Identification Of TBX2 And TBX3 Variants In Conotruncal Heart Defect Patients And Study Of Rare Copy Number Variants Associated With Pulmonary Atresia With Ventricular Septal Defect

Objective: Conotruncal heart defects(CTDs)are heterogeneous congenital heart malformations and are chief causes of infant death and childhood disability.Increasing studies demonstrated that genetic factors played primary roles in pathogenesis of CTDs,but the genetic determinants underlying CTDs remain unclear.We investigated CTDs genetic pathogenesis based on a large population.Furthermore,the roles of rare copy number variants(CNVs)in the pathogenesis of pulmonary atresia with ventricular septal defect(PA-VSD)were explored.Methods: In the first part,we screened for TBX2 and TBX3 variants in a large cohort of CTDs patients by target sequencing and the highly conservative damaging variants were genetically analyzed the expression and function of these variants,such as quantitative RT-PCR,western blot,immunofluorescence assay and luciferase assays.In addition,we explored the interesting downstream genes of TBX2 and TBX3.In the second part,we investigated rare CNVs in unrelated patients with PA-VSD using whole-exome sequencing,and performed the analysis of transcriptome array and gene networks to indicate novel candidate genes of interest.Results: In the first part,our data suggested that m RNA and protein expressions of TBX2 and TBX3 variants were altered compared with those of the wild-type.We screened HAS2,PEA3 and MEF2 C as novel downstream genes of TBX2 and TBX3,respectively.Functional analysis revealed that TBX2R608 W and TBX2R616 Q variant proteins further activated HAS2 promoter but failed to activate PEA3 promoter,and that TBX3A192 T and TBX3A562 V variant proteins showed a reduced transcriptional activity over MEF2 C promoter.In the second part,three CNVs were considered pathogenic or potentially pathogenic to PA-VSD: 16p11.2 del,5q35.3 del,5p13.1del.The analysis of transcriptome array revealed that PPP4 C,FLT4 and RICTOR were also significantly expressed in human embryonic heart.Meanwhile,the gene networks showed that PPP4 C,FLT4 and RICTOR had strong interaction with well-known cardiac genes relevant to heart or blood vessel development.Conclution: Our results indicated that the variants of TBX2 and TBX3 contributed to CTDs etiology.Moreover,we provided new insights into understanding for the pathogenesis of PA-VSD and helped elucidate critical genes for PA-VSD.