Discovery And Functional Study Of The Candidate DYNC2LI1 For Transposition Of Great Arteries

Objective The study aimed to explore novel candidate genes for transposition of the great arteries by genetic analysis and functional experiments,and to investigate the correlation between the cilia gene DYNC2LI1 and transposition of the great arteries,and explore the key role of the gene in heart development and outflow tract remodeling.Methods Whole exome sequencing was performed on 381 patients with sporadic translocation of the great arteries,and 125,748 individuals from the gnomAD database constituted a case-control study,and gene burden analysis was used to screen candidate genes.We carried out functional analysis for different mutations found in patients:in vitro expression plasmid was constructed and transfected into cells.Real-time quantitative PCR and Western blot were performed to detect the expression of DYNC2LI1 mRNA and protein in different mutants.The target gene was knocked down in NIH3T3 fibroblasts through siRNAs experiment,we extracted the mRNA and conducted pathway enrichment analysis on the expression profile.The Dync2li1 gene knockout mouse model was established to observe the embryonic heart phenotype and the morphology of cardiac ciliary,perform transcriptome sequencing on embryonic heart tissues of different genotypes and explore relevant molecules and signaling pathways..Results Compared with the population,the mutation burden of DYNC2LI1 gene in the transposition of the great artery group was significantly increased,suggesting that the mutation of DYNC2LI1 gene was necessarily related to the occurrence of the transposition of the great artery.Compared with NIH3T3 cells transfected with wild-type plasmids,the c.A239C,p.D80A mutation of Dync2li1 gene significantly reduced the expression levels of mRNA and protein,and the difference was statistically significant(P<0.0001:P<0.01).The mRNA expression level of c.T240A,p.D80E and c.T389C,p.L130R were also significantly decreased(P<0.0001;P<0.05),but there were no significant differences in protein expression(P>0.05).Analysis of the cell transcriptome after Dync2li1 knockdown revealed that genes for pseudopodium assembly and cell shape were significantly upregulated;genes related to pathways such as outflow tract and ventricular septum morphogenesis,and axis specification were down-regulated.The homozygous Dync21i1 knockout mice died before the embryonic stage about 11.5 days.The embryos of heterozygous and homozygous Dync2li1 knockout mice showed different degrees of growth and development defects,such as reversal of heart looping,and ballooning of the pericardial sac,neural tube closure defects and axial rotation defects.Dync2li1 knockout mice had shorter cilia in embryonic heart tissue.The transcriptome sequencing results of the heart tissues of different genotypes of embryos showed that the knockout of Dync2li1 affected the regulation of cardiac tissue metabolism and other biological processes,as well as the structure of cilia and the transport process involved in vesicles.Conclusions The DYNC2LI1 gene,which is related to ciliary structure and function,is a potential pathogenic gene for transposition of the great arteries.