Investigation of methylation at Hha I sites using the hypervariable probe M27 beta allows improved clonal analysis in myeloid leukaemia and demonstrates differences in methylation between leukaemic and remission samples.

The methylation-sensitive enzyme Hha I has been used to assess the differentially methylated patterns on active and inactive X-chromosomes at the DXS255 locus recognized by the hypervariable probe M27 beta. The X-chromosome inactivation ratios obtained from 37 haematologically normal females using PstI and HhaI and correlated well with results obtained using PstI Hpa II (r = 0.97), and in 19 individuals with values obtained probing for either phosphoglycerate kinase or hypoxanthine phosphoribosyl transferase (r = 0.92). At least one Hha I site was found to be unmethylated on all alleles on inactive X-chromosomes. A monoclonal or oligoclonal pattern could be obtained by digestion with Hha I in 18/22 (82%) patients with acute myeloid leukaemia who had previously shown hypermethylation of both alleles using Hpa II, although in six of these patients differences in methylation could still be demonstrated between leukaemic and remission samples.