Quantification of X-chromosome inactivation patterns in haematological samples using the DNA PCR-based HUMARA assay

Quantification of X-chromosome inactivation patterns (XCIPs) using PCR amplification of the human androgen receptor (HUMARA) locus is potentially valuable in a range of haematological disorders. Of 236 females screened, 203 (86%) were heterozygous. For quantitative XCIPs it was necessary to limit the number of PCR cycles to 20 to reduce preferential amplification of shorter alleles. The optimized PCR method was compared with Southern blotting results using either PGK, HPRT or M27beta in 51 haematologically normal females and blast cells from 27 patients with acute myeloid leukaemia (AML). Reproducible XCIP results were obtained in all 78 samples using digestion with Hpa II prior to amplification (median difference in duplicate values 3%, range 0-17%) and they correlated well with Southern blotting results, r=0.966. Greater variability was observed in the results using Hha I digestion (median difference 4%, range 0-48%). There were marked inconsistencies in repeated analyses of three AML samples and although the HUMARA-Hha I results correlated well overall with Southern blotting in the remaining 75 samples (r=0.922), in nine samples there were still discrepancies with > or = 20% difference between the two values. These results suggest that PCR analysis of the HUMARA locus in Hpa II-digested DNA is suitable for the quantification of XCIPs in haematological samples but results with Hha I should be treated with caution.