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 sub stitution at amino acid 314 (N314D), impairs 50% activity in the homozygous state in some patients but gives near normal activity in others. The forme r condition is called Duarte (D) and the latter, Los Angeles (LA). The D al lele is linked to hGALT polymorphisms including a deletion 5'to the transla tion start site (-119 to -116delGTCA), g1391G --> A and g1105G --> C. The L A allele is linked to a g1721C --> T transition. To investigate possible me chanisms for differences in hGALT activity between the D and LA alleles, we sequenced 3951 nucleotides of genomic DNA 5' to the hGALT translation star t site. Using a dual-luciferase reporter system to express deletion constru cts of the hGALT promoter, we noted both positive and negative regulatory r egions. Two putative positive regulatory domains overlap with the naturally occurring -119 to -116delGTCA linked to Duarte. One is an E-box motif (CAC GTG) 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 transf ected cell lines derived from human ovarian and liver neoplasms. Additional ly, human lymphoblasts derived from patients with the Duarte allele have re duced GALT mRNA. We conclude that the human GALT gene is regulated in the f irst -165 bp of its promoter region by positive regulators of GALT gene exp ression. The -119 to -116delGTCA reduces hGALT transcription resulting in r educed GALT activity in the Duarte allele. (C) 2001 Academic Press.