Ann-Marie Steen

Mutation analysis and prenatal diagnosis in a Lesch-Nyhan family showing non-random X-inactivation interfering with carrier detection tests

SummaryA nonsense mutation at the CpG-site in the codon for Arg(169) in the gene for hypoxanthine phosphoribosyltransferase (hprt) was identified by genomic polymerase chain reaction (PCR) and DNA sequencing in cultured fibroblasts from two brothers with Lesch Nyhans syndrome. The recurrence of mutation at this CpG-site in several unrelated Lesch-Nyhan families suggests that deamination of 5-methylcytosine is a possible mechanism for mutagenesis. The level of hprt-mRNA in the fibroblasts of the patients was similar to that in healthy controls, whereas hprt-enzyme activity was not detectable. The mutation in this family was also identified in five female relatives and prenatally in a male fetus. Unexpectedly, results from hair follicle analyses and fibroblast selection studies in 8-azaguanine and 6-thioguanine medium showed a non-carrier phenotype in three of the female heterozygotes, whereas X-inactivation mosaicism was demonstrated in one heterozygote. A possible explanation for the apparent non-random X-inactivation in this family is the co-existence of the hprt mutation with an undefined X-linked lethal mutation. This observation is of practical relevance for carrier detection in other Lesch-Nyhan families.

Hprt activities and RNA phenotypes in 6-thioguanine resistant human T-lymphocytes

The phenotypic effects of mutation in the hypoxanthine phosphoribosyltransferase (hprt) gene on hprt enzyme activity and hprt mRNA levels were studied in 6-thioguanine (TG) resistant human T-cell clones with various types of hprt mutation. The mean enzyme activity in 16 TG selected clones was less than 1% of the mean in unselected clones. The hprt mRNA levels, measured by a quantitative RNA/RNA solution hybridization assay, were within the normal range in 38% of the mutant clones. Reduced hprt mRNA levels were found in all of three nonsense mutations, four out of five splicing mutations, both of two clones with genomic alterations, three out of five missense mutations and one out of four frameshift mutations caused by 1-4-bp deletions. Intermediate and high enzyme activity and normal hprt mRNA levels were found in two TG selected clones where no hprt mutation was detected. Several clones with very low hprt mRNA levels were found to yield hprt cDNA by PCR amplification. These results show that hprt mutation leads to decreased steady state levels of hprt mRNA in a majority of TG resistant T-cell clones, and that many different types of hprt mutation can have this effect.

The fragile X mutation does not have any major effect on the expression of the hypoxanthine phosphoribosyltransferase (HPRT) locus in human fibroblasts

SummaryThe possible influence of the fragile X mutation at Xq27 on the expression of the neighbouring gene (at Xq26) for hypoxanthine phosphoribosyl transferase (HPRT) was studied by determination of the levels of HPRT-RNA and HPRT enzyme activity in fibroblast cell cultures from 7 fragile X patients. These levels were lower (although not statistically significantly lower) than in normal fibroblast cultures. Hence, these data do not support the notion of a major effect of the fragile X mutation on the expression of the HPRT gene.