Dihydropteridine reductase deficiency cloning of the dihydropteridine reductase gene analysis of mutant cells and prospects for genetic therapy

Ledley, F.D.; Lockyer, J.; Kaufman, S.; Milstein, S.; Woo, S.L.C.

Pediatric Research 21(4 Part 2): 344A

1987


ISSN/ISBN: 0031-3998
DOI: 10.1203/00006450-198704010-01059
Accession: 028105644

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Abstract
Deficiency of the enzyme dihydropteridine reductase (DHPR) causes a syndrome of hyperphenylalaninemia and mental retardation but is refactory to conventional dietary therapy. An antibody against sheep Dhpr was used to identify clones for human Dhpr from a human liver c Dna expression library. The full length c Dna clone comprises 1560 bases, codes for a protein of 244 amino acids and 25,774 daltons, and the predicted amino acid sequence matches an incomplete amino acid sequence of sheep DHPR. The c Dna was recombined in an eukaryotic expression vector and introduced into cultured cells by Dna mediated gene transfer. Cells transformed with the recombinant gene expressed Dhpr protein and enzymatic activity at levels 50% of human liver. Analysis of fibroblasts from five unrelated individuals genetically deficient in Dhpr by southern and northern blotting indicates that the Dhpr gene is grossly in tact and that Dhpr m Rna is transcribed. These results indicated that the mutations causing Dhpr deficiency are not large deletions of the Dhpr locus. These experiments also demonstrate the feasibility of reconstituting Dhpr activity by gene transfer of the recombinant clone and introduces the possibility of exploring somatic gene replacement therapy of Dhpr deficiency.