Identification And Functional Study Of Connexin45 Mutation Associated With Congenital Heart Disease

Background: Congenital heart disease(CHD)is a developmental defect in the structures of the heart and/or endothoracic major blood vessels,which is the most common type of birth malformation,accounting for about one-third of all major congenital anomalies.Genetic research shows that genetic risk factors are among the major causes of its pathogenesis.However,the molecular genetic mechanisms involved in the pathogenesis of CHD are still largely unknown.Previous studies have demonstrated that connexin(CX)is closely related to heart development.There are three main kinds of CX in human myocardium,encompassing CX40,CX43 and CX45,and to date it has been found that CX40 mutations are closely associated with CHD.Given that CX45 is the first CX gene expressed during embryonic cardiogenesis,and mice with deletion of CX45 are susceptible to CHD,it is justifiable to make a hypothesis that CX45 may be one of causative genes responsible for CHD.Objective: The study aimed at identifying a novel CX45 mutation associated with CHD,and revealing the molecular mechanism by which the mutation leads to CHD.Methods: A cohort of 162 unrelated patients with CHD and 300 unrelated,healthy individuals used as the controls were recruited.Clinical data and peripheral blood samples were collected,and genomic DNA was extracted from peripheral leucocytes.CX45 gene was screened for a novel mutation by direct polymerase chain reaction(PCR)-sequencing.A CX45 sequence mutation could be discovered by comparing the acquired CX45 sequences with those published in Gen Bank.The frequencies of CX45 mutation in CHD patients and healthy control individuals were analyzed,and alignment of multiple CX45 proteins across different species was performed by computer software in order to show whether the altered amino acid was conserved evolutionarily.The disease-causing potential of a CX45 mutation was predicted by online computer programs.The wild-type CX45 gene was cloned and the mutant CX45 gene was obtained by using site-directed mutagenesis technology.The eukaryotic expression vectors of wild-type CX45 gene and mutant CX45 gene were constructed,which were transfected into tool cells.Electrophysiological recordings bypatch clamp and laser scanning confocal microscopy were used to analyze the impact of the CX45 mutation on the electrophysiological characteristics of the gap junction and to evaluate the permeability of the gap junction to uncover the molecular mechanism by which the CX45 mutation predisposes to CHD.Results: A novel heterozygous missense mutation in the CX45 gene was detected in a patient with a ventricular septal defect.The mutation led to a substitution of glycine for arginine at amino acid position 184 of the CX45 protein(p.R184G),which was absent in 300 healthy control individuals.Alignment of multiple CX45 protein sequences across various species revealed that the arginine at amino acid 184 of CX45 was highly conserved in evolution process.The detected CX45 mutation was predicted to be pathogenic by on-line computer softwares.Studies by patch clamp revealed that this CX45 mutation resulted in impaired electrical current conduction of CX45 gap junction channels,however,didn't affect the permeability of the gap junction channels.Conclusions: A novel heterozygous missense mutations(p.R184G)in the CX45 gene is related to CHD.The novel loss-of-function mutation of CX45 discovered in this present study maybe an uncommon molecular etiology accounting for CHD.