Genetic Variants in Peroxisome Proliferator-ActivatedReceptor-g and Retinoid X Receptor-a Gene

and Type 2 Diabetes Risk: A Case-Control Studyof a Chinese Han Population

Ying Lu, B.S.,1,* Xinhua Ye, B.S.,2,* Yuanyuan Cao, M.S.,1 Qian Li, M.D.,3 Xiaofang Yu, B.S.,4

Jinluo Cheng, B.S.,2 Yanqin Gao, M.S.,4 Jianhua Ma, M.S.,3 Wencong Du, B.S.,1 and Ling Zhou, M.D.1

Abstract

Background: The serum levels of adiponectin are paradoxically decreased in obesity and may play importantroles in the development of type 2 diabetes mellitus (T2DM). Potentially functional polymorphisms in theperoxisome proliferator-activated receptor-g (PPAR-g) and retinoid X receptor-a (RXR-a) genes may alter T2DM risksby increasing the human adiponectin promoter activity in cells. Therefore, we hypothesized that single nucle-otide polymorphisms (SNPs) in PPAR-g and RXR-a were associated with risk of T2DM. To test this hypothesis,three potentially functional SNPs of PPAR-g and four of RXR-a with a minor allele frequency of �0.05 in theChinese Han population were identified from the National Center for Biotechnology Information dbSNPs da-tabase to evaluate their association with T2DM.Methods: Polymerase chain reaction–restriction fragment length polymorphism was performed to test the ge-notypes in T2DM patients (n¼ 540) and normal controls (n¼ 604).Results: The variant genotypes rs2920502CC, rs3856806CT, rs3856806CT/TT, and rs4240711AG/GG were as-sociated with T2DM. Furthermore, the prevalences of haplotype GTC and CTG in PPAR-g and GTAC in RXR-awere less frequent in cases (17.1%, 2.6%, and 2.4%, respectively) than in controls (22.3%, 3.8%, and 6.6%,respectively), whereas GTGT in RXR-a was more frequent in cases (6.9%) than in controls (4.4%) (P< 0.05 forboth two-sided w2 test and thousand times permutation tests). Patients with genotype CT/TT of rs3856806and genotype AG/GG of rs4240711 had higher levels of serum adiponectin than those with the genotype CCand genotype AA (P¼ 0.026 and 0.021, respectively). Model X2 X5 X6 X7 (rs3856806, rs3132291, rs4240711, andrs4842194) was the best model with the highest test balanced accuracy (0.5764) (cross-validation consistency¼10/10) in the multifactor dimensionality reduction method.Conclusions: The PPAR-g and RXR-a gene variants associated with the development of T2DM in this study mustbe investigated in a larger population to reveal any potential effects on metabolism.

Introduction

Type 2 diabetes mellitus (T2DM), which is characterizedby fasting and postprandial hyperglycemia and relative

insulin insufficiency, is one of the most common chronicdiseases and is associated with multiple environmental andgenetic factors.1 According to a press release of the Interna-tional Diabetes Federation, T2DM now affects a staggering246 million people worldwide, and the total number of people

living with diabetes will skyrocket to 380 million within 20years if nothing is done.2 Although the disease is widespread,its genetic determinants are still poorly defined.

Adiponectin, which has an inhibitory effect on prolifer-ation of vascular smooth muscle cells, is highly abundant inhuman serum and is secreted by adipose tissue.3 Its plasmalevels are paradoxically decreased in obesity and may playimportant roles in the development of T2DM.4,5 The cur-rent views indicate that thiazolidinediones, as peroxisome

1Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu, China.2Department of Incretion, Affiliated Changzhou 2nd Hospital of Nanjing Medical University, Changzhou, Jiangsu, China.3Department of Incretion, Affiliated Nanjing 1st Hospital of Nanjing Medical University, Nanjing, Jiangsu, China.4Department of Incretion, 3rd Affiliated Hospital of Nanjing Medical University, Yizheng, Jiangsu, China.*The first two authors contributed equally to this study.

DIABETES TECHNOLOGY & THERAPEUTICSVolume 13, Number 2, 2011ª Mary Ann Liebert, Inc.DOI: 10.1089/dia.2010.0122

157

proliferator-activated receptor-g (PPAR-g) agonists, havebeen shown to increase plasma adiponectin levels bytranscriptional induction in adipose tissues.5 The physio-logical relevance of PPAR-g in animal studies suggests arole of hepatic PPAR-g in the reduction of plasma triglyc-eride and glucose levels. 9-cis-Retinoic acid is the naturalligand of retinoid X receptor (RXR), and the ability of RXRto heterodimerize with many nuclear receptors, includingPPAR-g, indicates its important function within the nuclearreceptor family.4 In the adult liver, RXR-a is the mostabundant of the three RXRs (RXR-a, RXR-b, and RXR-g).Some recent research indicates that multiple metabolicpathways are coordinately dependent on RXR-a in themouse liver.6 The PPAR-g/RXR heterodimer, which di-rectly binds to the functional PPAR-responsive element(PPRE), could increase human adiponectin promoter ac-tivity in cells. The genes coding for PPAR-g and RXR-alocated in the adiponectin pathway are therefore candi-dates for T2DM and obesity.4,7

Single nucleotide polymorphisms (SNPs) in the PPAR-ggene have been associated with risk of diabetes.8–10 However,few studies of SNPs in the RXR-a gene have been published todate. Because only one SNP might not represent genetic var-iation in genes, we tested the hypothesis that multiple po-tentially functional SNPs of the PPAR-g and RXR-a geneswere associated individually or jointly with risk of T2DM in acase-control study with moderate sample size (540 cases and604 controls) in a Chinese Han population.

Materials and Methods

Subjects

We studied 540 T2DM patients and 604 T2DM-free controlswith their informed consent. All subjects were geneticallyunrelated ethnic Han Chinese and were from Nanjing cityand surrounding regions in southeast China. The patientswere consecutively recruited between 2008 and 2009 fromthe diabetes inpatient or outpatient clinic of the AffiliatedChangzhou 2nd Hospital of Nanjing Medical University(NJMU), the 3rd Affiliated Hospital of NJMU, and the Af-filiated Nanjing 1st Hospital of NJMU, without any restric-tions on age and sex. A diagnosis of T2DM required either afasting plasma glucose of �7.0 mmol/L (126 mg/dL) or a 2-hglucose of �11.1 mmol/L (200 mg/dL) after an oral glu-cose tolerance test. All the patients were tested by glutamicacid decarboxylase autoantibodies and islet cell antibodies(ICA512) (performed by the clinical laboratories in the threeaffiliated hospitals) to exclude patients with type 1 diabetes.T2DM-free controls were randomly selected from the outpa-tient clinic within the same geographical area and the periodof the cases. Controls received routine annual health exami-nations. These control subjects, who did not have diabetes asdetermined by an oral glucose tolerance test (75 g of glucose),performed according to World Health Organization criteria,had no history of T2DM and were frequency-matched to thecases for age (�5 years), sex, and residential area (urban orrural areas).

Each participant was scheduled for an interview afterwritten informed consent was obtained, and a structuredquestionnaire was administered by interviewers to collectinformation on demographic data and environmental expo-sure history.

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158 LU ET AL.

Measurements

Weight and height were measured by trained personnel,and body mass index (in kg/m2) was calculated. Bloodpressure was measured in the individual’s right arm after a10-min rest with a standard sphygmomanometer of appro-priate cuff size. After an overnight fast, venous blood sampleswere drawn and promptly centrifuged, and the plasma wasstored at �208C until the adiponectin assay was performed.Peripheral blood samples of 1,144 subjects were collected intubes containing the anticoagulant EDTA, immediatelyplaced on ice, and processed within a few hours.

All samples were assessed in the same assay. Serum adi-ponectin was measured by enzyme-linked immunosorbentassay (human adiponectin enzyme-linked immunosorbentassay kit; RapidBio Co., West Hills, CA). Fasting plasmaglucose was measured in the laboratories in the three affili-ated hospitals of NJMU with the glucose oxidase method.Total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides were de-termined in the three affiliated hospitals by an enzymaticcolorimetric method (Au5400; Olympus, Tokyo, Japan). DNAwas extracted from peripheral blood by the use of proteinaseK and phenol/chloroform. This study was approved by theResearch Ethics Committee of Nanjing Medical University.

SNP selection and genotyping

Three potentially functional SNPs of PPAR-g and four ofRXR-a with minor allele frequency of �0.05 in the ChineseHan population were identified from the National Centerfor Biotechnology Information dbSNPs database (www.ncbi.nlm.nih.gov/), including four at the coding region and threeat the 30 untranslated region. Genomic sequences were ob-tained from the Hapmap database. Primer version 5.0 andpolymerase chain reaction primer-introduced restrictionanalysis were used to design the seven primer sets. Thepolymerase chain reaction–restriction fragment length poly-morphism assay was used to genotype the seven SNPs (Table 1).Genotyping was performed without knowing the subjects’case or control status. Two research assistants read the gelpictures independently and repeated the assays if they did notreach a consensus on the tested genotype results. In addition,

10% of the samples were randomly selected to perform re-peated assays; the results were 100% concordant.

Statistical analysis

The distribution of the general characteristics and genotypefrequencies between the T2DM cases and T2DM-free controlswas compared by using two-sided w2 test and/or Student’st test. Differences between quantitative traits were comparedwith one-way analysis of variance and post hoc multiplecomparisons (using the Bonferroni method). Unconditionallogistic regression analyses were performed to estimate crudeand adjusted odds ratios (ORs) and 95% confidence intervals(95% CIs) as a measure of association of genotypes with therisk of T2DM. LDA software was used to calculate the linkagedisequilibrium (LD) of the seven SNPs. PHASE version 2.1programs was used to infer haplotype frequencies based onthe observed PPAR-g and RXR-a genotypes. The multifactordimensionality reduction method (MDR software, version1.0.1) was used to consider the joint effects of PPAR-g andRXR-a. We carefully controlled the potential influence ofmultiple comparisons using the thousand times permutationtest by using Stata software (version 9.2; StataCorp LP, Col-lege Station, TX). All the other statistical analyses were carriedout by Statistical Analysis System (version 9.1.3; SAS Institute,Cary, NC).

Results

Basic characteristics

Of the selected characteristics, significant differences ex-isted between cases and controls for blood pressure, bodymass index, triglycerides, high-density lipoprotein cholester-ol, low-density lipoprotein cholesterol, fasting plasma glu-cose, and adiponectin (Table 2).

Logistic regression analysis for the associationsbetween variant genotypes of PPAR-gand RXR-a and T2DM

The genotype distribution for all the SNPs did notshow any deviation from the Hardy–Weinberg equilibrium(all P> 0.05 in controls). Among the 1,144 subjects, the

Table 2. Basic Characteristics of the Case and Control Groups

Characteristic Cases (n¼ 540) Controls (n¼ 604) P value

Gender (male:female) 280:260 305:299 0.678Age (years) 57.74� 10.88 57.00� 10.35 0.237Systolic pressure (mm Hg) 139.84� 22.01 130.22� 17.27 < 0.001Diastolic pressure (mm Hg) 86.28� 12.40 79.92� 10.57 < 0.001BMI (kg/m2) 24.93� 3.57 24.23� 3.32 0.001Smoking (%) 170 (31.48) 177 (29.37) 0.440Drinking (%) 110 (20.37) 150 (24.83) 0.072Total cholesterol (mmol/L) 5.17� 1.37 5.05� 0.98 0.121Triglyceride (mmol/L) 2.77� 2.93 1.59� 1.03 < 0.001HDL-C (mmol/L) 1.16� 0.41 1.42� 0.37 < 0.001LDL-C (mmol/L) 2.88� 1.01 2.66� 0.85 < 0.001Fasting plasma glucose (mmol/L) 11.71� 3.79 5.18� 0.46 < 0.001Adiponectin (mg/L) 5.84� 1.89 7.90� 2.80 < 0.001

Data are mean� SD values except as marked. Significant differences are in bold type.BMI, body mass index; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.

GENETIC VARIANTS IN PPAR-c, RXR-a, AND T2DM 159

successfully genotyped rates for seven SNPs were all morethan 95%. The variant genotype CC (adjusted OR [95%CI]¼ 0.56 [0.34–0.92]) of rs2920502 was associated with asignificantly decreased risk of T2DM compared with theGG genotype. Similarly, the variant genotype CT (adjustedOR [95% CI]¼ 0.74 [0.58–0.95]) and the combined genotypeCT/TT (adjusted OR [95% CI]¼ 0.73 [0.58–0.93]) ofrs3856806 compared with the CC genotype were all asso-ciated with a significantly decreased risk of T2DM. Com-pared with the genotype AA homozygote, however, thecombined genotype AG/GG of rs4240711 was associatedwith a significant risk effect for T2DM (adjusted OR [95%CI]¼ 1.29 [1.00–1.66]) (Table 3).

Haplotype analysis

LDA software was used to calculate the LD by D0, by whichwe found that the LD among the three SNPs of PPAR-g rangedfrom 0.1 to 0.8 as well as the four SNPs of the RXR-a gene(Table 4). PHASE (version 2.1) was used to determine hap-lotypes. The total number was 1,114 of PPAR-g and 1,115 ofRXR-a, excluding the unsuccessfully genotyped subjects inthe SNPs. In the haplotypes of PPAR-g, haplotype GTC (17.1%and 22.3%, respectively) and CTG (2.6% and 3.8%, respec-tively) were all less common in cases than in the controlscompared with the most common haplotype, GCC. These twohaplotypes were associated with a significant protective effect

Table 3. Distribution of Genotypes in Case and Control Groups

Number (%) OR (95% CI)

Genotype Cases Controls Crude Adjusteda P value

rs2920502 531 (100) 599 (100)GG 303 (57.1) 337 (56.3) 1.00 1.00 —GC 201 (37.9) 209 (34.9) 1.07 (0.84–1.37) 1.07 (0.83–1.38) 0.609CC 27 (5.1) 53 (8.8) 0.57 (0.35–0.92) 0.56 (0.34–0.92) 0.023GC/CC 228 (43.0) 262 (43.7) 0.97 (0.77–1.23) 0.97 (0.76–1.23) 0.784C allele 255 (24.0) 315 (26.3) 0.231

rs3856806 538 (100) 601 (100)CC 323 (60.0) 309 (51.4) 1.00 1.00 —CT 181 (33.6) 243 (40.4) 0.71 (0.56–0.91) 0.74 (0.58–0.95) 0.020TT 34 (6.3) 49 (8.2) 0.66 (0.42–1.06) 0.68 (0.42–1.10) 0.110CT/TT 215 (39.9) 292 (48.6) 0.70 (0.56–0.89) 0.73 (0.58–0.93) 0.011T allele 249 (23.1) 341 (28.4) 0.005

rs1801282 534 (100) 594 (100)CC 470 (88.0) 526 (88.6) 1.00 1.00 —CG 64 (12.0) 67 (11.3) 1.07 (0.74–1.54) 1.01 (0.70–1.47) 0.958GG 0 (0.0) 1 (0.2) — — —CG/GG 64 (12.0) 68 (11.5) 1.05 (0.73–1.52) 0.99 (0.68–1.44) 0.974G allele 64 (6.0) 69 (5.8) 0.923

rs1045570 530 (100) 603 (100)GG 329 (62.1) 378 (62.7) 1.00 1.00 —GT 183 (34.5) 205 (34.0) 1.03 (0.80–1.31) 1.01 (0.79–1.31) 0.912TT 18 (3.4) 20 (3.3) 1.03 (0.54–1.99) 1.05 (0.54–2.02) 0.890GT/ TT 201 (37.9) 225 (37.3) 1.03 (0.81–1.31) 1.02 (0.80–1.30) 0.891T allele 219 (20.7) 245 (20.3) 0.880

rs3132291 532 (100) 603 (100)TT 224 (42.1) 253 (42.0) 1.00 1.00 —TC 239 (44.9) 282 (46.8) 0.96 (0.75–1.23) 1.01 (0.79–1.31) 0.921CC 69 (13.0) 68 (11.3) 1.15 (0.78–1.68) 1.17 (0.79–1.74) 0.420TC/CC 308 (57.9) 350 (58.1) 0.99 (0.79–1.26) 1.04 (0.82–1.33) 0.725C allele 377 (35.4) 418 (34.7) 0.733

rs4240711 527 (100) 597 (100)AA 184 (34.9) 232 (38.9) 1.00 1.00 —AG 270 (51.2) 285 (47.7) 1.20 (0.93–1.54) 1.29 (0.99–1.68) 0.054GG 73 (13.9) 80 (13.4) 1.15 (0.79–1.67) 1.27 (0.86–1.86) 0.227AG/GG 343 (65.1) 365 (61.1) 1.19 (0.93–1.51) 1.29 (1.00–1.66) 0.048G allele 416 (39.5) 445 (37.3) 0.304

rs4842194 526 (100) 603 (100)TT 235 (44.7) 261 (43.3) 1.00 1.00 —TC 238 (45.2) 285 (47.3) 1.03 (0.68–1.56) 0.98 (0.64–1.51) 0.948CC 53 (10.1) 57 (9.5) 0.93 (0.73–1.19) 0.95 (0.74–1.23) 0.710TC/CC 291 (55.3) 342 (56.8) 0.95 (0.75–1.20) 0.96 (0.75–1.22) 0.733C allele 344 (32.7) 399 (33.1) 0.881

Significant differences are in bold type.aAdjusted for age, gender, and body mass index.CI, confidence interval; OR, odds ratio.

160 LU ET AL.

for T2DM (adjusted OR [95% CI]¼ 0.73 [0.59–0.92] and 0.57[0.34–0.95], respectively) (Table 5). In the haplotypes of RXR-a, compared with the most common haplotype, GTAT, hap-lotype GTGT was more common in cases than in controls(6.9% and 4.4%, respectively). The GTGT haplotype was as-sociated with a 1.66-fold increased risk of T2DM (adjusted OR[95% CI]¼ 1.66 [1.14–2.43]). However, haplotype GTAC(2.4% and 6.6%, respectively) was less common in cases thanin controls compared with haplotype GTAT, as a significantprotective effect was associated with T2DM (adjusted OR[95% CI]¼ 0.35 [0.22–0.56]) (Table 6). All the significance stillexisted after the thousand times permutation test (Tables 5and 6).

Adiponectin level of the different genotypesrs2920502, rs3856806, and rs4240711 in caseand control groups

Because rs2920502, rs3856806, and rs4240711 had shownan association with T2DM in the logistic regression analysis,we calculated the adiponectin level of different genotypes ofthe three SNPs. As presented in Table 7, patients with theCT/TT genotype of rs3856806 had higher levels of serumadiponectin than those with the CC genotype (P¼ 0.026).Patients with the genotype AG/GG of rs4240711 also hadhigher levels of serum adiponectin than those with the ge-notype AA (P¼ 0.021).

Locus–locus interactions of PPAR-g and RXR-a

The genotypes rs2920502, rs3856806, rs1801282, rs1045570,rs3132291, rs4240711, and rs4842194 were renamed as X1, X2,X3, X4, X5, X6, and X7 in proper order. MDR software wasused to consider the joint effects of the seven SNPs in PPAR-gand RXR-a. As presented in Table 8, three models had sta-tistical significance (P< 0.05). The coefficient of variation ofconsistency of model X2, X2 X5 X7, and X2 X5 X6 X7 was 10/10.Model X2 X5 X6 X7 was the best model because of the highesttest balanced accuracy (0.5764).

Discussion

In this study, we selected three potentially functional SNPsof the PPAR-g gene and four of the RXR-a gene to investigatetheir associations with the risk of T2DM in a Chinese Hanpopulation with a moderate sample size of 540 T2DM casesand 604 T2DM-free controls.

In the single locus analyses, we observed statistically sig-nificant differences between case patients and control subjectsin genotype distributions of rs2920502CC, rs3856806CT, andrs3856806CT/TT of the PPAR-g gene (P¼ 0.023, 0.020, and0.011 respectively). Further study of haplotypes suggestedthat haplotype GTC, with variant allele T of rs3856806C/T,could decrease the risk for T2DM compared with themost common haplotype GCC (adjusted OR [95% CI]¼ 0.73

Table 4. Linkage Disequilibrium of Single Nucleotide Polymorphisms

in Peroxisome Proliferator-Activated Receptor-g and Retinoid X Receptor-a Gene Among 604 Type 2Diabetes Mellitus-Free Patients in Southeast China Based on Genotype Data

Gene Locus rs2920502 rs3856806 rs1801282 rs1045570 rs3132291 rs4240711 rs4842194

PPAR-g rs2920502 — 0.131 (0.002) 0.818 (0.114) 0.115 (0.010) 0.048 (0.002) 0.049 (0.001) 0.003 (0.000)rs3856806 — — 0.534 (0.044) 0.020 (0.000) 0.064 (0.001) 0.047 (0.001) 0.027 (0.000)rs1801282 — — — 0.111 (0.003) 0.334 (0.013) 0.370 (0.014) 0.038 (0.000)

RXR-a rs1045570 — — — — 0.794 (0.303) 0.644 (0.178) 0.420 (0.091)rs3132291 — — — — — 0.811 (0.587) 0.489 (0.223)rs4240711 — — — — — — 0.483 (0.194)rs4842194 — — — — — — —

Data are D0

(r2).PPAR-g, peroxisome proliferator-activated receptor-g; RXR-a, retinoid X receptor-a.

Table 5. Odds Ratios and 95% Confidence Intervals for the Association Between Inferred Peroxisome

Proliferator-Activated Receptor-g Haplotypes and Type 2 Diabetes Mellitus in the Case-Control Study

Chromosome numbers (%)

Haplotypea Cases (n¼ 526) Controls (n¼ 588) P valueb P valuec Adjusted OR (95% CI)

GCC 593 (56.4) 600 (51.0) — — —GTC 180 (17.1) 262 (22.3) 0.001 0.002 0.73 (0.59–0.92)CCC 200 (19.0) 225 (19.1) 0.348 0.363 0.92 (0.73–1.15)CTG 27 (2.6) 45 (3.8) 0.044 0.049 0.57 (0.34–0.95)Others 52 (4.9) 44 (3.8) 0.400 0.411 1.19 (0.78–1.82)

Significant differences are in bold type.aLoci of single nucleotide polymorphisms are written 50 to 30 and include the following single nucleotide polymorphisms: rs2920502,

rs3856806, and rs1801282.bAdjusted for age, gender, and body mass index.cThousand times permutation test.CI, confidence interval; OR, odds ratio.

GENETIC VARIANTS IN PPAR-c, RXR-a, AND T2DM 161

[0.59–0.92]). In fact, rs3856806T was responsible for the asso-ciation. Haplotype CTG, with variant allele C of rs2920502G/Cand T of rs3856806C/T, could decrease the risk for T2DMcompared with the most common haplotype GCC (adjustedOR [95% CI]¼ 0.57 [0.34–0.95]). Therefore, rs2920502C andrs3856806T were responsible for the association. PPAR-g, amember of the nuclear receptor family of ligand-activatedtranscription factors that heterodimerize with RXR to regulategene expression, is located primarily in the adipose tissue,lymphoid tissue, colon, liver, and heart and is thought toregulate adipocyte differentiation and glucose homeostasis.PPAR-g has been implicated in the pathology of numerousdiseases, including obesity, diabetes, atherosclerosis, and

cancer. The human gene encoding PPAR-g has been localizedto chromosome 3 (3p25). It was reported that polymorphismC1431T of exon 6 of PPAR-g (rs3856806) was associated withlower diabetes risks.11 The study of Tai et al.12 suggested adecreased risk of diabetes in carriers of the T allele of theC1431T polymorphism in exon 6 of the PPAR-g gene(rs3856806) in the whole Singaporean population that wasstronger in Indians. We also found the association betweenrs3856806C/T and diabetes in Chinese people. Currently,studies investigating the association between rs2920502 G/Cof the PPAR-g gene and the risk of T2DM have not been re-ported yet. We found rs2920502CC was associated with adecreased risk of T2DM compared with the genotypers2920502GG. The contribution of the PPAR-g rs1801282 SNPin the genetic risk for T2DM was suggested in different ethnicpopulations, including the French,13,14 Indians,12 Japanese,15

Mexican Americans,16 Singaporeans,12 Swedish,17 Taiwa-nese,18 and Tunisians.10 We failed to find the association in thesingle locus analyses. We considered that the rs1801282 SNPin PPAR-g may be not associated with risk of T2DM in theChinese Han population. The relationship between geneticvariation and the disease was different among various eth-nicities. These discrepancies might be related to genetic dif-ferences, such as different frequencies in genetic variation,and the general demographic characteristics of the subjects,including age and gender, living habits, and other environ-mental and regional differences. Recent research has indi-cated that the rs1801282 SNP does not raise the risk of type 2diabetes but may be a significant independent risk factor fordiabetic nephropathy in the Chinese Han population. Liu

Table 6. Odds Ratios and 95% Confidence Intervals for the Association Between Inferred Retinoid X

Receptor-a Haplotypes and Type 2 Diabetes Mellitus in the Case-Control Study

Chromosome numbers (%)

Haplotypea Cases (n¼ 520) Controls (n¼ 595) P valueb P valuec Adjusted OR (95% CI)

GTAT 529 (50.9) 599 (50.3) — — —TCGC 137 (13.2) 147 (12.4) 0.685 0.701 1.07 (0.82, 1.39)GCGC 125 (12.0) 118 (9.9) 0.198 1.000 1.27 (0.95–1.69)GTGT 72 (6.9) 52 (4.4) 0.018 0.019 1.66 (1.14–2.43)GTAC 25 (2.4) 79 (6.6) < 0.001 < 0.001 0.35 (0.22–0.56)Other 152 (14.6) 195 (16.4) 0.355 0.364 0.92 (0.72–1.18)

Significant differences are in bold type.aLoci of single nucleotide polymorphisms are written 50 to 30 and include the following single nucleotide polymorphisms: rs1045570,

rs3132291, rs4240711, and rs4842194.bAdjusted for age, gender, and body mass index.cThousand times permutation test.CI, confidence interval; OR, odds ratio.

Table 7. Adiponectin Level of the Different

Genotypes RS2920502, RS3856806, and RS4240711in Case and Control Groups

Cases Controls

rs2920502 531 599GG 5.75� 1.88 7.85� 2.81GC 5.96� 1.87 7.83� 2.74CC 6.08� 2.17a 8.74� 2.85GC/CC 5.97� 1.91 8.01� 2.78P value 0.388a NA

rs3856806 538 601CC 5.70� 1.93 7.75� 2.88CT 5.89� 1.63b 7.97� 2.81TT 7.01� 2.36 8.56� 2.17CT/TT 6.07� 1.81c 8.01� 2.72P value 0.803,b 0.026c NA

rs4240711 527 597AA 6.10� 2.07 8.09� 2.82AG 5.67� 1.70 7.71� 2.83GG 5.81� 2.08 7.94� 2.71AG/GG 5.70� 1.78d 7.77� 2.80P value 0.021d NA

Data are mean� SD values (in mg/L). Significant differences arein bold type.

aP¼ 0.388, versus GG genotype from analysis of variance in cases.bP¼ 0.803, versus CC genotype from analysis of variance in cases.cP¼ 0.026, versus CC genotype from t test in cases.dP¼ 0.021, versus AA genotype from t test in cases.NA, not applicable.

Table 8. Multifactor Dimensionality Reduction

Models for Locus–Locus Interactions

in Type 2 Diabetes Mellitus

Model

Trainingbalance

accuracy

Testbalance

accuracyCross-validation

consistency P

X2 0.5452 0.5452 10/10 0.0107X5 X7 0.5663 0.5259 8/10 0.0547X2 X5 X7 0.5924 0.5677 10/10 0.0107X2 X5 X6 X7 0.6248 0.5764 10/10 0.0010

162 LU ET AL.

et al.19 reported the rs1801282 genotype was significantlyassociated with diabetic nephropathy (OR [95% CI]¼ 2.30[1.18–4.45], P¼ 0.014). So the influence of the rs1801282 ge-notype may be on the development of diabetes complicationsrather than the risk of type 2 diabetes in the Chinese Hanpopulation.

In the single locus analyses, we observed statistically sig-nificant differences between case and control subjects in ge-notype distributions of rs4240711AG/GG of the RXR-a gene(P¼ 0.048). Furthermore, the study of haplotypes suggestedthat the four SNPs of RXR-a may jointly contribute to T2DMrisk. Compared with the genotype AA homozygote, thecombined genotype AG/GG of rs4240711 was associatedwith a significant risk effect for T2DM (adjusted OR [95%CI]¼ 1.29 [1.00–1.66]). In the study of haplotypes, haplotypeGTGT, with the variant allele G of rs4240711, could increasethe risk for T2DM compared with the most common haplo-type, GTAT. In fact, rs4240711 AG/GG was responsible forthe association. Interestingly, the SNP rs4842194T/C of theRXR-a gene indicated no association with T2DM in the singlelocus analyses, whereas haplotype GTAC, with the variantallele C of rs4842194, could reduce the risk for T2DM aftercomparing with the most common haplotype, GTAT. There-fore, the SNP rs4842194T/C may be associated with the risk ofT2DM in a protective role. RXRs are nuclear receptors thatmediate the biological effects of retinoids by their involve-ment in retinoic acid-mediated gene activation. The humangene encoding RXR-a has been localized to chromosome 9(9q34.3). Few studies have investigated the association be-tween the RXR-a gene and the risk of T2DM. Wan et al.20

reported the serum insulin-like growth factor-1 level wassignificantly increased in mice with hepatocyte RXR-a defi-ciency compared with wild-type mice; those mutant mice alsodevelop obesity, but they demonstrated the same degree ofinsulin sensitivity and the same level of serum insulin as thewild-type mice, even with an unexpected improvement ofglucose tolerance. In this study, we found the possible asso-ciation among rs4240711A/G, rs4842194T/C, and T2DM. Thehaplotype rs4842194C had a lower risk of T2DM comparedwith the haplotype rs4842194T (OR [95% CI]¼ 0.35 [0.22–0.56]). However, the SNP rs4240711A/G might be a risk factorin the development of T2DM (OR [95% CI]¼ 1.66 [1.14–2.43]).Those findings are preliminary and need to be validated infurther studies with a larger sample size.

Studies to date have identified a functional PPRE in thehuman adiponectin promoter. PPAR-g/RXR heterodimer di-rectly bound to the PPRE and increased the promoter activityin cells. In adipocytes, point mutation of the PPRE markedlyreduced the basal transcriptional activity and completelyblocked thiazolidinedione-induced trans-activation of adipo-nectin promoter.4 Treatment with thiazolidinediones, whichare PPAR-g agonists, increases adiponectin mRNA levels inadipocytes and adipose tissues. Because rs2920502, rs3856806,and rs4240711 had shown an association with T2DM in thelogistic regression analysis, we calculated the adiponectinlevel with different genotypes of the three SNPs. As pre-sented, patients with the CT/TT genotype of rs3856806 hadhigher levels of serum adiponectin than those with the CCgenotype (P¼ 0.026). Patients with genotype AG/GG ofrs4240711 also had higher levels of serum adiponectin thanthose with the genotype AA (P¼ 0.021). Thus, we consideredthat rs3856806 of PPAR-g and rs4240711 of RXR-a were as-

sociated with the serum adiponectin secretion. To analyze thejoint effects of the seven SNPs in PPAR-g and RXR-a, the MDRmethod was used. Model X2 X5 X6 X7 (rs3856806, rs3132291,rs4240711, and rs4842194) was the best model because of thehighest test balanced accuracy (0.5764) (cross-validationconsistency¼ 10/10). Notably, although all the SNPs exceptfor rs3856806 and rs4240711 were not associated with T2DMrisk in the single locus analysis, they might act in concert tomodulate the risk of T2DM. The genes coding for PPAR-g andRXR-a located in the adiponectin pathway are therefore can-didate for the serum adiponectin secretion and T2DM.

Like all other case-control studies, inherent biases existed.Because the cases were from hospitals and the controls wererandomly selected at outpatient clinics within the same geo-graphical area of the cases, the study subjects may not be fullyrepresentative of the general population. So this limitationmay influence the observed associations. Although less than5% of each loci of the DNA samples failed for genotyping, thismay cause some selection bias. The associations betweenPPAR-g/RXR-a variants and T2DM risk were estimated bycomputing the ORs and 95% CIs from logistic regression an-alyses with adjustment for age, sex, and BMI. Other limita-tions relate to the sample size of our study and to the lack ofconfirmation of our findings in other populations.

In conclusion, our study provided some evidence thatPPAR-g and RXR-a polymorphism may be two genetic sus-ceptibility markers for T2DM risk in a Chinese Han popula-tion. Our findings need to be validated by further functionalstudies as well as well-designed larger molecular epidemio-logical studies with diverse ethnic populations.

Acknowledgments

We would like to thank the National Natural ScienceFoundation of China for grant 30771858 and the JiangsuProvincial Natural Science Foundation for grant BK2007229.

Author Disclosure Statement

None of the authors declares any competing financial in-terests.

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Address correspondence to:Ling Zhou, M.D.

Department of Epidemiology and BiostatisticsSchool of Public Health

Nanjing Medical UniversityNanjing, Jiangsu, 210029 China

E-mail: lzhou@njmu.edu.cn

164 LU ET AL.