Diagnostic Yield Of Whole-Exome Sequencing And Identification Of Candidate Genes In Fetuses With Conotruncal Defects

Part 1:Diagnostic yield of whole exome data in fetuses aborted for conotruncal defectsBackground and Objective:Conotruncal defects（CTD）,caused by abnormal development of cardiac outflow tract,belong to a group of congenital heart defects（CHD）with similar molecular basis for embryonic development and with serious lesions that require early surgical correction.Whole-exome sequencing（WES）is an effective technique for prenatal genetic diagnosis.An accurate genetic diagnosis will help guide prenatal counseling and develop rational clinical strategies.This study aimed to assess the diagnostic value of WES in aborted CTD fetuses using a custom CHD-geneset,and to explore the impact of postmortem phenotyping through imaging and autopsy on genetic diagnosis.Methods:We sequentially analyzed copy number variation sequencing（CNV-seq）and WES data from 47 fetuses with CTD detected by prenatal ultrasonography.Fetuses with either a confirmed aneuploidy or pathogenic CNV were excluded from the WES analyses,which were performed following the American College of Medical Genetics and Genomics recommendations and a custom CHD geneset.Imaging and autopsy were applied to obtain postnatal phenotypic information about aborted fetuses.Results:CNV-seq identified 7/47 aneuploidy cases and 13/47 CNV-positive fetuses.Eighty-five rare deleterious variants in 61 genes（from our custom CHD gene set）were identified by WES of 27/47 karyotype-and CNV-negative fetuses.Of these,five（likely）pathogenic variants（PV/LPV）were identified in five fetuses,revealing a 10.6%incremental diagnostic yield.Furthermore,RERE:c.2461₂472delGGGA TGTGGCGA was reclassified as LPV based on postnatal phenotypic data.Conclusion:In this study,the genetic diagnosis of aborted fetuses with CTD was successfully performed using WES.Furthermore,more phenotypic information,obtained by postnatal autopsy and imaging,facilitate WES diagnostics.Part 2:Diagnostic yield of whole exome data in fetuses aborted for conotruncal defectsBackground and Objective:The most common and most studied conotruncal defect（CTD）is tetralogy of Fallot（TOF）.However,most of them are sporadic cases with complex genetic etiology,and only about 10%are caused by de novo mutation.Moreover,abnormal epigenetic modification is one of the important causes.All of these factors can cause the dysfunction of gene network,and lead to the occurrence and development of TOF.This part aims to use a weighted gene co-expression network analysis（WGCNA）to reveal the abnormal regulatory network of TOF and to identify candidate TOF genes.Methods:Based on GSE36761 transcriptome data,WGCNA was used to identify TOF-related WGCNA sub-networks,namely gene modules,and combined with protein-protein interaction（PPI）network to identify hub genes.Gene enrichment analysis was used to reveal the potential biological functions of hub genes.At the same time,a methylation dataset（GSE62629）and two single-cell datasets（EGAS00001003996,GSE126128）were used to analyze the changes of the hub gene at the methylation level and the single-cell transcription level in the embryonic heart.Finally,the TOF candidate genes were identified.Results:Eight hub genes were identified using WGCNA network and PPI network,and functional enrichment analysis found that GJA1,RUNX2,PTK7,PRICKLE1 and SFRP1 were involved in morphogenesis of an epithelium,and dysregulation of the signaling were also found in the other two TOF datasets.Furthermore,the study found that the promoters of GJAI,RUNX2,PTK7 and PRICKLE1 genes were hypermethylated,and that GJA1 and SFRP1 are highly expressed in mouse second heart field cells and neural crest cells,and the latter is expressed in human embryonic outflow tract cells.Since RUNX2 was not expressed in human and mouse embryonic hearts,GJA1,PTK7,PRICKLE1 and SFRP1 were finally identified as TOF candidate genes.Conclusion:Based on the WGCNA network and various bioinformatics analysis methods,we screened 4 TOF candidate pathogenic genes,and found that the signaling pathways related to morphogenesis of an epithelium may be involved in the pathogenesis of TOF.