CORRELATION BETWEEN POLYMORPHIC DNA HAPLOTYPES AT THE PHENYLALANINE HYDROXYLASE LOCUS AND CLINICAL PHENOTYPES OF PHENYLKETONURIA

Phenylalanine hydroxylase, the enzyme deficient in PKU, is synthesized in the liver but not in easy available cells like leucocytes or skin fibroblasts. However, the phenylalanine hydroxylase gene is part of our chromosome no. 12 and located in all nucleated cells. The structural organization of the gene has now been determined and ten polymorphic binding sites for restriction endonucleases identified. Any given individual in the population will be either homozygous or heterozygous with respect to the length of the DNA fragments obtained after digestion with a restriction endonuclease (Restriction Fragment Length Polymorphism, RFLP). The combination of the different lengthened fragments of DNA obtained after digestion of an individual's genomic DNA with each of 7 restriction enzymes and a cDNA copy of the phenylalanine hydroxylase gene as the probe, defines the RFLP haplotypes of the phenylalanine hydroxylase alleles of this individual. The RFLP haplotypes of 74 normal phenylalanine hydroxylase alleles and 74 mutant alleles have been determined in 37 Danish PKU-families. Of the 74 mutant alleles analysed, 67 (91%) were associated with only four haplotypes. Affected children, who were homozygous for two of these haplotypes, had classical PKU. On the other hand, children who had inherited mutant alleles associated with one of the two other haplotypes had a milder clinical course (mild PKU or benign hyperphenylalaninemia). Molecular cloning and sequence analysis have defined the specific mutations responsible for classical PKU associated with two of the common haplotypes, which comprise 60% of the PKU alleles in Denmark. Using mutant-specific oligonucleotide probes preliminary studies have demonstrated that these mutations are also present in other western European countries. The demonstration that the mutations responsible for classical PKU associated with two haplotypes are not present in mutant alleles of other haplotypes provides evidence that there are multiple mutations in the phenylalanine hydroxylase gene, and support the hypothesis that different combinations of mutant alleles are responsible for the clinical diversity of phenylketonuria.