Study of TCF7L2 Gene Polymorphism (Rs7903146) in Type Two Diabetes Mellitus of Iraqi Society

Journal of Babylon University/Pure and Applied Sciences/ No.(4)/ Vol.(23): 2015

Study of TCF7L2 Gene Polymorphism (rs7903146) in Type Two Diabetes Mellitus of Iraqi Society.

Ahmed Naseer Kaftan

Department of biochemistry, College of medicine, Kufa university

Abstract

Background: In Iraq, type 2 diabetes mellitus (T2DM) is increasing and accounts for a high proportion of medical costs. Recent genetic studies have shown polymorphism within the transcription factor 7-like 2 (TCF7L2) genes like rs7903146 were associated with type 2 diabetes (T2DM) and these polymorphisms can cause variations in body mass index(BMI) , lipid profile and insulin sensitivity.

Objectives: To study the association TCF7L2 gene (rs7903146) polymorphism with T2DM in Iraqi society.

Methodology: The study included 100 patients with T2DM referring to clinic and 80 healthy controls randomly selected based on WHO guidelines.BMI, FBS, lipid profile and insulin plasma levels were measured, DNA was extracted from blood and genotyped by PCR–RFLP with specific primers to amplify a fragment for digestion with restriction enzyme (RsaI). Multinomial logistic regression was applied to compare the proportions of genotypes or alleles. The odds ratio for risk of developing T2DM was calculated with and without adjustment for age, sex, and BMI.

Results: homozygous (TT) andheterozygous genotypes (CT) had failed to significantly increase the risk of T2DM when compared to the wild type (CC). In the same way genotypes of rs7903146 polymorphism exhibit no significant impact on BMI, lipid profile and insulin sensitivity.

Conclusion: TCF7L2 gene polymorphism (rs7903146) was not a risk factor for the development of T2DM in Iraqi society.

Recommendation: Includingof a large sample size, analyzing of more SNPs ofTCF7L2 and evaluating gene expression in the future studies.

Keywords: TCF7L2 rs7903146 Polymorphism RFLP diabetes Iraq

الخلاصة

المقدمة: في العراق هناك اعداد متزايدة من الإصابة بمرض السكري حيث تسبب نسب عالية من الخسائر، اثبتت الدراسات الوراثية الحديثة ان الطفرة الوراثية فيموروثة TCF7L2 مثل (7903146) متعلقة بمرض السكري. كما ان هذه الطفرات توثر علىمؤشر كتلة الجسم , مستوى الدهون و حساسية الانسولين.

الهدف: لدراسة علاقة التباين الجيني لموروثة TCF7L2(7903146) مع الداء السكري من النوع الثاني في المجتمع العراقي

المنهجية: شملت الدراسة 100 مريض بداء السكري و80 من الاصحاء تم اختيارهم عشوائيا استنادا الى المبادئ التوجيهية لمنظمة الصحة العالمية. تم قياس مؤشر كتلة الجسم، مستوى السكر، مستوى الدهون ومستوى الانسولين لكل شخص. الحامض النووي تم استخلاصه من الدم ثم تنميطه وراثيا باستخدام تقنية (RFLP-PCR) باستخدام ممهدات محددة لتضخيم جزء من الموروثة ثم تقطيعها باستخدام الانزيم القاطع (RsaI). تم استخدام الانحدار اللوجستي المتعدد لأجل مقارنة الأنماط الجينية وترددات الأنماط الوراثية. تم حساب نسبة الارجحية لخطر الإصابة بالسكري مع وبدون التعديل بالنسبة للعمر والجنس ومؤشر كتلة الجسم.

النتائج: تشير النتائج ان الأنماط الجينية متماثلة ومختلفة اللواقح قد فشلت في زيادة خطر الإصابة بالسكري بشكل مؤثر عند مقارنتها بالنوع البري. وفي نفس الاتجاه لم تظهر هذه الأنماط الوراثية تأثير مهم على مؤشر كتلة الجسم، مستوى الدهون وحساسية الانسولين.

الاستنتاجات: التباين الجيني لموروثة TCF7L2 (7903146) ليس عامل خطورة للإصابة بالسكري من النوع الثاني في المجتمع العراقي.

التوصيات:استخدام عينة أكبر، تحليل عدد أكبر من الطفرات الوراثية لهذه الموروثة وتقييم التعبير الوراثي.

الكلمات المفتاحية: الطفرة الوراثية لموروثةCF7L2 rs7903146 ، تقنية RFLP ، الداء السكري في العراق.

Introductin

One of the most challenging health problems of the twenty- first century was diabetes. Type 2 diabetes mellitus (T2DM), included 90–95% of those with diabetes,was ranging from predominantly insulin resistance with relative insulin deficiency to predominantly an insulin secretory defect with insulin resistance [IDF,2013]. 317 million people worldwide now affected with diabetes, but its prevalence was increasing rapidly because of increasing age of the society and surge of obesity in many countries [Shaw, 2009] including Iraq. Comprehensive survey result pointed out that the total prevalence of T2DM was 10.9% in Iraqi society. T2DM was a complex metabolic disease that results from the combination of genetic and environmental factors[Hansen, 2005]. The identification of causative genes that promotes susceptibility to T2DM could provide a better understanding of the molecular mechanisms underlying pathogenesis and may provide new information for treatment and prevention [Rathmann, 2000].Transcription factor 7-like 2 (T-cell specific, HMG-box) also known as TCF7L2 or TCF4 was a protein acting as a transcription factor, in humans, this protein was encoded by the TCF7L2 gene [Castrop, 1992].It was a member of the Want signaling pathway which was one of the key developmental and growth regulatory mechanism of the cell, encodes an enteroendocrine transcription factor that controls the transcription of the proglucagon gene that encodes both glucagon and glucagon like peptide-1 (GLP-1). It has been suggested that polymorphisms in TCF7L2 gene may affect the susceptibility to T2DM by indirectly altering GLP-1 levels [Yi, 2005] .In previous studies, TCF7L2 has shown the strongest known association with T2DM[Vaquero, 2012, Grant, 2006] . TCF7L2 wasexpressed in intestinal endocrine L cells, it enhance the expression of the proglucagon gene that leads to the production of the incretin hormone GLP-1[Shu, 2008].TCF7L2was also expressed in pancreatic β cells and it has been found by previous studies that TCF7L2 depletion resulted in a 5.1-fold increase in β-cell apoptosis, 2.2-fold decrease in β -cell proliferation, and 2.6-fold decrease in glucose-stimulated insulin secretion. In contrast, TCF7L2 overexpression protects islets from glucose- and cytokine-mediated apoptosis of pancreatic β –cells[Shu, 2008]. Mechanisms whereby altered TCF7L2 production or function may cause T2DM include:

•Decreased β-cell mass, decreased β-cell proliferation following TCF7L2 knockdown [Shu, 2008], decreased ‘maximal’ insulin secretion response to arginine at 28 mmol/l glucose[Pilgaard, 2009].

•Impaired insulin processing or release. Altered gene expression in mice with TCF7L2 knockdown [Xavier, 2009] , increased proinsulin.insulin ratio in rs7903146 T allele carriers [Schafer, 2007,Loos, 2007], decreased ‘maximal’ insulin secretion response to arginine at 28 mmol/l glucose [Nusse, 1992]

•Impaired GLP1 signaling in β -cells. Response to GLP1 impaired to greater extent than that to i.v. glucose [Schafer, 2007].

•Hepatic insulin resistance [Schafer, 2007].

There was a controversy about the direct relationship between TCF7L2 expression and glucose intolerance. While some rodent studies have demonstrated that reducing TCF7L2 expression results in diabetes others have found the opposite, in human islets, impaired glucose-stimulated insulin secretion has been reported following both TCF7L2 suppression and overexpression.So more complex, ‘‘humanized’’ models will be required to explore this [Mark, 2013].According to NCBI there was 5540 SNPs that map within the TCF7L2 locus in human, 78 of these SNPs were cited in PubMed [Sayers, 2008],two intronic single nucleotide polymorphisms (SNPs) within the transcription factor 7-like 2 (TCF7L2) gene rs7903146 and rs12255372, were found to substantially contribute to the risk of T2DM [Wilfred, 2012]. In SNP, rs7903146 C nucleotide was replaced by T allele [Sayers, 2008].

Objectives

1. To study the association TCF7L2 gene (rs7903146) with T2DM in Iraqi society.

2. To study the impact of this polymorphisms on BMI, lipid profile and insulin sensitivity.

Methods

Study subjects:

A case–control study of 180 subjects (100 T2DM and 80 control) was conducted to assess the association of SNP rs7903146 of TCF7L2 gene with T2DM in Iraqi society.

Patients:

The patient society included 100 subjects (41men and 59 women) with T2DM who attended the diabetes center in Al-Sader medical city, Najaf, Iraq from January 2014 to March 2014.

Inclusion criteria:

1. Those patients who were diagnosed by physicians as having type 2 diabetes, the criteria to diagnose diabetes were based on WHO guidelines.

2. A subject was said to have diabetes if his/her fasting glucose level was >126 mg/dl (7.0 mmol/l) +symptoms of diabetes.

3. Age of patients was >40 y.

Exclusion criteria:

1. those who were diagnosed with T1DM

2. those whowere taking insulin

Control group:

The control group includs 80 healthy subjects (36 men and 44 women). They were randomly selected from the people who attend the hospital for checkup also from relatives and colleagues.

The inclusion criteria:

1. Fasting plasma glucose<100 mg/dl.

2. No past medical history of type 2 diabetes.

3. No family history of diabetes in first-degree relatives

4. Matched to patients with regard to gender, age and geographical distribution.

5. Age at examination > 40 y

6. BMI < 30 kg/m2

7. TC < 200 mg/dl8. TG < 150 mg/dl

All cases answer a detailed questionnaire that includs information about age, sex, family history, drug history, medical history and other relevant information, for all subjects’ weight, height and BMI had measured. It should be noted that Najaf was one of the big cities of Iraq, and there was no much difference in genotyping distribution from one city to another, therefore our study society could represent the Iraqi society.Informed consent has been taken from all subjects. Kufa Medical College Ethical Committee has approved the study protocol.

Phenotypes data:

Phenotypes data included: BMI, FBS, fasting insulin and lipid profile.

Genotyping

Peripheral blood samples of T2DM and control groups were collected in EDTA-anticoagulated tubes, and then DNA was extracted from whole-blood samples using the Reliaprep genomic DNA extraction kit (Promega, U.S.A). Then DNA concentration and purity weremeasured by UV absorption at 260 and 280 nm (BioDrop, U.K).

Genotyping was performed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) for TCF7L2gene using thermocycler (Biometra, Germany). The sequence of primers used according to Parra, 2007was: forward 5-TTAGA GAGCTAAGCACTTTTTAGGTA-3 and reverse 5-ACTA AGTTACTTGCCTTCCCTG-3. Amplification was performed in a total volume of 25 μl contained 12.5 μl GoTaq Green Master Mix, (Promega Corporation, Madison, WI), 1.5 μl of each primer (1 Μm final concentration) (OneAlpha, U.S.A), 3.5 μl nuclease free water, and 6 μl of DNA template. Cycling condition was 95°C for 2min followed by 35 cycles of 94°C for 30s, 59.8°C for 30s, 72°C for 30s, and a final extension of 72°C for 4 min. Amplification product of TCF7L2 gene was 108bp bp. The product was digested with 10u of restriction enzyme (RsaI) (Promega) and run on 3% agarose gel. To determine genotyping error rate, we performed random duplication in 20% of the samples.

Statistical analysis

Phenotypes data expressed as mean ±SD and genotypes data expressed as frequencies, student T test and ANOVA test used to compare phenotypes data between control and T2DM groups and across genotypes using SPSS windows software (SPSS Inc., Chicago, IL). Genotype and allele frequencies in T2DM and control group weretested by multinominal logistic regression analysis with and without adjustment for age, sex and (BMI) using SPSS.

Results

Clinical and biochemical characteristics of the study subjectswere presented in Table 1, it exhibited significant differences in BMI, FBS, lipid profile and insulin sensitivity between T2DM and control group.

Table 1: Clinical and biochemical characteristics of study subjects:

Clinical Characteristics / Control Subjects / T2DMSubjects / P value
No (male/female) / 80 (36/44) / 100 (41/59) / 0.59
Age (years) / 55.66±9.13 / 53.57±9.71 / 0.14
BMI (kg/m2) / 22.6 ± 2.20 / 29.94±5.52 / 0.00
FBS (mg/dl) / 84.45±7.976 / 216.09±68.83 / 0.00
Cholesterol (md/dl) / 140.76±29.4 / 254.53±65.10 / 0.00
Triglycerides (mg/dl) / 113.6±26.08 / 248.42±77.1 / 0.00
VLDL (mg/dl) / 22.75 ± 5.25 / 49.68 ±15.42 / 0.00
LDL(mg/dl) / 65.34±29.94 / 153.41±63.67 / 0.00
HDL(mg/dl) / 75 ± 13.15 / 52.2 ± 14.68 / 0.00
Fasting plasma insulin μU/ml / 10.05 ± 4.68 / 28.13 ±19.98 / 0.00
HOMA-IR / 1.71 ± 1.15 / 15.6 ± 13.3 / 0.00

Genotyping:

Results of digestion with restriction enzyme (RsaI) for TCF7L2 gene (rs7903136)(figure 1) demonstrated two (80, 28 bp), one (108 bp) and three (108, 80, 28 bp) bands for those with wild type (CC), homozygous (TT) and heterozygous (CT) genotypes respectively.

Figure 1: Genotyping result for TCF7L2 gene (rs7903136)

DNA Marker: Line 1

CC genotype-80 bp/28 bp: Lines 3, 4, 7, 8, 9, 10, 13 &14

CT genotype-108bp /81 bp/28 bp: Lines 2, 5 & 12

TT genotype108 bp: Lines 6 &11

Genotype and allele frequencies of TCF7L2 gene were shown in table 2; the results demonstrate that TCF7L2 gene polymorphism (rs7903136) failed to significantly increase the risk of T2DM. The frequency of T allele was higher in T2DM subjects, may be overestimated due to small sample size.

Table 2:Genotype and allele frequency of rs7903146 polymorphism of TCF7L2 gene and association of this variant with T2DM in the study subjects.

Control
n=80 / T2DM
n=100 / Unadjusted OR
(95% CI) P value / Adjusted OR for Age ,Sex &BMI
(95% CI) P value
Genotype
rs7903146(C/T)
CC / 75 / 84 / Reference
CT / 4 / 12 / 2.67
(0.82-8.66) 0.1 / 2.97
(0.86-10.25) 0.08
TT / 1 / 4 / 3.57
(0.39-32.66) 0.26 / 2.92
(0.3-28.57) 0.35
Frequency of
T allele / 6
(3.75%) / 20
(10%) / 2.85
(1.11-7.28) 0.02

Clinical characteristics of study subjects according to TCF7L2 gene rs7903146 genotype were shown in table 3.It exhibited failure of rs7903146 to affect any of the measured clinical characteristics.

Table 3: Clinical characteristics of T2DM subjects according to TCF7L2 gene (rs7903136) genotype.

Genotype
Clinical characteristics / CC (n=84) / CT (n=12) / TT (n=4) / P
value
BMI (kg/m2) / 29.69 ± 5.17 / 29.84 ± 5.98 / 31.7 ±11.48 / 0.8
Cholesterol (mg/dl) / 254.25 ± 67.02 / 275.71± 56.68 / 254.02 ± 55.06 / 0.4
Triglycerides (mg/dl) / 243.62 ± 75.99 / 289.74 ± 46.4 / 265.46 ± 67.73 / 0.1
VLDL (mg/dl) / 48.72 ± 15.13 / 57.36 ± 9.45 / 53.37 ± 13.4 / 0.1
LDL(mg/dl) / 154.27 ± 66.72 / 161.13± 44.52 / 148.28 ± 42.53 / 0.8
HDL(mg/dl) / 52.16 ± 14.62 / 52.2 ±16.56 / 55 ± 9.62 / 0.9

Discussion

The identification of genetic variants influencing T2DM was a major focus of research to perceive the mechanisms underlying the pathogenesis of this disorder as well as related pathological consequences. Such attempt may improve the plans of protection, diagnosis and treatment of Iraqi society. Moreover the development of genome-wide association studies (GIS) have enabled the identification of a number of genes associated with T2DM risk. In this study, common genetic variant in TCF7L2 gene (rs7903146) was strongly suggested to be associated with T2DM [Marzi, 2007]. Thus, this SNP was investigated in the current study in Iraqi Arabic type 2 diabetic patients. For our knowledge, this study was the first one deals with the SNP of the TCF7L2 gene. Results of TCF7L2 gene polymorphism (rs7903146) do not reveal significant difference in the distributions among patients and the control group suggesting weak or even no association with T2DM in Iraq patients. These results were consistent with those studies reported previously in Arab society’s; Emirati [Saadi, 2008], Saudi [Alsmadi, 2008] and Tunisian[Kifagi, 2011], (except of the allelic model).They were also in agreement with past observations generated from Chinese [Ren, 2008, Zheng, 2012], Pima Indians [Guo, 2007], Indian [Mahurkar, 2008], Caucasian [Dabelea, 2011], Mexican-American [Parra, 2007]and African[Humphries, 2006]societys). Some studies suggests a significant association of rs7903146 with T2DM in Tunisian [Ezzidi, 2009, Turki, 2013] and Palestinian[Ereqat, 2010]Arab societys, Persian [Abbasali, 2012, Amoli, 2010] , Pakistani [Rees, 2008], Indian [Bodhini, 2007, Chandak, 2007] , Japanese [Hayashi , 2007,Horikoshi, 2007] Brazilians [Marquezine, 2008], and European [Saxena, 2010,Melzer, 2006]societys. Although there wasa clear association of rs7903146 polymorphism with T2DM in previous studies, the current investigation did not reveal such observation. Here, it was beneficial to suggest the possibility of other variants of TCF7L2 gene to be involved in the development of the disease in our society. Till now, it was not well elucidated how this intronic TCF7L2 variant increases susceptibility to the disease. Evidences suggested an important role in maintaining β cell mass and function. Thus, it seems to be critical for β cell proliferation, protection against apoptosis, stimulate insulin secretion [Loder, 2008,Shu, 2008] and involved in the expression of glucagon-like peptide 1[Drucker, 2006]. In addition, it was demonstrated that TCF7L2 expression in the endocrine pancreas of murine models of diabetes was correlated with β cell proliferation and regeneration [Shu, 2012]. Discrepancy of the current findings with those reported previously were possibly due to ethnic variations[Cauchi, 2007] and small sample size[Menashe, 2008], as ethnic and racial variations play an important role in genetic diversity among the societys [Edwards, 2003, Heyer, 2009].Analysis of the effect of TCF7L2 gene polymorphism (rs7903146) on phenotypic data do not reveal significant alterations, so it would be left for further studies.

Conclusion

TCF7L2 gene polymorphism (rs7903146) was not associated with increased risk of T2DM in Iraqi society and may be another SNP of that gene could be a risk factor.

Recommendations

1. Including a large sample size to get a sufficient genetic power

2. Analysing of more SNPs TCF7L2 gene to determine which one was more common in our society.

3. Evaluating gene expression to determine the effect of SNPs on the various phenotypic parameters.

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