Análise do gene do substrato do receptor da insulina 1 (IRS1) em pessoas com diabetes Mellitus tipo 2: identificação de variantes de risco

Abstract

Type 2 Diabetes Mellitus (DM2) is a multifactorial chronic disease characterized by hyperglycemia and due to its high incidence is of great importance for public health. Among the triggering mechanisms, the peripheral insulin resistance (RI) is highly relevant and it is characterized by an insufficient response of peripheral tissues target cells at normal circulating insulin levels, which is related to defects in cellular insulin signaling. The RI determining mechanisms are not fully understood yet, so the studies are based on the molecular structure and the functioning of insulin signaling pathway components. Therefore, the present study aimed to analyze the IRS1 gene, whose product, the IRS-1 substrate, is important for insulin signal transduction in peripheral tissues. The study sought to identify and evaluate risk variants for the triggering of DM2 present in the IRS1 gene. It included patients clinically diagnosed with DM2 and a questionnaire was applied to characterize each sample, obtaining data on socio-environmental aspects and the prevalence of comorbidities, clinical complications, conditions and risk behaviors associated with DM2. The molecular analysis was performed from the amplification and sequencing of the IRS1 gene coding region of the individuals of the sample. For the variant sequences identification, the sequences obtained from the human genomic bank were compared. The identified single nucleotide variant (SNV) was confirmed with the PCR-RFLP technique and evaluated according to family inheritance. For the analysis of the variant additive value, changes in genes associated with the insulin signaling pathway were verified using exome sequencing and variant prediction programs. Molecular modeling tests and phosphorylation were applied to check the impact of the variant on the IRS-1 substrate phosphorylation. The sample had a higher prevalence of females, with a mean age of 61.11 ± 14.573 and a predominant age ranging from 65 to 74 years old. High frequency of arterial hypertension, ophthalmic complications, overweight, obesity and family aggregation were observed. Regarding the behavioral variables, tobacco and alcoholic beverages consumption at some point in life and low practice of regular physical activity were noted. There was a significant statistical association between the variables: diagnosis time and clinical complications, comorbidities and clinical complications, dyslipidemia and more than one clinical complication, body mass index (BMI) and ophthalmic complications and BMI and more than one clinical complication. The screening for mutations in the IRS1 gene, which was confirmed with the PCR-RFLP technique, identified the SNV rs1801278 (c.2911G>A; p.Gly971Arg) in an individual of the sample whose family analysis revealed this variant in four generations, in symptomatic and asymptomatic individuals. The exome analysis of this individual revealed six non-synonymous variants in genes associated with the insulin signaling pathway, however variants prediction programs showed that only rs1801278 is harmful. The analysis of the mutant IRS-1 substrate (Arg971) revealed low interaction with PI3K in the molecular modeling and increased phosphorylation in serine in the in silica and in vitro assay. The results presented in this research confirm the risk association of the rs1801278 variant with insulin resistance and DM2. This research also shows the relevance of this variant in studies targeted to personalized medicine and to genetic counseling for DM2. Keywords: insulin resistance; IRS1 gene; IRS-1 substrate; rs1801278; serine phosphorylation.