Chapter 77

## Abstract

1. We describe an inborn error of metabolism called phenylketonuria (PKU; MIM No. 261600). The disease has been called an epitome of human biochemical genetics (Scriver and Clow, 1980a, 1980b). The disorder reflects a disadaptive interaction between nature and nurture. The component in nurture is an essential amino acid, L-phenylalanine; the one in nature is mutation in the phenylalanine hydroxylase gene (PAH) encoding the enzyme L-phenylalanine hydroxylase (EC 1.14.16.1). The discordance between nature and nurture leads to hyperphenylalaninemia (HPA), which can have a toxic effect on brain development and function. The "proximal" phenotype (phenylalanine hydroxylase dysfunction) is under the control of one locus encoding the phenylalanine hydroxylase enzyme and additional loci encoding several other enzymes necessary for synthesis and recycling of the tetrahydrobiopterin cofactor essential for the catalytic reaction; locus heterogeneity thus enters the interpretation of HPA. The intermediate (metabolic) and distal (cognitive) phenotypes of PKU disease both behave as complex traits that elude consistent interindividual genotype-phenotype correlations. The phenylalanine hydroxylase gene harbors great allelic diversity; several hundred mutations, both disease-causing and polymorphic, are recorded in PAHdb, a public-locus-specific mutation database (www.pahdb.mcgill.ca).

2. The HPAs are disorders of phenylalanine hydroxylation. The minimum requirements for the normal reaction, which occurs in both liver and kidney in human subjects, are phenylalanine hydroxylase enzyme (a monooxygenase, EC 1.14.16.1), oxygen, L-phenylalanine substrate, and the 6R isomer of the tetrahydrobiopterin (BH4) cofactor. For the pterin cofactor to function as a catalyst, BH4 must be regenerated from the carbinolamine byproduct (4a-hydroxytetrahydropterin) of the hydroxylation reaction. This is achieved by a recycling pathway in which 4α-carbinolamine dehydratase (formerly known as phenylalanine hydroxylase–stimulating protein) converts the carbinolamine to the quinonoid dihydropterin, which, as the substrate for dihydropteridine reductase in the presence of reduced pyridine nucleotide, is converted back to BH4. A pathway exists for biosynthesis of this obligatory cofactor involved both here and in the function of other aromatic monooxygenases and of nitric oxide synthase; the enzymes in the pathway are guanosine triphosphate cyclohydrolase, 6- pyruvoyltetrahydropterin synthase, and sepiapterin reductase. Diseases of BH4 synthesis and recycling are discussed in Chap. 78.

3. Hyperphenylalaninemia is defined as a plasma phenylalanine value greater than 120 µmol/liter (>2 mg/dl). Whether forms of HPA owing to altered integrity of the enzyme should be subdivided into different forms—notably phenylketonuria (plasma phenylalanine >1000 µmol/liter, diet phenylalanine tolerance < 500 mg/day) and non-PKU forms (plasma phenylalanine < 1000 µmol/liter, diet tolerance > 500 mg/day)—is a moot point. Evidence suggests that mild degrees of persistent untreated HPA (<600 µmol/liter) may not be harmful to cognitive development (as yet an unproven hypothesis). For purposes of diagnosis, counseling and correct treatment of the non-PAH enzyme deficiencies affecting BH4 homeostasis must be ruled out.

4. The human PAH gene covers approximately 100 kb of genomic DNA on chromosome 12, band region q23.2, and is embedded in a region of 1.5 Mbp harboring five other genes. The nucleotide sequences, both genomic (GenBank accession number AF404777) and cDNA (U49897.1), are now known (see www.pahdb.mcgill.ca); PAH has 13 exons and a complex 5' untranslated region containing cis-acting, trans-activated regulatory elements. The gene is rich in intragenic polymorphic markers, including biallelic restriction-fragment-length polymorphism (RFLP) and single-nucleotide polymorphism (SNP) alleles, a tetranucleotide short tandem repeat (STR) acting as a fast molecular clock in intron 3, and a variable number of tandem repeats (VNTRs) (30-bp-length cassettes) in the 3' untranslated region (UTR). The polymorphic sites are in linkage disequilibrium and describe a large series of extended and miniature haplotypes. The PAH gene also harbors several hundred disease-causing alleles associated with HPA, of which more than 60 percent are missense alleles. Only a half-dozen different alleles account for the majority of mutant chromosomes in Europeans or Orientals; the remainder are rare, even private alleles.

5. The human PAH gene has both developmental- and tissue-specific transcription/translation. Its translation product is a 452-amino-acid polypeptide homologous in several domains with the subunits of tyrosine and tryptophan hydroxylases. The catalytic domain of the human PAH polypeptide has been resolved at 2 Å for residues 117–427. The enzyme is homo-oligomeric and functions in alternating activated and deactivated states in dimeric and tetrameric conformations. The effect of mutant alleles is being studied by molecular modeling in silico by using the protein design algorithm FoldX to predict the energetic impact on native-state stability of the PAH enzyme of missense PAH alleles and by expression analysis in vitro. Missense alleles can cause misfolding of the PAH enzyme subunit, leading to aggregation and disposal by the proteasome.

6. The effects of disease-causing PAH mutations on the patient can be measured at three levels: proximal (enzymic), intermediate (metabolic), and distal (cognitive function). Enzyme dysfunction can be measured in vitro either directly by hepatic biopsy or indirectly by expression analysis when the mutation is expressed in a transgenic construct in mammalian or bacterial cell systems or in a cell free transcription/translation system. The latter enables hepatic PAH activity in vivo to be broadly predicted. Flux rates in vivo for phenylalanine hydroxylation/oxidation also can be measured by two different isotopic methods. All studies of genotype-phenotype correlations reveal reasonable correlations at the proximal (enzyme) level; however, at intermediate (metabolic) and distal (cognitive) levels, the phenotypes have emergent properties and behave as complex traits in which the effects of PAH, the major locus, is modulated by "modifiers."

7. Pathogenic PAH alleles produce their effects on PAH enzyme by various mechanisms and behave in broad terms as null (no activity), Vmax altering (reduced activity), kinetic (altered affinity for substrate or cofactor), unstable (as a result of misfolding and increased turnover and loss of PAH protein), and BH4-responsive. Findings occasionally have been taken to signify negative allelic complementation as an additional mechanism of mutant genotype expression. An important subset of missense alleles that do not map to the BH4-binding region confers a 6R-BH4-responsive phenotype in vivo by mechanisms that include stabilizing a misfolding subunit by chaperone-like therapy and by overcoming unfavorable BH4-binding kinetics by saturation.

8. Newborn screening for PKU occurs in many societies and is a potent resource for ascertainment and sampling of mutant PAH genes. Prevalence data for HPA (5–350 cases per 1 million live births) and mutation analysis together reveal nonuniform distribution of patients and alleles in human populations. Human genetic diversity at the PAH locus complements data from analysis of mitochondrial DNA, the Y chromosome, and classic autosomal polymorphisms. The distribution and types of PAH alleles indicate how migration, genetic drift, natural selection (perhaps), recurrent mutation, and intragenic recombination over the past 100,000 years might account for the present-day incidence of PKU, the observed mutation-haplotype associations, and the nonuniform distribution of cases and major alleles in modern human populations. The prevalence rates for PKU in persons of African descent appear to be an order of magnitude lower than those for persons in European, Chinese, and Korean populations, where prevalence is similar (10–4) in these populations.

9. Pathogenesis of the most important clinical (disease) phenotype (cognitive and neurophysiologic impairments) is undoubtedly complex, but there is an emerging consensus that phenylalanine itself, at elevated concentrations, is the harmful molecule that starts the process of allostasis. Several strains of mice mutagenized by N-ethyl-N-nitrosourea, with documented PAH gene mutations and deficient enzyme activity, are orthologous resources to study pathogenesis, as well as treatment to control the phenotypic effects of the mutant genotype.

10. Newborn screening with measurement of blood phenylalanine is the most reliable method for early detection of HPA. The classification of phenotype includes severe and less severe forms of PAH deficiency, of BH4-responsive and BH4-nonresponsive primary deficiency, and of primary BH4 deficiency. Classification requires measurements of phenylalanine, pterins, and neurotransmitter derivatives in urine, plasma, and cerebrospinal fluid (CSF), along with specific protocols and various assays of enzyme activity (see Chap. 78). If it is requested, prenatal diagnosis for PKU is feasible by DNA analysis of mutations and haplotypes.

11. Treatment of HPA requires restoration of blood phenylalanine to values as near normal as possible as early as possible in postnatal life and for as long as possible—perhaps for a lifetime. At present, it seems that any deviation from this policy may incur a cost in structure and function of brain in the PKU patient. Among the modalities of treatment, the low-phenylalanine diet is still paramount, but it needs to be improved in organoleptic properties and in nutrient composition—notably of the essential fatty acids and the relative ratios of amino acids. The BH4-responsive PAH alleles may require only pharmacologic therapy (e.g., 10 mg BH4/kg per day). Alternative modalities include the possibility of enzyme substitution with engineered recombinant phenylalanine ammonia lyase (promising) and gene therapy (in a holding pattern for human subjects but showing efficacy in the mouse model).

12. Maternal HPA, a toxic embryopathy/fetopathy, causes congenital malformations, microcephaly, and permanently impaired cognitive development. It is a consequence of intrauterine phenylalanine excess in the fetal compartment derived from a positive transplacental gradient. All females of reproductive age with HPA should receive reproductive counseling, social support, and continued or renewed treatment to restore euphenylalaninemia before conception and throughout pregnancy. Meticulous treatment of maternal HPA is compatible with a normal outcome for the fetus.

13. Virtually all the major themes and issues now considered to be important in PKU were recognized by the human geneticist Lionel Penrose half a century ago. The fundamental questions about PKU are the same then and now; only the tools and opportunities to address them have changed.

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