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Phenylketonuria: Paradigm for a Treatable Genetic Disease...?

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The content in this publication was current at the time it was published, but it is not being updated. The publication is provided for historical purposes only.​

By Charles R. Scriver, M.D.

Phenylketonuria (PKU) is a Mendelian recessive inborn error of metabolism in which phenotype can be modified by restriction of dietary phenylalanine (Phe) (Scriver, 1994; Scriver, Kaufman, 2000). PKU is the prototype for early diagnosis and treatment to prevent a major disease phenotype (impaired cognitive development). Early diagnosis of postnatal hyperphenylalaninemia, the metabolic hallmark of the trait, is critical, hence the importance of a newborn screening test to identify an affected person (incidence ~ 10 -4).

  • The disease has “a cause.” The ultimate cause is phenotype-modifying allelic variation in the phenylalanine hydroxylase gene (symbol PAH); the proximate cause is dietary intake of the essential amino acid L-Phe.
  • Pathogenesis of PKU “disease” has both biochemical and physiologic components. The former involves extreme loss of Phe hydroxylation as served by hydroxylase enzyme function (Scriver, 1998). The catalytic reaction requires an intact homotetrameric enzyme, substrate (L-Phe), equimolar molecular oxygen, and catalytic amounts of tetrahydrobiopterin. Impaired synthesis or recycling of tetrahydrobiopterin cofactor impairs the aromatic hydroxylases (for Phe, tyrosine, and tryptophan) and nitric oxide synthase. The term “PKU” is reserved for primary dysfunction of PAH enzyme due to mutations in the PAH gene.

PAH enzyme disfunction results in metabolic dishomeostasis; Phe levels rise above the maximum normal level (0.125mM), and tyrosine levels may fall (because of impaired product formation). There is a subsequent imbalance in the distribution of many amino acids across cellular membranes and across the blood-brain barrier.

Historically, there were many other manifestations of PKU which today have little significance. The current focus of the applied knowledge is prevention of mental retardation.

The Gene

The human PAH locus is on chromosome region 12q24.1. The locus spans approximately 100kb. The gene comprises 13 exons, which take up no more than 3 percent of the total nucleotide sequence. Only the cDNA has been cloned (GenBank U49897), but a full genomic sequence is anticipated soon. The 5’ regulatory region of the gene has been fully characterized (Konecki, Wang, Tretz, et al., 1992). A Web site External Web Site Policy, provides a knowledge base, including a full description of the gene, its alleles, and a large range of annotations on every allele (Scriver, Waters, Sarkissian, et al., 2000). The PAH locus harbours well over 400 alleles, the majority of which are disease-causing mutations; 50 percent of these are missense mutations, the majority of which do not map to critical residues in the catalytic region of the protein. There are 28 “polymorphic” alleles in the PAH gene, of which 2 are multiallelic (STR and VNTR), 8 are RFLPs, and the remainder are so-called SNPs.

PAH Gene Expression

The gene is both transcribed and translated into polypeptide in only two human tissues: liver (hepatocytes) and kidney (mainly proximal renal tubule epithelial cells) (Lichter-Konecki, Hipke, Konecki, 2000). The significance of PAH gene expression in kidney is not fully known, but it may contribute to net reabsorption of this essential amino acid by the process of metabolic run out; it also implies that renal transplantation might benefit an affected patient.

The Enzyme

PAH enzyme is a homotetramer; its crystal structure is known (Erlandsen, Stevens, 1999). The monomer contains 452 amino acids (~ 51kD mass) and has 3 domains: an N-terminal regulatory region, a central catalytic domain, and a C-terminal tetramerization domain. Mutations in any region can affect function. The enzyme functions in vivo as a homotetramer permitting allosteric modulation in the presence of substrate and cofactor; activity is modulated by phosphorylation of an N-terminal serine; the catalytic center requires an iron atom in each subunit. Recent work (Waters, Parniak, Akerman, et al., 2000) shows that missense mutations not mapping to key residues in the catalytic domain cause misfolding of the protein and diversion to proteolytic intracellular pathways. The study of missense PAH mutations suggests paradigms for the effect of missense mutations elsewhere in the human genome on other metabolic processes.

Metabolic Homeostasis

In the presence of normal dietary Phe intake, the obligatory response is accumulation of Phe in body fluids when PAH enzyme function is impaired. The “pathogenic” molecule is (apparently) L-Phe itself; in excess, it will disturb transport of other critical amino acids across the blood-brain barrier and across neuronal membranes themselves and will impair synthesis of neurotransmitters; the relevance of mediated Phe transport at the blood-brain barrier has been well documented (Pietz, Kries, Rupp, et al., 1999).

There was much interest earlier in the pathogenic significance of Phe metabolites (for example, phenylpyruvic acid and its derivatives). Studies in mutagenized mouse models of hyperphenylalaninemia/PKU (Sarkissian, Boulais, McDonald, et al., 2000) show that these derivative metabolites are not present at significant concentrations in brain to account for impaired cognitive development (Sarkissian, Scriver, Mamer, 2000).

Genotypes and Phenotypes

  • Genotypes and alleles. Only one-quarter of the mutant human PAH genotypes identified so far are homoallelic. On a worldwide basis, about 10 different PAH mutations account for about two-thirds of the relative frequencies of mutations in human population; the remainder are rare, many even private. The distribution of mutations in human populations is nonrandom. It reflects an out-of-Africa hypothesis and independent distributions into Oriental and Caucasian populations; PKU alleles are also good markers of European range expansion over the past 500 years. It may be said that “the history of the population is (very frequently) the history of the PKU allele.” Both the disease-causing and the polymorphic PAH alleles contribute to these conclusions (Scriver, Kaufman, 2000).
  • Phenotype correlations. There was a hope that knowledge of PAH genotype would have predictive value for severity of phenotype. In broad terms, this is true (Kayaalp, Treacy, Waters, et al., 1997; Guldberg, Rey, Zschocke, 1998). Genotypes containing a pair of null alleles confer a severe form of hyperphenylalaninemia (HPA). Missense mutations conferring considerable residual activity (measured by in vitro expression analysis [see External Web Site Policy] tend to confer non-PKU HPA when paired (homo- or heteroallelic) in a mutant genotype. A range of intermediate HPA phenotypes is associated with other mutant genotypes. The metabolic phenotype (HPA) is itself the result of many events other than Phe hydroxylation activity (Scriver, 1998; Scriver, Waters, 1999); hence, it is not surprising that discordance between genotype and metabolic phenotype is found.

There is also recognized discordance between metabolic and cognitive phenotypes; PKU does not always cause mental retardation (as recognized long ago by Penrose). One explanation involves events at the blood-brain barrier; siblings with identical mutant genotypes can have different cognitive phenotypes by virtue of differences in brain Phe levels that have been modulated by differences in Phe flux at the the blood-brain barrier.

A case has been made for looking at PKU and other Mendelian (metabolic) disorders as complex traits nested in Mendelian disorders (Scriver, Waters, 1999; Dipple, McCabe, 2000).


PKU is an orphan disease. However, four decades of dietary therapy coupled with early postnatal diagnosis reveal that an approximation of mild HPA is compatible with near-normal cognitive development. The benefits of treatment have been seen in large cohorts of affected probands and also in smaller but convincing numbers of women with HPA who receive preconception and intrapartum treatment. Guidelines (Medical Research Council Working Party on Phenylketonuria, 1993; Recommendations on the dietary management of phenylketonuria, 1993) now recommend more aggressive treatment: earlier in onset, more stringent in restoring euphenylalaninemia, and longer in duration—perhaps for life. Premature termination of therapy is frequently associated with neurophysiologic and psychological dysfunction and perhaps a decline in cognitive function.

Compliance with the new guidelines will benefit from new approaches to therapy, for example, to improve the organoleptic properties of current diet treatment products, to develop new low-Phe proteins (perhaps by modifying milk proteins in lactating animals), and to provide enzyme substitution therapy (Sarkissian, Shao, Blain, et al., 1999). Each of these options is being investigated while PKU remains the flagship for the armada of treatable genetic disease.


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