Quantification of X-chromosome inactivation patterns using RT-PCR of the polymorphic iduronate-2-sulphatase gene and correlation of the results obtained with DNA-based techniques.
Most studies using X-chromosome inactivation patterns (XCIPs) to determine clonality in females have used polymorphic DNA loci, but interpretation may be complicated by the complexity of the differential methylation patterns necessary to distinguish active and inactive X-chromosomes. Recent description of polymorphisms within the transcribed region of three X-chromosome genes has enabled XCIP analysis of allele expression at the RNA level, which should circumvent this problem. We report here a quantitative RT-PCR assay for one of these loci, the iduronate-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 polymorphism. Reproducible results were obtained from clonal analysis of 56 RNA samples covering the complete spectrum of XCIP ratios. The values correlated closely with those obtained from the corresponding DNA samples analyzed using either PGK or HUMARA (r=0.98) provided that the number of amplification cycles was limited to 25 to reduce heteroduplex formation. However, sample purity is of greater importance than in the DNA-based assays as contaminating red cells and platelets will still contain the relevant transcripts and could influence the result.
Harrison
C N
CN
Department of Haematology, University College London Medical School, UK.
Gale
R E
RE
Linch
D C
DC
D001483:Base Sequence; D001854:Bone Marrow Cells / Q000378:metabolism; D019070:Cell Lineage; D017931:DNA Primers; D004303:Dosage Compensation, Genetic; D005260:Female; D006580:Genetic Carrier Screening; D006801:Humans; D007066:Iduronate Sulfatase / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D007066:Iduronate Sulfatase