一新的遗传性痉挛性截瘫致病基因SMEK1在疾病发生中的作用及分子病理机制研究

Hereditary spastic paraplegias (HSPs) are a group of genetically heterogeneous neuro-degenerative disorders with the cardinal feature of lower extremity spastic weakness. Despite the significant progress in the genetics of HSPs, lots of people with the diseases do not have a genetic diagnosis. The identification of the remaining disease genes is expected to yield important insights in the disease pathways and pathophysiology associated with axonal degeneration. .We initially recruited several families and sporadic cases with HSP, after excluding the known disease causing genes using linkage analysis combined with Sanger sequencing or MLPA, we got a HSP family possibly caused by a novel gene mutation. Using whole genome scan, we mapped the disease locus in a 12Mb region on 14q32 with a Lod Score=2.92, then whole exome sequencing identified an only candidate variant p.Lys572Thr in SMEK1 gene in the HSP family. This missense variant cosegregate with the phenotype in the family and is conservative across different species. Preliminary results also suggest that the mutant gene affect BMP pathway and expressed in Purkinje cells of mouse cerebellum..In this proposed project, we aimed to confirmed the candidate causing gene by 1) screening more disease families and sporadic cases to find other mutations in the SMEK1 gene, 2) in vitro sdudies to provide the functional supportive evidences for the mutations, 3)phenotypic resemblance in zebrafish using morpholino knocking down the candidate disease genes and phenotype rescue by human wide-type mRNA injection, 4) phenotype assay for the knock-in mouse model. In addition, in vivo and in vitro functional studies of identified disease-causing genes will be carried out to investigate their critical roles in the pathogenesis of HSP. Identification of more genes implicated in the neuro-degenerative disorders will provide us a better understanding of the molecular pathology and help find potential treatments.

课题组前期利用全基因组扫描将一个新的遗传性痉挛性截瘫（Hereditary Spastic Paraplegia, HSP)致病基因定位于14q32 ，全外显子组测序发现SMEK1基因内的 p.Lys572Thr突变与表型共分离，并且影响了BMP信号通路，免疫组化结果显示该基因表达于小脑皮层的浦肯野细胞。根据前期结果及文献复习我们提出假说：SMEK1基因作为一个新的HSP致病基因，可能通过影响BMP通路改变了微管稳定性进而影响了突触生长从而导致了HSP的发生。为明确SMEK1基因在遗传性痉挛性截瘫发生中的分子机制，课题组利用基因打靶技术构建了Smek1 K572T突变的knock in 小鼠模型，结合斑马鱼模型，通过表型分析、分子生物学及电生理等实验检测小鼠的神经元等是否发生改变来分析SMEK1导致HSP发生的分子机制，为遗传性痉挛性截瘫及其他神经退行性疾病的防治进行积极的探索。

本项目发现一遗传性痉挛性截瘫家系并发现了其致病候选基因。在后续分析过程中发现该疾病为脊髓小脑共济失调3型（SCA3)。SCA3是一类具有显著遗传异质性的神经退行性疾病，其致病变异是ATXN3基因异常的CAG三碱基重复扩增。通过三名患者的全外显子组测序数据，我们发现SMEK1 c.1715A>C为可能的修饰变异。利用小鼠模型发现携带Smek1K572T/+修饰变异的MJD84.2小鼠具有更为严重的运动障碍，与患者的临床表征类似。病理上，在患者和携带修饰变异小鼠中，都观察到突变的SMEK1蛋白与ATXN3以及Ub在沉积物处共定位，且沉积物的分布广泛。进一步分析揭示，SMEK1的变异导致缺失了一个泛素化修饰位点，蛋白降解异常。随年龄增长，神经元表现为蛋白质量控制系统的紊乱、持续的内质网应激以及神经元凋亡。在这个过程中，冗余的SMEK1蛋白倾向于异常累积，与polyQ expanded ATXN3以及其他的蛋白质质量控制组分共同参与了聚集物的形成，导致病理特征的加重以及广泛分布，本项目发现了一个SCA3的修饰基因，为理解其发生的分子机制提供了一个重要的线索。