Screening Of Risk Genes Associated With Chinese Families Of Non-syndromic Cleft Lip And/or Palate By Whole Exome Sequencing And Construction Of Mouse Model With Mutant LAR Family Genes By Using CRISPR/Cas9 Technology

Objective:1.Whole exon sequencing was performed on the families with non-syndrome cleft lip and/or palate to obtain gene sequencing results,blood samples were collected clinically and DNA were extracted from blood samples;2.The bioinformatics technology was used to analyze the results of whole exons sequencing and screen candidate pathogenic genes and loci;3.In view of the screened candidate pathogenic genes and loci,related genetic engineering animal models were constructed to explore the phenotypic effects of mutations in candidate genes or loci on animal models,and preliminary exploration was made on the pathogenesis of candidate pathogenic genes and loci.Methods:1.On the premise of fully obtaining the informed consent of patients and their family members,epidemiological investigation was conducted on family members.And members who were to participate in the project have to conduct a comprehensive physical examination and imaging examination in order to exclude the existence of the syndrome cleft lip and/or palate.Blood samples of non-syndrome cleft lip and/or palate genetic families were collected,and DNA was extracted for whole exon sequencing;2.Based on the human genome reference sequence hg19,the mutation sites were obtained by GATK,FastQC,PICARD,BWA and other software;SIFT,SnpEff,Clinvar,DAVID,SeattleSeq and CADD were used to annotate mutation sites.As well as filtering through NCBI dbSNP,HapMap,ESP,ExAC and other databases.The candidate genes with high correlation with non-syndrome cleft lip and/or palate were screened according to the bioinformatics characteristics of the mutated genes and loci;3.UniProt Browser,Mutationassessor and other software were used to score the biological functions of the candidate mutation sites.PSIPRED and SWISSMODEL were used to bulid models to predict the related protein structures.Structural changes of models were compared after mutations,and we also evaluated the deleterious effect caused by mutations;4.For the candidate pathogenic genes obtained after screening and evaluation,CRISPR/Cas9-mediated gene knockout and point mutation were used to construct the genetic engineering animal model.In this study,by constructing sgRNA vector and Cas9 vector,the mouse model of two homozygous gene strains,including a gene knockout model and a point mutation model,were obtained by superovulation,microinjection,embryo transfer,animal passage and genotype identification of target animals;5.By comparing the homozygous mice with the wild-type mice born in the same generation,phenotypic of abnormal development visible to the naked eyes were confirmed and recorded by photographs.The developmental difference between homozygous adult mice and wild-type homozygous mice was investigated by imaging examinations such as MRI.After the homozygous male and female mice were obtained,the homozygous mouse embryos were obtained after mating,and the tissue sections of the obtained embryos were made by using the histoembryology experimental technology.Results:1.From September 2018 to October 2019,a total of 5 non-syndrome cleft lip and/or palate genetic pidegrees were collected and whole exon sequencing among 3 genetic families with relatively complete genetic data and clear epidemiological situation were performed;2.Site annotation,analysis and screening were performed on the results of whole exons sequencing of 3 families to obtain several candidate disease-causing genes and loci,IRF6,CDH1,PTPRF and PTPRD;3.Bioinformatics analysis was conducted on the candidate pathogenic sites,and prediction and modeling analysis were conducted on the structure of related proteins.Mutation sites of IRF6,CDH1 and PTPRD had deleterious effects on the protein structure;4.As for the genetic engineering animal model for the candidate pathogenic genes PTPRF and PTPRD,we constructed the mouse model with Ptprf gene knockout and the mouse model withPtprd point mutation.At last,genotype of the homozygous mouse model was comformed by DNA agarose gel electrophoresis and first-generation sequencing;5.Cranialfacial phenotypes in homozygous mouse models included cleft palate and abnormal facial development.Phenotypes of abnormal facial development included asymmetry of eyeball development,unilateral facial soft tissue atrophy,abnormal urinary development,etc;6.Homozygous embryo study,imaging examination and tissue section revealed cleft palate,unilateral ocular deletion,abnormal development of urinary tract and incomplete death of mouse embryos.Conclusion:1.This study screened out several candidate pathogenic genes and loci,IRF6,CDH1,PTPRF and PTPRD,which may be the risk pathogenic genes of non-syndrome cleft lip and/or palate in Chinese population;2.This study found abnormal craniofacial development in animal models with knock out of candidate disease-causing gene Ptprf,including abnormal eye development,cleft palate and incomplete death.This gene may be one of the pathogenic genes for cranialfacial birth defects in China.