Identifcation Of Susceptibility Genes In Non-syndromic Cleft Lip With Or Without Cleft Palate Using Whole-exome Sequencing

Objective(1)To search for the candidate virulence genes of the non-syndromic cleft lip with or without cleft palate(NSCL/P)by whole-exome sequencing.(2)To determine the virulence gene of NSCL/P by verifying the candidate virulence genes.(3)To explore the role of virulence genes in the pathogenesis of NSCL/P,and to lay a foundation for future gene diagnosis and gene therapy.Methods(1)The Oral and Maxillofacial Surgery,in the affiliated hospital of Qingdao University,to carry out the exon sequencing with 30 children who has been suffer from the NSCL/P.(2)Through filter out variant sites of thousand-person genome database,and retain the variant sites with frequencies below 0.01 in 1000G;Preserving variation at the exon or splice sites;The mutation,which does not affect the function of the gene,is removed by the synonym mutation,and only the mutations of the non-synonymous mutations and the shear sites that affect the function of the gene are retained.The follow software,SIFT,Poly Phen,MutationTaster,CADD were used to evaluate the sites harmful effects.(3)Refer to ACMG's evidence to classify the hazard of mutation sites,and reduce the number of candidate genes.(4)Further screening of gene function to predict the possible virulence mutation/gene of NSCL/P.(5)The PCR sequencing method was used to verify the mutation sites detected by exon sequencing in 30 children and whether functional mutations were detected in the exon of the candidate virulence gene in another 100 children with NSCL/P.Results(1)After the whole exome sequencing,506,144 single nucleotide variants(nucleotide variants,SNVs)sites were found.(2)The number of mutation sites that were evaluated as harmful by software was 4637 with the remaining 8,459 snvs after filtering thousands of human databases and synonymous mutation sites without affecting gene function.(3)The mutation number of the classification for pathogenicity is 44.(4)The gene function was then analyzed and the remaining sites were 21,located in the following 9 genes:EDN3(rs11570255),LAMA5(rs559318635,rs145192286,rs201130283,rs375094493),FDFT1(rs79708434,rs80310078),HERC1(63901334,rs149457868,rs759604048,63966826,64019868),SSH2(rs139830628),ERC1(rs61740169,rs74740082),MPP2(rs149791694),NISCH(rs150644559,rs117164680),ASTN2(rs138209428,rs142855762,rs3818503).(5)The PCR sequencing method was used to verify the 24 mutation sites in the sequenced children,which showed a high mutation rate at the rs145192286 sites of the Gene Lama 5(LAMA 5).(6)In another 100 children with NSCL/P,the rs145192286 sites of the gene were detected as mutations,thinking that it may be the a pathogenetic gene of NSCL/P.Conclusions(1)The NSCL/P has a complex genetic characteristics,this study uses the whole exome sequencing method,and the first successfully discovers the virulence gene LAMA5 of NSCL/P and its mutation locus rs145192286.(2)rs145192286 may be a risk factor for children with atypical cleft lip and palate,which may increase the risk of NSCL/P.(3)This study further confirms that WES is an effective strategy for revealing the etiology of NSCL/P.