Abstract: Objective To screen mutations in the coding region of DACT1 gene in patients with congenital heart disease (CHD) and to study their biological functions.Methods The subjects of study were from Chinese Han population in Shandong Province. CHD samples were collected from sporadic CHD patients diagnosed at the Cardiovascular Disease Institute in General Hospital of Jinan Military Region from Aug. 2008 to Jan. 2011. The samples collected from the patients with other syndromes or familial heart diseases were excluded. The control samples were collected from healthy children without CHD or familial heart diseases in the same hospital during the same time. ①Genomic DNAs were extracted from the CHD and control samples. The second-generation sequencing technology was used for target sequencing of 404 samples of CHD and 213 healthy controls for the exons of DACT1 gene. Bioinformatics methods were used to screen case-specific new missense mutations or rare missense mutations, which were further verified by Sanger Sequencing. ②The plasmids expressing wild type or mutant DACT1 genes were transfected into HEK293T cells and the effects of the mutants on the level of DACT1 protein were examined by Western blot. ③The effects of the mutations on canonical Wnt signaling and PCP signaling were evaluated by luciferase reporter assay.Results There were 404 CHD cases at the age of (2.9±2.7) years and 213 controls at the age of (7.1±3.7) years. The ratios of male to female for CHD and control samples were 1.2:1 and 1:1 respectively. Four novel or rare missen mutations, c.1486C>T (p.S441L), c.1598C>A (p.S478R), c.1659G>C (p.E499Q) and c.1891T>A (p.M576K), (thereafter called DACT1^S441L, DACT1^S478R, DACT1^E499Q and DACT1^M576K, respectively) were identified from three CHD samples. The four mutations were verified by Sanger sequencing. The mutations of DACT1^S478R and DACT1^E499Q occurred at highly conserved sites and the mutations of DACT1^S441L and DACT1^E499Q were predicted to be harmful. The four mutations have no effects on the protein levels of DACT1 mutant protein themselves, respectively. However, the mutation of DACT1^E499Q attenuated the degradation of DVL2 protein and reduced the inhibition of the canonical Wnt signaling pathway and PCP signaling pathway.Conclusion DACT1^E499Q mutation may contribute to the development of CHD by decreasing the inhibitation of Wnt signalings.

Key words: Congenital heart disease, DACT1, Missense mutation, Wnt pathway