1Phenylketonuria (PKU)- classic “inborn error of metabolism”- autosomal recessive disease characterized by mutations in the liver enzyme, phenylalanine hydroxylase, encoded by the PAH gene PAH converts phenylalanine to tyrosine(reaction requires O2 and co-factor BH4)- HPA or non-PKU hyperphenylalaninemia are related disorders of phenylalanine hydroxylation involving several enzymes necessary for the synthesis and recycling of co-factor for PAH, tetrahydrobiopterin (BH4)- incidence: 1 in 10,000History of PKU1934: Asbjorn Folling described an inherited metabolic disorder characterized by severe intellectual impairment, motor problems and skin abnormalities - affected individuals identified by abnormal excretion of phenylpyruvic acid (high frequency of consanguinity in parents of PKU patients; Mendelian disease)1950s: PKU patients shown to have deficient activity of PAH1960s: treatment of PKU with low phenylalanine diet shown to be effective1980s: mapping and cloning of PAH gene1990s: PKU more than a simple Mendelian trait; also behaves as complex,multifactorial disorder2000s: Non-dietary treatments for PKU developed2PKU has a multifactorial cause: mutation in PAH gene (genetic) exposure to dietary phenylalanine (environmental)Clinical features of PKUenzyme deficiency is a primarily hepatic phenotype but major clinical presentation is abnormal brain development and function- severe mental retardation will result in untreated cases (estimated that 1% of patients in mental institutions have PKU)- reduced higher-brain abilities (executive functions)- neuropsychological dysfunction (imbalance of neurotransmitters)- emotional disturbance and behavioral problems (clinical depression)3dopamine(neurotransmitter)defects in these enzymes leadto albinism4Subtypes of PKU, phenylalanine levels & clinical outlook fold increase blood [Phe] clinical picture treatment subtype (over normal) (brain dysfunction) required? classic PKU >20 severe mental yes (untreated) retardation mild PKU 10-15 cognitive loss yes (untreated) non-PKU 2-8 normal maybe mild HPANewborn screening for PKU- done with a simple blood test, screening is standard in many developed countries- resource for sampling of mutant PAH genes- prenatal diagnosis is possible- classification of severe and less severe forms as well as non-PKU HPA requires Phe and BH4 measurements in several body fluids5Maternal PKU- pregnant mothers with untreated PKU can give birthto children with severe defects- congenital malformations- microcephaly- severe mental retardation - careful treatment with diet is compatible with normaloutcome for fetusPathogenic PAH alleles• null alleles or gene deletions (no activity)• Vmax alleles (reduced activity)• kinetic alleles (altered affinity for substrate or cofactor)• unstable alleles (increased turnover and loss of PAH protein)majority of mutations6Effects of disease-causing PAH mutations on a patient can be measured at three levels:• proximal (enzymatic) : in vitro assay • intermediate (metabolic) : plasma phenylalanine levels• distal (cognitive function) : IQ testsgenotype-phenotype correlations show good correlations at the proximal levelsat intermediate and distal levels, phenotypes behave as complex traits suggesting the presence of “modifiers”Pathophysiology of PKU- metabolites of PKU (i.e. phenylpyruvate) not present in high enough concentrations to be toxic- is phenylalanine the neurotoxic agent? 1) brain protein synthesis 2) transport processes and neurotransmitter biosynthesis (tyrosine (Tyr) and tryptophan (Trp) are transported across blood-brain barrier for synthesis of the neurotransmitters, dopamine and serotonin, respectively)7- hypotyrosinemia: low [tyrosine], low neurotransmitters(loss of biogenic amines at critical stages in postnatal brain maturation)- decreased protein synthesis in brain (weak evidence)- defective brain myelination (chronic and irreversible)Phe has higher affinity for transporter compared to Tyr and Trp blood-brain barrierPheTyrTrpPotential problems with the low tyrosine theory...• postnatal tyrosine supplementation without reduction of phenylalanine intake does not prevent mental retardation in PKU• no consistent or pathological reduction in plasma tyrosine content in untreated PKU patients• tyrosine supplements during treatment of PKU sufficient to increase plasma tyrosine levels do not improve neurophysiological parameters8Treatment: dietary restrictionsadherence to a phenylalanine-free diet postnatally can prevent mental retardation and improve behavior in children with PKUsynthetic dietary supplement needed to avoid malnutritionPhenyl-free: Phe-free amino acid mixture, vitamins, minerals, fat (marketed by Mead Johnson) - offensive in odor and taste - must be continued for life - emotional stress in PKU families - high cost (“patient years”)9Aspartame (NutrasweetTM) is an amino acid sweetener, with two constituent amino acids, aspartic acid and phenylalanine, both commonly found in food.phenylalaninesynthetic diet not perfect...- produces several biological side effects due to periodic nutrient deficiencies- needs improvement in organoleptic properties (essential fatty acids) and nutrient composition (ratios of amino acids)treatment alternatives:- gene therapy (not yet applicable)- enzyme replacement therapy (PAL and PEG-PAL papers)PAL: non-mammalian enzyme; degrades Phe to ammonia and trans-cinnamic acid (harmless metabolite)10treatment of mild PKU with tetrahydrobiopterin (BH4) loading- several recent studies suggest that BH4 can be a treatment alternativeto dietary restriction of phenylalanineTetrahydrobiopterin as an alternative treatment for mild phenylketonuria N Engl J Med. 2002 Dec 26;347(26):2122-32• out of 38 with PAH deficiency, 87% showed responsiveness to BH4 (i.e. had lower blood phenylalanine levels)• no response in 7 patients with classic PKU• long-term treatment with BH4 in 5 patients increased daily phenylalanine tolerance enough to discontinue Phe-restricted diet• mutations connected to BH4 responsiveness predominantly in the catalytic domain of the protein and were not directly involved in cofactor bindingTreatment of classical PKU with BH4- recent reports indicate that BH4 loading