RESUMO
Epimerase deficiency galactosemia is an autosomal recessive disorder that results from partial impairment of UDP-galactose 4'-epimerase (GALE), the third enzyme in the Leloir pathway of galactose metabolism. Clinical severity of epimerase deficiency ranges from potentially lethal to apparently benign, likely reflecting the extent of GALE enzyme impairment, among other factors. We report here a case study of monozygotic twins identified by newborn screening with elevated total galactose and normal galactose-1P uridylyltransferase (GALT). Follow-up testing revealed partial impairment of GALE in hemolysates but near-normal activity in lymphoblasts; molecular testing identified a missense substitution, R220W, apparently in the homozygous state. The twins were treated with dietary galactose restriction for the first 18 months of life. During this time, independent testing revealed concurrent diagnoses of Williams Syndrome in both twins, and cytomegalovirus (CMV) infection in one. Expression studies of R220W-hGALE in a null-background strain of Saccharomyces cerevisiae demonstrated a very limited impairment of V (max) for UDP-galactose (UDP-Gal) and K (m) for UDP-N-acetylgalactosamine (UDP-GalNAc), but a galactose challenge in vivo failed to uncover any evidence of impaired Leloir function. Similarly, both twins demonstrated normal hemolysate galactose-1-phosphate (Gal-1P) levels following normalization of their diets at 18 months of age. While these studies cannot rule out a negative consequence from some cryptic GALE impairment in a specific tissue or developmental stage, they suggest that the substitution, R220W, is mild to neutral, so that any GALE impairment in these twins is likely to be peripheral and therefore unlikely to be the cause of the negative outcomes observed.
RESUMO
Galactose is metabolized in humans and other species by the three-enzyme Leloir pathway comprised of galactokinase (GALK), galactose 1-P uridylyltransferase (GALT), and UDP-galactose 4'-epimerase (GALE). Impairment of GALT or GALE in humans results in the potentially lethal disorder galactosemia, and loss of either enzyme in yeast results in galactose-dependent growth arrest of cultures despite the availability of an alternate carbon source. In contrast, loss of GALK in humans is not life-threatening, and in yeast has no impact on the growth of cultures challenged with galactose. Further, the growth of both GALT-null and GALE-null yeast challenged with galactose is rescued by loss of GALK, thereby implicating the GALK reaction product, gal-1P, for a role in the galactose-sensitivity of both strains. However, the nature of that relationship has remained unclear. Here we have developed and applied a doxycycline-repressible allele of galactokinase to define the quantitative relationship between galactokinase activity, gal-1P accumulation, and growth arrest of galactose-challenged GALT or GALE-deficient yeast. Our results demonstrate a clear threshold relationship between gal-1P accumulation and galactose-mediated growth arrest in both GALT-null and GALE-null yeast, however, the threshold for the two strains is distinct. Further, we tested the galactose-sensitivity of yeast double-null for GALT and GALE, and found that although loss of GALT barely changed accumulation of gal-1P, it significantly lowered the accumulation of UDP-gal, and also dramatically rescued growth of the GALE-null cells. Together, these data suggest that while gal-1P alone may account for the galactose-sensitivity of GALT-null cells, other factors, likely to include UDP-gal accumulation, must contribute to the galactose-sensitivity of GALE-null cells.
