DNA methylation differences within <i>INS</i>, <i>PTPN22</i> and <i>IL2RA</i> promoters in lymphocyte subsets in children with type 1 diabetes and controls

We elucidated the effect of four known T1D-susceptibility associated single nucleotide polymorphism (SNP) markers in three genes (rs12722495 and rs2104286 in <i>IL2RA</i>, rs689 in <i>INS</i> and rs2476601 in <i>PTPN22</i>) on CpG site methylation of their proximal promoters in different lymphocyte subsets using pyrosequencing. The study cohort comprised 25 children with newly diagnosed T1D and 25 matched healthy controls. The rs689 SNP was associated with methylation at four CpG sites in <i>INS</i> promoter: −234, −206, −102 and −69. At all four CpG sites, the susceptibility genotype AA was associated with a higher methylation level compared to the other genotypes. We also found an association between rs12722495 and methylation at CpG sites −373 and −356 in <i>IL2RA</i> promoter in B cells, where the risk genotype AA was associated with lower methylation level compared to the AG genotype. The other SNPs analyzed did not demonstrate significant associations with CpG site methylation in the examined genes. Additionally, we compared the methylation between children with T1D and controls, and found statistically significant methylation differences at CpG −135 in <i>INS</i> in CD8+ T cells (<i>p</i> = 0.034), where T1D patients had a slightly higher methylation compared to controls (87.3 ± 7.2 vs. 78.8 ± 8.9). At the other CpG sites analyzed, the methylation was similar. Our results not only confirm the association between <i>INS</i> methylation and rs689 discovered in earlier studies but also report this association in sorted immune cells. We also report an association between rs12722495 and <i>IL2RA</i> promoter methylation in B cells. These results suggest that at least part of the genetic effect of rs689 and rs12722495 on T1D pathogenesis may be conveyed by DNA methylation.本研究采用焦磷酸测序（pyrosequencing）技术，阐明了3个基因中4个已知的1型糖尿病（Type 1 Diabetes, T1D）易感单核苷酸多态性（Single Nucleotide Polymorphism, SNP）标记——即白细胞介素2受体α链（IL2RA）基因中的rs12722495与rs2104286、胰岛素（INS）基因中的rs689、蛋白酪氨酸磷酸酶非受体型22（PTPN22）基因中的rs2476601——对不同淋巴细胞亚群（lymphocyte subsets）中其近端启动子（proximal promoter）区域CpG位点（CpG site）甲基化（DNA methylation）水平的影响。本研究队列纳入25名新诊断T1D患儿与25名匹配的健康对照者。rs689 SNP与INS基因启动子区域的4个CpG位点（-234、-206、-102与-69）的甲基化水平显著相关；在这4个CpG位点中，易感基因型AA的甲基化水平均高于其他基因型。我们还在B细胞（B cell）中发现，IL2RA基因启动子区域的-373与-356两个CpG位点的甲基化水平与rs12722495存在关联，其中风险基因型AA的甲基化水平低于AG基因型。其余纳入本研究分析的SNP与所检测基因的CpG位点甲基化水平均未呈现显著关联。此外，本研究对比了T1D患儿与健康对照者的甲基化水平，发现CD8阳性T细胞（CD8+ T cell）中INS基因的CpG-135位点甲基化水平存在统计学显著性差异（p=0.034），T1D患者的甲基化水平略高于对照者（87.3±7.2 vs. 78.8±8.9）；其余分析的CpG位点甲基化水平无显著差异。本研究结果不仅验证了既往研究中报道的INS基因甲基化与rs689的关联，还在分选获得的免疫细胞中证实了这一关联；同时本研究还首次报道了rs12722495与B细胞中IL2RA启动子甲基化的关联。上述结果提示，rs689与rs12722495对T1D发病机制的遗传影响，至少有一部分可通过DNA甲基化介导。