Department of Biotechnology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India.
Department of Internal Medicine, Yenepoya Medical College, Yenepoya Deemed to be University, Derlakatte, Mangalore, Karnataka, 570018, India.
Lab Invest. 2020 Aug;100(8):1090-1101. doi: 10.1038/s41374-020-0422-7. Epub 2020 Apr 1.
The interaction of genetic and epigenetic mechanisms is one of the underlying causes of phenotypic variability in complex diseases such as type 2 diabetes (T2D). To explore the influence of genetic and epigenetic changes in T2D, we examined the effect of methylation of CpG-SNP sites on allele-specific expression (ASE) in one-carbon metabolism pathway genes in T2D. Case-control study was conducted on 860 individuals (430 T2D and 430 controls). CpG-SNPs shortlisted through in silico analysis were genotyped using tetra ARMS PCR and validated using Sanger DNA sequencing. Global DNA methylation was carried out using RP-HPLC. Promoter DNA methylation and CpG site-specific methylation were carried out using bisulfite sequencing. mRNA expression and ASE were examined by SYBR green and TaqMan assay, respectively. Four exonic CpG-SNPs of MTHFD1, MTRR, and GGH genes were identified in folate pathway genes. Among these, MTHFD1 rs2236225 showed significant association with T2D independent of obesity, displayed ASE, and correlated with CpG-SNP site-specific methylation when compared with controls. Our results demonstrate that SNP rs2236225 in the CpG site of MTHFD1, which regulates allele-specific gene expression in PBMCs is methylation dependent and may perturb one-carbon metabolism pathway in T2D subjects.
遗传和表观遗传机制的相互作用是 2 型糖尿病（T2D）等复杂疾病表型变异性的根本原因之一。为了探讨 T2D 中遗传和表观遗传变化的影响，我们研究了在 T2D 中，一碳代谢途径基因中 CpG-SNP 位点的甲基化对等位基因特异性表达（ASE）的影响。对 860 名个体（430 例 T2D 和 430 例对照）进行病例对照研究。通过计算机分析筛选出的 CpG-SNPs 采用四引物扩增阻碍突变系统 PCR 进行基因分型，并通过 Sanger DNA 测序进行验证。采用 RP-HPLC 进行全基因组 DNA 甲基化检测。采用亚硫酸氢盐测序法进行启动子 DNA 甲基化和 CpG 位点特异性甲基化检测。通过 SYBR 绿色和 TaqMan 测定法分别检测 mRNA 表达和 ASE。在叶酸代谢途径基因中鉴定到 MTHFD1、MTRR 和 GGH 基因中的四个外显子 CpG-SNPs。其中，MTHFD1 rs2236225 与肥胖无关，与 T2D 显著相关，与对照组相比，还显示出 ASE，并与 CpG-SNP 位点特异性甲基化相关。我们的研究结果表明，MTHFD1 基因 CpG 位点的 SNP rs2236225 调节 PBMC 中等位基因特异性基因表达，这种调节依赖于甲基化，并可能破坏 T2D 患者的一碳代谢途径。
