Simple, Rapid Nonradioactive Method to Detect the Three Most Prevalent Hereditary Fructose Intolerance Mutations

Abstract

Hereditary fructose intolerance (HFI) is an autosomal recessive disorder, the true incidence of which is not known but may be estimated as high as 1 of 20 000 (1)(2). It is caused by any of several molecular abnormalities of the human aldolase B gene (3). Twenty-one mutations have been reported worldwide (2). Three mutations (A149P, A174D, and N334K) of the aldolase B gene account for 95% of the defective alleles in Europe (2 , 4) . They are investigated by various methods, including restriction enzyme digestion, direct sequencing, use of an amplification refractory mutation system, and allele-specific oligonucleotide hybridization (4). Unidentified HFI patients are at high risk if inadvertently given an infusion of fructose, sorbitol, or inverted sugar (5)(6). Three methods are used for diagnosis. The measurement of blood variables in response to intravenous fructose tolerance test and assay for aldolase B activity on liver biopsy samples are both invasive methods and do not allow heterozygote diagnosis (7). The third method, allele-specific oligonucleotide hybridization (4), circumvents these issues but is a radiobioassay restricted to certified laboratories. We have developed a simple and rapid method for detecting simultaneously the three most prevalent mutations in Europe by PCR-mediated, site-directed mutagenesis and restriction analysis. We investigated the diagnostic value of this method in patients from independent families. Thirty-five HFI patients from 30 independent families were studied. HFI presented with acute symptoms (hypoglycemia, vomiting, and liver failure) before 6 months of age in 12 patients. In a second group of 23 patients, HFI presented with symptoms related to chronic exposure (hepatomegaly, growth delay, and aversion toward foods containing fructose) between 1 and 6 years of age. Diagnosis was assayed by conventional methods: intravenous fructose tolerance tests or aldolase B activity assays in liver biopsy samples. Genomic DNA was obtained from …