QUANTIFICATION OF X-CHROMOSOME INACTIVATION PATTERNS USING RT-PCR OF THE POLYMORPHIC IDURONATE-2-SULFATASE GENE AND CORRELATION OF THE RESULTS OBTAINED WITH DNA-BASED TECHNIQUES

Most studies using X-chromosome inactivation patterns (XCIPs) to deter mine clonality in females have used polymorphic DNA loci, but interpre tation may be complicated by the complexity of the differential methyl ation patterns necessary to distinguish active and inactive X-chromoso mes. Recent description of polymorphisms within the transcribed region of three X-chromosome genes has enabled XCIP analysis of allele expre ssion at the RNA level, which should circumvent this problem. We repor t here a quantitative RT-PCR assay for one of these loci, the iduronat e-2-sulphatase (IDS) gene, using a mismatch primer to introduce a Bc/l cutting site in one of the alleles. DNA samples from 150 females were screened and 61 (41%) were found to be informative for the polymorphi sm. Reproducible results were obtained from clonal analysis of 56 RNA samples covering the complete spectrum of XCIP ratios. The values corr elated closely with those obtained from the corresponding DNA samples analyzed using either PGK or HUMARA (r = 0.98) provided that the numbe r of amplification cycles was limited to 25 to reduce heteroduplex for mation. However, sample purity is of greater importance than in the DN A-based assays as contaminating red cells and platelets will still con tain the relevant transcripts and could influence the result.