Phenylketonuria Jonathan Baghdadi and Evan Marlin

Transcription

1 Phenylketonuria Jonathan Baghdadi and Evan Marlin Hyperphenylalaninemia (HPA) was first connected to certain types of mental retardation in 1934 by Asjborn Folling. Just a few years later it was understood that HPA was a trait inherited in an autosomally recessive manner and that its resulting mental retardation could be prevented. By the 1980s, the gene for hepatic phenylalanine hydroxylase (PAH), which is responsible for metabolizing phenylalanine to tyrosine had been mapped and cloned (1). Phenylketonuria is the most common genetic error of amino acid metabolism with an incidence of approximately 1:10,000 in Caucasians, and approximately 1:200,000 in Ashkenazi Jews (2). Today, it is understood that PAH deficiency can lead to three different classification of HPA as a result of dietary intake of phenylalanine, all of which lead to a life threatening intolerance of dietary phenylalanine (Phe). Classic PKU is due to a defect in the phenylalanine hydroxylase enzyme that leads to decrease or nonactivity. Children with Classic PKU will suffer extreme and permanent mental retardation without rigorous dietary restrictions. Non-PKU HPA is associated with a much decreased risk of mental retardation without strict dietary restrictions. Finally, Variant or Intermediate PKU has a decrease of phenylalanine metabolism in between Non-PKU HPA and Classic PKU. A more infrequent cause of HPA is the in ability to correctly produce a coenzyme required for phenylalanine metabolism, tetrahydrobiopterin (BH 4 ). Genetics The inheritance pattern of phenylalanine hydroxylase deficiency is autosomal recessive, thus based on simple Mendelian genetics the siblings of an affected child have a 25% chance of being affected, 50% chance of being asymptomatic carriers, and a 25% of being non-affected non-carriers. The PAH gene is located on the long arm of chromosome 12. Normally, the PAH gene is 90 kilobases and encoded for thirteen exons. This gene has had thirty-one neutral polymorphisms identified as having no affect on the 2.4 kb mature protein mrna. The mature mrna will encode for a protein containing 452 amino acids. Of the mutations found in the PAH gene, 67% are missense, 14% deletions, 12% splicing errors, and 6% nonsense mutations. Currently, targeted mutation analysis is a panel of four to fifteen of the most common point mutations and small deletion mutations; this method of testing has a 30% to 50% rate of detection. Mutation scanning for all known point mutations is also possible, but only complete sequence analysis has a mutation detection rate of 99% (1). Biochemistry/Molecular Biology In healthy humans, phenylalanine oxidation by PAH is the normal route for the production of tyrosine. From the production of tyrosine further biosynthetic connections exist to neurotransmitter catecholamines, melanin used for skin pigmentation, and other hormones. In cases of abnormally increased Phe concentrations the excess Phe can be metabolized via its normal route of oxidation to tyrosine or by transamination to phenylpyruvate (3).

2 Figure 1: Normal Phenylalanine Oxidation In vivo, the PAH enzyme is able to exist as both a tetramer and dimmer acting in equilibrium. PAH will use the cofactor tetrahydrobiopterin, which will get oxidized to dihydrobiopterin (BH2) in the course of the reaction. In order for proper Phe metabolism to take place BH4 must be regenerated from BH2 in a separate sequence of reactions, failure of this will be discussed below. When PAH is functioning in a correct manner the transamination of Phe to phenylpyruvate occurs at a negligible rate; however a lack of function of PAH causes the transamination pathway to increase in activity. As the serum levels of phenylalanine increase the transamination reactions, which are usually negligible, also increase. Byproducts such as phenlypryuvate, phenylacetate, phenyllactate, and phenylethylamine are produced and excreted in the urine. Phenylpyruvate is an alpha-keto acid and the major byproduct excreted in urine, and therefore the reason PAH deficiency is called phenylketonuria (3). Figure 2 (above): Transamination of Phenylalanine to Phenylpyruvate. Figure 3 (right): Excreted byproducts due to excess Phe. [Figures courtesy of University of Illinois at Chicago] In 2% to 3% of patients suffering from PKU the defect is not in the PAH gene, but rather a defect involving the cofactor tetrahydrobiopterin (4). Case in which the cause is not PAH, but rather BH4 are sometimes termed malignant because a change in diet will not alleviate the excess concentration of Phe. When phenylalanine is oxidized to tyrosine BH 4 is also oxidized by PAH to BH 2. For continued oxidation to take place it is

3 necessary to regenerate BH 4 through a separate series of reactions requiring the enzyme dihydropteridine reductase (DHPR) (5). Beyond a defect in DHPR there may be a defect in de novo synthesis of BH 4. Of the four enzymes required in tetrahydrobiopterin synthesis two have been isolated as frequently defective within this pathway: GTP Cyclohydrolase I and Pyruvoyl Tetrahydropterin Synthase. Though a completely different cause for HPA then Classic PKU, the symptoms involved in BH 4 defects are the same and must be early identified as cofactor deficiencies by the concentrations of pterins found in urine and CSF. In either defect the increase of Phe causes catastrophic problems for the nervous system due to phenylalanine s critical role in neurotransmitter synthesis. It will halt serotonin production, gamma-amino butyric acid (GABA) production, and 3, 4- dihydroxyphenylalanine (DOPA) production, which in return halts melanin and catecholamine biosynthesis. Inhibition of biosynthesis of these molecules is in return responsible for the fair complexions of PKU patients and the neurological deficits they suffer. The lack of neurotransmitters does not allow for the brain to develop or function properly therefore causing a quick decline in IQ and permanent mental retardation (3). Figure 4: Production of Neurotransmitters from phenylalanine. Diagnosis Phenylketonuria manifests itself in newborns as elevated phe levels in the blood. Since the 60s, as a matter of routine, all US newborns are tested two or three days after birth to determine if their serum phe levels are normal. In most cases, the test given is the Guthrie heel prick test. The Guthrie test is a simple bacterial inhibition assay: blood taken from the pricked heel is allowed to soak into a filter paper, a small piece of which is cultured on medium containing growth-inhibiting factor and dormant bacteria. If the blood contains more phe than is usual in a healthy individual, the bacteria in the medium is able to grow and a white ring will develop on the edge of the piece of filter paper. Normal individuals usually have phe levels between 3 and 4 mg/dl; the Guthrie test will register a positive if the phe level is above 4 mg/dl, and will provide a reasonable

4 estimate of the actual value up to about 5 mg/dl based on proportional growth of the white ring (6). The Guthrie test is reliable and inexpensive but is prone to giving false positives. For this reason, any newborn that is tested positive for elevated phe levels should be kept under appropriate dietary restrictions for a couple weeks and then tested again. If the second test confirms the presence of elevated phe levels, the newborn will undergo further testing to determine the correct method of treatment, which will differ depending on the severity of the HPA and whether the source of the problem is based on a PAH deficiency of a problem with BH 4. Other neonatal tests for HPA include fluorometric analysis and tandem mass spec. Fluorometric analysis is automated and produces less false positives than the Guthrie test but requires expensive lab equipment. Tandem mass spec offers the benefit of being able to observe the levels and ratios of various metabolites at once, but has not yet proven to be cost effective (7). For unaffected relatives of individuals with PKU, carrier status can be confirmed or rejected by either genetic testing or biochemical analysis of phe levels and phe / tyr ratios. Prognosis If diagnosed at birth and kept under dietary restriction, individuals with PKU will undergo normal development. However, the diet, in most cases maintained throughout life, can itself become a problem. It stresses the system both physically and emotionally and has been shown in some individuals to lead to neuropsychological dysfunction (8). Nonetheless, many individuals with PKU live full, healthy lives. If untreated, on the other hand, a child with PKU will undergo permanent brain damage resulting in mental retardation, as well poor development and microcephaly. The degree of damage is proportionate to the severity of the HPA. Because the diet is so onerous, there is some debate over whether individuals with PKU may be able to safely relax their dietary restrictions upon reaching adolescence. Though some say the effects of relaxing the diet are not dramatic, others point to a reduction in cognitive abilities (9). Adults with PKU who relax their diet have also been shown to undergo neurological deterioration (10). Possible Treatments Currently, the main treatment for PKU is to maintain low levels of phenylalanine in the blood via a highly restrictive diet. Individuals on the diet are not allowed to ingest meat, dairy, bread or some vegetables. In order to avoid malnutrition, many individuals supplement the natural foods they can eat with synthetic mixtures reminiscent of soylent green (11). As explained before, though effective at combating the disorder, the pherestricted diet has its own drawbacks and side effects which under optimal circumstances could be relieved. For this reason, there are currently several techniques under investigation that could lessen the gravity of the HPA associated with PKU and thus render the restrictive diet more manageable. One such potential treatment is the addition of phenylalanine ammonia lyase (PAL) to the diet. PAL is an enzyme that degrades phenylalanine into ammonia and benzoic acid. By administering PAL within semipermeable microcapsules to mice, researchers have been able to decrease serum phe levels by 30-40%. This reduction is about half what would be needed to treat PKU on its own (12). Many

5 individuals, including even some with classic PKU, may also benefit from supplements of BH 4. Though much effort has gone into researching potential PAH enzyme therapy and somatic gene therapy, neither is yet feasible (12). Risk of Other Family Members PKU is an autosomal recessive disorder. This qualification has several ramifications for family members of an affected individual. First, parents of the proband are obligate carriers. However, being a carrier does not put them directly at risk since heterozygotes do not manifest symptoms. Siblings of an affected individual have a 1/4 chance also inheriting the disorder, a 1/2 chance of being carriers, and a 1/4 chance of inheriting two functional alleles. If one parent is affected and the other is a carrier, the unborn child has a 1/2 chance of also being affected. Carrier testing for spouses of affected individuals can be performed on a limited basis biochemically, so that knowledge of the specific mutation is not necessary (1). One of the most serious issues regarding PKU is maternal HPA / PKU. For pregnant women with PKU, phenylalanine is a strong teratogen. A woman on the regular diet restriction and consequently low phe serum levels will have no problems and her baby will develop normally. For this reason, women who want to become pregnant but do not observe the diet need only start if they want to give birth to a healthy baby. However, if a woman who does not observe the diet becomes pregnant unexpectedly and does not reduce her phe levels within the first couple months of pregnancy, the baby will be put at serious risk of undergoing congenital heart defects, intrauterine and postnatal growth defects, microcephaly and mental retardation. As always, defects are proportional in severity to the severity of the mother's PKU (1). References 1. Mitchel JJ, Scriver CR. GeneTests: Phenylalanine Hydroxylase Deficiency < =1ZKKWPDKzlal9&gry=&fcn=y&fw=4NW1&filename=/profiles/pku/index.html>. 2. Scriver CR, Kaufman S. Tge hyperphenylalaninemias. IN: Scriver CR, et al. The Metabolic and Molecular Bases of Inhered Disease, 8 ed. McGraw Hill 2001; Chapter Champe PC, Harvard RA, Ferrier DR. Biochemistry: Defects in Amino Acid Metabolism. Biochemistry, LWW 2005; Waters PJ. How PAH gene mutations cause hyper-phenylalaninemia and why mechanism matters: insights from in vitro expression. Hum Mutat 2003;21(4): Berg JM, Tymoczko JL, Stryer L. Amino Acid Degredations. Biochemistry, Freeman 2002; Gelehrter TD, Collins FS, Ginsburg D. Principles of Medical Genetics, 2nd Ed. Williams & Wilkins 1998; Chapter Pandor A, Eastham J, Beverly C, Chilcott J, Paisley S. Clinical effectiveness of neonatal screening for inborn errors of metabolism usin using tandem mass spectrometry: a systematic review. Heath Technol Assess Mar 8:iii,

6 8. Smith I, Beasley M G, Wolff O H, Ades A E. Behavior disturbance in 8-year-old children with early treated phenylketonuria. Report from the MRC/DHSS Phenylketonuria Register. J Pediatr. 1988;112: Azen C, Koch R, Friedman E G, Berlow S, Coldwell J, Krause W, Matalon R, McCabe E, O Flynn M, Peterson R, et al. Intellectual development in 12-year-old children treated for phenylketonuria. Am J Dis Child. 1991;145: Villasana D, Butler IJ, Williams JC, Roongta SM (1989) Neurological deterioration in adult phenylketonuria. J Inherit Metab Dis 12: Prince A P, McMurry M P, Buist N R M. Treatment products and approaches for phenylketonuria: improved palatability and flexibility demonstrate safety, efficacy and acceptance in US clinical trials. J Inherited Metab Dis. 1997;20: Levy, H.L. (1999). Phenylketonuria: Old disease, new approach to treatment. Proc. Natl. Acad. Sci. USA 96,