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'Molecular and Metabolic Bases of Tetrahydrobiopterin-Responsive Phenylalanine Hydroxylase Deficiency'

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'Molecular and Metabolic Bases of Tetrahydrobiopterin-Responsive Phenylalanine Hydroxylase Deficiency' Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2012 Molecular and metabolic bases of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency Heintz, Caroline Other titles: Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-65952 Dissertation Originally published at: Heintz, Caroline. Molecular and metabolic bases of tetrahydrobiopterin-responsive phenylalanine hydrox- ylase deficiency. 2012, University of Zurich, Faculty of Science. MOLECULAR AND METABOLIC BASES OF TETRAHYDROBIOPTERIN- RESPONSIVE PHENYLALANINE HYDROXYLASE DEFICIENCY Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Caroline Heintz aus Luxemburg Promotionskomitee Prof. Dr. François Verrey (Vorsitz) Prof. Dr. Nenad Blau (Leitung der Dissertation) Prof. Dr. Beat Thöny Prof. Dr. Marc Donath Zürich, 2012 Abstract Phenylketonuria (PKU) is the most common inborn error of human amino acid metabolism. The disorder is characterized by elevated levels of phenylalanine (Phe) in the blood, due to a deficiency in phenylalanine hydroxylase (PAH), the hepatic enzyme oxidizing Phe to tyrosine (Tyr) in the presence of O2 and the cofactor tetrahydrobiopterin (BH4). The accumulated Phe is neurotoxic, leading to CNS Tyr deficiency and disturbed neurotransmitter synthesis. If not treated early in life by dietary restriction of essential Phe, the metabolic phenotype is severe resulting in disturbances in brain development and cognition. PKU is inherited in an autosomal recessive manner with a broad mutational spectrum leading to heterogeneity among PKU patients. Many patients are compound heterozygous with the genotype being the main determinant for the phenotype and not the single allele alone. The variation of dietary protein combined with genetic alteration lead to a multifactorial cause for hyperphenylalaninemia. However, there are many unsolved aspects between the patient’s genotype, its predicted effect on enzyme function, and the associated metabolic phenotype. Oral supplementation of the PAH natural cofactor BH4 is the first pharmacological therapy for patients with PKU, but there is still limited information on the molecular basis of BH4 responsiveness. About 20 - 30% of PKU patients are known to respond to BH4 administration by a significant reduction of their blood phenylalanine levels, i.e. they can be treated with BH4 instead of a low Phe diet or in combination with the diet. In this work, we aimed at gaining further insight into the metabolic and molecular mechanisms of determining BH4 responsiveness. The thesis project outlines a molecular, in vitro analysis of several common, and also new PKU mutations and testing of their response to BH4 in a mammalian cell system. In order to gather more genotype information and select mutation combinations for analysis, two patient studies are described. They include the large-scale genotyping of PKU patients previously tested for BH4 responsiveness. Phe levels as well as loading test data were available from most of the 750 patients from Turkey and Croatia. The large dataset permitted the division into several smaller groups to discriminate between distinct BH4 loading test protocols and BH4 formulation used. Based on these data we calculated correlations of the genotypes with response to BH4, phenotype, and with in vitro residual PAH activities. The correlations showed a relative high I residual PAH activity is required for response to BH4 supplementation. Among the Turkish PKU patients, a relatively high proportion (22%) of potential candidates for the BH4 therapy was detected. Furthermore, the studies highlighted that single allele mutations are not reliable for the selection of potential PKU candidates for pharmacological therapy with BH4. Considering both studies, twenty-four novel mutations were detected. The assembly of more than 700 genotypes, phenotypes and BH4 loading tests data further extended the BIOPKU database. The database is public and new patients with documented non-responsive genotype may not need to undergo BH4 testing. The quantification of PAH activity expressed in cultured cells or in animal tissues is performed by a novel tandem mass spectrometry assay. Mass spectrometry allows the use of stable isotopes for Phe and Tyr quantification and PAH activity measurement. Among the discordances reported with effective prediction of a mutation’s effect on enzyme function and residual PAH activity, is the use of various assay protocols and quantification methods. Therefore, a validated, reproducible, and more specific method compared to previously used approaches for determining in vitro PAH activity was established. LC-MSMS is a powerful technique where low detection limits were found for both amino acids and thus it requires lower sample amounts for accurate determination of PAH activity. Furthermore, several particular mutations found in Turkish PKU patients were selected for detailed molecular analysis. The study included investigating the association of an intronic mutation in PAH intron 10 with BH4 responsiveness and two other mutations in PAH exon 11. Discordant PAH activity results were found for these mutations, as well as their location in important splicing regulatory elements pointed to an implication on PAH exon 11 mRNA processing. PAH minigene constructs and EBV-transformed PKU patient cell lines harboring these mutations were established and analyzed. More than one transcript was detected upon amplification of exons 10 to 12 from the minigenes and the patient cells carrying the mutations of interest. Sequencing confirmed skipping of PAH exon 11 during mRNA processing. Affinity purifications showed abolished binding to specific SR proteins of RNA oligonucleotides, carrying the two missense mutations investigated. PAH exon 11 is vulnerable and recognition of exon 11 is subtle with mutations in splicing regulatory sites. Only the combination of the two mutant PAH alleles that determines residual PAH activity in vivo, and the individual mutations of a patient should not be viewed by themselves. The final section of this thesis presents the setup and validation of a mammalian cell test system with engineered plasmid vectors. The system allows transient co-expression of wild type or mutant PAH variants of previously selected genotypes and measurement of II PAH activity by LC-MSMS and expression levels, in presence or absence of exogenous BH4. The selection of genotype combinations was done referring to the previously mentioned patient studies and the BIOPKU database. The co-expression of two distinct PAH mutant alleles revealed possible dominance effects (positive or negative) by one of the mutations on residual activity as result of interallelic complementation. Treatment of the transfected cells with sepiapterin as BH4 precursor showed an increase in residual PAH activity with several mutations co-expressed. In summary, the results presented herein provide additional information and elucidation on PKU genotypes, phenotypes, and response to BH4 as a reference available for clinicians, health care professionals, and researchers for diagnosis and establishment of tailored treatment of patients. III Zusammenfassung Die Phenylketonurie (PKU) gehört zu den häufigsten, angeborenen Störungen im menschlichen Stoffwechsel der Aminosäuren. Durch einen Defekt im Leberenzym Phenylalanin Hydroxylase (PAH) wird die essentielle Aminosäure Phenylalanin (Phe) unvollständig zu Tyrosin (Tyr) umgewandelt. Dieser Vorgang führt, unter der Beteiligung von Tetrahydrobiopterin (BH4, Kofaktor) und O2, zur Anreicherung von Phe im Blut. Daraus resultieren Störungen im Tyrosinstoffwechsel sowie auch in der anschließenden Neurotransmittersynthese. Bei strenger, von Anfang an eingehaltener Phe-reduzierter Diät können die neurotoxischen Schäden von Phe und der Mangel an Folgemetaboliten in der Hirn- und Wahrnehmungsentwicklung vermindert werden. Die Vererbung von PKU erfolgt autosomal rezessiv mit einem breiten Mutationsspektrum und führt zu einer Vielfalt an metabolischen Phänotypen. Die meisten Patienten haben einen gemischt heterozygoten Genotyp, welcher für die Ausprägung des Phänotyps im Gegensatz zum einzelnen mutierten Allel bestimmend ist. Die Schwankungen in der Nahrungsproteinaufnahme in Kombination mit der Genänderung sind multifaktorielle Ursachen für Hyperphenylalaninämie. Diese Faktoren führen zu verschiedenen Unstimmigkeiten in der Vorhersage des assoziierten metabolischen Phänotyps, aufgrund des Einflusses der Mutationen und deren vorhergesagten Effekts auf die Enzymfunktion. Die erste pharmakologische Behandlung für PKU Patienten besteht in der Einnahme von BH4, dem natürlichen Kofaktors von PAH. Es gibt allerdings nur wenig Information über die molekularbiologischen Hintergründe bezüglich des Ansprechverhaltens auf BH4. Etwa 20 bis 30% der Patienten reagieren auf diese Einnahme durch eine Abnahme der Blutphenylalaninkonzentration und können somit mit BH4 anstelle der niedrigen Phe-Diät behandelt werden. Das Ziel der vorliegenden Arbeit ist die weitere Aufklärung der metabolischen und molekularbiologischen Mechanismen der BH4-Sensitivität. Das Projekt behandelt die molekulare, in vitro
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