Molecular analysis of PKU-associated PAH mutations: a fast and simple genotyping test

Abstract: Neonatal screening for phenylketonuria (PKU, OMIM: 261600) was introduced at the end of the 1960s. We developed a rapid and simple molecular test for the most frequent phenylalanine hydroxylase (PAH, Gene ID: 5053) mutations. Using this method to detect the 18 most frequent mutations, it is possible to achieve a 75% detection rate in Italian population. The variants selected also reach a high detection rate in other populations, for example, 70% in southern Germany, 68% in western Germany, 76% in Denmark, 68% in Sweden, 63% in Poland, and 60% in Bulgaria. We successfully applied this confirmation test in neonatal screening for hyperphenylalaninemias using dried blood spots and obtained the genotype in approximately 48 h. The method was found to be suitable as second tier test in neonatal screening for hyperphenylalaninemias in neonates with a positive screening test. This test can also be useful for carrier screening because it can bypass the entire coding sequence and intron–exon boundaries sequencing, thereby overcoming the questions that this approach implies, such as new variant interpretations

In vitro study of uptake and synthesis of creatine and its precursors by cerebellar granule cells and astrocytes suggests some hypotheses on the physiopathology of the inherited disorders of creatine metabolism

<p>Abstract</p> <p>Background</p> <p>The discovery of the inherited disorders of creatine (Cr) synthesis and transport in the last few years disclosed the importance of blood Cr supply for the normal functioning of the brain. These putatively rare diseases share a common pathogenetic mechanism (the depletion of brain Cr) and similar phenotypes characterized by mental retardation, language disturbances, seizures and movement disorders. In the effort to improve our knowledge on the mechanisms regulating Cr pool inside the nervous tissue, Cr transport and synthesis and related gene transcripts were explored in primary cultures of rat cerebellar granule cells and astrocytes.</p> <p>Methods</p> <p>Cr uptake and synthesis were explored in vitro by incubating monotypic primary cultures of rat type I astrocytes and cerebellar granule cells with: a) D₃-Creatine (D₃Cr) and D3Cr plus β-guanidinopropionate (GPA, an inhibitor of Cr transporter), and b) labelled precursors of Guanidinoacetate (GAA) and Cr (Arginine, Arg; Glycine, Gly). Intracellular D3Cr and labelled GAA and Cr were assessed by ESI-MS/MS. Creatine transporter (<it>CT1</it>), L-arginine:glycine amidinotransferase (<it>AGAT</it>), and S-adenosylmethionine:guanidinoacetate N-methyltransferase (<it>GAMT</it>) gene expression was assessed in the same cells by real time PCR.</p> <p>Results</p> <p>D3Cr signal was extremely high in cells incubated with this isotope (labelled/unlabelled Cr ratio reached about 10 and 122, respectively in cerebellar granule cells and astrocytes) and was reduced by GPA. Labelled Arg and Gly were taken up by the cells and incorporated in GAA, whose concentration paralleled that of these precursors both in the extracellular medium and inside the cells (astrocytes). In contrast, the increase of labelled Cr was relatively much more limited since labelled Cr after precursors' supplementation did not exceed 2,7% (cerebellar granule cells) and 21% (astrocytes) of unlabelled Cr. Finally, <it>AGAT, GAMT </it>and <it>SLC6A8 </it>were expressed in both kind of cells.</p> <p>Conclusions</p> <p>Our results confirm that both neurons and astrocytes have the capability to synthesize and uptake Cr, and suggest that at least in vitro intracellular Cr can increase to a much greater extent through uptake than through <it>de novo </it>synthesis. Our results are compatible with the clinical observations that when the Cr transporter is defective, intracellular Cr is absent despite the brain should be able to synthesize it. Further research is needed to fully understand to what extent our results reflect the in vivo situation.</p

The spectrum of phenylalanine variations under tetrahydrobiopterin load in subjects affected by phenylanine hydroxylase deficiency.

A fall in blood phenylalanine (Phe) after tetrahydrobiopterin (BH4) administration is a common trait in phenylalanine hydroxylase (PAH, EC 1.14.16.1) deficiency (McKusick 261600). To explore the extent and biological correlates of this phenomenon we studied: (a) the spectrum of BH4 response in patients with PAH deficiency; (b) the variability of BH4 response according to the severity of the biochemical phenotype; and (c) the variability of the response to BH4 in subjects with the same genotype. Fifty PAH-deficient subjects (age 1 month–35 years) were enrolled for the study (5 with mild hyperphenylalaninaemia (MHPHE), 15 with mild phenylketonuria (MPKU) and 30 with classic phenylketonuria (CPKU) and underwent an identical schedule of blood samplings 24 h before and after oral BH4 challenge (6(R)-BH4, 20 mg/kg per day), leaving Phe intake unchanged. The effect ofBH4 on blood Phe concentration was evaluated according to the percent decrease of Phe during the 24 h following the challenge (criterion a), and as variation exceeding the individual variability of blood Phe (criterion b). The number of BH4-responders according tocriterion b was 31 (including all the 14 detected by criterion a): 17 out of 30 CPKU (57%), 9 out of 15 MPKU (60%), and all the MHPHE subjects (?2 = 3.45, df = 2, p = 0.178). The effect of BH4 showed a large interindividual variability unrelated to diagnostic classification, basal value of blood Phe, maximum percentage of Phe reduction, Phe intake, and genotype. Some inconsistencies were found in patients with identical genotype. The first responsive case homozygous for the severe R408W mutation was found. Two new mutations, Y387X and G352C, were identified (the former was BH4- responsive), and the responsiveness of three already reported mutations (R261Q, D338Y, T92I) was substantiated