Assessment of FTO Gene Polymorphism and its Association with Type 2 Diabetes Mellitus in North Indian Populations
Kahkashan Naaz1 • Anil Kumar2 • Ipsita Choudhury3
Abstract FTO gene polymorphism related to type 2 dia- betes and obesity was studied in this north Indian popula- tion. This study was done, due to a continuous increase in the risk of obesity and type 2 diabetes in north Indian population, because of lifestyle and genetic variations. Clinically diagnosed subjects of type 2 diabetes mellitus (as per ADA criteria) were taken as cases and age and sex matched subjects without any associated illness were taken as controls. Obesity was estimated by calculating waist circumference and BMI in the study cases and controls. For genetic variation, DNA was isolated with Quaigen kit method and isolated DNA was amplified with PCR. Amplified DNA was resolved in 1% agarose gel elec- trophoresis. The Hardy–Weinberg equilibrium, OR, CI and P value were calculated using standard protocols. FTO gene polymorphism (SNP 9940128) was found to be sig- nificantly correlated with type 2 diabetes mellitus and obesity. The AG genotype frequency was observed to be higher (13.09%) with (P \ 0.0001) in the cases as com- pared to controls. Logistic regression analysis was con- ducted for AG and GG genotypes with respect to AA. In this novel study genetic co-relation was observed between FTO gene polymorphisms and type 2 diabetes and obesity in the north Indian population.
Introduction
Obesity is a common disease occurring in humans at any stage of life but is more frequent at middle age. Obesity is the forerunner for many cardiovascular diseases like hypertension and more commonly diabetes. This disease is increasing globally with its prevalence and common in developing countries [1]. In the report of International Diabetes Federation (IDF), there were 382 million people suffering with diabetes worldwide in 2013, and this number is estimated to augment to 592 million by 2035 [2]. Most of the obese and diabetic population are suffering due to rapid changes in lifestyle, environmental factors along with genetic factors. The standard global prevalence of obesity and overweight is defined by a BMI as 30 (Body Mass Index) and 25 kg/m2, respectively which act as the main risk factors for the development of type 2 diabetes [3].
FTO associated protein also known as alpha-ketoglu- tarate-dependent dioxygenase, is an enzyme encoded by the FTO gene and is located on chromosome 16 belonging to the AlkB family of proteins. Several variants of the FTO gene are known which has correlation with obesity. In many studies, it has been reported that polymorphisms within the FTO gene are strongly concerned to obesity. FTO gene is one of the best examples of diabetogenic gene mediating its effect through obesity. FTO was first identi- fied as susceptibility gene for obesity and later body mass index (BMI) dependent association with type 2 diabetes was revealed in European population [4].
Genome-wide association studies (GWAS) have identi- fied 32 loci which are concerned for body mass index (BMI) [5]. Till date, only some variants have been effec- tively replicated in the populations of different ethnicities. The most familiar genetic variant (rs9939609) in the first intron of FTO locus has acknowledged strong connection with obese phenotype in individuals of European popula- tion [6–8], however, these results have been found changeable amongst Asians, (Indians). The accurate role of FTO genes relating obesity still remains blurred while several studies have demonstrated that FTO might have a prospective role in modifiable energy balance, appetite and adipocyte lipolysis. In the reports on FTO gene polymor- phism among Asian Indians [9], confirmation with type 2 diabetes mellitus has been found independent of BMI. Later studies have showed that larger data sets were either incapable to replicate the FTO-type 2 diabetes mellitus risk [10–12]. Population-based studies mainly as the Indian migration study [13], the Indian health study [14] have given confirmation related to FTO variants with obesity. Indians have been known as thin-fat phenotype and because of their unique higher body fat and lower incline mass content at any given BMI. Due to differences of their body fat distribution, Indians are being more centrally obese as compared to other ethnic groups [15]. In several genome-wide studies it has been exposed that single nucleotide polymorphisms (SNPs) of the FTO gene are strongly connected factors for obesity and diabetes
Materials and Methods
This study was conducted at Rama Medical College and Research Centre, Kanpur (India), a tertiary health care centre in North India. All the reagents and chemicals were purchased from standard companies such as Sigma Aldrich, Fermentas and Quaigen. For this study, total 220 samples (110 cases and 110 controls) were taken at sterile conditions. Before taking the samples a written consent form was obtained. While collecting the samples a standard questionnaire form was filled from each patients and con- trols. This study was conducted and entire protocol was approved by the Ethics Committee of the Rama University. Clinically diagnosed cases of type 2 Diabetes Mellitus (as per ADA criteria) were taken as cases as compared to age and sex matched controls without any associated illness. Patients who were not willing to participate for this study were excluded from the study. Sample size was calculated with assuming a minimum power up to 80% and 95% significance level using this formula: 2(p)(1 — p)(Zb + Za/2) [16–22] and obesity is the fore most risk factor for type 2 n = diabetes [23]. (p1 — p2)2.
Recently, it is estimated that common variants of FTO gene in East Asian population has their association with obesity and type 2 diabetes [24]. Though, involvement of widespread variants of FTO gene in Indian population is still questionable. In the two studies conducted at Pune and Mysore [25], it has stated that north Indian Sikhs [3] has shown BMI-independent association of rs9939609 with type 2 diabetes, but unsuccessful to replicate its connection with obesity. In other study conducted at south Indian population in Chennai [26] it has demonstrated a BMI- dependent connection of rs8050136 with type 2 diabetes and also with obesity. From the above contradictions, it was the need of the hour to conduct further studies in Indian population.
In the present study, it was tried to estimate a connection of FTO gene polymorphism with obesity and type 2 dia- betes in North Indians population. This study is novel and not reported in literature. This study on FTO gene variation on quantitative traits may improve our understanding about genetical alterations to fat mass and could incline to type 2 diabetes and other obesity-related diseases. Here p—prevalence of the disease (diabetes mellitus and obesity), (p1 – p2)2 or d2 was the difference to detection of specified power and level of confidence. Zb was power of statistical minimised by 80% for which is Zb is 0.84. Za/ 2 was the level of confidence chosen up to 95% confidence Za/2 = 1.96. p indicated the incidence of the clinical con- ditions e.g., type 2 diabetes. The calculated minimum sample size for patients and control was 102 for each group.
For anthropometric measurements height, weight, waist diameter, hip diameter, systolic and diastolic blood pres- sure were considered as per standardized protocols. BMI and waist-to-hip ratio were calculated. Biochemical mea- surements containing levels of fasting plasma glucose, two hours post-load plasma glucose, fasting plasma insulin, total cholesterol (TC), high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, triglyceride, were performed using standard laboratory assays [21]. Single- nucleotide polymorphisms (SNP) from intron 1 rs9940128 which was earlier known to be linked with obesity/type 2 diabetes were chosen. Furthermore, SNP (rs9940128) was taken from the database (NCBI) to confirm its purity.
Fresh blood (2 ml) was collected to isolate DNA by using Quaigen kit with standard protocol. Purity of DNA was checked on 1% agarose gel. Primers were synthesized from Chromous Biotech Pvt. Ltd, Bengaluru. DNA were amplified with PCR (Bio-RAD) and the amplified DNA were separated with 1.2% agarose gel containing ethidium bromide. Gel photographs were documented in gel docu- mentation system (Bio-RAD).
Statistical analyses were conducted using PLINK ver- sion 1.07 and SPSS version 17.0 (SPSS, USA).Statistical power of the study was estimated under log-additive model, assuming 10% population risk by Quanto. Hardy– Weinberg equilibrium for genotypes was checked by v2 analysis. Obesity and type 2 diabetes was calculated by logistic regression analysis assuming log-additive model. A P value of \ 0.01 (a = 0.05/5) was considered significant after Bonferroni correction. Mean and standard deviation were presented in inverse normal units of the parameters in the tables. For quantitative traits association, a P value of \ 0.00053 (a = 0.05/(5 9 19) was estimated to be highly significant. Association with obesity and quantitative traits was performed only in control subjects. Meta-analysis of stage 1 and 2 results were performed by combining sum- mary data of two study population both under fixed and random models. Similarly, the summary data of previous studies on Indians for association with type 2 diabetes [17–19] and this study were combined for meta-analysis. Association of variants with type 2 diabetes, obesity and quantitative traits were also conducted by mixing the data for two study population and adjusting for study popula- tion. Allele frequencies of cases and controls of two study population were compared by equality of proportions Z test. Entire data analyses were adjusted for age, sex and BMI as required. The odds ratio (OR) and 95% confidence interval (CI) were calculated with respect to minor alleles in the study.
Results
The clinical and biochemical data was obtained and repre- sented on the Table 1. The clinical parameters such as BMI, waist circumference, fasting plasma glucose, and systolic and diastolic blood pressures were having significant P value \ 0.0001, which was found significantly greater in cases as compared to controls (Table 2). Involvement of FTO gene polymorphisms with type 2 diabetes (Table 3) was demon- strated by adding the genotype and allele frequencies of the selected FTO SNP (9940128) in the study population. The genotype frequencies of the FTO gene in cases with type 2 diabetes were found by Hardy–Weinberg equilibrium. The rs9940128 A/G gene polymorphism of the FTO gene was recorded with significant involvement with type 2 diabetes, and the AG genotype was noted to be significantly associated with type 2 diabetes and obesity. Logistic regression analysis was conducted for AG and GG genotypes with respect to AA, which was assumed as a reference genotype (Figs. 1, 2, 3). To estimate the effect of the genotypes on the disease, logistic regression analysis was performed. The comparison between the AG and GG genotypes gained an unadjusted OR of 1.69, which was statistically significant (0.021), furthermore significance was retained after adjusting for age, sex, and diabetes (Table 3). When we compared the AA genotype with the GG genotype, the OR remained significant, conferring 1.50 times higher risk towards obesity, even after adjusting for age, sex, and diabetes. Thus genotype and allele frequencies of the rs9940128 A/G polymorphisms were found significantly higher dif- ference between the obese and non-obese patients.
Discussion
Many genome-wide concerned studies have shown a con- nection of the FTO gene variants with obesity and type 2 diabetes in many Caucasian [7, 27, 28] and Asian [25, 29, 30] populations. The selected SNP (rs9940128 A/G) was found in intron 1 of FTO gene. This SNP was chosen because of their positive association with obesity and type 2 diabetes which has been mentioned in various populations [29, 30] The rs9940128 A/G SNP has been reported by Scuteri et al. [7] which demonstrated a sig- nificant association with type 2 diabetes and obesity. In our study the obtained GG genotype indicated two fold higher risk of type 2 diabetes. With the adjustment of age, sex, and BMI the GG genotype frequency was found high. Our results corroborated with the results of Chinese and Malay populations [31]. While, no significant association related with obesity and FTO gene polymorphism was found in this study a significant association of obesity with FTO gene polymorphism has been reported in Japanese popu- lation [29]. Furthermore, several reports have been acknowledged about the significant association of the FTO variants with obesity within intron 1. The first intron of the FTO gene was noted to be highly conserved across species. The flanking region of intron 1 consist of FTO gene variants mainly rs9940128 which was considered in our studied population [32]. To our knowledge, this is the first study to report on FTO gene variants (rs9940128 A/G) in north Indian pop- ulation. In the complex genetic connection, the FTO gene has been showed as a potential role in energy homeostasis, which is the main regulating factor that regulates complex disorders such as type 2 diabetes and obesity. In our sense, such type of studies are needed to know the strength and exact nature of the genetic changes to determine which of the variant within a haplotype cluster could be functionally related to obesity or type 2 diabetes [33]. To conclude, the polymorphisms rs9940128 A/G of the FTO gene was found concerned with type 2 diabetes in our study for north Indian populations. This study also observed the significant connection with obesity in the studied population.
Acknowledgement The Authors are thankful to the principal of Rama Medical College, Dac51 Hospital and Research Centre, Kanpur (India) for the permission to do this original research work at this institute and for the financial support.