Identification and functional analysis of three distinct mutations in the human galactose-1-phosphate uridyltransferase gene associated with galactosemia in a single family.

We have identified three mutations associated with transferase-deficiency galactosemia in a three-generation family including affected members in two generations and have modeled all three mutations in a yeast-expression system. A sequence of pedigree, biochemical, and molecular analyses of the galactose-1-phosphate uridyltransferase (GALT) enzyme and genetic locus in both affected and carrier individuals revealed three distinct base substitutions in this family, two (Q188R and S135L) that had been reported previously and one (V151A) that was novel. Biochemical analyses of red-blood-cell lysates from the relevant family members suggested that each of these mutations was associated with dramatic impairment of GALT activity in these cells. While this observation was consistent with our previous findings concerning the Q188R mutation expressed both in humans and in a yeast-model system, it was at odds with a report by Reichardt and colleagues, indicating that in their COS cell-expression system the S135L substitution behaved as a neural polymorphism. To address this apparent paradox, as well as to investigate the functional significance of the newly identified V151A substitution, all three mutations were recreated by site-directed mutagenesis of the otherwise wild-type human GALT sequence and were expressed both individually and in the appropriate allelic combinations in a GALT-deficient strain of the yeast Saccharomyces cerevisiae.(ABSTRACT TRUNCATED AT 250 WORDS)