Screening And Functional Analysis Of GATA-4 Mutations In Congential Cardiac Septal Defects

Congenital heart disease (CHD) are the most common developmental anomaly as well as the leading non-infectious causes of mortality in newborns with an estimated incidence of 4‰～50‰. More than 150 thousand live births with CHD were born each year in China, seriously harming infants' health.As a complex disease,CHD is due to the embryo's abnormal development that results from the interaction of genetic factor and environmental agent during the vulnerable period of cardiac development.CHD is a complex genetic disorder with heritability estimated from 55 % to 65 %,But the model of inheritance and penetrance of CHD is unclear,the predisposing genes of which is still unknown.With the development of molecular biology and molecular genetics, it will provide further insight into the development and mechanisms of congenital cardiovascular heart disease. Heart formation which integrates different structures and cell types is a complex process that involves a network of genes regulated by transcription factors. Proper spatiotemporal expression of these factors ensure the highly needed tight control of each step in organogenesis. A mistake at any step from cell-commitment to valve formation will have a major impact on heart morphogenesis and function leading to CHD. Then, special transcription factors have been become molecular hotspots of CHD. The transcription factors GATA-4,TBX5,ZIC3,NKX2.5,TFAP2B,TBX1 and FOG2 were associated with CHD; Furthermore, NKX2.5,TBX5 and GATA-4 were relative to cardiac septal defects(CSD).Recently, GATA-4 is one of the most important novel transcription factors in cardiac development process. The human GATA-4, which is located on chromosome 8p23.1-p22, spans 50kbp, and is composed of 442 amino acids with seven exons. GATA-4, one of the earliest markers of heart development , is known as a monogenic contributor to non-syndromic CHD along with NKX2.5,MYH6,NOTCH1 and others. GATA-4 mutations have found to be associated with atrioventricular septal defect, atrial septal defect, ventricular septal defect, patent ductus arteriosus,Tetralogy of Fallot, pulmonary stenosis, hypoplastic right ventricle, partial anomalous pulmonary venous connection,and so on. However, so far inconsistency has been shown to exist between GATA-4 gene mutation and its phenotype; the relationship between GATA-4 and other transcription factors and a network of genes regulated by transcription factors are uncertain.Therefore, further investigations are required. The present study aim to screen germline GATA-4 mutations in congenital CSD, The study of transcription factor GATA-4 mutant function provided information for its roles in CHD development. Such understanding promises to lead to improved early diagnosis and novel therapeutic strategies.Part 1 Analysis of GATA-4 mutations in Han ancestry patients with congenital cardiac septal defectsObjective: To elucidate the mutations of GATA-4 gene and to analyze the correlation between genotype and phenotype in Han ancestry patients with congenital atrial and ventricular septal defects.Methods: Fifty Han ancestry patients with congenital atrial and ventricular septal defects and 100 normal subjects of the same ethnical background were studied. A total of six exons and the intron-exon boundaries of GATA-4 were amplified by the polymerase chain reaction(PCR). The PCR products were purified and directly sequenced with an ABI PRISM 3730 Automatic DNA Sequencer. Using bioinformatic methods predicts whether GATA-4 gene point mutation affects its protein function.Results:1. rs1139244,rs1062215 and rs804280 were found, which were reported in the SNP database. Interestingly, we found that a G→A transition at nucleotide 196 in the GATA-4 gene was predicted to result in a substitution of alanine for threonine at codon 66 (Ala66Thr), whereas a G→C transversion at nucleotide 196 in the GATA-4 gene was predicted to result in a substitution of alanine for proline at codon 66 (Ala66Pro), which was reported in the SNP database (c.196G>C as rs1139244).2. Two novel heterozygous mutations were discovered in the GATA-4 gene, His28Tyr in exon 2 and His436Tyr in exon 7 of GATA-4 respectively, which were neither found in the control population nor reported in the SNP database.3. Alignment of GATA-4 gene amino acid sequences indicated Histidine residues at position 28 and 436 were highlighted.4. Two novel heterozygous GATA-4 mutations (His28Tyr and His436Tyr) are both expected to affect its protein function by SIFT and PolyPhen programs.Conclusions: Our findings suggest that the mutations of GATA-4 gene may be related to congenital atrial and ventricular septal defects in Han ancestry patients.Part2 Functional analysis of GATA-4 mutations in Han ancestry Patients with Congenital Cardiac Septal DefectsObjective: To explore the functional analysis of two novel mutations of GATA-4 gene in Han ancestry patients with congenital atrial and ventricular septal defects.Methods: Expression mutation vector in the GATA-4 gene was introduced into the wild type construct by Quik Change?Lightning site-directed mutagenesis Kit and verified by DNA sequencing. DNA for GATA-4-wt or GATA-4-mut were cotransfected Hela cells with DNA for the ANF luciferase reporter gene in vitro; Luciferase activity was measured by the Lumat LB 9507 48h after transient transfection.Results:1. Successful incorporation of the mutation GATA-4 expression vector, confirmed by automated sequencing bidirectionally.2. Relative Luciferase activity assays in Hela cells cotransfected with different plasmids. Mutation His28Tyr was (112±6.9)% with ANF luciferase reporter when compared to wild-type (P-value > 0.05); Nevertheless, mutation His436Tyr was (53.8±6.6)% with ANF luciferase reporter when compared to wild-type (P-value < 0.01).Conclusions:1. Successful incorporation of the mutation GATA-4 expression vector.2. The mutation c.82C→T of GATA-4 gene didn't affect its transcriptional activation of downstream target genes;Whereas, the mutation c.1306C→T of GATA-4 gene impaired the activation of the downstream target, which suggested that His436Tyr mutation in the C-terminal region of GATA-4 prevented its biological function.