The Role Of CTDSP2 In The Occurrence Of Congenital Heart Disease

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Screening of susceptible genes in congenital heart disease trios by whole exome sequencingObjective The second-generation sequencing technology was applied to sequence the families of children with congenital heart defect(CHD),we aimed to detect the rare mutations and determine the candidate genes for functional research.Methods All exomes were sequenced in clinically diagnosed families with CHD(a total of 29 members in 8 families,including 13 children with CHD).After compared with the public databases,the common mutations were filtered out,and the rare variants and genes carried by children with CHD were screened from the de novo model,the recessive model and the composite heterozygous model,respectively.The pathogenicity assessment,protein sequence conservation analysis of the variant functions,literature and database mining were used to identify candidate genes.The IMPC database retrieves the phenotype of candidate susceptibility knockout mice.The candidate gene's protein interaction network was constructed by STRING database.The expression of candidate genes was analyzed by transcriptome sequencing data of mouse heart development in ENCODE database.The expression of candidate genes in developing hearts was detected by immunofluorescence staining.Results A total of 42 candidate gene lists were obtained by whole exome sequencing in 8 CHD families.The IMPC database results suggest that CTDSP2 knockout mice have a phenotype associated with cardiac defects.Immunofluorescence staining reveals the protein of CTDSP2 was widely expressed in the fetal rat heart(E14.5).The protein interaction network shows that CTDSP2 is closely related to the Smad in the TGF? signaling pathway.We found that rare mutations in the CTDSP2 gene were identified in three children with CHD.The mutation p.R101 T is shared by two children with CHD in family 5,and the mutation p.V193 L and p.R205 H are present in family6.All three mutation are de novo mutations,and located in the FCP1 functional domain.Pathogenicity analysis indicates these three mutations are deleterious mutation.Sequence conservation analysis also suggests that the amino acid sequences of the three mutation sites are highly conserved between different species.The ENCODE database transcriptome data hints that CTDSP2 has its expression at different stages of mouse heart development(E10.5-D0).Conclusion We found that CTDSP2 was a new susceptibility gene for congenital heart disease by the second-generation sequencing,combined with database mining and histological analysis,and might participate in cardiac development through the TGF?/Smad pathway.Part ? Functional study of CTDSP2 in the process of endothelialmesenchymal transitionObjective Endothelial-to-mesenchymal transition(End MT)is the most characteristic event in cardiac development.This part of the experiment explored the role and influence of CTDSP2 gene in the process of endothelial mesenchymal transition.Methods Stimulation of human umbilical vein endothelial cells(HUVEC)by TGF? is a feasible method to simulate the endothelium-to-endothelial transformation process during cardiac development in vitro.si RNA technology was used to effectively knock down CTDSP2 in HUVEC cells,and the effect of CTDSP2 knockdown on HUVEC cell migration ability was detected by wound healing assay,invasion ability was detected by traswell assay.q RT-PCR was applied to detect the change of genes in HUVEC cells during End MT,and Western Blot assay was used to detect the change of Smad2 phosphorylation level.The CTDSP2 plasmid carrying the mutation site of CHD was constructed and overexpressed in HUVEC cells.The effect of mutation site on the phosphorylation of CTDSP2 was detected by Western Blot.The effect of CTDSP2 knockdown on the proliferation of HUVEC cells was examined by Ed U assay.The apoptosis of HUVEC cells was measured by Flow cytometry,and the expression of cleaved-caspase3 was detected by Western Blot.RNA-Seq was applied to show differential genes in the End MT process after CTDSP2 knockdown.Gene ontology and pathway function analysis were performed.The effect of CTDSP2 on endothelial mesenchymal transition was also investigated at the transcriptome level.Results Wound healing assay showed that the migration ability of HUVEC cells during End MT was weakened,and transwell assay showed that the invasive ability was also weakened.The expression of End MT factors(SNAI1,SLUG,TWIST1)was significantly decreased by q RT-PCR,suggesting that the effect of End MT process was weakened,which was consistent with the reduced phenotype of migration ability. Western Blot indicated that knockdown of CTDSP2 could elevate the phosphorylation level of Smad2 in TGF? pathway,overexpression of CTDSP2 reduced its phosphorylation level,and mutation p.R101 T attenuated the dephosphorylation level of CTDSP2.Ed U assay and Flow cytology apoptosis assay showed that the proliferation of HUVEC cells was weakened after CTDSP2 knockdown,the apoptosis rate increased,and the expression of cleaved-caspase3 was upregulated.RNA-Seq analysis revealed differential gene enriched in cell proliferation,cell adhesion,and extracellular matrix formation that are closely related to the End MT.Conclusion CTDSP2 knockdown impaired End MT process in endothelial cells,elevated phosphorylation level of Smad2 in TGF? signaling pathway,and impaired endothelial cell migration and invasion ability.The knockdown of CTDSP2 reduced endothelial cell proliferation and induced apoptosis.Significant changes in pathways such as cell proliferation,intercellular adhesion,and extracellular matrix formation were detected.The results suggested that CTDSP2 might be involved in the End MT process of endothelium,which is associated with the development of cardiac defects in knockout mice.Part ? The effect of CTDSP2 in Cardiac Differentiation of Human Induced Pluripotent Stem CellsObjective Human induced pluripotent stem cells(hi PSC)can be induced to differentiate into human cardiomyocytes in vitro.In this part of the study,we aimed to explore the role and influence of CTDSP2 in cardiac differentiation of human induced pluripotent stem cells.Methods SgRNA was designed online for the CTDSP2 CDS region based on CRISPR/Cas9 technology.And the truncation mutation was induced to knock-out the protein.The human induced pluripotent stem cell line ND2.0.6 from the i PSC Center of National Institutes of Health,National Heart,Lung,and Blood Institute,was selected for CRIPR/Cas9 editing.The monoclonal i PS cell line was screened and verified.Karyotype identified chromosome abnormalities and finally the CTDSP2 knockout i PS cell line was obtained.q RT-PCR was applied to detect the expression changes of cardiac specific transcription factors GATA4,NKX2-5,TBX3 and TBX5,as well as the expression changes of stem cell pluripotency genes OCT4,SOX2 and NANOG.The expression of NANOG was also detected by immunofluorescence.The normal group and the knockout group i PSC were simultaneously subjected to standardized differentiation induction for 10 days.The morphology and beat frequency of each componentized cardiomyocyte were evaluated.The expression of myocardial specific genes(c TNT,c TNI,MYH6,MYH7,MLC2 a and MLC2v)was measured by q RT-PCR.Results CRISPR/Cas9 generated the insertion of base A in CTDSP2 Exon5,resulting in a frameshift mutation and a truncation at the 124 th amino acid of the protein.Western Blot and Karyotype results indicated that CTDSP2 knockout i PSCs were successfully constructed.The q RT-PCR assay found that knockdown of CTDSP2 caused an upregulated expression in GATA4,NKX2-5,TBX3,and TBX5,with the expression of TBX5 increasing significantly.The q RT-PCR and immunofluorescence assay showed that NANOG expression was elevated,and there was no significant changes in OCT4 and SOX2.Furthermore,there was no significant difference in differentiation efficiency and myocardial cell morphology between the two groups.No significant changes in myocardial beat frequency and myocardial specific gene expression was observed.Conclusion Knockout of CTDSP2 resulted in increased expression of key transcription factors GATA4,NKX2-5,TBX3,TBX5 in heart development and stem cell pluripotency factor NANOG in i PSCs.Deletion of the CTDSP2 gene did not affect the differentiation of i PSCs into cardiomyocytes.