谷氨酰半胱氨酸连接酶催化亚单位基因甲基化而非多态性与慢性阻塞性肺疾病相关

Methylation, but not polymorphisms of glutamate-cysteine ligase catalytic subunit gene is associated with chronic obstructive pulmonary disease

目的 探讨谷氨酰半胱氨酸连接酶催化亚单位（GCLC）基因多态性和甲基化与慢性阻塞性肺疾病（COPD）易感的相关性.方法 收集166例COPD患者为COPD组,同期与上述COPD患者相似的非COPD人群166例为对照组.使用基因测序法检测两组人群GCLC基因启动子区域的单核苷酸多态性（SNP）,分析各SNP位点的单倍体型与COPD发病的关系.使用甲基化DNA免疫共沉淀芯片（MeDIP-chip）检测两组人群GCLC启动子区域甲基化水平,进行两组人群GCLC mRNA表达水平和血谷胱甘肽（GSH）浓度的比较.结果 通过直接测序法在GCLC启动子区域鉴定出12个SNP,其中4个SNP,即-2137MT、-129C/T、＋27591C/G和＋37764MG在COPD组和对照组人群中的发生率超过10％.-129C/T与-2137MT、＋27591C/G、＋37764MG均处于连锁不平衡.COPD组和对照组人群上述4个SNP的等位基因频率和基因型频率差异均无统计学意义（均P＞0.05）.MeDIP-chip检测显示COPD组GCLC启动子区域甲基化水平明显高于对照组（P＜0.001）.COPD组肺组织标本GCLC mRNA表达水平明显低于对照组（3.71±0.48比5.16±0.39,P＜0.05）,血谷胱甘肽（GSH）水平也低于对照组[109.72±32.38）mg/L比（179.87±46.23） mg/L,P＜0.05].结论 中国人群GCLC启动子区域甲基化而非GCLC基因多态性与COPD相关.

Objective To investigate the association between susceptibility to chronic obstructive pulmonary disease （COPD） and the polymorphisms and methylation of glutamate-cysteine ligase catalytic subunit （GCLC） gene.Methods One hundred and sixty-six patients with COPD （COPD group） and 166subjects without COPD （control group） were recruited in this study.The single nucleotide polymorphisms （SNPs） of GCLC promoter region of two groups were investigated by DNA sequencing,which entailed the analysis on the association between haplotypes of individual SNP locus of GCLC gene and the risk of developing COPD.The level of GCLC promoter methylation was determined by using methylated DNA immunoprecipitation chip （MeDIPchip）.The GCLC mRNA expressions and the levels of serum glutathione （GSH） were examined and compared between two groups.Results Twelve SNPs in GCLC gene promoter region were identified by direct DNA sequencing.Only 4 SNPs （-2137MT,-129C/T,＋27591C/G,＋37764MG） were found to harbor 10％ incidence in both groups.The-129C/T was in linkage disequilibrium with-2137A/T,＋27591C/G and ＋ 37764A/G.However,no difference in GCLC SNP allele frequency or genotype frequency was observed between COPD patients and controls （all P＞0.05）.Compared with the control group,the methylation level of GCLC promoter was significantly increased in COPD patients （P＜0.001）,as evidenced by the MeDIP-chip assay,and the expression of GCLC mRNA was down-regulated in the lung tissues （3.71±0.48 vs 5.16±0.39,P＜0.05） and serum GSH levels decreased [（109.72±32.38） mg/L vs （179.87±46.23） mg/L,P＜0.05] in COPD patients.Conclusion Methylation,but not polymorphisms of GCLC promoter is associated with the susceptibility to COPD in Chinese population.