Whole Exome Sequencing Identifies PTPN21 As A Novel Candidate Gene For Congenital Heart Disease And Its Mechanism Research

Objective:Congenital heart disease refers to cardiovascular malformations caused by abnormal development of the cardiovascular system during embryonic development.At present,the mainstream view is that the interaction between environment and genetics plays an important role in the pathogenesis of congenital heart disease,but there are differences between specific cases,and the genetic mechanism is still unknown in 80% of cases.Understanding the genetic mechanism of congenital heart disease may provide important targets for prenatal diagnosis,early intervention,and even gene therapy.In the early stage of the project,a pedigree with secundum atrial septal defect combined with pulmonary valve stenosis was included,and a novel candidate gene PTPN21 was detected by whole exome sequencing and bioinformatics analysis.This thesis will further validate and analyze the PTPN21 mutation carried by the pedigree,and explore the relationship between PTPN21 and heart development or congenital heart disease at the population level,the cell level,and the animal level.Materials and Methods:1.Validated whether each member of the pedigree had carried the PTPN21 frameshift mutation by polyacrylamide gel electrophoresis and Sanger sequencing.Analyzed the conservation of the PTPN21 mutation site and the domain of PTPN21 protein and its mutant with NCBI;2.Included 20 children with secundum atrial septal defect combined with pulmonary valve stenosis.Detected harmful mutations of PTPN21 in sporadic cases by whole exome sequencing and bioinformatics analysis.Validated the harmful mutation of PTPN21 by Sanger sequencing.Analyzed the conservation of the harmful mutation site of PTPN21 with NCBI;3.Used the primary human pulmonary artery endothelial cells（HPAEC）as the object,silenced PTPN21 expression with siRNA,and detected the knockdown efficiency by RT-q PCR and Western blot.Detected cell proliferation after siRNA transfection by CCK8,and detected cell migration after siRNA transfection with scratch test.Explored the differentially expressed genes and signal pathways related to heart development by mRNA sequencing and GO,KEGG analysis;4.Analyzed the feasibility of using mice to construct model animals: compared the mouse and the human PTPN21 protein sequence with NCBI,detected the expression of Ptpn21 mRNA in mouse heart from wild-type embryo to postnatal day by RT-q PCR,and analyzed the domain of mouse PTPN21 mutant.Constructed gene knock-in mice with CRISPR/Cas9 in collaboration with Professor Gao Fei from the Institute of Zoology,Chinese Academy of Sciences.Designed primers to identify the mouse genotype,and measured the oxygen uptake rate of mutant mouse and wild type mouse with measurement device.Results:1.Polyacrylamide gel electrophoresis and Sanger sequencing confirmed that all diseased members of the pedigree carried the PTPN21 frameshift mutation（c.2345₂346ins CCGCAGA）,which was a heterozygous mutation,and all healthy members did not carry this mutation.The validation result was consistent with the result of whole exome sequencing.Further analysis showed that the mutation initiation site 782 E was highly conserved in 8 representative species.PTPN21 frameshift mutation resulted in a premature stop codon,leading to the loss of the catalytic domain PTP_DSP_Cys;2.A harmful heterozygous PTPN21 mutation（c.G984 C,p.R328S）was detected in the sporadic case AP4 among 20 sporadic cases by whole exome sequencing and bioinformatics analysis.The validation result of Sanger sequencing was consistent with the result of whole exome sequencing.Further analysis showed that the mutation site 328 R was highly conserved in 10 representative species.The harmful mutation frequency of PTPN21 in the included sporadic cases with atrial septal defect combined with pulmonary valve stenosis was 5%;3.After siRNA transfection,compared with the control group（NC group）,the expression level of PTPN21 mRNA in the experimental group（Si group）decreased by 44.38±4.18%（P<0.0005）,and the expression level of PTPN21 protein in Si group decreased by 44.89±15.40%（P<0.05）.The result of CCK8 experiment showed that the proliferation of Si group at 48 h and 72 h after transfection was significantly higher than that of NC group respectively（P<0.0005）.The result of scratch experiment showed that the relative mobility of Si group was significantly lower than that of NC group at 24 h after scratching（P<0.0001）.The mRNA sequencing showed that PTPN21 silencing had changed the expression level of 24 genes related to heart development,and showed that GO and KEGG pathway related to heart development were significantly enriched;4.Using mice as the experimental animal to simulate PTPN21 frameshift mutation in the pedigree was feasible,based on: the mouse and the human PTPN21 protein had the same domains and they were highly homologous,the Ptpn21 mRNA expression peaked at E12.5 in embryonic mouse heart（E12.5 is the key time point for the development of the secundum atrial septum and pulmonary valve）,and simulating PTPN21 mutation of the pedigree in the mouse would also result in a premature stop codon,leading to the loss of the catalytic domain PTP_DSP_Cys.After constructing the Ptpn21 frameshift mutant mice by CRISPR/Cas9,it was found that the oxygen uptake rate of homozygous mutant mouse（0.038±0.005）was lower than that of wild type mouse（0.038±0.005）（P<0.005）.Conclusion:1.The pedigree with secundum atrial septal defect combined with pulmonary valve stenosis carried the PTPN21 frameshift mutation（c.2345₂346ins CCGCAGA;p.E782fs）.This mutation would cause the loss of catalytic domain PTP_DSP_cys of PTPN21 protein,which was likely the genetic factor for congenital heart disease of the pedigree.2.The harmful mutation frequency of PTPN21 in the included sporadic cases with atrial septal defect combined with pulmonary valve stenosis 5%,which provided further evidence for PTPN21 to become a novel candidate gene for the screening,genetic counseling,diagnosis and treatment of congenital heart disease.3.PTPN21 silencing could promote the proliferation and inhibit the migration of endothelial cells,and induce the expression level changes of genes related to heart development,such as SMAD4,BMPR2,and WNT11.The disorder of endothelial cell proliferation and migration caused by PTPN21 was likely the pathological basis of secundum atrial septal defect and pulmonary valve stenosis.4.Using mice as the experimental animal to simulate PTPN21 frameshift mutation in the pedigree was feasible.After constructing Ptpn21 mutation knock-in mice with CRISPR/Cas9,it was found that the oxygen uptake rate of the mutant mouse was lower than that of the wild-type mouse,suggesting that the Ptpn21 mutation had an impact on the mouse cardiopulmonary function.