IDENTIFICATION AND FUNCTIONAL-ANALYSIS OF 3 DISTINCT MUTATIONS IN THEHUMAN GALACTOSE-1-PHOSPHATE URIDYLTRANSFERASE GENE ASSOCIATED WITH GALACTOSEMIA IN A SINGLE-FAMILY

We have identified three mutations associated with transferase-deficie ncy galactosemia in a three-generation family including affected membe rs 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-l-phosphate uridyltransferase (GALT) enzyme and genetic locus in both affected and carrier individuals revealed th ree distinct base substitutions in this family, two (Q188R and S135L) that had been reported previously and one (V151A) that was novel. Bioc hemical analyses of red-blood-cell lysates from the relevant family me mbers suggested that each of these mutations was associated with drama tic impairment of GALT activity in these cells. While this observation was consistent with our previous findings concerning the Q188R mutati on expressed both in humans and in a yeast-model system, it was at odd s with a report by Reichardt and colleagues, indicating that in their COS cell-expression system the S135L substitution behaved as a neutral polymorphism. To address this apparent paradox, as well as to investi gate the functional significance of the newly identified V151A substit ution, all three mutations were recreated by site-directed mutagenesis of the otherwise wild-type human GALT sequence and were expressed bot h individually and in the appropriate allelic combinations in a GALT-d eficient strain of the yeast Saccharomyces cerevisiae. The results of these yeast-modeling studies were fully consistent with the patient da ta, leading us to conclude that, at least within the context of the ce ll types studied, in the homozygous state Q188R is a mutation that eli minates GALT activity, and S135L and V151A are both mutations that imp air GALT activity to <6% of wild-type values.