Mutation Analysis On DNAI1 And DNAH5 Gene In Children With Heterotaxy Syndrome And The Role Of MMP21 Mutation In The Pathogenesis Of Heterotaxy

Congenital heart disease(CHD)is one of the most common congenital malformations in live births.Complex congenital heart malformations,such as double outlet of right ventricle(DORV),transposition of great artery(TGA),pulmonary atresia/ventricular septal defect(PA/VSD),are important causes of high mortality in neonates,and are also one of the hotspots of current research.Heterotaxy syndrome is often associated with complex congenital heart malformation,which affects the prognosis of surgery.The normal visceral position is determined by the asymmetric differentiation of the embryonic body left-right axis,and is regulated meticulously by many genes during differentiation.Heterotaxy Syndrome Part I.Mutation analysis on DNAI1 and DNAH5 gene in Chinese Han Children withObjective To analysis the role of DNAI1 and DNAH5 on the pathogenesis of heterotaxy syndrome in Chinese Han patients.Methods 83 children with confirmed heterotaxy and 92 normal children with outpatient health examination were recruited.DNA was extracted from peripheral leukocyte cells for whole exome sequencing.The mutations of DNAI1 and DNAH5 genes were detected and validated by Sanger sequencing.The affection of the mutations on wild-type protein was analyzed by in silico softwares.Results After excluding 2 unqualified data from patient group,170 exome sequencing data were used to analysis.3 DNAI1 and 3 DNAH5 mutations were found in the patients group,with the frequencies of 4.94%(4/81)and 2.50%(2/81)in patients group,respectively.Neither of the mutations was found in the control group.Bioinformatic analysis suggests that these mutations may affect wild-type protein function.Conclusion DNAI1 and DNAH5 gene mutations may be associated with heterotaxy syndrome in Chinese Han children.Part II.To explore the role of matrix metalloproteinase 21 gene(MMP21)in the pathogenesis of HeterotaxyObjective To explore the role of matrix metalloproteinase 21 gene(MMP21)in the pathogenesis of Heterotaxy.Methods 83 children with identified heterotaxy syndrome and 92 normal children with outpatient physical examination were recruited.DNA was extracted from peripheral leukocyte cells for whole exome sequencing.The mutations of MMP21 were detected and validated by Sanger sequencing.The affection of the mutations on wild-type protein was analyzed by in silico softwares.Cell transfection was used to analyze the changes of the expression of the mutant m RNA and protein.Using splice-blocking Morpholino(MO)to knock down the expression of MMP21 in zebrafish,and the wild-type or mutant human m RNA were used to rescue the morphant.Results Among the exon sequencing results of 81 patients,three MMP21 mutant sites c.G731A(p.G244E),c.C829T(p.L277F)and c.A1459G(p.K487E)were detected.Bioinformatics software predicted that both mutations could affect protein function.The transfection experiment indicated that the expression level of mutant MMP21 mRNA decreased,but the protein level did not decrease significantly.Zebrafish experiment suggested that most MMP21 morphant embryos exhibited deformity of left-right axes differentiation,which was significantly different from the control group(p<0.0001),and mutant RNA c.G731 A and c.A1459 G could not effectively rescue the deformity phenotype caused by MO.Conclusion MMP21 mutations C.G731A(p.G244E),C.C829T(p.L277F)and C.A1459G(p.K487E)can lead to the decline of protein function and the occurrence of heterotaxy.