Molecular Mechanism Of 15q11.2 Microdeletion Leading To Abnormal Pulmonary Myocardial Dysplasia

AbjectiveTotal anomalous pulmonary venous connection(TAPVC)is a congenital cardiovascular disease,accounting for about 2% of congenital heart disease,and its pathogenic mechanism remains unknown.In this paper,TAPVC cases were tested for copy number variation,and the mechanism of TAPVC occurrence caused by copy number variation was further studied through a cell model to explain the occurrence of TAPVC from the perspective of copy number variation.Methods:1.231 TAPVC patients and 200 healthy control samples were detected by using CNVplex? technology to investigate the incidence of CNVs in TAPVC patients and its relationship with related clinical phenotypes was explored.2.iPS cell lines were induced by using iPS technology from 15q11.2 BP1-BP2 deleted TAPVC core family,a cell research model was established by inducing differentiation into cardiomyocytes in vitro with iPS.Transcriptome sequencing on cells during differentiation was performed to detect the signal pathways affected and the morphological function.Meanwhile,differentiation markers at every stage of cardiomyocytes were detected.The differences in morphology of cardiomyocytes,differentiation efficiency,expression levels and localization of markers at different stages of cardiomyocytes between patients and normal subjects were studied.3.Through the deletion of related genes in the 15q11.2 BP1-BP2 region,the effect of15q11.2 BP1-BP2 deletion on the differentiation and development of cardiomyocytes was studied,and the relationship between 15q11.2 BP1-BP2 deletion and TAPVC was clarified to enrich the understanding of the pathogenesis of TAPVC.Results:1.Among the 231 patients with TAPVC,22 patients carried copy number variations that could cause disease.Six cases of unknown clinically significant copy number variation were detected in 200 healthy controls.Statistical analysis shows that 15q11.2proximal microdeletions are more likely to occur in TAPVC patients.2.The iPS cell line model of TAPVC core family with 15q11.2 BP1-BP2 deletion was established by iPS technology.It was found that the iPSCs of proband with deletion had different markers and protein encoding genes in 15q11.2 region during myocardial differentiation.The expression levels,differentiation efficiency,myocardial cell morphology,beating frequency,and contraction strength were decreased compared to the normal control group,and the results are statistically different.Transcriptome sequencing has identified many differentially expressed genes in this family,and the results indicate that TAPVC pathogenic genes(such as PITX2,NKX2-5 and ANKRD1)were significantly overexpressed in the proband with TAPVC.3.The expression of TUBGCP5 and NIPA1 in the stem cell stage of the father's iPSCs washeterogeneous,and the iPSCs after TUBGCP5 knockout were differentiated at different stages of differentiation during the process of inducing differentiation of cardiomyocytes.Compared with the normal control group,the beat frequency and contraction intensity were decreased,and the results are statistically different.Conclusions:This study found that 15q11.2 microdeletions were significantly enriched in TAPVC patients.Cell model studies showed that the gene expression of 15q11.2 deletion carriers in TAPVC was significantly affected,the 15q11.2 deletion affected the expression of pathogenic genes for TAPVC(Such as PITX2,NKX2-5 and ANKRD1)during the differentiation of cardiomyocytes,these genes were highly expressed in the proband with TAPVC.It affected the normal differentiation of cardiomyocytes and was an important molecular mechanism leading to TAPVC.