Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography–electrospray ionization tandem mass spectrometry

Heintz, Caroline; Troxler, Heinz; Martı́nez, Aurora; Thöny, Beat; Blau, Nenad

doi:10.1016/j.ymgme.2011.12.025

Cited by 34 publications

(22 citation statements)

References 38 publications

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“…7,9 A number of mutations are more frequently found in BH4 responders, whereas BH4 nonresponders harbor more severe mutations, with less or little residual PAH activity. 38 All of the 85 genotypes identified within our survey and the 8 mutations detected on only 1 allele were reported to be associated with substantial (.30%) residual PAH activity (www.biopku.org).…”

Section: Figurementioning

confidence: 80%

Long-term Follow-up and Outcome of Phenylketonuria Patients on Sapropterin: A Retrospective Study

et al. 2013

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WHAT'S KNOWN ON THIS SUBJECT: Pharmacologic treatment with sapropterin dihydrochloride (6R-tetrahydrobiopterin; BH4) has been an effective option for some phenylketonuria patients since its approval by the US Food and Drug Administration in 2007 and the European Medicines Agency in 2008. WHAT THIS STUDY ADDS:This retrospective multicenter study revealed the long-term effects of sapropterin on metabolic control, dietary tolerance, and the outcome of BH4-responsive phenylketonuria patients harboring specific phenotypes and genotypes. It also confirmed that the minor adverse events disappeared by lowering the dose. abstract OBJECTIVE: Sapropterin dihydrochloride, the synthetic form of 6R-tetrahydrobiopterin (BH4), is an approved drug for the treatment of patients with BH4-responsive phenylketonuria (PKU). The purpose of this study was to assess genotypes and data on the long-term effects of BH4/sapropterin on metabolic control and patient-related outcomes in 6 large European countries. METHODS:A questionnaire was developed to assess phenotype, genotype, blood phenylalanine (Phe) levels, Phe tolerance, quality of life, mood changes, and adherence to diet in PKU patients from 16 medical centers. RESULTS:One hundred forty-seven patients, of whom 41.9% had mild hyperphenylalaninemia, 50.7% mild PKU, and 7.4% classic PKU, were followed up over #12 years. A total of 85 different genotypes were reported. With the exception of two splice variants, all of the most common mutations were reported to be associated with substantial residual Phe hydroxylase activity. Median Phe tolerance increased 3.9 times with BH4/sapropterin therapy, compared with dietary treatment, and median Phe blood concentrations were within the therapeutic range in all patients. Compared with diet alone, improvement in quality of life was reported in 49.6% of patients, improvement in adherence to diet was reported in 47% of patients, and improvement in adherence to treatment was reported in 63.3% of patients. No severe adverse events were reported. CONCLUSIONS: Our data document a long-term beneficial effect of orally administered BH4/sapropterin in responsive PKU patients by improving the metabolic control, increasing daily tolerance for dietary Phe intake, and for some, by improving dietary adherence and quality of life. Patient genotypes help in predicting BH4 responsiveness.

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Section: Figurementioning

confidence: 80%

Long-term Follow-up and Outcome of Phenylketonuria Patients on Sapropterin: A Retrospective Study

et al. 2013

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“…In mice, Pah is mainly expressed in liver but there is also considerable expression in kidney 29 ( Supplementary Figure S3 ). We analyzed Pah -mRNA, as well as PAH protein and enzyme activity in Pah enu2/+ mice subjected to different treatment regimes.…”

Section: Resultsmentioning

confidence: 99%

“…PAH enzyme activity in liver extracts was measured according to a highly sensitive and quantitative assay using isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MSMS) as described. 29 Briefly, assay conditions included preincubation at 25 °C with L-Phe (1 mmol/l) for 4 minutes, then Fe(NH 4 ) 2 (SO 4 ) 2 (100 ìmol/l) was added, and incubation was continued for one more min. After 5 minutes total preincubation time, BH 4 (75 ìmol/l) was added to start the reaction.…”

Section: Methodsmentioning

confidence: 99%

A Sensitive Assay System To Test Antisense Oligonucleotides for Splice Suppression Therapy in the Mouse Liver

Gallego

Viecelli

Pérez

et al. 2014

Molecular Therapy - Nucleic Acids

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We have previously demonstrated the efficacy of antisense therapy for splicing defects in cellular models of metabolic diseases, suppressing the use of cryptic splice sites or pseudoexon insertions. To date, no animal models with these defects are available. Here, we propose exon skipping of the phenylalanine hydroxylase (Pah) gene expressed in liver and kidney to generate systemic hyperphenylalaninemia in mice as a sensitive in vivo assay to test splice suppression. Systemic elevation of blood L-Phe can be quantified using tandem MS/MS. Exon 11 and/or 12 skipping for the normal PAH gene was validated in hepatoma cells for comparing two oligonucleotide chemistries, morpholinos and locked nucleic acids. Subsequently, Vivo-morpholinos (VMO) were tested in wild-type and in phenotypically normal Pahenu2/+ heterozygous mice to target exon 11 and/or 12 of the murine Pah gene using different VMO dosing, mode of injection and treatment regimes. Consecutive intravenous injections of VMO resulted in transient hyperphenylalaninemia correlating with complete exon skipping and absence of PAH protein and enzyme activity. Sustained effect required repeated injection of VMOs. Our results provide not only a sensitive in vivo assay to test for splice-modulating antisense oligonucleotides, but also a simple method to generate murine models for genetic liver diseases.

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“…PAH activities were determined using a novel mass spectrometry method for quantification of Phe and Tyr in cell lysates [19]. Mutations in the human PAH cDNA sequence in pCMV-FLAG-PAH were introduced by site-directed mutagenesis using QuikChange XL II kit from Agilent Technologies (Santa Clara, CA, USA) and confirmed by DNA sequence analysis.…”

Section: Expression Of Pah Proteins and Activity Assaymentioning

confidence: 99%

Splicing of phenylalanine hydroxylase (PAH) exon 11 is vulnerable: Molecular pathology of mutations in PAH exon 11

Heintz

Dobrowolski

Andersen

et al. 2012

Molecular Genetics and Metabolism

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In about 20-30% of phenylketonuria (PKU) patients, phenylalanine (Phe) levels can be controlled by cofactor 6R-tetrahydrobiopterin (BH(4)) administration. The phenylalanine hydroxylase (PAH) genotype has a predictive value concerning BH(4)-response and therefore a correct assessment of the mutation molecular pathology is important. Mutations that disturb the splicing of exons (e.g. interplay between splice site strength and regulatory sequences like exon splicing enhancers (ESEs)/exon splicing silencers (ESSs)) may cause different severity of PKU. In this study, we identified PAH exon 11 as a vulnerable exon and used patient derived lymphoblast cell lines and PAH minigenes to study the molecular defect that impacted pre-mRNA processing. We showed that the c.1144T>C and c.1066-3C>T mutations cause exon 11 skipping, while the c.1139C>T mutation is neutral or slightly beneficial. The c.1144T>C mutation resides in a putative splicing enhancer motif and binding by splicing factors SF2/ASF, SRp20 and SRp40 is disturbed. Additional mutations in potential splicing factor binding sites contributed to elucidate the pathogenesis of mutations in PAH exon 11. We suggest that PAH exon 11 is vulnerable due to a weak 3' splice site and that this makes exon 11 inclusion dependent on an ESE spanning position c.1144. Importantly, this implies that other mutations in exon 11 may affect splicing, since splicing is often determined by a fine balance between several positive and negative splicing regulatory elements distributed throughout the exon. Finally, we identified a pseudoexon in intron 11, which would have pathogenic consequences if activated by mutations or improved splicing conditions. Exonic mutations that disrupt splicing are unlikely to facilitate response to BH(4) and may lead to inconsistent genotype-phenotype correlations. Therefore, recognizing such mutations enhances our ability to predict the BH(4)-response.

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Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography–electrospray ionization tandem mass spectrometry

Cited by 34 publications

References 38 publications

Long-term Follow-up and Outcome of Phenylketonuria Patients on Sapropterin: A Retrospective Study

Long-term Follow-up and Outcome of Phenylketonuria Patients on Sapropterin: A Retrospective Study

A Sensitive Assay System To Test Antisense Oligonucleotides for Splice Suppression Therapy in the Mouse Liver

Splicing of phenylalanine hydroxylase (PAH) exon 11 is vulnerable: Molecular pathology of mutations in PAH exon 11

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