Functional analysis of variants found in classic galactosemia patients using a novel cell-free translation method.

Classic galactosemia is an autosomal recessive disorder caused by deleterious variants in the galactose-1-phosphate uridylyltransferase () gene. GALT enzyme deficiency leads to an increase in the levels of galactose and its metabolites in the blood causing neurodevelopmental and other clinical complications in affected individuals. Two variants NM_000155.3:c.347T>C (p.Leu116Pro) and NM_000155.3:c.533T>G (p.Met178Arg) were previously detected in Filipino patients. Here, we determine their functional effects on the GALT enzyme through analysis and a novel experimental approach using a HeLa-based cell-free protein expression system. Enzyme activity was not detected for the p.Leu116Pro protein variant, while only 4.5% of wild-type activity was detected for the p.Met178Arg protein variant. Computational analysis of the variants revealed destabilizing structural effects and suggested protein misfolding as the potential mechanism of enzymological impairment. Biochemical and computational data support the classification of p.Leu116Pro and p.Met178Arg variants as pathogenic. Moreover, the protein expression method developed has utility for future studies of variants.