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Diagnosis, classification, and genetics of phenylketonuria and tetrahydrobiopterin (BH4) deficiencies

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Abstract

This article summarizes the present knowledge, recent developments, and common pitfalls in the diagnosis, classification, and genetics of hyperphenylalaninemia, including tetrahydrobiopterin (BH4) deficiency. It is a product of the recent workshop organized by the European Phenylketonuria Group in March 2011 in Lisbon, Portugal. Results of the workshop demonstrate that following newborn screening for phenylketonuria (PKU), using tandem mass-spectrometry, every newborn with even slightly elevated blood phenylalanine (Phe) levels needs to be screened for BH4 deficiency. Dried blood spots are the best sample for the simultaneous measurement of amino acids (phenylalanine and tyrosine), pterins (neopterin and biopterin), and dihydropteridine reductase activity from a single specimen. Following diagnosis, the patient's phenotype and individually tailored treatment should be established as soon as possible. Not only blood Phe levels, but also daily tolerance for dietary Phe and potential responsiveness to BH4 are part of the investigations. Efficiency testing with synthetic BH4 (sapropterin dihydrochloride) over several weeks should follow the initial 24–48-hour screening test with 20 mg/kg/day BH4. The specific genotype, i.e. the combination of both PAH alleles of the patient, helps or facilitates to determine both the biochemical phenotype (severity of PKU) and the responsiveness to BH4. The rate of Phe metabolic disposal after Phe challenge may be an additional useful tool in the interpretation of phenotype–genotype correlation.

Section snippets

Newborn screening

Phenylketonuria (PKU) is identified through national newborn screening programs [1]. The first efficient test for hyperphenylalaninemia (HPA) was a bacterial inhibition assay developed by Robert Guthrie [2]. The test was based on Bacillus subtilis, which requires phenylalanine (Phe) for growth. The Guthrie test was very useful for mass screening as the dried blood spot (DBS) can be obtained in the hospital or a doctor's office using a standardized filter paper (“Guthrie card”) and mailed to

Phenylalanine loading test

Phenylalanine loading tests were applied since 1956 for the detection of heterozygotes in PKU families [27], until, in the late 1980s, this approach was replaced by molecular analysis of PAH gene haplotypes and mutations [28]. Phenylalanine loading tests gained further interest when Guthrie card mass screening uncovered not only the expected cases of classic PKU but also variants of PKU [29]. Because these variants were initially thought not to require dietary treatment, a reliable

PAH mutations and PKU genotypes (incl. databases)

As already stated, knowing whether a patient has residual PAH enzyme activity can be relevant for the therapeutic approach, the likely Phe tolerance, and the expected response to BH4. Delineation of the mutations of the PAH gene was initiated immediately after the cloning of the gene in 1983 [28]. Initially the most prevalent mutations in the Western European population were identified and characterized with regard to the in vitro residual enzyme activity associated with the respective mutation

Acknowledgments

Authors would like to thank Serono Scientific International Foundation (SSIF) for supporting organization of the workshop on “Diagnosis, classification and genetics of PKU”. This work was supported in part by the Swiss National Science Foundation grant no. 31003A-119982 (to NB). UL is grateful to the German Collaborative Study on PKU for providing the study data whose analysis resulted in Ref. [36] and Fig. 3 of the present communication.

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