Functional and In-silico analysis of ATP8A2 and other P4-ATPase variants associated with human genetic diseases.

P4-ATPases flip lipids from the exoplasmic to cytoplasmic leaflet of cell membranes, a property crucial for many biological processes. Mutations in P4-ATPases are associated with severe inherited and complex human disorders. We have determined the expression, localization, and ATPase activity of four variants in ATP8A2, the P4-ATPase associated with the neurodevelopmental disorder known as cerebellar ataxia, mental retardation, and disequilibrium syndrome 4 (CAMRQ4). Two variants, Gly447Arg and Ala772Pro, harboring mutations in catalytic domains, expressed at low levels and mislocalized in cells. In contrast the Glu459Gln variant in a flexible loop displayed wild-type expression levels, Golgi-endosome localization, and ATPase activity. The Arg1147Trp variant expressed at 50% wild-type levels, but showed normal localization and activity. These results indicate that the Gly447Arg and Ala772Pro mutations cause CAMRQ4 through protein misfolding. The Glu459Gln is unlikely causative, whereas the Arg1147Trp may display a milder disease phenotype. Using various programs that predict protein stability, we show that there is a good correlation between the experimental expression of the variants and in silico stability assessments suggesting that such analysis is useful in identifying misfolded disease-associated variants.