PAHdb 2003: What a locus-specific knowledgebase can do

Abstract

For the PKU Special IssuePAHdb, a legacy of and resource in genetics, is a relational locus-specific database (http:// www.pahdb.mcgill.ca). It records and annotates both pathogenic alleles (n = 439, putative diseasecausing) and benign alleles (n = 41, putative untranslated polymorphisms) at the human phenylalanine hydroxylase locus (symbol PAH). Human alleles named by nucleotide number (systematic names) and their trivial names receive unique identifier numbers. The annotated gDNA sequence for PAH is typical for mammalian genes. An annotated gDNA sequence is numbered so that cDNA and gDNA sites are interconvertable. A site map for PAHdb leads to a large array of secondary data (attributes): source of the allele (submitter, publication, or population); polymorphic haplotype background; and effect of the allele as predicted by molecular modeling on the phenylalanine hydroxylase enzyme (EC 1.14.

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

“…The results of each mutant, expressed as percent of wild-type PAH, are well in accordance with previously published data for the same mutations [33]. The slight differences found were due to the fact that previously reported PAH activity measurements were performed under different assay conditions and using several substrates with different affinities (e.g.…”

State-of-the-art 2003 on PKU gene therapy

“…Breeding such mice to the Pah enu2 strain for homozygosity resulted in offspring lacking PAH activity in the liver but expressing Pah in the skeletal muscle. These animals had significantly elevated serum phenylalanine levels, which decreased only when BH 4 was supplemented by intraperitoneal injections, as the skeletal muscle lacks cofactor biosynthesis. Thus, PAH gene therapy in muscle might become a feasible approach provided that enough BH 4 cofactor is supplied.…”

“…Primary human keratinocytes or skin fibroblasts, which both do not produce BH 4 endogenously, were engineered by transducing two independent retroviral vectors expressing PAH and GTPCH separately. GTPCH is the rate-limiting enzyme in de novo BH 4 biosynthesis in these cells, and co-transduction of human keratinocytes or fibroblasts with retroviral vectors carrying the PAH and the GTPCH genes resulted in phenylalanine clearance in vitro without additional BH 4 supplementation [48,49].…”

“…It is caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH; EC 1.14.16.1), responsible for converting phenylalanine to tyrosine, using molecular oxygen and tetrahydrobiopterin (BH 4 ) as a necessary cofactor to perform its catalytic activity [1,2]. This conversion is the rate-limiting step in phenylalanine catabolism in the liver.…”

“…HPA can also be caused by the absence of the BH 4 cofactor due to deficiency of cofactor biosynthesis and regeneration, but this group of neurometabolic diseases will not be discussed here [3]. The biochemistry and genetics of PKU are well characterized (see for instance http://www.pahdb.mcgill.ca/ [4]). The clinical phenotype is defined and treatment is also available.…”