DNA甲基化异常在多系统萎缩发病机制中的研究

Multiple system atrophy (MSA) is a fatal, adult-onset, neurodegenerative movement disorder with unknown etiology. Accumulative evidences suggest that genetic and environmental factors may contribute to the development of MSA. However, the underlying molecular mechanism of the disease-associated environmental, particularly the interaction with genetic factors in the pathogenesis of MSA, remains unknown. It is thought that epigenetic changes are intermediary between genetic factor and environmental toxins. Therefore, this study will explore the pathogenesis of MSA based on the epigenetic DNA methylation. The whole blood samples from patients including MSA, Parkinson Disease (PD), Alzheimer Disease (AD) and healthy controls will be tested in this study. First, the whole genome DNA methylation and the expression of mRNA in the blood sample of six patients with MSA and four healthy controls have been analyzed by microarray microarray and compared to obtained abnormal methylated genes of MSA patients. The candidate genes, MECC, HMGCL1 and PTS, which are closely related to the neurodegeneration of neuron have been chosen from the finding of the first step study. Second, the DNA methylation and the mRNA levels of candidate genes of 20 patients with MSA, PD or AD have been determined by Bisulfite Sequencing PCR and real-time PCR, respectively. The specific methylated genes related to MSA are probably MECC, HMGCL1 and PTS, and later will be identified it by enlarging the population. Finally, the methylation level of specific DNA methylated genes related to MSA at different stages of patients with MSA will be compared and to reveal the specific genes which are related to the development and progression of MSA and further we will establish a cell model of MSA and study the pathogenic mechanism of the target genes. Overall, the results of the present project will propose the abnormal DNA methylation involved in the pathogenesis of MSA and can provide a new clue or target for prevention and treatment of MSA.

多系统萎缩(Multiple system atrophy，MSA)是一种慢性进行性、成年发病的复杂性神经系统变性病。遗传与环境因素在其发生发展过程中起了重要的作用，表观遗传学可能是两者相互作用的桥梁。我们前期的研究，发现MSA患者的外周血DNA存在异常甲基化的基因，并通过生物信息学软件分析，筛选出可能参与MSA发病的候选基因。同时通过甲基化测序和RT-PCR方法，在大样本中初步确定了与MSA致病相关的DNA甲基化程度改变的基因MCEE、HMGCL及PTS。后期我们拟在扩大的样本中进一步验证上述实验结果，构建MSA体外细胞模型，进一步对上述发现的候选基因可能的致病机制进行探讨；动态比较不同疾病时期患者这些特异性基因甲基化的差异，结合环境暴露与临床数据，发现与疾病发生或进展相关的特异性基因甲基化。本项目成果将有助于对MSA发病机制的研究提供新的思路和方法，并为新的干预治疗策略提供理论依据。

多系统萎缩(multiple system atrophy, MSA)是一种严重的、进行性加重的神经退行性疾病，目前该病的病因和发病机制尚不十分明确，研究显示遗传与环境因素在疾病的发生发展中发挥了重要作用，而表观遗传学可能是连接两者的重要桥梁。本项目通过芯片筛查MSA患者外周血基因甲基化情况及表达情况，并通过生物信息学分析及甲基化与表达量相关分析，发现MCEE、HMGCL及PTS三个差异基因均参与代谢相关通路，可能是参与MSA发病的重要因子。进一步我们利用大样本队列，通过Massarray和RT-qPCR的方法验证了上述三个基因启动子区域甲基化及表达情况。结果显示在HMGCL基因启动子区，HMGCL_58 _CpG_2位点的甲基化率均值在MSA-C组较对照组降低, 而HMGCL_58_CpG_12、HMGCL_30_CpG_23位点的甲基化率均值则显著升高；在MCEE基因启动子区，MCEE_9_CpG_14.15在MSA-C组的甲基化率均值高于对照组；在PTS基因启动子区，PTS_25_CpG_26.27位点在MSA-C组的甲基化率均值低于对照组, 而PTS_61_CpG_5.6位点在MSA-C、PTS_25_CpG_31.32位点在MSA-P组的甲基化率均值均高于对照组。在mRNA表达水平上，HMGCL和MCEE的表达量在MSA-P和MSA-C患者组均显著高于与健康对照组，而PTS的表达量则未见明显改变。进一步关联分析显示HMGCL_58_CpG2位点的甲基化程度与HMGCL的表达量呈负相关（r=-0.464，P=0.003），提示HMGCL可能在MSA发病中发挥重要作用。在未来工作中，进一步探究HMGCL异常甲基化与MSA发病机制有望为MSA发病机制的研究提供新的思路和方法，并为疾病的诊疗提供新的靶点。