腓骨肌萎缩症1A（CMT1A）PMP22基因启动区域的调控研究

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common form of the Hereditary Motor and Sensory Neuropathy which is caused by a 1.5 Mb duplication on chromosome 17p11.2 which contains the peripheral myelin protein-22 (PMP22) gene. The phenotype of CMT1A is quite different according to the divers mRNA and protein levels of PMP22 in patients. To elucidate how the same duplication can lead to the diverse mRNA and protein levels and even the clinical phenotype disparity is a key point in the study of CMT1A. The expression levels of PMP22 is affected by the some elements in the promote region, however, the crucial element to determine the transcription and translation of PMP22 is still ambiguous. In this study, to explicit the critical region participating and regulating the process of transcription of the PMP22 gene, CRISPRa/CRISPRi will be adopted to activate or inhibit the elements of the PMP22 promote region in the normal Schwann cells. Next, the corresponding promote region of iPS cells will be interfere by CRISPRi. After intervention, the expression levels of PMP22 mRNA and protein and the myelination of Schwann cell would be observed and the possibility of curing the CMT1A by CRISPRi would further be explored.

腓骨肌萎缩症1A型（CMT1A）是临床常见的遗传性周围神经病，由位于染色体17p11.2周围神经髓鞘蛋白22（PMP22）基因区域1.5Mb片段重复突变引起。CMT1A临床表型差异极大，这种差异与PMP22 mRNA表达水平和蛋白表达量相关。同样的基因片段重复为何导致其mRNA及蛋白表达量差异，甚至影响临床表型，是本病的重要科学问题。PMP22基因表达受启动区域多个位点调节，但影响转录和蛋白翻译效率的关键位点尚不明确。本项目将采用CRISPRa/CRISPRi技术，对正常Schwann细胞PMP22启动区域各位点分别激活或抑制，以明确参与转录调控的关键位点；继而对患者的iPS细胞PMP22启动区域相应位点进行CRISPRi干扰，观察下调后PMP22 mRNA水平、蛋白表达量以及髓鞘形成情况，探索CRISPRi干扰启动区域对CMT1A治疗的可能。

腓骨肌萎缩症1A型是最常见的遗传性周围神经病，其致病原因是17p12区域内PMP22基因杂合重复引起的，本项目通过收集中国南方人群脱髓鞘CMT发现，CMT1A是最常见的脱髓鞘型CMT，并且CMT1A临床表型差异较大。本项目建立了CMT1A患者的iPS细胞并鉴定该细胞株具有胚胎干细胞特性，并且具有多项分化潜能,为疾病发病机制提供了良好的细胞模型。进一步将CMT1A患者iPS细胞成功分化为Schwann细胞。同时针对PMP22基因启动区域进行CRISPRa与CRISPRi干预发现，NF1与CREB结合区域影响PMP22基因表达增加，但不影响PMP22基因表达减少。项目资助发表SCI论文2篇，培养博士2名。项目投入直接经费17.5万元，支出15.769963万元，各项支出基本与预算相符。剩余经费2.605037万元，剩余经费拟计划用于本项目后续研究。