Sequence Variants of ADIPOQ and Association with Type 2 Diabetes Mellitus in Taiwan Chinese Han Population

Diabetes is a serious global health problem. Large-scale genome-wide association studies identified loci for type 2 diabetes mellitus (T2DM), including adiponectin (ADIPOQ) gene and transcription factor 7-like 2 (TCF7L2), but few studies clarified the effect of genetic polymorphisms of ADIPOQ and TCF7L2 on risk of T2DM. We attempted to elucidate association between T2DM and polymorphic variations of both in Taiwan's Chinese Han population, with our retrospective case-control study genotyping single nucleotide polymorphisms (SNPs) in ADIPOQ and TCF7L2 genes both in 149 T2DM patients and in 139 healthy controls from Taiwan. Statistical analysis gauged association of these polymorphisms with risk of T2DM to show ADIPOQ rs1501299 polymorphism variations strongly correlated with T2DM risk (P = 0.042), with rs2241766 polymorphism being not associated with T2DM (P = 0.967). However, both polymorphisms rs7903146 and rs12255372 of TCF7L2 were rarely detected in Taiwanese people. This study avers that ADIPOQ rs1501299 polymorphism contributes to risk of T2DM in the Taiwanese population.


Introduction
Global prevalence of type 2 diabetes mellitus (T2DM) is 6% but is expected to rise due to aging population [1]; 2012 saw some 1.6 million patients (∼7% of total population) in Taiwan suffering from it, with ∼90% of these cases diagnosed as T2DM, non-insulin-dependent diabetes characterized by impaired insulin secretion in peripheral tissues. It accounts for up to 11.5% of healthcare costs in Taiwan annually. Factors such as age, weight, diet, lifestyle, and family history are linked therewith [1]; higher concordance rate among monozygotic versus dizygotic twins and sibling risk manifested itself, and prevalence differs among racial groups [2], suggesting genetic contribution to risk of T2DM.
Genome-wide studies identify diabetes susceptibility loci on chromosomes 3q27 and 10q25.3, where adiponectin genes (ADIPOQ or APM1) and TCF7L2 are located, respectively [3]. Human adiponectin, encoded by ADIPOQ gene, is an adipose-specific secretory protein involved in many metabolic processes: for example, glucose modulation and 2 The Scientific World Journal Values represented mean ± SD (standard deviation).

Results
Two SNPs of ADIPOQ and two SNPs of TCF7L2 genes were genotyped in T2DM patients and controls, using allelespecific polymerase chain reaction analysis (Table 3). Distributions of ADIPOQ and TCF7L2 genotypes are plotted in Table 4, and genotype frequencies were evaluated by chi-square test. The method of Benjamini and Hochberg (threshold = 0.1) served to correct for multiple testing of each tested gene. Results showed significant difference ( = 0.042) in ADIPOQ (rs1501299 (G276T)) polymorphism after Benjamini and Hochberg correction (FDR < 0.05). Yet no differences of genotype frequencies in ADIPOQ (rs2241766 (T45G)) and TCF7L2 (rs7903146) polymorphisms were detected. Surprisingly, genotypes of rs12255372 for TCF7L2 were the only GG genotype appearing in both healthy and T2DM subjects: that is, rs12255372 is not informative among the Taiwanese population. Genotype distributions of all SNPs do not deviate from HWE in either T2DM patients or controls (data not shown). Similar results were observed in allele frequency: minor allele frequency of rs1501299 SNP in ADIPOQ being 28.5% higher among T2DM patients compared to healthy controls (20.6%, = 0.028, FDR < 0.05) ( Table 5). The odds ratio (OR) showed T allele of rs1501299 SNP as the risk factor for T2DM in our subjects (OR = 0.59, 95% confidence interval (CI): 0.01-0.24, = 0.032), with no difference apparent in other polymorphisms. Collectively, these results suggest genetic variation of ADIPOQ, in particular rs1501299 SNP, may have a role in prevalence of T2DM among the Taiwanese population. We likewise compared distribution of genotypes for ADIPOQ rs1501299 SNP among Taiwanese T2DM and control subjects    The Scientific World Journal

Discussion
Novel associated genetic diseases identified by genome-wide association studies are needed for further testing in different populations. Association study is the most important tool for identifying genes conferring susceptibility to complex disorders. Genetic studies on many complex disorders like diabetes and psychiatric traits confirm many risk-associated genetic variants contributing to a subtle effect [2]. Our casecontrol study analyzed association of T2DM with ADIPOQ and TCF7L2 polymorphism in the Taiwanese. Interestingly, significant correlation was noted between ADIPOQ rs1501299 polymorphism and T2DM subjects, with T allele shown as a risk factor for prevalence of T2DM.
Polymorphisms of TCF7L2. TCF7L2 is involved in betacatenin-dependent Wnt signaling pathway, which employs its physiological functions in pancreatic and intestinal endocrine cells [20,21]. Previous investigations correlated TCF7L2 with risk of T2DM in extensive populations [31,32], though not including the Taiwanese. We rated association of TCF7L2 polymorphisms rs7903146 and rs12255372 with T2DM prevalence in Taiwan. However, minor allele frequencies of rs7903146 and rs12255372 are 0.023 and unknown, inconsistent with prior study of the Chinese population [33]. Data hinted these SNPs are not polymorphic in our population and were removed from the final analyses. Although polymorphisms of TCF7L2 were rare in Taiwanese population, it is noteworthy to observe linkage between rs7903146 in TCF7L2 and T2DM in ethnic groups [31,[34][35][36][37]. Genetic markers well represented in one population but showing significantly different allele frequencies, even almost total absence in another allele across ethnicities, are frequently observed [38]. These phenomena might result from heterogeneity among ethnic groups; their effects on susceptibility to T2DM are unclear and merit follow-up study.
Polymorphism of ADIPOQ. Adiponectin, adipocytokine encoded by ADIPOQ, is central to insulin sensitivity and resistance and other metabolic syndrome traits in diverse populations [39,40]. In addition, plasma level of adiponectin was proposed as a biomarker for T2DM, hypertension, and obesity [41,42]; ADIPOQ SNPs were linked with plasma adiponectin level among the Caucasian population [11,12] and metabolic syndrome [9,10]. ADIPOQ polymorphisms and T2DM or diabetic nephropathy had also been evaluated from many populations: for example, the European-derived [17,18], the Asian [14, 16,40], the non-European South African [43], and the African American [44] populations. Still, no difference appeared between these SNPs and T2DM in the Taiwanese population [45]. Gao et al. indicated G allele frequency of ADIPOQ SNP (rs2241766) as prevalent in metabolic syndrome patients versus control groups in the Chinese Han population [10], which demonstrably augments risk of T2DM in the Chinese population [10]. We analyzed genetic association between ADIPOQ polymorphisms and T2DM in 149 patients and 139 normal controls in Taiwan (study power = 0.7); genotype frequency of SNP rs2241766 showed no difference between T2DM and controls. Since rs2241766 is a silent mutation for Gly15, this SNP may link with other functional SNPs. We focused on other SNPs of ADIPOQ, rs1501299, to find rs1501299 SNP definitely associated with T2DM ( = 0.045). High prevalence of T allele ( = 0.028) and risk of T2DM in the Taiwanese population was observed ( = 0.032), findings that are consistent with studies performed in populations of southern India [14], Japan [15,16], and Mainland China [46]. Also, Yang et al. reported SNP rs1501299 linked with obesity, metabolic syndrome, and diabetes in the elderly Taiwanese [47], which suggest that T allele of SNP rs1501299 increases risk of diabetes among the Chinese population, consistent with our data. However, these results conflicted with other studies of Chinese people [48][49][50][51], suggesting that SNP rs1501299 may not be a functional SNP but most likely be in linkage disequilibrium with other functional variants [47]. Moreover, it suggests genetic background of the Taiwanese may be unique compared to the Mainland Chinese. Prevalence of TT genotype in SNP rs1501299 noted among Taiwanese was compared with controls from Europe, China, and Nigeria in HapMap database. Results indicate most groups (Taiwanese, European, Japanese, and Nigerian) retain high levels of GG content, albeit not Mainland China (Table 6), suggesting genetic background of Chinese people is heterogeneous [46]. This portends a greater number of subjects from different populations to evaluate in the future.
Study Limitation. One limitation of this study was small sample size due to cost considerations. While genetic analysis of SNP rs1501299 proved significant, we could not entirely exclude a possibility of false positives because of sampling limitations and other factors (confounders with other medical conditions, subject assessment lacking support by structured interviews). Yet we added statistics, FDR < 0.05, as a true positive association to support our conclusions.

Conclusions
Our case-control study linked T2DM with ADIPOQ and TCF7L2 polymorphism in Taiwan's Han Chinese population: strong correlation emerged between ADIPOQ rs1501299 and T2DM subjects in Taiwan. Taken together, this verifies that ADIPOQ is associated with T2DM risk in Taiwanese population, which may prove useful clinically as a genetic marker. Results suggest ADIPOQ as a key genetic contributor to T2DM that may serve as a clinical biomarker.