Functional analysis of missense mutations in GLI2 and GLI3 involved in congenital heart disease

Objective: In this study, we aimed to explore the biological functions of 10 rare case-specific missense mutations in GLI2 and 4 in GLI3, which were previously screened in a cohort of 412 patients with congenital heart disease (CHD) and 213 normal controls from Shandong Province, China. Methods: A dual-luciferase reporter assay was used to assess the effects of these mutations in GLI2 and GLI3 on the activity of the sonic Hedgehog signaling pathway in HEK293T cells. Differences in protein levels between mutant and wild-type GLI2 and GLI3 were detected in HEK293T cells using Western blotting. Results: The dual-luciferase reporter assay showed that compared to the wild-type GLI2 protein, p.A1113V significantly increased activation of the sonic Hedgehog signaling pathway, whereas p.H78P and p.I1451S did not have a significant effect. The other mutations largely reduced the activation effect. Compared with the wild-type GLI3 protein, only p.A286V, among the four mutations, significantly reduced the activation effect on the SHH signaling pathway. Western blotting data showed reduced expression of GLI2 p.G716V, GLI2 p.K736N, GLI2 p.I1451S, and GLI3 p.A286V, whereas the remaining mutations had no significant effects. Conclusion: The mutations GLI2 c.2147G>T (p.G716V), GLI2 c.2208G>C (p.K736N), and GLI3 c.857C>T (p.A286V) involved in CHD affect the regulation of the sonic Hedgehog signaling pathway; thus, these rare missense mutations in GLI2 and GLI3 might increase the risk of CHD.