QUANTIFICATION OF X-CHROMOSOME INACTIVATION PATTERNS IN HEMATOLOGICALSAMPLES USING THE DNA PCR-BASED HUMARA ASSAY

Quantification of X-chromosome inactivation patterns (XCIPs) using FOR amplification of the human androgen receptor (HUMARA) locus is potent ially valuable in a range of haematological disorders. Of 236 females screened, 203 (86%) were heterozygous. For quantitative XCIPs it was n ecessary to limit the number of PCR cycles to 20 to reduce preferentia l amplification of shorter alleles. The optimized PCR method was compa red with Southern blotting results using either PGK, HPRT or M27 beta in 51 haematologically normal females and blast cells from 27 patients with acute myeloid leukaemia (AML). Reproducible XCIP results were ob tained in all 78 samples using digestion with Hpa II prior to amplific ation (median difference in duplicate values 3%, range 0-17%) and they correlated well with Southern blotting results, r=0.966. Greater vari ability was observed in the results using Hha I digestion (median diff erence 4%, range 0-48%). There were marked inconsistencies in repeated analyses of three AML samples and although the HUMARA-Hha I results c orrelated well overall with Southern blotting in the remaining 75 samp les (r=0.922), in nine samples there were still discrepancies with gre ater than or equal to 20% difference between the two values. These res ults suggest that PCR analysis of the HUMARA locus in Hpa Ii-digested DNA is suitable for the quantification of XCIPs in haematological samp les but results with Hha I should be treated with caution.