节点文献
WES筛查RP致病基因PRPF31新位点及RP候选基因EMC9的功能研究
Whole Exome Sequencing Identifiy Novel PRPF31 Mutations in RP and Function Study of EMC9 of RP Causitive Gene
【作者】 谢丹;
【导师】 鲁芳;
【作者基本信息】 电子科技大学 , 生物化学与分子生物学, 2018, 硕士
【摘要】 视网膜色素变性(retinitis pigmentosa,RP)是一种遗传性眼部疾病,它最终会导致失明。目前已鉴定了超过70个RP基因,但仍有35%45%的RP病人无法用已知基因解释,根据该疾病的相关研究提示还有很多未知RP基因等待鉴定。基于我们对RP复杂发病机制的认识不清晰,目前尚缺乏并且公认有效的干预手段来防治RP。对RP致病基因新突变及新型候选基因的发现是首要目的,对RP分子诊断、治疗方向和分子遗传学研究具有重要意义。然而传统Sanger测序已经无法满足需求,其费用高额、耗时巨大、且无法获取未知致病基因,随着二代测序技术的发展,全外显子测序(whole exome sequencing,WES)运用在疾病研究中成功案例越来越多,它们逐渐成为疾病研究中极为常用的筛查手段,它们具有高的灵敏度,低成本投入等优点,值得一提的是WES可以检测到未知致病基因。在此研究中,我们利用WES对收集到的家系及散发患者筛查,得到RP已知致病基因的一个新致病突变位点、一个候选致病突变位点和一个常染色体隐性遗传(arRP)候选基因:(1)在中国人群中,我们检测到常染色体显性遗传(adRP)基因PRPF31一个新型移码突变c.12261227insA(p.T410Dfs*65),与一个候选终止突变c.1015C>T(p.Q339*)。在1000例正常人中没有这些突变位点。(2)对前期筛选出的arRP候选基因EMC9及其突变位点c.59G>A(p.R20Q)的初步功能研究。EMC9在正常小鼠视网膜、睾丸、脊髓、小脑、大脑呈现高表达。我们成功构建了EMC9质粒pCDNA3.1-EMC9-FLAG-WT和pCDNA3.1-EMC9-FLAG-MUT(59G>A),与WT组相比较,pDNA3.1-EMC9-FLAG-MUT细胞蛋白表达量降低90%以上,且其蛋白定位呈缺失状态。这些均与基因突变的致病性一致。下一步我们打算构建EMC9基因敲除小鼠,EMC9基因对视网膜功能的影响有待进一步研究。综上所述:(1)我们通过WES,发现了中国人群RP患者中已知adRP基因PRPF31的新型突变,扩大了RP致病基因突变谱,给产前诊断提供新思路。(2)对新发现的RP基因EMC9及其新位点c.59G>A(p.R20Q)进行了初步的功能研究,其突变导致了蛋白不稳定表达,从而可能导致疾病。
【Abstract】 Retinitis pigmentosa(RP)is a hereditary eye disease and it ultimately leads to blindness.At present,more than 70 RP genes have been identified,but there are still 35%-45% of RP patients cannot be explained by known genes.According to the disease related studies suggest there are many unknown waiting for identification of RP genes.Because of our unclear understanding of the complex pathogenesis of RP,there is a lack of effective intervention to prevent RP.It is the primary objective for the discovery of novel mutations and novel candidate genes.And it is a great significance for the diagnosis of RP,the direction of treatment,and the study of molecular genetics.However,traditional Sanger sequencing has been unable to meet the demand,which need the high cost,time-consuming and unable to obtain unknown disease genes.As more and more successful cases in the study of diseases by Next Generation Sequencing and Whole-exome sequencing,and they have gradually become very common screening tool.They have the advantages of high sensitivity,reduced cost investment,etc.It is worth mentioning that WES technology can detect unknown pathogenic genes.We used WES to screen the collected family and sporadic patients to obtain a novel mutation site of the known pathogenic genes of RP,a candidate site and a candidate gene of autosomal recessive retinitis pigmentosa(arRP)in this study.(1)We detect a novel mutation site and a candidate site of the autosomal dominant retinitis pigmentosa(adRP)PRPF31 gene in Chinese persons: a frameshift mutation c.12261227insA(p.T410Dfs*65);a candidate terminating mutation c.1015C>T(p.Q339*),and there are no the mutation sites for 1000 normal persons.(2)We verify the function of arRP candidate gene EMC9(ER membrane protein complex subunit 9).We first studied the expression of EMC9 gene in mouse: EMC9 gene shows high expression in the retina,testicles,spinal cord,cerebellum and brain for normal mices.Next,we successfully constructed the wild plasmid for EMC9 pCNA3.1-EMC9-FLAG-WT and mutant plasmid pCDNA3.1-EMC9-FLAG-MUT(59G>A).We found that the expression of cell protein of pCDNA3.1-EMC9-FLAG-MUT was ninety percent down,moreover the expression of cells’ localization couldn’t be detected and showed a lack of state.These are consistent with the pathogenicity of genetic mutations.Next we intend to build the knockout mouse of EMC9 gene.And we will have a further study about effects of EMC9 gene on retinal function.To sum up:(1)We have discovered three novel mutations of PRPF31 in RP patients for Chinese population by WES.It extends the RP gene mutation spectrum and provides new ideas for antenatal diagnosis.(2)We have primary function study of the discovered candidate EMC9 gene for RP and its novel mutation c.59G>A(p.R20Q).The mutation of c.59G>A(p.R20Q)led to the unstable expression of the EMC9 protein,which may cause morbidity.