GENETIC SUSCEPTIBILITY OF TRANSCRIPTION FACTOR 7-LIKE 2 GENE VARIANT AND RISK OF TYPE 2 DIABETES IN ASIAN INDIANS

Methods: The present case–control study included a total of 638 human subjects (318 T2DM subjects and 320 healthy controls). Various anthropometric, biochemical, and genetic parameters were studies in all the subjects. Genotyping of TCF7L2 gene was carried out using allele-specific polymerase chain reaction method. Results: The results of this study indicate significantly higher values of body mass index, waist circumference, waist-to-hip ratio, and body fat (%) in T2DM subjects than controls (p≤0.001). Dyslipidemia represented by higher levels of triglycerides and reduced values of high-density lipoprotein was more predominant in diabetic subjects compared to healthy subjects. The frequency of risk genotype (TT) frequency was significantly higher in T2DM subjects (16.4%) compared to controls (11.6%). The “T” allele was more dominant in diabetic subjects than controls. Logistic regression analysis of the data revealed a significant association of TT genotype with 2-fold (odds ratio with 95% of confidence interval; 2.09 [1.29–3.42] p=0.003) and CT genotype with 1.7-fold (1.73 [1.23–2.44] p=0.002) increased risk of developing T2DM. Conclusions: The present study demonstrated a significant association of rs7903146 (C/T) variant in TCF7L2 with the augmented risk of T2DM in North Indian population.


INTRODUCTION
Type 2 diabetes mellitus (T2DM) is one of the most challenging problems of the 20 th century. T2DM is characterized by hyperglycemia, impaired insulin secretion or insulin resistance, is the most common type of diabetes accounting 90-95% of total diabetic cases. The prevalence of T2DM is rising rapidly across the globe posing significant socioeconomic burden in the form of lost productivity and stress on health-care system, both in developed and developing countries [1]. At present, T2DM affects 285 million people all over the world and is predicted to rise to 642 million by 2040 globally [2]. At present, India has 85 million individuals suffering from T2DM; however, this number is expected to rise to 109 million by 2035 [3]. India has become diabetic capital of the world. About 80% of the diabetic population lives in low-and middle-income countries. T2DM is a very complex metabolic disease in which both genetic and environmental factors play an important role in their pathophysiology [4]. The recent advancement in technology had made it possible to identify new genetic loci as well as genes associated with the risk of the development of T2DM. These genetic determinants can be used to better understanding of the pathogenesis of a disease, help in the development of policies to counter the economic burden, and provide a new way for improved and preventive therapeutic measures. Transcription factor 7-like 2 (TCF7L2) gene is located on the long arm of chromosome 10q25.3 and involved in Wnt signaling pathway which plays a pivotal role in cell development and growth regulation [5]. The previous studies established TCF7L2 gene as possible determinants of type 2 diabetes [6,7]. Although several single-nucleotide polymorphisms (SNPs) in TCF7L2 gene have been replicated in different population and ethnicities, few studies have been done in Indian population. Furthermore, genetic association of TCF7L2 gene has not yet been explored in North Indian population. Hence, the present study was planned to investigate the association of rs7903146 (C/T) variant in TCF7L2 with the risk of T2DM in North Indian population.

Study population
The present study included 638 participants (318 T2DM patients and 320 healthy controls) recruited from North Indian population. The diagnosis of T2DM was done using criteria established by the American Diabetes Association as follows: A medical record indicating either a fasting glucose levels >7.0 mmol/l or >126 mg/dl after a minimum 12-h fast or 2-h post-glucose level (oral glucose tolerance test or 2-h) >11.1 mmol/l or >200 mg/dl on more than one occasion with symptoms of diabetes. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This study was ethically approved by the Institutional Ethics Committees of Post Graduate Institute of Medical Education

Kaur et al.
and Research, Chandigarh and Panjab University, Chandigarh, India. A model consent form adhering to Indian, and International guidelines regarding the use of human subjects were used along with detailed questionnaire of details regarding demographic and socioeconomic characteristics. The participant's self-reported age, sex, educational status, physical activity, dietary habits, family history of the disease and individual's smoking and alcohol use, etc., were recorded.

Anthropometric measurements
Standard anthropometric measurements were performed including stature, weight, and waist and hip circumferences. Body mass index (BMI) was calculated according to Quetelet equation (BMI = weight in kilograms/height in meters squared). Waist-to-hip ratio (WHR) was calculated as ratio of abdomen to hip circumferences. Blood pressure (BP) was measured using Omron's BP machine in a sitting position, from the left arm resting on the table, with legs uncrossed and feet flat.
The abdominal obesity was measured according to the new cutoffs proposed for South Asian Indians as mentioned in our previous study [8], i.e., WHR >0.89 for men and >0.81 for women. BMI <23 kg/m 2 has been proposed for low risk, 23-27.5 kg/m 2 for increased risk, and ≥27.5 kg/m 2 for high risk for developing weight-related diseases in Asian populations.

Clinical parameters
Venous blood samples were extracted from each subject after 12 h of fasting. A serum sample was analyzed for fasting serum glucose, creatinine, and lipid profile (triglycerides [TG], total cholesterol [TC], high-density lipoprotein cholesterol [HDL-C], and low-density lipoprotein cholesterol [LDL-C]). Fasting and random blood glucose levels were measured using a portable glucometer (Abbott Optium Xceed, USA).

Genotyping of TCF7L2 gene
The genomic DNA was isolated from the blood using salting out method [9]. DNA yield was measured by absorbance at 260 nm and purity was checked by calculating A 260 /A 280 . Amplification of rs7903146 polymorphism in TCF7L2 gene was carried out using allele-specific polymerase chain reaction (PCR) as described in an earlier study [10]. Genotyping was based on differential amplification due to the presence of mismatches. In rs7903146 genotyping, two forward primers with a mismatch in their last 3' nucleotide such a way that each is specific for one of the two variants of the polymorphism and a common reverse primer was used. The forward primers also contain a second mismatch at the third nucleotide from the 3′ end to enhance PCR specificity. The forward primers (rs7903146 C or rs7903146 T) specific for allele C detection: 5`GAACAATTAGAGA GCTAAGCACTTTTTAGAAAC 3` and forward primer for allele T detection: 5`GAACAATTAGAGAGCTAAGCACTTTTTAGAGAT 3`, and common reverse primer (rs7903146 R) 5` AGATGAAATGTAGCAGTGAAGTGC 3` were combined in two parallel PCR reactions one with primers rs7903146 C and rs7903146 R (PCR C) and a second with primers rs7903146 T and rs7903146 R (PCR T). For each sample, two PCR reactions were run in parallel, one with primers rs7903146 C and rs7903146 R (PCR C) and a second with primers rs7903146 T and rs7903146 R (PCR T) each containing 200 ng genomic DNA, 1X Taq polymerase buffer, 1.5 mM MgCl 2 , 10 pmol of each primer, 200 μmol/l dNTPs, and 1U of Taq DNA polymerase (Thermo) in a final volume of 25 μl. DNA amplification was carried out on thermal cycler (Eppendorf Mastercycler Nexus Gradient) with an initial denaturation for 3 min at 94°C, followed by 32 cycles of 1 min at 94°C, 1 min at 50°C, and 1 min at 72°C and final extension for 5 min at 72°C. Fig. 1 shows the amplified PCR products separated on 1.5% agarose gel electrophoresis.

Statistical analysis
Results were expressed as mean ± standard deviation. Chi-square analysis was applied to test the significance of differences in genotypic and allelic frequencies. Group comparisons were done using unpaired t-tests. p<0.05 (two tailed) was considered as statistically significant difference. Logistic regression analyses were performed to calculate odds ratio (OR) and 95% confidence intervals (CIs) for each risk factor. Statistical analysis was performed using IBM-SPSS for Windows, version 20 (SPSS, Inc., Chicago, IL).  32.7±9.1). Although TC levels fall under the limit of borderline, there is no significant difference between diabetic and non-diabetic controls. Significantly higher values of TG and reduced values of HDL were reported in diabetic patients than controls. Along with abdominal obesity evidenced by higher BMI and WHR, 12% of patients were having dyslipidemia and were on lipid-lowering drugs. Alterations in clinical and anthropometric measurements pretend a risk for the development of cardiac diseases as demonstrated by higher TC/HDL, LDL/HDL, and TG/HDL ratio in diabetic subjects compared to healthy controls ( Table 1).

Association of TCF7L2 gene polymorphisms with T2DM
The distribution of genotype and allelic frequencies for rs7903146 polymorphisms of TCF7L2 gene are shown in Table 2. TCF7L2 rs7903146 gene polymorphism analysis demonstrated that the frequency of risk genotype TT genotype was significantly higher in diabetics than in controls (16.4% vs. 11.6%). The frequency of the "T" allele was significantly higher in diabetic subjects (42%) compared with that in the healthy control subjects (33%). Furthermore, CT genotype frequency was more predominant in T2DM subjects (51.3%) than control subjects (42.2%  Table 3 summarizes the comparison of clinical and biochemical characteristics of T2DM and control subjects according to different genotypes of TCF7L2 rs7903146. There were no significant differences in metabolic characteristics such as glucose, BMI, WC, WHR, body fat (%), systolic BP, diastolic BP, TC, TG, HDL, LDL, very LDL, and creatinine among T2DM as well as control subjects carrying CC, CT, and TT genotypes of rs7903146 polymorphisms in TCF7L2 gene.

DISCUSSION
India is currently experiencing an epidemic of DM. Both environmental and genetic factors contribute to the development of insulin resistance and type 2 diabetes [11,12]. It is evident from the previous studies that several genetic determinants are associated with increased risk of type 2 diabetes, but their conclusive role is still unclear [13]. TCF7L2 gene is considered one of the most important candidate genes for T2DM, playing a key role in blood glucose homeostasis and beta-cell function [14]. TCFL2 encodes a basic helix-loop-helix TCF-4, which acts as a nuclear receptor for the Wnt/β-catenin pathway [15] and can preferentially bind to Wnt-responsive elements in genes induced by β-catenin [16]. It is well known that the β-catenin/TCF-4 complex participates in various biological events. Particularly, the complex has been found to have an important role in pancreas islet cell proliferation and differentiation and thus contributes to T2DM initiation and progression.
Following the initial report by Grant et al. [17] showing that TCF7L2 variants were strongly associated with T2DM risk, several other studies consistently replicated this association in different ethnicities [6]. The present case-control study established the association of 2-fold increased diabetes risk with TT homozygous, while CT heterozygous carried 1.7-fold increase in T2DM risk when compared with CC homozygous of rs7903146 polymorphisms in the TCF7L2 gene. Bodhini et al. observed a significant association between the T allele of rs7903146(C/T) SNPs and T2DM in South Indians [18]. Chandak et al. also observed a strong association of rs7903146 polymorphism with T2DM (OR=1.46) [19]. A previous study revealed a positive significant association between TT genotype of rs7903146 (C/T) variant of TCF7L2 gene and diabetes-related complications in Indian population [20]. Several studies conducted in other parts of the world demonstrated significant relationship between TCF7L2 gene and T2DM in British [21], the US [22], Finnish [23], Amish [24], Scandinavian [25], Polish [25], French [26], Dutch Breda [27], European Whites, migrant Asian Indian, Afro-Caribbean [28], Northern Swedish [29], and Japanese populations [30].
A large meta-analysis study confirmed the association of TCF7L2 gene with T2DM in different ethnicities [31]. Our results are similar with the results from the overall meta-analysis of the rs7903146 polymorphism wherein heterozygous genotype CT carried over a 1.4-fold increased risk for T2DM, while TT homozygous carried near a 2.0-fold increase in T2DM risk when compared with CC homozygous. Another metaanalysis study examining 66 studies also confirmed the association of the rs7903146 SNPs with T2DM in 66 studies (OR = 1.41, 95% CI 1.37-1.46 for the T allele) [32]. A similar meta-analysis study in Chinese population also reflected the role of rs7903146(C/T) SNP towards  [19]. Also, no significant difference in respect to metabolic parameters between different genotypes of rs7903146(C/T was observed [36]. Contrary, many studies show the association of TCF7L2 gene polymorphism with many metabolic traits [6,17,[35][36][37][38]. Cauchi et al. found that the T allele of rs7903146 predicts hyperglycemia in French population [26]. Saxena et al. found that individuals homozygous for the rs7903146 risk allele had a significant reduction in insulinogenic index and insulin disposition index [25]. Damcott et al. found significant association with insulin sensitivity index, acute insulin response to glucose in non-Amish Caucasian subjects [24]. Our results showed that the rs7903146 T allele of the TCF7L2 gene was positively correlated with an enhanced risk of T2DM in codominant and dominant models of inheritance.

CONCLUSIONS
The present study confirms the association of rs7903146 (C/T) polymorphism in TCF7L2 gene with risk of T2DM in North Indian population. However, no significant influence of this polymorphism was observed with cardiometabolic traits.