An Exploratory Study On The Relationship Between NOTCH1 Compound Heterozygosity And Tetralogy Of Fallot

Congenital heart disease?CHD?is an abnormal heart anatomical structure caused by the abnormality of the heart tube during the embryonic development,or the channel which should be automatically closed after birth is not closed.CHD is the most common heart disease in children and one of the leading causes of infant and child death worldwide.Epidemiological data show that the prevalence of CHD is about 0.6%¹.2%.The main subtypes of CHD including Ventricular septal defects?VSDs?,Atrial septal defects?ASDs?,Atrioventricular septal defects?AVSD?,Patent ductus arteriosus?PDA?,Tetralogy of Fallot?TOF?,Tight ventricular outflow tract?RVOT?,et al.TOF is one of the most serious cyanotic congenital heart disease.It is caused by abnormal development of conus arteries during embryonic period.The prevalence is estimated to be 1 in 3,000 live births worldwide,accounting for 5%of CHD.With the development of advanced medical technology,the early mortality rate of TOF is not high,but patients will be accompanied by long-term sequelae,such as arrhythmia,ventricular dysfunction and even permanent disability.The etiology of CHD is complex and generally considered to be related to genetic and environmental factors.The main environmental factors are maternal exposure during pregnancy,including harmful environmental exposure,drug exposure,and nutritional factors.A large number of genetic studies have confirmed that the occurrence of CHD is associated with genetic factors,including chromosomal abnormalities,single gene genetic defects,multiple gene defects,copy number variation and so on.As the most common cyanotic congenital heart disease,TOF is promoted by various conditions,chromosomal abnormalities such as 22q11.2 microdeletion?DiGeorge syndrome?and trisomy 21?Down syndrome?account for 15%and 7%of the incidence of TOF,respectively.Genetic variants such as TBX1,NKX2-5,GATA4,ZFPM2,GATA6,GDF1,and JAG1 would cause TOF.However,the genetic factors of most TOF patients is not clear.Previous studies have mostly focus on gene-coding regions,except for coding mutations,numerous researchs have reported that non-coding mutations in distal regulatory elements could be pathogenic factors,Smemo S,et al.demonstrated that mutations in TBX5 enhancer may cause CHD associated with TBX5 dysfunction,through a combination of genomics,bioinformatics,and genetically engineered mouse model.Sankaran VG,et al.studied three families with unusual patterns of hemoglobin expression,and identified an intergenic region near the?-globin gene which is necessary for fetal hemoglobin silencing.however,the potential role of non-coding mutations in complex human diseases?including congenital heart disease?remains poorly understood.With the development of non-coding region research projects such as the Roadmap Epigenomics Roadmap Project and the Encyclopedia of DNA Elements?ENCODE?,non-coding region mutations have received increasing attention recently.With the development of second-generation sequencing technology,the existing data can be used to study the new pathogenic genes and pathogenic sites of TOF through genome-wide sequencing?WGS?and bioinformatics tools,which can help reveal the molecular mechanism of TOF deeply.In this study,we performed whole genome sequencing?WGS?on two Non-syndromic TOF trios?two non-affected parents and one sick child?,and the data were analyzed through a screening procedure designed for the coding region,non-coding region and compound heterozygous mutation,to try to explore the genetic etiology of Non-syndromic tetralogy of Fallot.We characterized the annotated deleterious coding mutations,including de novo and inherited mutations,and impaired noncoding mutations in regulatory elements by various data analysis.It was found that there were no pathogenic mutations in the coding region that met the American Medical Genetics and Genomics?ACMG?guidelines?categorized as Tier 1?.Then we predicted deleterious coding mutations categorized as Tier 2,and identified potential deleterious mutations in the Integrated Methods for Predicting Enhancer Targets?IM-PET?annotated regulatory elements in the non-coding region.The composite heterozygous mutations in the same gene are grouped into Tier 3.The ToppGene software was used to prioritize the target genes of the complex heterozygous mutations.Finally a compound heterozygous pattern with pathogenic coding and noncoding mutations was identified in probands.In proband 1,biallelic mutations?chr9:g.139409115A>T,p.Asn685Ile;chr9:g.13944949 C>A?in NOTCH1exon and its regulatory element were detected.In proband 2,we also found compound heterozygousmutations?chr2:g.216235029C>T,p.Val2281Met;chr2:g.216525154A>C?which potentially regulated the function of FN1 gene.NOTCH1 is a classic causative gene of congenital heart disease.The functional effects of the NOTCH1 underlying regulatory regions and non-coding mutations were further verified through the CRISPR/Cas9 gene-editing system and dual-luciferase reporter gene assay.The results of CRISPR/Cas9-mediated genome editing experiments showed that the expression of NOTCH1 gene was significantly higher in heterozygous knockout and homozygous knockout HEK293T cell clones(Region^+/-,Region^-/-)than in wild-type clones(Region^+/+),indicating that this potential regulatory region functions as a silencer.Moreover,the expression of NOTCH1 in homozygous knockout clone(Region^-/-)was the highest,indicating that there was a certain dose-sensitive effect.The dual luciferase reporter gene assay showed that the level of luciferase activity of the plasmid carrying the non-coding mutant sequence was significantly lower than that of the wild-type,indicating that non-coding mutations in regulatory elements increase the activity of the silencer,resulting in the further decreased expression of NOTCH1.Combined with the CRISPR/Cas9 knockout results,it revealed that the noncoding mutation in the silencer region reduced the expression of NOTCH1.In summary,this study identified the variations in coding region and non-coding functional region by performing WGS of two Non-syndromic TOF trios.Trying to explore the relationship between the heterozygous genetic model of non-coding mutations/coding mutations and the pathogenesis of Non-syndromic TOF.Additionally,functional assays were conducted to validate the function of regulatory elements and noncoding mutations.The results indicated that the compound heterozygous pattern may increase the penetrance of TOF and provide a deeper understanding of TOF genetic architectures.