INVESTIGATION OF THE SIRT1 GENE SINGLE NUCLEOTIDE POLYMORPHISM (rs7069102) IN OVERWEIGHT PATIENTS WITH TYPE 2 DIABETES MELLITUS OF THE EAST UKRAINIAN POPULATION

Abstract

Introduction. Sirtuin-1, SIRT1, known as the gene of longevity, among other things, protects cells from oxidative stress and promotes DNA stability. Genetic studies show that sirtuins can be involved in the development of obesity in humans, as it has been found that some of their genetic variants are associated with weight gain and metabolic complications. One of the SIRT1 single-nucleotide polymorphisms (SNP), which is actively studied in relation to metabolic disorders in different populations, is the C>G polymorphism in intron 4 (rs7069102). It is determined that it is linked to several mutations in the promoter of the gene and with two coding SNPs in exon 1. It is the different degree of linkage which explains that the results relative to its functional role are significantly different between populations.

The aim of the study was to evaluate the association of polymorphic variants of the sirtuin-1 gene rs7069102 with functional and metabolic characteristics in type 2 diabetes and overweight patients of the East Ukrainian population.

Materials and methods. The study was conducted in accordance with international and domestic ethical and moral and legal requirements and approved by the Institute Medical Ethics Committee. All patients were examined during their stay at the Institute's clinic. A retrospective analysis of clinical and biochemical parameters obtained for patients whose samples are included in the DNA collection of patients with type 2 diabetes mellitus (T2DM). 61 patients with T2DM (f/m 27/34) were selected for analysis (53.35±1.38 years old, duration of diabetes 5.33±0.67 years, glycosylated hemoglobin (Hb_A1c) level 7.74±0.19 %, body mass index 33.28±0.89 kg/m², waist-to-hip ratio 0.99±0.01). Antidiabetic therapy included sulfanilamides, biguanides or their combination. Biochemical and immunoassays were conducted on the basis of the National Institute of Public Health and Environment (Bilthoven, the Netherlands) within the framework of scientific cooperation. Insulin resistance (IR) was characterized by НОМА-IR, НОМА-ІR/adiponectin, adiponectin/leptin, НОМА-ІR/leptin and Adipo-IR. Determination of cytosine replacement with guanine in intron 4 SIRT1 rs7069102 (C>G) was carried out by a polymerase chain reaction with two pairs of opposed primers. Electrophoretic fractionation allowed us to determine the following genotypes by SIRT1 rs7069102 (С>G): СС – 391/277 b.p.; CG – 391/277/167 b.p.; GG – 391/167 b.p. Allele frequencies for the genotype studied were calculated. The normality of distribution of variables was determined by the criterion of Kolmogorov-Smirnov. To compare the indicators characterized by a normal distribution, used the odd two-sided Student’s T-test, to compare the parameters with the abnormal distribution, the Mann-Whitney U-test. The χ² was used to statistically evaluate the differences observed between empirical and theoretical frequencies of the variation series.

Results. Despite a small number of observations, the frequency of alleles in men and women did not differ and there was a noticeable discrepancy between the distribution of genotype frequencies compared to the theoretical distribution by Hardy-Winberg equilibrium. In both men and women, a significant deviation was observed of the actual series of expected frequencies towards the accumulation of heterozygotes (χ²=10,43, Р=0,005; χ²=11,76, Р=0,003, respectively). The analysis of clinical parameters revealed that this SNP did not affect the age of diabetes manifestation, the degree of its compensation, blood pressure and the degree of obesity. At the same time, the indicators that characterize the resistance to insulin associated with adipose tissue (HOMA-IR/adiponectin, HOMA-IR/leptin, adiponectin/leptin) have undergone distinct changes. Despite some trend of greater levels of triglycerides and total cholesterol in the carriers of the CC-genotype, the above-mentioned IR-indices were statistically increased in the carriers of the minor G-allele. At the same time, they revealed a clear gender difference in manifestations of adipose tissue resistance to insulin, which was accompanied by statistically elevated tumor necrosis factor alpha (TNF-α) levels. Namely, men had much worse than women, indexes Adipo-IR (139.59±17.44 vs 85.18±10.48, respectively, Р<0.05) and adiponectin/leptin (0.23±0.06 vs 0.12±0.0, respectively, Р<0.05) against the background of statistically constant parameters of carbohydrate and lipid metabolism, as well as the same degree of obesity, while the high TNF-α levels in the group of G-allele carriers were associated with male gender (4.73±1.30 vs 1.92±0.46 ng/L in women, respectively, 0.05<P<0.1).

Conclusion. The results suggest that the C>G rs7069102 polymorphism is functional in the East Ukrainian population, since there is a shift in the frequencies of genotypes towards the accumulation of heterozygotes, and the alleles themselves are associated with metabolic differences in the studied patients. Against the background of the comparable degree of obesity and compensation of carbohydrate metabolism the G-allele carriers of both genders are characterized by a greater degree of insulin resistance of adipose tissue. Statistically significant intensity of low-grade inflammation has been revealed in men-carriers of G-allele with type 2 diabetes, which requires further research.