Phenylketonuria (PKU) the Biochemical Basis. Biol 405 Molecular Medicine

Transcription

1 Phenylketonuria (PKU) the Biochemical Basis Biol 405 Molecular Medicine

2 PKU a history In 1934 Følling identified a clinical condition - imbecillitas phenylpyruvica. Mental retardation associated with this condition was associated with persistent post-natal hyperphenylalaninemia. This phenotype was recognised as the consequence of a deficiency of the enzyme phenylalanine hydroxylase. phenylalanine hydroxylase The deficiency was shown to have a genetic basis i.e. the inheritance of a pair of autosomal recessive mutations from healthy parents. The frequency of phenylketonuria (PKU) was shown to vary in different populations.

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5 In 1946 Penrose renamed the disease phenylketonuria (PKU). Dietary control of serum phenylalanine was demonstrated and this led to the prevention of post-natal mental retardation.

6 The roles of tetrahydrobiopterin and dihydropteridine reductase in the phenylalanine hydroxylation system were established. Mutations can affect any of the enzymes involved in either hydroxylation or cofactor homeostasis.

7 GTP GTP cyclohydrolase 7,8-Dihydroneopterin triphosphate 5,6,7,8-Tetrahydrobiopterin 6-pyruvoyl-tetrahydrobiopterin synthase 6-Pyruvoyl tetrahydrobiopterin sepiapterin reductase dihydropteridine reductase q-dihydrobiopterin pterin-4a-carbinolamine dehydratase phenylalanine hydroxylase 4a-peroxy-tetrahydropterin phenylalanine hydroxylase pterin-4a-carbinolamine The enzymes of biopterin metabolism

8 Defects in cofactor synthesis may lead to impaired production of dopamine and serotonin. Dietary treatment in such patients does not prevent brain damage. These patients require cofactor and/or neurotransmitter therapy.

9 Homozygous mutations One heterozygous mutation Classic PKU 60% 5% 35% Moderate PKU 19% 13% 68% Mild PKU 10% 14% 76% Mild hyperphenylalaninemia 11% 25% 64% Two (different) heterozygous mutations

10 Neonatal screening programs were established for the detection of PKU and other hyperphenylalaninemic conditions. Blood-spot screening has, from the outset, detected about double the number of case of PKU as expected. Milder cases may not need treatment. Screening was possible because there were simple methods for the detection of elevated phenylalanine concentrations in small blood samples. As a result, programs for screening, follow-up, diagnosis and dietary treatment were established. The success of the screening and treatment program led to the identification of maternal hyperphenylalaninemia.

11 In the 1980's Woo cloned a human cdna then isolated and charaterised the human phenylalanine hydroxylase gene. The gene, which consists of 13 exons, is on the long arm of chromosome 12 where it spans approximately 90,000 base pairs. Several intronic polymorphic restriction sites have been identified. These polymorphisms which are the result of nucleotide substitutions, have no phenotypic consequences and can only be detected by the use of restriction endonucleases. A composite profile of individual RFLP's on a given allele is defined as an RFLP haplotype. The development of allele-specific oligonucleotide hybridisation techniques has offered an improved tool for carrier detection compared with phenylalanine loading tests.

12 1990s - It has since been possible to identify over 600 different mutations of the phenylalanine hydroxylase gene that result in phenylketonuria. All the phenylalanine hydroxylase mutations characterised so far have affected specific exons or intron-exon splice junctions. Exon 7 is the one most affected by phenylketonuric mutations.

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15 Mutant Residue (No) Residue Change Site Immunoreactivity Enzymic Activity 243 arg-->term. Exon 7 <1% <1% 281 pro-->leu Exon 7 <1% <1% 408 arg-->trp Exon 12 <1% <1% -- IVS-12 Intron 12 <1% <1% 280 glu-->lys Exon 7 <3% <3% 158 arg-->gln Exon 5 100% 10% 261 arg-->gln Exon 7 30% 30% 414 tyr-->cys Exon 12 50% 50%

16 The future It would be surprising if PKU mutations affected only exons or mrna editing. Some of the, as yet, uncharacterised mutations probably affect DNA sequences regulating the liver-specific expression of the gene. A novel transcriptional regulatory element has recently been found in intron 8. Still unanswered is the reason for the high frequency of mutations of the phenylalanine hydroxylase gene. It is not clear if there is, or ever was, any selective advantage for heterozygotes.

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18 Gene therapy? A bacteriophage integrase system has been successfully used to achieve site-specific genome integration of murine phenylalanine hydroxylase cdna in livers of PKU mice. Following integration, the hyperphenylalaninemic phenotype was significantly decreased.

19 Summary PKU - persistent post-natal hyperphenylalaninemia leading to mental retardation is a consequence of an inherited deficiency of the enzyme phenylalanine hydroxylase. Mutations can affect any of the enzymes involved in either hydroxylation or cofactor homeostasis. Dietary control of serum phenylalanine was demonstrated and this led to the prevention of post-natal mental retardation. Neonatal screening programs were established for the detection of PKU and other hyperphenylalaninemic conditions. It has since been possible to identify over 400 different mutations of the phenylalanine hydroxylase gene that result in PKU.

20 References Erlandsen, H. & Stevens, R. C. (1999) Mol. Gen. Metab. 68, Structural basis of PKU. Harding, C. O. (2008) Clin. Gen. 74, cell-directed therapy for phenylketonuria van Spronsen, F. J. (2010) Nature Reviews Endocrinology 6, PKU a review. Mitchell, J. J. et al., (2011) Genetics in Medicine 13, PKU a review. Staudigl M. et al., (2011) Hu. Molec. Gen. 20, treatment of PKU. - the PAHdb World Wide Web site.