A human purine nucleoside phosphorylase deficiency caused by a single base change

Purine nucleoside phosphorylase (PNP) deficiency in humans is associated with a severe defect in T-lymphocyte function. The mutant gene was cloned from one PNP-deficient patient who was the offspring of a consanguineous mating. The exons and intron/exon boundaries of the mutant PNP gene were sequenced and compared with the wild-type cDNA sequence. A single base difference was found in the coding region of the mutant gene, a G to A transition in the third exon. This single base mutation alters the codon at position 89 from Glu to Lys, a result which is consistent with previously published peptide mapping data. The patient was demonstrated to be autozygous for the single base mutation on the basis of hybridization of synthetic oligomers to genomic DNA digests. A mammalian expression vector was constructed containing the entire mutant gene under the transcriptional regulation of its own promoter. In another construction, the single base mutation was reverted to the wild-type sequence by in vitro mutagenesis. An isoelectric focusing gel containing extracts of the cells transfected with the mutant and reverted PNP gene was stained histochemically for PNP activity. The proteins from a similar gel were blotted on a nitrocellulose membrane, and immunoreactive human PNP protein was visualized. Cells transfected with the mutant gene contained no human PNP activity, but expressed immunoreactive PNP which focused at an abnormally alkaline pI. Cells transfected with the reverted gene expressed human PNP activity which co-focused with human PNP from a HeLa cell control, proving that the observed single base change was responsible for the loss of catalytic function.