A Series Of Studies On Prenatal Investigation And Diagnosis Of Genetic Etiology With Congenital Cleft Lip And Palate

BackgroundCleft lip and palate(CL/P)is the most common maxillofacial deformity and one of the most common birth defects in the world.There are over 500 Mendelian syndromes,including those arising secondarily from chromosomal or teratogenic effects associated with CL/P.Due to the pathogenic mechanism of congenital cleft lip and palate is complex and difficult to understand,including genetic and environmental factors,not abiding by the Mendelian ratio.Nowadays there are mainly two kinds of its inheritance pattern: polygenic threshold and main pathogenic gene.Prenatal diagnosis is an important way to manage the birth of cleft lip and palate and guide the prevention and treatment of postpartum defective child.Thereforce,we acquired the actual situation of prenatal cleft lip and palate with epidemic survey and try to use advanced molecular technology mainly for CNVs and SNVs to on puerperal cleft lip and palate to diagnose pathogenic genes in our article.ObjectivesFrom the prenatal research on congenital cleft lip and palate and application of array comparative genomic hybridization(CMA)with genome-wide high-resolution and next generation sequencing(NGS),our research can analyse the genetic etiology of CL/P.There are three parts :(1)To discuss diagnosis,prognosis and intervention strategy of prenatal cleft lip and palate,and to investigate its dynamic incidence and epidemiological characteristics.(2)To explore the value of the application of whole-genome high resolution chromosome microarray analysis(CMA)investigated16 cases with congenital cleft lip and cleft palate.Compare the differences of pathogeneic copy number variants(CNVs)detection rate among the cases with isolated cleft lip(CL),isolated cleft palate(CP)and cleft lip with cleft palate(CLP).(3)Combined with NGS platform for the detection of the CL/P,we can estimateeffectiveness of genetic diagnosis in clinical practice.Materials and methods1.Review on 147 prenatal cases of cleft lip and palate in prenatal diagnosis center of Guangzhou women and children's medical center from January 2011 to December 2016,gestational age ranging from 12.1 to 38.7 weeks,we paid regular visit on its whole perinatal period including antepartum,postpartum,postoperation.Based on the information of the subjects,classified into disease type,diagnosis rate,outcome and risk factors and so on,we made some analysis and conclusions.2.A total of 16 cases from 10 pedigrees referred for cleft lip and cleft palate from the Department of Prenatal Diagnosis and Oral and Maxillofacial Surgery in Guangzhou Women and Children's Medical Center from October 2012 to December2015.The DNA samples were run on CytoScan TM HD or 750 Array using the manufacturer's protocol.The CNVs detected were further analyzed with known CNVs listed in databases publically available online,including database of DECIPHER,OMIM,DGV,UCSC,CAGdb and ISCA.3.A total of 28 individuals from 9 pedigrees with CL/P were enrolled.The DNA samples were run on Hiseq 2500 of Illumina Corporation using the manufacturer's protocol.The SNP detected were further analyzed in database publically online including Exac?ClinVar?HGMD?Mutation Taster?SIFT and PloyPhen.Results1.According to investigation on 147 cases of prenatal cleft lip and palate,we found CLO diagnostic sensitivity(detection rate)had achieved 100%,the lowest73.3% in isolated CLP,the sensitivity of NSCL/P(78.5%)less than SCL/P(80%);In the majority of SCL/P associated with other malformations were craniofacial and cardiovascular system,individually accounted for 10.9% and 8.8%;During 2011 to2016 years,major prenatal diagnosis time was pre-28 w,increasing year after year,whose average is 25.4±3.5W;According to the outcome of 147 cases ranking large from small proportion were induced labour,childbirth and lost.Induced labour causes consists of objective factors like SCL/P confirmed and subjective factors like culture,economy;We found preparation for pregnancy,abnormal childbearinghistory and maternal age have more and more influence on CL/P malformation through investigation and evaluation of high risk factor of the abnormality.2.High resolution array comparative genome hybridisation(CMA): A total of16 cases from 10 familial pedigrees with CL/P were performed on CMA successfully.There is a pathogenic CNV were detected,both familial and hereditary in 2 cases of this study.3.After analyzing of SNVs and indels,2 cases of pathogenic gene were found and 3 cases of possible pathogenic mutations were detected,and the other 6 are considered uncertainty.All SNV were confirmed by Sanger sequencing.Conclusions1.The hot spot of prenatal work is the birth defects of cleft lip and palate,mean to improve prenatal diagnostic rate,to reduce the adverse consequences of subjective factors bringing to family and social,to improve the quality of children life and birth population for reference.2.We should pay attention to conservation work and carry out three stages measures to avoid or reduce the impact of high-risk factors in cleft lip or cleft palate,decreasing the incidence of cleft lip and palate.3.Whole-genomic microarray technique is of great application value in the etiological diagnosis of congenital cleft lip and palate,which detected the abnormal CNVs the traditional G-banding karyotype had not found.4.Exon sequencing is of great application value in the etiological diagnosis of cleft lip and palate,accurate and efficient to detect comprehensively the pathogenic SNVs.5.By High-resolution chromosome microarray analysis(CMA)combined with high-throughput next-generation sequencing(NGS),whole genome get the comprehensive,accurate and efficient genetic screening,found new candidate pathogenic CNVs and SNVs locus in congenital cleft lip and palate.