Molecular basis for Duarte and Los Angeles variant galactosemia.

Human orythrocytes that are homozygous for the Duarte enzyme variant of galactosemia (D/D) have a characteristic isoform on isoelectric focusing and 50% reduction in galactose-1-phosphate uridyltransferase (GALT) enzyme activity. The Duarte biochemical phenotype has a molecular genotype of N314D/N314D. The characteristic Duarte isoform is also associated with a variant called the "Los Angeles (LA) phenotype," which has increased GALT enzyme activity. We evaluated GALT enzyme activity and screened the GALT genes of 145 patients with one or more N314D-containing alleles. We found seven with the LA biochemical phenotype, and all had a 1721C-->T transition in exon 7 in cis with the N314D missense mutation. The 1721C-->T transition is a neutral polymorphism for leucine at amino acid 218 (L218L). In pedigree analyses, this 1721C-->T transition segregated with the LA phenotype of increased GALT activity in three different biochemical phenotypes (LA/N, LA/G, and LA/D). To determine the mechanism for increased activity of the LA variant, we compared GALT mRNA, protein abundance, and enzyme thermal stability in lymphoblast cell lines of D and LA phenotypes with comparable genotypes. GALT protein abundance was increased in LA compared to D alleles, but mRNA was similar among all genotypes. When LA/D and D/D GALT biochemical phenotypes were compared to N/N GALT phenotypes, both had 50%, as compared to 21%, reduction in GALT activity in the wild type (N/N) after exposure at identical initial enzyme activity to 50 degrees C for 15 min. We conclude that the codon change N314D in cis with the base-pair transition 1721C-->T produces the LA variant of galactosemia and that this nucleotide change increases GALT activity by increasing GALT protein abundance without increasing transcription or decreasing thermal lability. A favorable codon bias for the mutated codon with consequently increased translation rates is postulated as the mechanism.