TCF7L2 Gene Polymorphisms and Susceptibility to Type 2 Diabetes Mellitus, A Pilot Study

Published by Oriental Scientific Publishing Company © 2018 This is an Open Access article licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-nc-sa/4.0/ ), which permits unrestricted Non Commercial use, distribution and reproduction in any medium, provided the original work is properly cited. TCF7L2 Gene Polymorphisms and Susceptibility to Type 2 Diabetes Mellitus, A Pilot Study

Diabetes mellitus is a major public health problem worldwide and its prevalence has been increasing rapidly in developed and developing countries. 1 This prevalence is suspected to increase by 55% in 2035 to reach 591.9 million of adult's affected. 2 Type 2 diabetes mellitus (T2DM) represents the most common form of diabetes. The most likely explanation for the dramatic increase in T2DM prevalence observed over the past two decades is changing patterns of diet and physical activity. However, it has been assumed that these factors may cause T2DM only in the existence of genetic susceptibility. 3 Genome-wide association studies (GWAS) have identified over 70 loci associated with T2DM including Transcription factor 7-like 2 (TCF7L2) gene. 4 TCF7L2 is located on chromosome 10q25.3. 5 TCF7L2 plays a master role in regulating insulin biosynthesis, secretion, and processing . 6 Moreover, TCF7L2 through the Wnt signaling pathway is essential for proliferation of the pancreatic epithelium and islet proliferation. 7 Recently, TCF7L2 was found to protect pancreatic cells against interleukin-1 and interferon induced cell apoptosis, stimulates cell proliferation and mediates glucose stimulated insulin secretion. 8 rs7903146, rs12255372 variants are among the commonest single nucleotide polymorphisms (SNPs) of TCF7L2 that are connected to T2DM risk. Multiple studies among European, North Indian and African populations have confirmed the association of TCF7L2 variants to T2DM risk. [9][10][11] However, TCF7L2 variants had no association to T2DM in Euro-Brazilian individuals and in Arabic subjects . 12,13 The present study was conducted to analyze the distribution of TCF7L2 rs7930146 and rs12255372 polymorphisms in a group of Egyptian patients with T2DM and healthy controls in an attempt to find an association that might exist between these two polymorphisms and the risk of developing T2DM and their effect on different laboratory parameters.

Subjects
This case-control cross sectional study included 60 patients with T2DM (group 1) attending the Endocrinology and Diabetic Clinic at kasr al Ainy Hospitals, Cairo University and 60 normal unrelated subjects of the same age and sex taken as control (group 2) .The study protocol was approved by Institutional Ethical Committee of Kasr Al Ainy Hospital. All participants provided informed consent to participate in this study. The study protocol and procedures conform to the ethical guidelines of the 1975 declaration of Helsinki.
The patients were diagnosed based on to American Diabetes Association criteria for diabetes. Patients between 40-70 years and receiving oral hypoglycemic drugs were included in the study. Patients with type 1diabetes and diabetes secondary to endocrinopathies, pancreatic diseases or drugs have been excluded.
All patients and control subjects were subjected to thorough medical evaluation including determination of age, gender, blood pressure, anthropometric measures [weight, height and body mass index (BMI)]. Biochemical tests including: fasting blood glucose, fasting insulin, glycosylated hemoglobin (HbA1c), total cholesterol, high density lipoprotein cholesterol (HDL), Low density lipoprotein cholesterol (LDL), triglycerides. Insulin resistance status was determined using the HOMA-IR calculation, from the following equation: fasting insulin (microU/L) x fasting glucose (nmol/L)/22.5. 14 Detection of TCF7L2 polymorphisms (rs 7930146, rs12255372) was done by real time PCR.

Sample collection and biochemical assay
Eight ml of blood were collected from each patient and divided as follows: Two ml of plasma were withdrawn by aseptic venipuncture to a pre-chilled violet top EDTA vacutainer tubes for genomic DNA study, DNA samples were stored at -20°C to be used for TaqMan real time PCR; Two ml of blood were withdrawn to EDTA tube for measuring HbA1C;Two ml of blood were withdrawn to a red-topped serum separator tube, serum was harvested by centrifugation, samples were analyzed for serum insulin and lipid profile; and Two ml of blood was withdrawn into fluoride tube for measuring serum fasting glucose. Clinical chemistry analysis was done on Dimension RxL Max (Siemens,USA), while serum insulin was assayed on Cobas e 411(Rosh diagnostics, Germany).

Isolation of Genomic DNA and SNP genotyping
DNA extraction and analysis was done using Gene JET Whole blood Genomic DNA Purification Mini Kit (Thermo Fisher, USA). The quantity of DNA was assessed on the basis of Qubit dsDNA BR assay kit 10 with the use of Qubit 2.0 fluorometer (Invitrogen, UK).
Genotyping of TCF7L2 polymorphisms (rs 7930146, rs12255372) was determined using RT-PCR allelic discrimination assays that were designed using Taq-Man SNP Genotyping Assays by using Step One Real-Time PCR systems (Applied Biosystems, Foster City, USA).The probes were labeled using the fluorescent dyes VIC and FAM. PCR reactions were run in a 25 UL final volume containing 12.5 UL of TaqMan Universal PCR Master Mix (2x), 1.25 UL of TaqMan SNP Genotyping Assay (20x) and 20 ng of genomic DNA. Cycling conditions were 95°C for 10 min, 50 cycles of 92°C for 10 s and 60°C for 1 min. Controls were included in each run. Data analysis for allele discrimination was performed with the Applied Biosystems Real-Time PCR System software (Applied Biosystems, Foster City, USA).

Statistical analysis
Data were coded and entered using the statistical package SPSS version 23. Data was summarized using mean, standard deviation, median, minimum and maximum for quantitative variables and frequencies (number of cases) and relative frequencies (percentages) for categorical variables. Comparisons between groups were done using unpaired t test when comparing 2 groups and analysis of variance (ANOVA) with multiple comparisons post hoc test when comparing more than 2 groups. For comparing categorical data, Chi square (c2) test was performed. Exact test was used instead when the expected frequency is less than 5. Genotype and allele frequencies were compared between the disease and the control groups using logistic regression. Odds ratio (OR) with 95% confidence intervals was calculated. P-values less than 0.05 were considered as statistically significant.

Characteristics of studied groups
The mean age of the studied patients was (55.83+10.9) years and 57.5% of them were female .The age and gender of participants didn't differ between patients and healthy control. Different clinical and laboratory characteristics of patients and healthy control are shown in table (1). rs7903146 genotypes and alleles in the two studied groups The frequency of rs7903146 genotypes in group 1 was (CT: 48.3%; CC: 31.7%; TT: 20%), while in group 2 was (CT: 36.7%; CC: 20%; TT: 43.3%). The TT genotype was associated with lower risk of diabetes (OR = 0.291, 95% CI = 0.108-0.788, P =0.015).
The frequency G allele was not significantly different between group 1 (60.8%) and group 2 (48.3%).also; T allele frequency didn't differ between both groups and it was not associated with increased diabetes risk (OR = 0.602, 95% CI = 0.361-1.005, P = 0.052).
When we classified the diabetic patients according to their rs12255372 genotypes (GG, GT, TT), there was no significantly difference between the 3 genotypes regarding any of the clinical or laboratory parameters (Table 4).

DISCuSSION
Diabetes is a growing heath problem of multifactorial origin, understanding its genetic background might help in early detection and disease prevention. TCF7L2 genetic variants have been closely linked to T2DM risk, yet this association in the Egyptian population needs further evaluation. [9][10][11] The aim of the current study was to investigate the relationship between TCF7L2 polymorphisms rs 7903146, rs 12255372 and susceptibility to Type 2 diabetes mellitus in a group of Egyptian population and healthy control.
Based on our findings, T allele of TCF7L2 rs7903146 was associated with lower risk of T2DM, and TT genotype frequency was higher in healthy subjects. Different studies yielded controversial results in different ethnic groups. In agreement with the current work; the T allele was protective against diabetes in North Indian population and Cameroon population. 10,11 conversely, in Sudanese and a Turkish population the T allele at rs7903146 was strongly associated with T2DM risk. 15,16 In addition, there was no association between rs 7903146 variant and T2DM in Euro-Brazilian individuals. 17 A recent meta-analysis by Guan et al. revealed that the rs7903146 T allele of the TCF7L2 gene was positively linked with higher T2DM susceptibility. 18 Same results were obtained by another meta-analysis involving 10 studies related to the rs7903146 loci and risk of T2DM; this included 3404 cases of T2DM patients and 6473 control cases. 19 Also, the frequency of TT genotype in our study was significantly lower in diabetic patients compared to healthy control. Verma et al. reported the same finding in subjects from North India and same and results obtained in Aferican population. 10,11 As regards TCF7L2 (rs 12255372), in agreement with the current work, a Venezuelan study found that the genotypic and allelic frequencies for the TCF7L2 rs12255372 polymorphism did not differ significantly between the diabetic patients and healthy subjects. 20 In contrast to our results, T allele of rs12255372 was found to be a risk factor for diabetes in a Cameroonian population and a Chines population. 21,22 While, rs12255372 G allele was the risk allele in Chinese Hui population. 23 A meta-analysis of 40 studies showed an effect of rs12255372 on type 2 diabetes risk (OR: 1.33, 95% CI: 1.27-1.40), Whereas in some ethnic populations including African, American, Pima Indian, Mexican American and Arab no significant association was found. 24 Several studies investigated both SNPs (rs7930146, rs12255372) together in relation to T2DM. A study by Barros et al., in northern Brazil showed that the genotype and allele frequencies of rs7903146 and rs12255372 were not significantly associated with T2DM risk. 25 In contrast, a study by Shokouhi et al. done in a sample of the Kurdish population of Iran found that the T-allele of rs12255372 and rs7903146 polymorphisms of TCF7L2 was risk allele for T2DM. 26 To date, no major GWAS for diabetes has been reported for an Arab population. Replication studies conducted in Arabs have given mixed results. 27 For example, a study of Saudi Arabian and a study for United Arab Emirates population reported weak or no association of rs7903146 and rs12255372 with T2DM, while a study of Arab Tunisian showed association of T-allele of rs7903146 and rs12255372 with T2DM. 13,28,29 Likewise our results, a Brazilian study showed that the T allele was significantly associated with T2DM risk, while no significant difference was found among the three rs7903146 SNP genotypes regarding blood pressure, BMI, lipid profile, and HbA1c. 30 The association of TCF7L2 with T2DM independently of BMI, insulin resistance and other metabolic indices confirms that impaired insulin secretion is a result of genetic defect of TCF7L2. 31 Similar to our findings, In T2DM Iranian population, no significant difference was found in any of the clinical or biochemical parameters between GG and GT + TT genotypes of rs12255372. 26 In contrast to our study, CT + TT genotypes were significantly associated with the lower levels of total cholesterol the CC genotype of rs7903146 in Iranian Kurdish ethnic population. 26 Also, in a group of Italian elderly population, the TCF7L2 rs7903146 polymorphism was associated with lower insulin levels, and lower risk lipid profiles. 32 The reason for these discrepancies between the studies, including our study might be related to different factors such as the study design, sample size, population heterogeneity, and environmental factors. 33

CONCLuSIONS
TCF7L2 was related to T2DM in the studied Egyptian population and both T allele and TT genotype of rs7903146 were associated with lower risk of T2DM. However, no significant association was found to any of rs12255372 genotypes or alleles. Moreover, there was no significant difference in any of metabolic parameters between different genotypes of rs7930146 and rs12255372.