Identification And Function Of Novel HAND1 Gene Mutations Associated With Congenital Ventricular Septal Defect

Background: Congenital heart disease(CHD)is a developmental defect in the structures of the cardiovascular system,the most common type of birth malformation,which accounts for about one-third of all major congenital anomalies.Genetic studies find that genetic risk factors are the major causes of its pathogenesis.However,the genetic mechanisms involved in the pathogenesis of CHD are still largely unknown.Previous researches have demonstrated that HAND1 is related to heart development.To date it has been found that HAND1 mutations lead to CHD,it is justifiable to make a hypothesis that HAND1 mutation may be one of causative genes responsible for CHD.Objective: The study aimed at identifying a novel HAND1 mutation associated with VSD,and revealing the molecular genetics mechanism by which the mutation leads to CHD.Methods: A cohort of 125 unrelated patients with ventricular septal defect(VSD),and 210 unrelated,healthy individuals used as the controls were recruited.Peripheral blood samples and clinical data were collected,and the genomic DNA was extracted from peripheral leucocytes.By direct polymerase chain reaction(PCR)-sequencing,HAND1 gene was screened for a novel mutation.A HAND1 sequence mutation could be discovered comparing the acquired HAND1 sequences with those published in GenBank.The frequencies of HAND1 mutation in healthy control individuals and CHD patients were analyzed,and alignment of multiple HAND1 proteins across different species was carried out by computer software in order to find whether the altered amino acid was conserved evolutionarily.The disease-causing potential of a HAND1 mutation was predicted by online computer software programs.The wild-type HAND1 gene was cloned and the mutant HAND1 gene was obtained using site-directed mutagenesis technology.The eukaryotic expression vectors of wild-type HAND1 gene and mutant HAND1 gene were constructed,which were transfected into tool cells.The functional characteristics of the mutant HAND1 were analyzed by a dual-luciferase reporter assay system.Results: A novel heterozygous nonsense mutation in the HAND1 gene was detected in a patient with VSD.The mutation led to a substitution of termination codon for glutamate at amino acid position 119 of the HAND1 protein(p.E119X),which was absent in 210 healthy control individuals.Alignment of multiple HAND1 protein sequences across various species revealed that the glutamate at amino acid 119 of HAND1 was highly conserved in evolution process.The detected HAND1 mutation was predicted to be pathogenic by on-line computer softwares.Functional analysis revealed that the mutant HAND1 lost the ability to transactivate a target gene.Conclusions: A novel heterozygous nonsense mutation(p.E119X)in the HAND1 gene is related to VSD.The novel loss-of-function mutation of HAND1 discovered in this this study maybe an uncommon molecular genetics etiology accounting for VSD.