Alan S. Lidsky

Correlation between polymorphic DNA haplotypes at phenylalanine hydroxylase locus and clinical phenotypes of phenylketonuria

Eight polymorphic sites for seven restriction endonucleases have been reported at the human phenylalanine hydroxylase locus. The composite profile of the presence or absence for each of the eight polymorphic sites within an allele defines the haplotype of the corresponding allele. Twelve such haplotypes associated with normal and mutant phenylalanine hydroxylase alleles have been identified in 33 Danish families with children with phenylketonuria. Of the 66 mutant alleles analyzed, 59 (89%) were associated with only four haplotypes. The identification of individual phenylalanine hydroxylase alleles by haplotype analysis enables correlation of the hyperphenylalaninemic phenotypes of the patients with their genotypes. Patients who were either homozygous or heterozygous for the mutant alleles of haplotypes 2 and 3 had a severe clinical course. Patients who had a mutant allele of either haplotype 1 or 4 usually had a less severe clinical phenotype. The recent demonstration that the mutation responsible for classic phenylketonuria associated with haplotype 3 is not present in mutant alleles of other haplotypes provides unambiguous evidence that there are multiple mutations in the phenylalanine hydroxylase gene and supports the hypothesis that different combinations of mutant alleles may be responsible for the clinical diversity of phenylketonuria.

POLYMORPHIC DNA HAPLOTYPES AT THE PHENYLALANINE HYDROXYLASE LOCUS IN PRENATAL DIAGNOSIS OF PHENYLKETONURIA

Restriction fragment length polymorphisms (RFLPs) detected in the human genome by means of a cDNA clone of phenylalanine hydroxylase (PAH) segregate concordantly with phenylketonuria (PKU) in several families. To establish the usefulness of these DNA polymorphisms for prenatal diagnosis of PKU the genotypes and chromosomal haplotypes of eight RFLPs at the PAH locus were determined in 46 families, each with one or more affected children. 34 of these families were informative for linkage analysis, giving a maximum probability of 10(9):1 in favour of tight linkage between PKU and the PAH RFLP haplotypes. Thus the deficiency of hepatic PAH in PKU patients is caused by mutations in the PAH gene itself, and the RFLPs could be used effectively for prenatal diagnosis and carrier detection of PKU in affected families. Use of the PKU haplotypes based on these eight RFLPs would establish disease status in approximately 87% of siblings at risk, although three RFLPs alone (detected with the restriction enzymes Pvu II, Xmn I, and EcoR R) are nearly as effective.

1225 PKU AND MILD HYPERPHENYLALANINEMIA (MHP) IN SIBLINGS: BIOCHEMICAL CHARACTERIZATION AND MOLECULAR RFLP ANALYSIS OF THE PHENYLALANINE HYDROXYLASE (PAH) GENE

Mutations in the PAH gene can cause either classical phenylketonuria (PKU) or MHP. The genetic relationship between these different forms of PAH deficiency is complex. We report biochemical characterization and restriction fragment length polymorphism (RFLP) analysis of the PAH gene using cloned PAH in two families in which some siblings have PKU and others have MHP. In both families the mother has MHP and the father is biochemically normal. The first family has one child with PKU, one child with MHP, and two normal children. The second family has one child with PKU, three children with MHP, and one normal child. Haplotype analysis of the PAH gene using RFLPs demonstrates that in each family one allele from the father segregates with all the affected children (both PKU and MHP) while one of the maternal alleles segregates with PKU and the other with MHP. This analysis identifies several mutant PAH alleles which can cause either PKU or MHP. These findings demonstrate that, at least in these families, there is extensive overlap between those mutations causing PKU and those causing MHP. The two distinctly different clinical phenotypes reflect the effects of pairs of mutant alleles, presumably representing a variety of different mutations, which together have distinctive biochemical and pathophysiological consequences.