Study On The Promoter And Interactional Protein Of TBX5

Congenital heart disease (CHD) is a common newborn defect, the morbidity of which is from 4% to 8%.The clinical data and epidemiological study both indicated that genetic factors play an important role in the pathogenesis of CHD, and the heritability is from 55% to 65%.Identifying genes involved in the cardiac development from the molecular level is very important to work out the mechanism of CHD and design a better therapeutic program. TBX5 is a transcription factor that plays a critical role during cardiac development. TBX5 gene was identified and cloned in 1997 from Holt-Oram syndrome (HOS) autosomal dominant inherited disease characterized by congenital heart defects and upper limb abnormalities. Previous studies have proved that TBX5 gene mutations can lead to HOS but it is still unknown whether mutations and abnormal expression of TBX5 gene may cause human simple congenital heart disease.We had detected mutation and expression of TBX5 gene in human simple congenital heart disease, but didn't found the mutation in 8 exons of TBX5 gene.However we found that the expression of TBX5 gene in human simple congenital heart disease declined, expecialy in ASD.Therefore, we investigated the mutation of the regulatory region(1200bp upstream of transcriptional start site ) and methylation situation of the promoter of TBX5 gene in human simple congenital heart disease and at the same time we used co-immunoprecipitation to gain the TBX5 interactional protein, which may establish foundation for understanding of heart development and etiological research of congenital heart disease. Methods1. Denatured High-Performance Liquid Chromatography (DHPLC) was used to detect mutations of regulatory sequence of TBX5 geneWe investigated the mutation in the regulation region (1200bp upstream of transcriptional start site) of TBX5 in 100 CHD patients .2. Methylation-sensitive restriction enzyme was used to detect methylation in promoter region of TBX5Using software, we predict two CpG island in the promoter region of TBX5, and both have CGCG repeats. Two primers were designed for these regions. Methylation sensitive restriction enzyme, Bstu I was then used to detect recognize specification methylation situation in 50 cases CHD patients.3. Using Immunoprecipitation to gain the interactional proteinNucleoprotein was extracted from 20 weeks fetal, TBX5 antibody was used to precipitate its interactional protein.Results1. Mutation screeningNo mutations were detected in 1200bp regulation region of TBX5 gene in all CHD patients.2. Methylation in promoter region of TBX5There were methylation in two CpG island in promoter region of TBX5 in 5 mormal heart tissue and 50 CHD heart tissue, no stastistical difference was found.3. Co-ImmunoprecipitationUsing Co-Immunoprecipitation we obtained a TBX5 interactional protein, which was confirm as MYH7 by mass spectrometric analysis.Conclusion1. Down-regulated of TBX5 gene in CHD patients may have no association with mutation in regulation region of TBX5 and methylation in promoter region of TBX5 gene.2. TBX5 and MYH7 have interaction in heart development , which may be take part in the CHD happened.