The molecular characterisation of HPRT CHERMSIDE and HPRT COORPAROO: two Lesch-Nyhan patients with reduced amounts of mRNA.

A complete deficiency of the purine salvage enzyme, hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8), in man results in the Lesch-Nyhan (LN) syndrome. Two unrelated patients with the full LN syndrome showed no evidence of a major alteration to the gene encoding HPRT (HPRT) by restriction endonuclease analysis, but exhibited negligible levels of HPRT mRNA on Northern blots. DNA from these patients was characterised further. Amplification, by the polymerase chain reaction (PCR), of individual HPRT-exon fragments from genomic DNA followed by nucleotide (nt) sequence analysis using automated technology, revealed single-base mutations in each patient. One patient has an insertion of a T within exon-2, which places a stop codon in frame, presumably resulting in premature termination of translation of the HPRT mRNA. The other patient has a G----A base substitution at the 5' end of intron-6, at the junction of exon-6 and intron-6. Although dot blot analysis indicated negligible HPRT mRNA in lymphoblast cells from both patients, we were successful in amplifying HPRT cDNA using PCR. Direct nt sequence analysis of the amplified cDNA confirmed the insertion of a T in exon-2 in the one patient and revealed a complete deletion of exon-6 in the other patient, the latter event presumably arising due to aberrant splicing of primary message. Both mutations were also confirmed by hybridisation of amplified genomic DNA with allele-specific oligodeoxyribonucleotide probes. This study illustrates two approaches for analysing DNA mutations at the molecular level and demonstrates the power of PCR technology in the study of genetic diseases.(ABSTRACT TRUNCATED AT 250 WORDS)