DNA甲基化在SCA3/MJD分子发病机制中的作用研究

SCA3/MJD is one of the most common polyglutamine (polyQ) diseases. The virulence genes ATXN3 with CAG repeats dynamic mutation in coding region of 3'terminal, encodes protein product- - ataxin-3, which is causative due to its cytotoxicity caused by polyQ expansion mutations. It is supposed that epigenetics may participate in the modulation of CAG repeats dynamic mutation and ataxin-3 mutant expressio, causing genetic and clinical heterogeneity. This study is to apply DNA methylation chips to screen interesting genes from brain tissues of SCA3/MJD transgenic mice and patients; to apply bisulfite sequencing and ELISA methods to investigate whether DNA methylation modification of interesting genes and the expression levels of serum proteins can act as a potential biomarkers for diagnosising and reflecting duration of SCA3/MJD; to apply RT-PCR, HPLC to study the mechanism of abnormal DNA methylation of interesting genes caused by ataxin-3 mutant; to apply gene overexpression and RNAi approach to discuss the affects of interesting genes on cell growth and apoptosis processes; to apply SCA3/MJD transgenic cell and mice models to illustrate relationship between DNA methylation and CAG repeats dynamic mutation; to apply optogenetic tools to unravel relationship between DNA methylation and ATXN3 expression. This study will provide a new insight into the molecular pathogenesis of SCA3/MJD.

SCA3/MJD是多聚谷氨酰胺（polyQ）病中最常见的一种，致病基因ATXN3的3'端编码区内CAG重复序列发生动态突变，其编码蛋白ataxin-3发生polyQ扩展突变而具致病性。表观遗传可能参与调控CAG重复序列动态突变和ataxin-3蛋白表达而导致遗传和临床异质性。本研究拟应用DNA甲基化芯片从SCA3转基因小鼠和患者脑组织筛选感兴趣基因；应用重亚硫酸盐测序和ELISA探讨感兴趣基因DNA甲基化修饰和血清表达水平能否成为诊断和反映病程进展的标记物；应用RT-PCR、HPLC等探讨ataxin-3蛋白致感兴趣基因DNA甲基化异常的机制；应用基因过表达、RNAi等探讨感兴趣基因表达异常对细胞生长、凋亡的影响；应用SCA3转基因细胞和小鼠探讨DNA甲基化与CAG重复序列动态突变的关系；应用光遗传学技术探讨DNA甲基化与ATXN3表达的关系，为揭示该病的分子发病机制提供了新思路。

SCA3/MJD 是多聚谷氨酰胺（polyQ）病中最常见的一种，致病基因 ATXN3 的 3’端编码区内 CAG 重复序列发生动态突变，其编码蛋白 ataxin-3 发生 polyQ 扩展突变而具致病性。表观遗传可能参与调控 CAG 重复序列动态突变和 ataxin-3 蛋白表达而导致遗传和临床异质性。本项目聚焦于DNA甲基化，通过SCA3/MJD患者、SCA3/MJD转基因细胞和小鼠模型探讨DNA甲基化与SCA3/MJD发病机制的关系。在SCA3/MJD患者及健康对照中，通过重亚硫酸盐测序、RT-PCR结合启动子信息分析，发现SCA3/MJD病例组较正常对照组ATXN3基因启动子区的DNA甲基化水平升高，且该区域的甲基化水平升高与SCA3/MJD患者发病年龄提前和CAG重复序列动态突变有关；应用飞行时间质谱及PCR技术在SCA3/MJD病例组与正常对照组中检测了DNA 甲基化相关基因单核苷酸多态性（SNP），发现MBD1基因rs12957023可能与CAG动态突变相关，DNMT3A基因rs13420827及DNMT3L基因rs7354779可能与患者发病年龄相关。在转基因细胞模型中验证了DNA甲基化对表达的调控及主要修饰位点，并成功构建了转基因小鼠模型，探讨了SCA3/MJD小鼠的行为学，以及DNA甲基化差异对小鼠的影响。在SCA3/MJD转基因小鼠研究中挑选出19个差异甲基化基因及50个基因表达存在差异的基因进行焦磷酸测序及RT-PCR验证，联合二者结果发现其中Shh通路及Wnt信号通路可能参与SCA3/MJD发病过程，后续蛋白质生化及细胞生物学实验证实其分子机制的工作正在进行。我们的研究表明了，DNA甲基化可能通过影响ATXN3基因的功能或其他分子信号通路等过程而参与了SCA3/MJD的发病机制，这为SCA3/MJD等一类polyQ疾病的研究提供了新的方向，也为这一类疾病的治疗带来了新的前景。

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