Human congenital cataract mutation in MYH9 alters F-actin organization and cell functions

To explore the role of a previously-found MYH9 tail domain mutation (p.E1384Q) in the pathogenesis of congenital cataract.

The cell experiments were conducted in vitro. Wild-type (WT) MYH9 and p.E1384Q mutant fragments were constructed, which was then transiently transfected into Hek293T cell lines. Western blotting and quantitative real time polymerase chain reaction (qRT-PCR) were used to analyze the protein and mRNA level of non-muscle myosin IIA (NM IIA) and F-actin in transfected cells, and fluorescence microscopy was applied to explore the subcellular localization of NM IIA and F-actin. ‌Cell counting kit-8 (CCK8), wound-healing and double staining flow cytometry assays were performed to evaluate the proliferation, migration and apoptosis function of transfected cells, respectively. Transmission electron microscope was conducted to observe the alteration of organelle structure.

The transiently-transfected WT and p.E1384Q mutant Hek293T cell lines was constructed. Western blot demonstrated that, comparing with MYH9 ^WT group, the relative protein amount of NM IIA and F-actin significantly decreased in MYH9 ^E1384Q cells (P<0.001). qRT-PCR analysis revealed that the relative mRNA amount of NM IIA and F-actin also significantly reduced in MYH9 ^E1384Q cells when compared with MYH9 ^WT. The immunofluorescence microscopy showed that the fluorescence signal of NM IIA and F-actin significantly decreased in E1384Q cells. The diffuse cytoplasmic distribution of NM IIA in MYH9 ^WT was changed to be clumped distribution, presenting a "speckled" pattern characterized by aggregates of small size in MYH9 ^E1384Q. Functional study revealed that the E1384Q mutation significantly inhibited cell proliferation (P=0.003) and migration (P<0.001), and promoted apoptosis (P<0.001). Electron microscope showed that the mutation remarkably decreased the number of mitochondria (P<0.001) and changed the phenotype of mitochondria.

The missense gene mutation in MYH9 (p.E1384Q) causing congenital cataract results in decreased amount and altered subcellular distribution of NM IIA and F-actin, accompanied by decreased cell proliferation and migration, promotes apoptosis and mitochondrial alteration.