Study On Pathogenic Gene Of Congenital Posterior Subcapsular Cataract

Objective To identify the clinical phenotype in a Chinese family with congenital cataract(CC),to exclude the known causative gene of this family by linkage analysis,and to find the causative gene with whole exome sequencing(WES).Methods 1.Clinical study: Blood samples were collected from family members after informed,a history-taking and comprehensive clinical examination were performed for all members.2.Linkage analysis: The microsatellite markers,which are closely linked to the candidate genes of CC,were selected for linkage analysis,the markers were amplified with M13 by Polymerase Chain Reaction(PCR).Fragments were separated by ABI3130 automatic sequencing,and the fragment sizes were identified using Gene Mapper software according to a size standard.Pedigree was used to observed if the fragment was co-segregated with the disease phenotype,so as to the corresponding candidate genes were found.The disease-causing mutation were found by direct sequencing of the candidate gene,and sequence comparison was performed with DNAStar software.3.WES: Agilent’s liquid chip capture system was used to build libraries,enrich and capture all exome of DNA samples.Followed by high-throughput,high-depth sequencing on Illumina platform.After sequencing,the results of WES was subjected to biological information analysis.Firstly,the non-causative polymorphic loci were excluded by filtering the data from the human genetic database,and then causative genes were screened out according to the genetic model and mutation type.Then SIFT and Polyphen-2 software were used to predict the deleterious protein while the known candidate genes of CC were carried out to screen in the results of WES.Validated the disease-causing mutations by Sanger sequencing,and sequence comparison was performed also with DNAStar software.Results 1.This family was autosomal dominant congenital cataract,and the clinical phenotype was posterior subcapsular cataract.There were no other eye disease and systemic disease except cataract in this family.2.38 candidate genes for CC in this study were found.Linkage analysis with 66 microsatellite markers on chromosomes at 1,2,3,4,6,8,10,11,12,13,14,15,16,17,18,19,20,21 and 22 were performed respectively.The results of linkage analysis showed that markers of D19S902 and D19S904 could be co-segregated with the disease phenotype in the family while the others not.Direct sequencing was performed on the gene of OPA3 and FTL,but there was no mutation of these two genes were found after compared with the normal gene sequences.3.Six suspected genes were found after the screening of bioinformation of WES,they were BFSP2(c.G1219A),WFS1(c.A41G),RPE65(c.C1154T),CRYBG3(c.A8620C),EPHA2(c.C1532T)and FYCO1(c.C2036T),respectively.But no causative genes were found after compared the sequences results of direct sequencing of these genes with those of normal gene database.The results that no causative genes were identified in the known congenital cataract candidate gene after WES was consistent with the results of linkage analysis.Conclusion 1.The pedigree of CC was simple autosomal dominant posterior subcapsular cataract.2.Linkage analysis excluded the 38 candidate genes on chromosomes at 1,2,3,4,6,8,10,11,12,13,14,15,16,17,18,19,20,21 and 22,suggesting that the causative gene of this pedigree may be a new gene.3.This research narrowed the study range of the causative gene of this family,provided an idea for the follow-up experiment of this pedigree,and also offered a new research method for the study of causative genes of other pedigree with CC.