Identification and characterization of UDP-galactose 4'-epimerase (GALE) mutations in GALE-deficient patients identified by newborn screening

Abstract

UDP-galactose 4'-epimerase (GALE)-deficient galactosemia is an autosomal recessive disorder with clinical manifestations ranging from asymptomatic, where enzyme deficiency is restricted to peripheral blood, to severe, with a generalized GALE deficiency affecting multiple tissues. It has been postulated that different point mutations in the GALE gene account for this clinical and biochemical heterogeneity. Two mutations, K257R and G319E, have previously been identified in an African American GALE-deficient patient, an ethnic population with a ten-fold higher incidence of the asymptomatic form than non-African Americans. It is hypothesized that K257R and G319E alleles are common among African American GALE-deficient patients and exhibit a subtle effect on GALE function. The main objectives of this study were (1) to identify mutations in GALE-deficient patients identified through newborn screening, (2) to determine the distribution of these mutations in different ethnic backgrounds, and (3) to characterize the function of these GALE alleles using a yeast expression system. Through mismatch PCR and restriction enzyme digestion, K257R was determined to be the most common allele, identified in 93% of African American patients and comprising 52% of their alleles, yet it was not detected in non-African Americans (seven patients or 121 controls). G319E, the second most common allele, was identified in both African Americans and Hispanics. Together, these two alleles represent 50% of African American patient genotypes. Modeling studies on activity, kinetic effects, and stability were consistent with the predictions of mutations identified in asymptomatic patients exhibiting a subtle effect on GALE function, but did not rule out the alternate interpretation that they represent polymorphisms closely associated with GALE-deficiency. Five novel mutations were identified through sequencing, including three missense substitutions, D246N, P293L, and R335H, and two deletions, Int7 5'del and L326-R328del. Modeling studies for activity and steady-state abundance demonstrated drastically reduced GALE activity and abundance for L326-R328del and D246N and near-normal GALE activity for R335H. These results indicate that both severe and mild GALE mutations exist in patients identified by newborn screening, suggesting the potential for clinical complications in patients inheriting two severe mutations. These studies also demonstrate the potential for implementing molecular testing as a follow-up to newborn screening to identify patients at risk for adverse clinical effects.