Association Analysis Of CSNP And Studies On Mutation And Expression Of GATA4 Gene In CHD

ObjectivesCongenital heart disease ( CHD) is an inborn abnormality seriously harming infants'health. The morbidity is from 0. 3% to 1% in live births while up to 10. 2 in abortus. As a complex disease, CHD is due to the embryo & abnormal development that results from the interaction of genetic factor and environmental agent during the vulnerable period of cardiac development. GATA4 is one of the transcription factors closely related to heart development. In 1995, GATA4 gene was located on human chromosome 8. In 1996, it's further located to 8p23. 1 -p22 by fluorescence in situ hybridization (FISH). Some researches showed that several kinds of meaningful mutations in GATA4 gene were discovered in simple CHD pedigrees. We selected four cSNPs (Coding -region single nucleotide polymorphisms) that could result in changing of amino acids in coding region of GATA4 gene and performed cSNP association analysis and haplotyping in 62 Congenital heart disease nuclear pedigree. It was also studied that the mutation and expression of GATA4 gene in simple CHD. We hoped to find the potential mechanism of GATA4 gene causing the CHD.Methods1. We selected four cSNPs that could result in changing of amino acids in SNP database and performed PCR - RFLP in 186 members from 62 Congenital heart disease nuclear pedigree.2. The Association analysis of individual SNP GATA4 with simple CHD was analyzed using ETDT software.3. Haplotype was built utilizing TRANSMIT software and whether there wasdifference in haplotype frequency or not was statistically analyzed.4. Seven exons primers of GATA4 gene were designed and a [GC] clamp was added to the upper primer 5' And the mutations of GATA4 gene were examined in 66 CHD patients by polymerase chain reaction - denaturing gradient gel electrophoresis (PCR - DGGE).5. Using non -CHD patients'myocardium as normal control and (3 - actin as internal control, the expression of GATA4 genes mRNA level in myocardiums of 41 simple CHD patients (including ASD, VSD and F4) was detected by reverse transcription - polymerase chain reaction ( RT — PCR ).ResultsPolymorphism was not be detected at rs 1139240 among the four cSNP. Significant association with CHD was observed at rsl 139241, rsl 139244 and rs3729856. Linkage disequilibrium analysis showed that there existed linkage disequilibrium between rsl 139241 and rsl 139244. However, the further haplotype analysis was of no significance ( P <0. 05 ).No mutations were detected in 7 exons of GATA4 gene in all 66 samples by PCR - DGGE. The mRNA expression levels of GATA4 gene in simple CHD patients showed descending tendency compared with normal controls.ConclusionPolymorphism was not detected at rsl 139240 among the Northeastern People of China. There was significant association of rsl 139241, rsl 139244 and rs3729856 with simple CHD.Mutations in coding region of GATA4 gene was not found in our selected simple CHD samples.The transcriptional abnormality of GATA4 gene may be a potential mechanism causing human simple CHD.