Mechanism Of Outflow Tract Malformation And Development

Cardiac outflow tract malformation is one of the most common congenital heart diseases,caused by abnormal development of the outflow tract,which is threatened the life of human beings seriously.Abnormal separation and remodeling of the outflow tract leads to most of the structural malformations of the outflow tract,such as tetralogy of Fallot,transposition of the great arteries(TGA)and double outlet of the right ventricle.Both environmental factors and genetic factors could be related to the outflow tract malformation.However,the mechanisms leading to the occurrence of outflow tract malformation is not clear.The study of cell diversity,differentiation trajectory and cellular regulatory network during the development of the normal outflow tract is very important in dissecting the etiology of outflow tract malformation.Therefore,a cohort of patients with TGA was used to study the genetic etiology in human sample.Besides,our study intends to analyze the development process of the outflow tract and its key cell pedigree using single-cell RNA sequencing technology comprehensively and unbiasedly.In the study of genetic etiology of human outflow tract,we recruited 249 patients with TGA(66 trios)and performed the whole exome sequence.The occurrence rate of patent ductus arteriosus in complete TGA was significantly higher than other subtypes(52.7%).And the occurrence rate of dextrocardia/mesocardia in congenital corrected TGA was significantly higher than other subtypes(32.8%).The prevalence of bicuspid pulmonic valve was high in patients with TGA(9.6%).Through a series of bioinformatics screening,we obtained 82 candidate genes which contain loss of function,de novo,compound heterozygosity or X-linked variants.Some causal genes of congenital heart disease were identified,such as FOXH1 and CITED2.Interestingly,we also found 19 cilia related genes that contain rare,potentially destructive mutations,for example,DYNC2L11 containing de novo variants.The significantly enrichment of cilia genes demonstrates the role of cilia in the pathogenesis of TGA.Overall,up to 33%of TGA probands carried one more potentially damaging variation,indicating that there may be oligogenic or polygenic inheritance patterns in some patients of TGA.Next,we sequenced 55,611 single cells of three key stages of outflow tract development and found 17 cell subtypes and 6 cell lineages.The endothelial-mesenchymal transition was observed which had been reported in the literature.Our study further reported both cardiomyocytes and mesenchyme could be transformed into vascular smooth muscle cell(VSMC)for the first time.It showed that one cluster of mesenchymal cells(Penk+)was undergoing a transition from mesenchyme to VSMC,which was related to the fusion of outflow tract.Then,we reveal the dynamics of potential cellular state transition and key transcriptional regulators through pseudo-time series analysis.Cardiac neural crest cells(CNCC)played an important role in the development of outflow tract.Ablation of CNCC caused a variety of outflow tract deformities.To study the contribution of CNCC to the heart and its developmental process,we sequenced the single-cell transcriptome of 34,131 CNCC-derived cells from 8 different developmental stages:E10.5 to postnatal day 7.Single-cell transcriptome analysis and single-molecule fluorescence in situ hybridization confirmed the existence of CNCC derived mural cells.Besides,we found the transition from CNCC-derived pericytes to micro-VSMC,and identified the regulatory genes in the transformation process by pseudo-time analysis.Our data showed that CNCC derivatives have differentiate into specific lineages when they migrate to the heart.It was also observed that the molecular characteristics of the neural crest were gradually lost with the development of CNCC derivatives.Finally,we describe some special subgroups of CNCC derivatives during their development.For example,Penk+ cells are mainly occupied in the cushions of aorticopulmonary septum,and they were all derived from CNCC.The analysis of clinical characteristics of human outflow tract defect TGA is of great significant for the stratification of patients with TGA.The results of genetic analysis highlight the pathogenicity of cilia related genes and the complex genetic structure behind TGA.We hope our results can help to improve the prenatal diagnosis and treatment management of patients with TGA.We described a reference landscape of the normal development of outflow tract and CNCC,and demonstrate the multi-original developmental characteristics of the VSMC in the root of the great artery and the existence of myocardial trans-differentiation into VSMC for the first time.These findings provided an important resource for the research of congenital heart disease,which were of great importance in cell therapy of congenital heart diseases.