Abstract: Objective To screen disease-specific mutations in the coding region of TGFBR3 gene in congenital heart disease (CHD) and to study the biological functions of the mutations.Methods The study subjects were from Chinese Han population of Shandong province, composed of 404 sporadic CHD samples collected from CHD patients diagnosed during the period from Aug.2008 to Jan.2011. A total of 213 control samples were collected from healthy children without CHD or familial heart diseases in the same hospital during the same time period. The genomic DNAs were extracted from the peripheral blood of CHD and control samples and the target sequencing for the exons of TGFBR3 of the patients and controls was performed. Bioinformatics methods were used to screen case-specific new missense mutations or rare missense mutations, which were further verified by Sanger Sequencing. Wild-type and mutant TGFBR3 expression vectors were constructed and were then transfected into HEK293T cells for evaluating TGFBR3 protein levels by Western Blot and the effects of the mutants on TGF-β signaling pathway by luciferase reporter assay.Results Three disease-specific heterozygous mutations were identified from three different CHD patients separately, TGFBR3^K685R is a newly discovered mutation, while TGFBR3^A791V and TGFBR3^A804S were rare mutations and were predicted to be deleterious mutations by SIFT and PolyPhen-2 software. The protein levels of TGFBR3^K685R and TGFBR3^A804S mutants were significant lower than that of wild type TGFBR3 and all three mutations significantly impaired their function to inhibit TGF-β signaling.Conclusion Mutations TGFBR3^K685R, TGFBR3^A791V and TGFBR3^A804S may participate in the development of congenital heart disease by reducing their inhibition of TGF-β signaling.

Key words: Congenital heart disease, TGFBR3, Missense mutation, TGF-β pathway