk alleles comprised 640 subjects (143/497). The ORs for each group were estimated relative to the group with two risk alleles by logistic regression with a model assuming equal additive effects of the risk alleles. The ORs for type 2 diabetes increased with 1.14/risk allele (test for trend, P = 0.003).

 
The separate effect of the KCNJ11 E23K variant on measurements of insulin secretion was analyzed in genotype-quantitative trait studies of the 4273 glucose-tolerant subjects from the population-based Inter99 cohort. The carriers of the homozygous form of the polymorphism displayed a significant 4% decrease in insulin secretion, as estimated by the insulinogenic index for serum C peptide compared with subjects carrying the variant in the wild-type or heterozygous form (P = 0.007; Table 5Go). In carriers of the homozygous form, other estimates for insulin secretion were also decreased (Table 5Go); however the differences did not reach statistical significance.


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  		TABLE 5. Clinical and biochemical characteristics of 4273 glucose-tolerant Danish Caucasian participants from the population-based Inter99 cohort stratified according to the KCNJ11 E23K polymorphism and analyzed applying an additive (add), a recessive (rec), and a dominant (dom) model

 
In genotype-quantitative trait analyses of the separate influence of the PPARG P12A polymorphism on measurements of insulin resistance and serum lipids among the 4470 glucose-tolerant subjects from the population-based Inter99 cohort (Table 6Go), carriers of the variant in the homozygous or heterozygous forms were more insulin sensitive, with 5% decreased levels of fasting serum insulin and HOMA IR compared with carriers of the wild-type form (P = 0.002 and P = 0.01, respectively). Additionally, carriers of the variant in the homozygous and heterozygous forms had a 4% decrease in levels of fasting serum triglycerides (P = 0.004) and a 2% increase in levels of fasting serum high density lipoprotein cholesterol (P = 0.004).


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  		TABLE 6. Clinical and biochemical characteristics of 4470 glucose-tolerant Danish Caucasian participants from the population-based Inter99 cohort stratified according to the PPARG P12A polymorphism and analyzed applying an additive (add), a recessive (rec), and a dominant (dom) model

 
Of the total of 4733 glucose-tolerant subjects with genotypes for both the KCNJ11 E23K polymorphism and the PPARG P12A polymorphism, 4223 subjects were recruited from the population-based Inter99 cohort. In studies of a potentially synergistic interaction between the KCNJ11 E23K and PPARG P12A polymorphisms on prediabetic quantitative traits among these 4223 glucose-tolerant subjects, we found no evidence for a significant interaction (data not shown). The same 4223 glucose-tolerant subjects were also categorized into the five groups according to the number of risk alleles. Analyses of differences in prediabetic quantitative traits among the five groups of subjects showed a nonsignificant tendency toward increased levels of plasma glucose both in the fasting state and after an OGTT with increasing number of risk alleles (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
By expanding our previous case-control study of the KCNJ11 E23K polymorphism (28), we found an OR for type 2 diabetes of 1.19 for the minor K allele, which is comparable with published meta analyses showing OR of 1.12–1.23 (2, 26, 30). Moreover, the genotype-quantitative traits analyses of a potential effect of the KCNJ11 E23K polymorphism on insulin secretion in a population-based study sample of 4273 middle-aged, glucose-tolerant subjects showed that carriers of the KCNJ11 E23K variant in the homozygous form were characterized by a subtle decrease in insulin secretion, as estimated from the insulinogenic index for serum C peptide. This result is in agreement with data from our previous study demonstrating that the KCNJ11 E23K polymorphism was associated with decreased levels of serum insulin during an OGTT and decreased insulinogenic index values for serum insulin among 519 glucose-tolerant subjects from a different study population than that enrolled in the genotype-quantitative trait analyses in this present study (28). A more recent study of 674 glucose-tolerant subjects showed that the KCNJ11 E23K variant was associated with a more substantial decrease in the insulinogenic index for serum insulin of about 20% (30). The modest alterations in circulating insulin during an OGTT observed in the present study may partially explain the association of the KCNJ11 E23K polymorphism with type 2 diabetes. However, some studies of the KCNJ11 E23K variant have shown associations with minor alterations in other prediabetic phenotypes, such as increased body mass index (28) and decreased suppression of circulating glucagon in response to hyperglycemia (36), whereas other studies have failed to support these findings (34, 37). These inconsistent associations with minor alterations in various prediabetic traits may be related to the widespread expression of KIR6.2 in tissues such as the pancreas, heart, small intestines, kidney, glucose-sensing hypothalamic neurons, and skeletal and smooth muscles (38). Thus, besides the involvement in regulating pancreatic secretion of insulin and glucagon, KIR6.2 may additionally be implicated in the regulation of food intake via hypothalamic neuron responses to ambient glucose levels and regulation of glucose-induced glucagon-like peptide-1 release from the intestine (39). Furthermore, the increased ability of insulin to lower circulating glucose levels in mice lacking the KIR6.2 gene indicates that KIR6.2 may be involved in regulating glucose uptake in skeletal muscles (21).

Hence, the KCNJ11 E23K polymorphism has now shown consistent association with type 2 diabetes in several studies (4, 25, 26, 27, 28, 29, 30); furthermore, a functional impact of the polymorphism, which may confer increased susceptibility to type 2 diabetes, has been suggested (31). Interestingly, several polymorphisms of the adjacently positioned ABCC8, which encodes SUR1, have also shown association with type 2 diabetes and/or altered insulin secretion (40, 41, 42, 43, 44, 45). However, a recent study analyzed the haplotype block structure of a 207-kb region covering both ABCC8 and KCNJ11 among CEPH pedigrees with Utah Caucasian individuals, and found a 75-kb haplotype block of strong linkage disequilibrium that included the totality of KCNJ11 and the 3' end of ABCC8. Among several examined SNPs in this region, the KCNJ11 E23K polymorphism was the best candidate explaining the association with type 2 diabetes, although its effect could not be distinguished genetically from the ABCC8 A1396S polymorphism (30).

Even though we expanded our previous case-control study of the PPARG P12A polymorphism (15) with 807 type 2 diabetic patients and 4244 glucose-tolerant control subjects, adding up to a total of 1461 type 2 diabetic patients and 4986 glucose-tolerant control subjects, we failed to demonstrate a significant separate effect of the PPARG P12A polymorphism on risk of type 2 diabetes. However, among the selected age- and sex-matched group of control subjects, who were, on the average, approximately 10 yr older than the total group of glucose-tolerant control subjects, the P allele was less frequent than in the total population of control subjects. This finding is in line with a previous study showing a lower frequency of the P allele among elderly control subjects (8). The frequency of a potential diabetogenic gene variant may theoretically be decreased among elderly control subjects, because carriers of the polymorphism presumably may have contracted type 2 diabetes at an earlier age. When comparing the type 2 diabetic patients with the age- and sex-matched group of control subjects, the P allele of the PPARG P12A polymorphism was borderline significantly more frequent among the type 2 diabetic patients. The estimated difference in minor allele frequency and the OR are smaller, but still comparable to what was shown in recent meta analyses (2, 13).

The diabetogenic impact of the PPARG P12A variant is assumed to be mediated through an increased transcriptional activity of the PPAR-{gamma}2, resulting in increased expression of adipocyte-specific genes involved in lipid storage and adipocyte differentiation, which eventually will increase adipose tissue mass and thereby decrease insulin sensitivity (3). Coherently and in accordance with previous studies (3, 14, 15, 16), we observed a subtly, but significantly, decreased insulin sensitivity among glucose-tolerant subjects carrying the P allele in the homozygous form (measured as both fasting serum insulin level and HOMA IR). The subjects carrying the P allele in the homozygous form also displayed increased levels of fasting serum triglycerides and decreased levels of fasting serum high density lipoprotein cholesterol.

Altogether, there is accumulating evidence that the widespread P allele of the PPARG P12A polymorphism confers a modestly increased risk of type 2 diabetes. However, the nonsignificant effect of the variant demonstrated in the present study may possibly be considered a false negative result due to an inadequate sample size, which underlines the need for large case-control studies with thousands of individuals in each group to quantify the impact of genetic risk factors in the pathogenesis of type 2 diabetes (13, 46). Intriguingly, a recent study has also suggested that the PPARG P12A polymorphism may affect susceptibility of type 1 diabetes (47), indicating the presence of common genetic risk factors for both type 1 and type 2 diabetes.

Evidence for synergistic gene-gene interactions (epistasis) in the pathogenesis of several complex diseases or traits such as Alzheimer’s disease (48), sporadic breast cancer (49), ischemic stroke (50), coronary heart disease (51), myocardial infarction (52), hypertension (53), obesity (54, 55, 56), obesity and/or prediabetic traits (57, 58, 59), and type 2 diabetes (60, 61) has been reported. In this study we investigated a potential synergistic gene-gene interaction between the KCNJ11 E23K polymorphism and the PPARG P12A polymorphism on risk of type 2 diabetes among a total of 5897 type 2 diabetic patients and glucose-tolerant subjects. The results indicated that the KCNJ11 E23K and the PPARG P12A variants act in an additive manner to increase risk of type 2 diabetes, and we found no evidence for a significant synergistic interaction between the two polymorphisms. However, we cannot rule out a more modest synergistic interaction, because the OR from a multiplicative model with increasing number of risk alleles is 0.99 (95% CI, 0.80–1.24; P = 0.847). In comparison, the PPARG P12A variant has been shown to interact synergistically with polymorphisms in other genes encoding the ß3-adrenergic receptor (49), PPAR-{alpha} (57), and adiponectin (61) on increasing the risk of obesity, type 2 diabetes, and/or prediabetic phenotypes. These interactions are between genes encoding proteins participating in the same metabolic pathways or that are expressed in the same tissues making the proposed interactions plausible. KIR6.2 and PPAR{gamma}-2, in contrast, share no known common biological pathways, and consistently, the diabetogenic impacts of the KCNJ11 E23K and PPARG P12A variants appear to be additive.

Analysis of the additive impact of the KCNJ11 E23K and PPARG P12A polymorphisms showed that the OR for type 2 diabetes was increased by 1.14/risk allele. Because the risk alleles are relatively frequent, with 10% of the individuals in the present study having four risk alleles, a rather large estimated population-attributable risk for type 2 diabetes (28%) was estimated for the additive effects of the KCNJ11 E23K and PPARG P12A polymorphisms.

In analyses of a potentially synergistic gene-gene interaction of the KCNJ11 E23K polymorphism and the PPARG P12A polymorphism on prediabetic quantitative traits among 4223 glucose-tolerant subjects from the population-based Inter99 cohort, we also failed to demonstrate any significant synergistic interactions. However, categorization of the same 4223 glucose-tolerant subjects into the five groups according to the number of risk alleles showed a nonsignificant tendency toward increasing levels of plasma glucose both in the fasting state and after an OGTT with increasing number of risk alleles, which may suggest a slight impairment of glucose regulation in subjects carrying an increased number of risk alleles, but still displaying normal glucose tolerance. Clearly, the results from our quantitative trait analyses of gene-gene interplays are preliminary and are exploratory in nature; therefore, our results should definitely be interpreted with caution while awaiting replication in even larger population-based studies of glucose-tolerant subjects.


   Acknowledgments
 
We thank Annemette Forman, Lene Aabo, Inge-Lise Wantzin, and Marianne Stendal for technical assistance, and Grete Lademann for secretarial support. We also thank Birger Thorsteinsson, M.D., D.M.Sc., for collecting data for some of the type 2 diabetic patients. Finally, we gratefully acknowledge Lars Hansen, M.D., D.M.Sc., for his help with data interpretation.


   Footnotes
 
This work was supported by grants from the Danish Diabetes Association, the Danish Medical Research Council, the Danish Heart Foundation, the European Economic Community (BMH4-CT98-3084 and QLRT-CT-1999-00 546), and the Velux Foundation.

First Published Online March 29, 2005

Abbreviations: BMI, Body mass index: CI, confidence interval; HOMA IR, homeostasis model assessment of insulin resistance; KATP, ATP-sensitive potassium; LNA, locked nucleic acid; OGTT, oral glucose tolerance test; OR, odds ratio; P12A, Pro12Ala; PPAR-{gamma}2, peroxisome proliferator-activated receptor-{gamma}2; RFLP, restriction fragment length polymorphism; SNP, single nucleotide polymorphism.

Received October 1, 2004.

Accepted March 17, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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G. Sesti, E. Laratta, M. Cardellini, F. Andreozzi, S. Del Guerra, C. Irace, A. Gnasso, M. Grupillo, R. Lauro, M. L. Hribal, et al.
The E23K Variant of KCNJ11 Encoding the Pancreatic {beta}-Cell Adenosine 5'-Triphosphate-Sensitive Potassium Channel Subunit Kir6.2 Is Associated with an Increased Risk of Secondary Failure to Sulfonylurea in Patients with Type 2 Diabetes
J. Clin. Endocrinol. Metab., June 1, 2006; 91(6): 2334 - 2339.
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