A partially active mutant aldolase B from a patient with hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is a potentially fatal autosomal recessive disease of carbohydrate metabolism. HFI patients are deficient in aldolase B, the isozyme expressed in fructose-metabolizing tissues. The eight protein coding exons, including splicing signals, of the aldolase B gene from one American HFI patient were amplified by the polymerase chain reaction (PCR). Single-strand conformational polymorphism (SSCP) analysis and direct sequence determination were applied to the amplified fragments. The mutations in the patient's alleles were identified as a nonsense mutation (R59op) in exon 3 and a missense mutation (C134R) in exon 5. These mutations were confirmed by sequence determination of cloned PCR-amplified exons 3 and 5 from the patient. Allele specific oligonucleotide (ASO) hybridizations of amplified exons 3 and 5 showed the Mendelian inheritance of both mutations. Site-directed mutagenesis was used to generate an expression plasmid for the C134R mutation, and the mutant enzyme was expressed in bacteria. Assays of partially purified enzyme preparations showed that this missense mutation results in an apparently unstable enzyme that retains partial activity. This is the first evidence for a partially active aldolase B from an HFI individual with an identified mutation, and supports the hypothesis that adequate gluconeogenesis/glycolysis is maintained in HFI patients by the presence of partially active enzymes.