Pathogenicity analysis of three SCA14-associated missense mutations in PRKCG gene of Chinese patients with ataxia

Abstract

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant disorder characterized by progressive cerebellar dysfunction and neurodegeneration. To date, it is rarely reported in China. SCA14 is caused by mutations in the PRKCG gene, which encodes protein kinase C gamma (PKCγ). Although nearly eighty distinct mutations of PRKCG gene have been identified, the pathological mechanisms of SCA14 remain unclear. In this study, we performed whole exome sequencing to screen causative genes in patients with unexplained progressive cerebellar ataxias, and identified three PRKCG mutations (c.302A > G, p.H101R, c.520C > G, p.H174D and c.2063C > G, p.P688R) that have not been previously reported in Chinese patients with SCA14. To explore the pathogenicity and function of these SCA14-associated PRKCG mutations, HEK293T and HeLa cells were transfected with the plasmids of empty vector, wild-type PRKCG and indicated PRKCG mutants. Protein stability, aggregation propensity, phosphorylation status, mitochondrial function and cytotoxicity were then measured. We found that H101R mutant PKCγ protein is unstable, prone to aggregate, exhibits reduced basal phosphorylation, and is resistant to agonist-mediated dephosphorylation. Also, H101R mutant PKCγ protein could result in increased apoptosis and reduced cell viability. These findings are similar to other pathogenic mutations. Additionally, cellular mitochondrial dysfunction was observed for the first time in cells expressing mutant PKCγ. Together, we identified three PRKCG mutations, expanding the mutation spectrum of PRKCG in China. The c.302A > G, p.H101R variant is pathogenic and mitochondrial dysfunction is suggested involved in the pathogenesis of SCA14.