Whole Exome Sequencing Identifiy Novel PRPF31 Mutations In RP And Function Study Of EMC9 Of RP Causitive Gene

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.