Hereditary fructose intolerance: Metabolic implications and analysis of aldoB mutations

Hereditary fructose intolerance (HFI) is a potentially fatal inborn error of fructose metabolism caused by a deficiency of the glycolytic enzyme, aldolase B, in the liver and kidney. Over 40 disease-causing mutations in the protein-coding region of the aldolase B gene (aldoB) are known. Analysis of the previously uninvest gated promoter region of several HFI patients reveals single base mutations. First, a novel promoter mutation at an evolutionarily-conserved position located 132 bp upstream of the start of transcription (g.-132G>A) causes decreased transcription factor binding as determined by electrophoretic mobility shift analysis (EMSA) and a significant decrease in transcription as determined by luciferase reporter analysis. Second, a mutation at the donor splice site (IVS1 +1G>C) abolishes normal splicing with retention of the entire 4.8 kb first intron as shown by cDNA analysis. Using a reporter plasmid with luciferase fused downstream of this mutation, a significant decrease in activity is shown. Follow-up analysis of the prevalence of these two mutations along with seven other common HFI-causing alleles (A149P, A174D, N334K, R59Op, Delta4E4, L256P, and A337V) is analyzed in the American HFI population (153 patients) using allele-specific oligonucleotide hybridization. The combined frequency of these mutations is 67% among American HFI patients, which is distinctly different from worldwide frequencies. The American population shows increased prevalence of the "null" alleles, R59Op and Delta4E4. Further analysis of the aldoB promoter and enhancer reveals single nucleotide polymorphisms (SNPs). One SNP (g.-129T>A) is examined for its effect on the HFI phenotype. EMSA indicate a 5-fold decrease in transcription factor binding, but only a 25% decrease in luciferase reporter transcription in vivo. While not an HFI-causing allele, this SNP could predispose to problems with fructose metabolism. An enigma of HFI is that gluconeogenesis is unaffected by the loss of aldolase B in the liver. Investigation by bioinformatics analysis and RNA in situ hybridization for gluconeogenesis-specific genes suggests the cerebellum may be an alternative site of gluconeogenesis. Lastly, progress towards an aldo2-knockout mouse model for HFI is described using recombination-mediated genetic engineering of a targeting vector, which successfully targets mouse ES cells at the aldo2 locus.