Identification Of A Novel Mutation In The SAG Gene In A Chinese Family With Autosomal Dominant Retinitis Pigmentosa

Objective:Retinitis pigmentosa （RP） is a set of retinal diseases that feature degeneration of rod and cone photoreceptor cells, which will lead to visual loss eventually. RP is one of the main reasons causing blindness in the worldwide. RP is a monogenic disease and has obvious genetic heterogeneity. The number of disease-causing genes of RP is enormous and each gene has one or more disease-causing mutations. So far, the identified genes of non-syndromic RP can explain only 60% of all non-syndromic RP patients. The aim of our study was to find more disease-causing genes of RP, and to better understand the relations between genotype and phenotype of RP and pathogenic mechanism of RP. In this article, we started the research object based on a Chinese family with Autosomal Dominant Retinitis Pigmentosa. Our goal was to identify the disease-causing gene of the family, and establish the genotype-phenotype relationship and establish the pathogenic mechanism of RP.Methods:Firstly, We got a Chinese family with Autosomal Dominant Retinitis Pigmentosa and negative controls, then collected information of the family and pictured pedigree chart. All available members of the family were inquired about their medical history and received some ophthalmic examinations. Their blood samples were collected and genomic DNAs were extracted. We employed the whole-exome sequencing to screen genes associated with inherited retinal dystrophies（IRDs） in the proband by blasting with reference genome of UCSC hg19,selecting and rating data by BWA, detecting and annotating variants by SAMTools, predicting the mutation’s function by SIFT and removing known variants by dbSNP & 1000G database. Then we performed Sanger sequencing to analyze the intra-familial co-segregation and validate the putative variant in 200 unrelated controls. Biological information of the targeted gene would be analyzed. Immunofluorescence was used to detect the expression and location of the targeted gene in the retinal tissue of mouse. Then we constructed an eukaryotic expressing vector carrying the targeted gene for the further functional analysis of targeted gene. We detected the expression of both vector was examined through Western blot. Then confocal microscopy was used to determine the expression and location of the wild type and mutant type of the targeted gene.Results:The Retinitis Pigmentosa family was inherited by autosomal dominant.27 putative variants with IRDs were initially identified in proband （Ⅲ-14） through the whole-exome sequencing and data screening. After Sanger sequencing and analyses of intra-familial co-segregation, SAG（NM₀00541.4） c.A257G was the only putative pathogenic variant which was absent in 200 unrelated normal controls. SAG is the specific gene about degeneration of photoreceptors. S-arrestin which is the product of SAG is mainly expressed in rod photoreceptor cells of retina. Eukaryotic expression vector carrying the target gene was successfully constructed. The vectors carrying the wild type and mutant type SAG gene were introduced into 293T cells. The distribution of the proteins is different between the wild type and the mutant Sag gene, observed by confocal microscopy.Conclusion:1.SAG:c.A257G may be the disease-causing gene of the RP family.2. The diffenrence of expression between wild type and mutant type SAG gene on cultured cells in vitro may be the result from mutant type SAG.