Genetic and microrna polymorphisms in young South African Indians with coronary artery disease.

Abstract

The global burden of cardiovascular disease (CVD) is on the increase with coronary artery disease (CAD) estimated to become the leading cause of mortality worldwide by 2020. The age of onset of this chronic disorder is on the decline, particularly in the South African Indian population. Indians in South Africa (SA) have a higher prevalence of premature CAD compared to other ethnic groups in SA. Coronary artery disease is a lifestyle and genetic disease, and the inheritance of genetic variation from one or both parents plays an important role in the risk of an individual developing CAD. Genetic and epigenetic studies are being explored as potential tools for therapeutic interventions against CVDs. The role of single nucleotide polymorphisms (SNP) in microRNAs (miR, miRNA) and molecules regulating epigenetic pathways remains poorly understood. This study investigated SNPs in candidate genes; methylenetetrahydrofolate reductase (MTHFR), sirtuin (SIRT) 1, miR-499, and miR-146a; in young SA Indians with CAD. The study population included 106 SA Indian male CAD patients, 100 sex- and age-matched Indian, and 84 sex- and age-matched Black controls. The MTHFR, miR-146a, and miR-499 SNPs were investigated by PCR-RFLP, whilst a TaqMan SNP Genotyping assay assessed the SIRT1 SNPs. MiR-146a expression was measured by qPCR and western blot was used to assess the expression of NF-κB, IRAK-1, and TRAF-6. Interleukin (IL)-6 levels were analysed using an ELISA. All clinical parameters were obtained from pathology reports. Methylenetetrahydrofolate reductase is involved in folate metabolism and methylation pathways. The MTHFR rs1801133 has been associated with increased levels of homocysteine, a well known risk factor for CAD. Sirtuin 1, histone deacetylase, has been identified as a candidate molecule affecting the epigenetic mechanisms of CAD. Two common SNPs in the SIRT1 gene, rs7895833 and rs1467568, have been associated with several well-established risk factors for CAD. MicroRNAs (miRNAs) are small noncoding RNA molecules that inhibit messenger RNA (mRNA) translation or promoting mRNA degradation. MiR-499 and miR-146a are inflammatory-associated miRNAs. Two miRNA SNPs, miR-146a rs2910164 and miR-499 rs3746444, have been implicated in chronic inflammatory diseases. There was a significant association between the MTHFR variant (T) allele and CAD patients compared to Indian controls (p=0.0353, OR=2.105 95% CI 1.077–4.114). Indian controls presented with a higher frequency of the T allele compared to Black controls (7% vs. 2% respectively, p=0.0515 OR=3.086 95% CI 0.9958–9.564). The variant allele for the two SIRT1 SNPs occurred more frequently in the total Indian group compared to the total Black population (rs1467568: 41% vs. 18.5% respectively, p<0.0001, OR=3.190 95% CI 2.058-40943 and rs7895833: 41% vs. 22%

respectively, p<0.0001, OR=2.466 95% CI 1.620–3.755). Indian controls presented with a higher frequency for both SNPs compared to Black controls (rs1467568: 40% vs. 18.5% respectively, p<0.0001, OR=2.996 95% CI 1.850–4.853 and rs7895833: 41% vs. 22% respectively, p<0.0001, OR=2.513 95% CI 1.578–4.004). No difference was seen in the distribution of both SNPs between CAD patients and either control group. The MTHFR and SIRT1 SNPs were not associated with any clinical parameters in CAD patients and controls. The miR-499 variant (G) allele was found at a higher frequency in the total Indian group (34%) compared to the total Black population (22%) (p=0.0070, OR=1.796 95% CI 1.182–2.730). Indian cases presented with higher frequency of the rs3746444 G allele compared to Indian controls (38% vs. 29%, p=0.059, respectively). No differences in genotypic frequency for rs2910164 was found (GG: 45 vs. 47 %, GC: 46 vs. 41 %, CC: 9 vs. 12 %) in controls and patients respectively (odds ratio=1.025; 95 % confidence interval 0.6782–1.550; p=0.9164). The lowest levels of NF-κB and C-reactive protein (hsCRP) were found in patients with the homozygous C allele compared to the heterozygous GC and wildtype variants. Higher levels of miR-499 targets, hsCRP and IL-6, were observed in CAD patients with the variant genotypes compared to those with the wild type genotypes (8.92±1.91 vs. 6.73±0.87 mg/L; p=0.299, 3.02±0.77 vs. 2.18±0.57 pg/mL; p=0.381 respectively). A 6.25-fold increase in miR-146a levels was observed in CAD patients with the CC genotype (relative to controls and patients with the wildtype variant, p<0.0001). These (CC genotype) patients had significantly lower levels of miR-146a targets, IRAK-1 (0.38±0.02; p=0.0072) and TRAF-6 (0.44±0.02; p=0.0146). Taken together, this study provides the frequency distribution of the SNPs in four candidate genes for CAD in young South African Indians compared to Indian and Black controls. The frequency of variant alleles of rs1801133, rs3746444, rs7895833 and rs1467568 was greater in the Indian population compared to Black South Africans. Although no difference in frequency was observed for rs2010164, our results suggest a role for miR-146a in toll-like receptor (TLR) signalling via a negative feedback mechanism involving the attenuation of NF-κB by downregulation of IRAK-1 and TRAF-6. Thus miR-146a can act as a target for therapy towards lowering inflammation in CAD patients.