Identification And Functional Analysis Of A Novel NOTCH1 Mutation Causing Left-sided Congenital Heart Defects In Fetus

Background and Objective:Congenital structural abnormalities play an important role in perinatal fetal death.Congenital heart disease（CHD）is the most common congenital malformation and significantly causes mortality of infants worldwide.Congenital structural abnormalities are mostly caused by genetic or environmental factors.Genetic factors generally contain chromosomal aberrations,copy number variants,and gene sequence variants.However,with the use of karyotype and chromosomal microarray analysis,the etiology of more than 50%of the fetuses with sonographic abnormalities remains unknown.Whole exome sequencing has been gradually applied in practice to further detect the causative genes for genetic diseases.Left-sided congenital heart defects（LS-CHD）encompass a group of heritable malformations including bicuspid aortic valve,coarctation of the aorta,and hypoplastic left heart syndrome.The clinical manifestation and exact inherited pathogenesis of LS-CHD remain elusive.The NOTCH signaling pathway plays a crucial role in heart development and NOTCH1 haploinsufficiency or reduced dosage caused by variants in genes can lead to the occurrence of LS-CHD.The penetrance of cardiac malformations in NOTCH1 mutated carriers is incomplete with variable expressivity.The purpose of this study was to explore the application value of prenatal trio-whole exome sequencing technology in structural-abnormal fetuses with normal karyotype and CMA results.We also analyze the clinical features and molecular mechanisms related to genetic mutation in a family affected with LS-CHD,which facilitates genetic counseling.Methods:1.A total of 81 congenital structural-abnormal fetuses with normal karyotype and CMA results and their parents were received from the Department of prenatal diagnosis in Guangdong General Hospital during December 2018 through January2022.The Trio-WES was used to access the prevalence of the monogenic diseases in different groups divided according to the affected organs,and the inheritance pattern of variants was analyzed.2.We report the members of a nuclear pedigree affected by LS-CHD.The clinical features and cardiac sonographic results of the members were collected.Whole exome sequencing was carried out to identify the variants associated with clinical expression.Sanger sequencing was performed to confirm the validation of candidate variants identified by Trio-WES.3.We further evaluate the effect of NOTCH1 novel mutation on LS-CHD in vitro.The wild-type Notch1 or mutant Notch1^Q1864*vectors were inserted into p B513B and then transfected into 293T cells.We examine the impact of NOTCH1 mutation on NOTCH signaling activity,downstream signaling and EMT process to demonstrate a potential pathogenic effect.Results:1.18 of 88 fetuses were detected with positive molecular diagnosis.Trio-WES provides an additional diagnostic yield of 22.2%in fetus with structural anomalies.The positive diagnosis rates varied between different phenotypic groups,with the highest genetic diagnostic yield in the group of cardiovascular abnormalities（25%）,especially complex cardiac malformations（26.8%）.Autosomal dominant disease（15/18）accounted for most of the overall 18 positive diagnoses.2.The proband with complex cardiac malformations harbored a heterozygous variant in NOTCH1（NM＿017617）gene were identified by Trio-WES.A novel nonsense variant of NOTCH1 gene（c.5590C>T（p.Q1864*））was detected,which was inherited from its BAV mother.The loss of function mutated amino acid locates in a conserved region of NOTCH1.The c.5590C>T（p.Q1864*）was classified as likely pathogenic（PVS1,PM2）based on the ACMG Standards and Guidelines.3.The expression level of NOTCH1 and its“canonical Notch1 pathway”cleaved-NICD were much lower in Notch1^Q1864*-transfected cells compared to the Notch1^WT-transfected cells.And the canonical NOTCH1 signaling was affected and the expression level of downstream target genes was markedly reduced in the cells transfected with Notch1^Q1864*mutant vectors.Conclusion:1.Prenatal Trio-WES technology plays an important role in structural-abnormal fetuses with negative conventional genetic test results and the positive additional diagnosis rates are high,which varied between different phenotypic groups.2.Notch1^Q1864*mutation contributes to the LS-CHD phenotype beyond valve formation and negatively impacts cardiogenesis.3.Novel Notch1^Q1864*nonsense mutation reduced canonical NOTCH1 signaling and the expression level of downstream target genes,as well as caused reduced status of EMT.