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

Abstract

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 BclI 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 HUM-ARA (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.

Skewing of X-inactivation ratios in blood cells of aging women is confirmed by independent methodologies

“…9 These observations led the authors to conclude that the skewed XCI patterns observed with HUMARA were not an accurate reflection of the proportion of cells bearing a particular X chromosome in the active state but rather reflected age-related dysregulation of methylation patterns. 9 Although this explanation is a plausible one for these data, it is at odds with previously published reports validating the HUMARA assay against transcription-based assays at the HUMARA or IDS loci [10][11][12] and also with data showing concordance of skewing at the unlinked HUMARA and phosphoglycerate kinase (PGK) loci. 2 To address the disparity suggested by the implementation of the suppressive PCR assay, we analyzed blood cells of aging women by using (1) HUMARA, (2) a TaqMan SNP assay at the IDS locus, and (3) the suppressive PCR assay.…”

Clonal hemopoiesis and risk of thrombosis in young female patients with essential thrombocythemia

“…More recently, other studies introduced analysis of different DNA polymorphisms, such as phosphoglycerate kinase (PGK), hypoxanthine phosphoribosyl transferase (HPRT), and the human androgen receptor (HUMARA), which enable analysis of X-chromosome inactivation patterns (X-CIPs) in the majority of female patients [8]. The analysis of RNA expression of three polymorphic genes, iduronate-2-sulphatase (IDS), palmitoylated membrane protein p55, and G6PDH, allowed further evaluation of clonality on platelets and subpopulation of cells [9].…”