Snp genotyping within ins, insr, tcf7l2 and ppr- y genes associated with type 2 diabetes in india

Alireza Shahrjerdi,1,* Mohsen najafi,2

1. Razi vaccin &Serum Research Institute, Agricultural Research, Educatuion and Extension (AREEO), Tehran, Iran
2. School of Medicine, Faculty of Medical Sciences, IAU, Sari, Iran

Abstract

Introduction

A pilot population-based study was carried out to evaluate linkage between singlenucleotide polymorphisms within the known risk candidate genes of type 2 diabetes in western indian population. the study population was comprised of 25 normal glucosetolerant individuals (12 males and 13 females) and 25 type 2 diabetes patients (13 males and 12 females). the genes and their corresponding single-nucleotide polymorphisms that we screened were ins (rs5505), insr (rs10500204), tcf7l2 (rs7903146) and ppar-g (rs1801282). the risk alleles were more frequent in the subjects with type 2 diabetes. copyright ª 2013, jpr solutions; published by reed elsevier india pvt. ltd. all rights reserved.

Methods

2.1. human subjects the study subjects were a part of an ongoing insulin resistance study being undertaken by department of life sciences, university of mumbai in association with medical genetics study centre, geneombio technologies, india. the study was directed towards a sub-population living in the state of maharashtra (india) and suffering from t2d. blood, serum and dna samples of 25 t2d cases (13 males and 12 females) and 25 normal glucose tolerant (ngt) (12 males, 13 females) individuals were studied. all blood samples were obtained at the baseline visit and all participants provided a written informed consent for investigations. the recruited members of the resource population were above the age of 25 years with an average of (mean  sd) 44.6  10.42 and 49.6  12.5 years for control and t2d group respectively. the diagnosis of t2d was confirmed by analyzing medical records for symptoms, use of medication, and measuring of fasting glucose levels following the guidelines of american diabetes association (diabetes care, december 29, 2009; january 2010, supplement). primary inclusion criteria comprised a medical record indicating either 1) a fasting plasma glucose level of 126 mg/ dl or 7.0mmafter aminimum of 12 h fasting or 2) a 2-h postglucose level [2-h oral glucose tolerance test (ogtt)] of 200 mg/dl or 11.1 mm on more than one occasion with symptoms of diabetes. impaired glucose tolerance was defined as a fasting plasma glucose level of 100 mg/dl (5.6 mm) but 126 mg/dl (7.0 mm) or a 2-h ogtt of 140 mg/dl (7.8 mm) but 200 mg/dl (11.1 mm). in cases where a medical report was not readily available, self-reported t2d cases were confirmed by performing a 2-h ogtt. the 2-h ogtts were performed according to the who criteria (75 g oral load of glucose). body mass index (bmi) was computed as weight (kg)/height (meter) while waist-tohip ratio (whr) was calculated as the ratio of abdomen or waist circumference to hip circumference. details of the ngt and t2d population mentioned in tables 1 and 2. the ngt subjects that participated in this study were from the same subpopulation group from maharashtra. all protocols were reviewed and approved by the project authorities at geneombio technologies in pune and a memorandum of understanding and material transfer agreements for sample sharing were signed between the two collaborating institutes. 2.2. metabolic assays quantification of hba1c was done from whole blood. hba1c levels were determined by turbidometric inhibition immunoassay (tina quant; roche). the homeostatic model assessment (homa) was used to quantify insulin resistance and beta-cell function. homa-ir value of t2d population was 4.6  0.75 as compared to control group 2.7  0.44. the homa-b mean value in control and diabetic population was 196.6  180.17 and 28.7  7.15 respectively. thus indicating insulin resistance and reduction in beta-cell function in t2d population. 2.3. snp genotyping dna was extracted from blood cells using standardized sdsephenol/chloroform method described by sambrook et al (1989).7 genotyping of samples for single-nucleotide polymorphisms (snps) within ins (rs5505), insr (rs10500204), tcf7l2 (rs7903146) and ppar-g (rs1801282) were done according to halsall et al (2004), bennermo et al (2004), marquezine et al (2008), and romeo et al (2001) respectively.8e11 for quality control, 2 replicates of positive controls and 1 replicate of negative controls were included in each pcr run to match the concordance. the discrepancy in the concordance was <0.01%. genotyping success ratewas 100%for all the investigated snps. statistical analysis the hardyeweinberg equilibrium was used with a one-degree of freedom goodness-of-fit test separately among cases and controls with the help of the pearson chi-square test. allelic frequencies between test and control samples were done using the chi-square test or the fisher exact probability test, wherever appropriate. unconditional logistic regression was used, before and after adjusting for gender, age and other variants for statistical analysis of genetic effects measured by the odds ratio (or) and its corresponding 95% confidence limits. association analyzes were performed for each polymorphism using the ‘snpassoc’ software.

Results

All samples, including those with t2d (n ¼ 25) and normal glucose tolerant (n ¼ 25), were genotyped for 4 snp within 4 genes of interest. a total of 4 genes and 4 snps were identified for genotyping analysis within each of the samples from the resource population. the details of the gene name, snp identification number (reference snp or the ‘rs’ number), position of the snp on the chromosome as indicated by genome build version 37.1 (the fasta sequence of the human chromosomes; build 37; national council of biological information, usa) and frequency of occurrence of each of the snps in the resource population are summarized in tables 3 and 4. the genes and their snps indicate strong association with conditions of t2d. ins: rs5505 with risk allele ‘t’ was observed in the present study population. the risk allele ‘t’ was found 58% in t2d cases (or ¼ 1.52) compared to 38% in the control group thus showing a strong link with decreased insulin level. among the t2d group, 13 cases had the risk allele ‘t’ as compared to only 5 cases in control group. same risk allele ‘t’ was also reported by boesgaard et al (2010) in danish and czech populations.13 the insulin gene variable number tandem repeat (insevntr) has been extensively studied and is proposed to exert pleiotropic effects on birth weight and diabetes susceptibility.14 however, evidence for this has been conflicting and a role for ins in type 2 diabetes predisposition has not been definitively established.

Conclusion

The present study provided insight into the association of snps linked tot2d. the above findings suggest that there is a co-relation between the risk alleles and susceptibility to t2d in the present pilot study population. the data raises the prospects of developing an snp-based genetic prediction test for detecting genetic predisposition towards this important lifestyle disease and aid to design better management ideas to defer or prevent the onset of t2d.

Keywords

Snp ins insr tcf7l2 ppar-y