Functional analysis of the human galactose-1-phosphate uridyltransferase promoter in Duarte and LA variant galactosemia.

Human galactose-1-phosphate uridyltransferase (hGALT) is an evolutionarily conserved enzyme central to D-galactose metabolism. The impairment of hGALT causes galactosemia. One missense mutation, an aspartate to asparagine substitution at amino acid 314 (N314D), impairs 50% activity in the homozygous state in some patients but gives near normal activity in others. The former condition is called Duarte (D) and the latter, Los Angeles (LA). The D allele is linked to hGALT polymorphisms including a deletion 5'to the translation start site (-119 to -116delGTCA), g1391G --> A and g1105G --> C. The LA allele is linked to a g1721C --> T transition. To investigate possible mechanisms for differences in hGALT activity between the D and LA alleles, we sequenced 3951 nucleotides of genomic DNA 5' to the hGALT translation start site. Using a dual-luciferase reporter system to express deletion constructs of the hGALT promoter, we noted both positive and negative regulatory regions. Two putative positive regulatory domains overlap with the naturally occurring -119 to -116delGTCA linked to Duarte. One is an E-box motif (CACGTG) at -117 to -112 bp. The second is an AP-1 motif (TCAGTCAG) at -124 to -119 bp. The delGTCA mutation confers reduced luciferase activity to transfected cell lines derived from human ovarian and liver neoplasms. Additionally, human lymphoblasts derived from patients with the Duarte allele have reduced GALT mRNA. We conclude that the human GALT gene is regulated in the first -165 bp of its promoter region by positive regulators of GALT gene expression. The -119 to -116delGTCA reduces hGALT transcription resulting in reduced GALT activity in the Duarte allele.