Methylation of the DXS255 hypervariable locus 5' CCGG site may be affected by factors other than X-chromosome activation status.

Differences in the methylation status of certain cytosine residues between active and inactive X chromosomes can be used to determine X-inactivation in females heterozygous for X-linked restriction fragment length polymorphisms. We have studied methylation patterns in 105 females heterozygous at the DXS255 locus by Southern blotting of PstI and MspI or HpaII double digests and hybridization with the probe M27B. Unequivocal patterns of unilateral or bilateral X-inactivation were obtained in 15/64 and 49/64 cases, respectively. In the remaining 41 cases the results were unclear due to the absence of HpaII digestion of one or both PstI fragments. In 7 samples an unequivocal digestion pattern was demonstrated on repeat analysis, suggesting that the initial ambiguous pattern was due to incomplete HpaII digestion. In certain individuals, methylation at the 5' CCGG DXS255 locus may be affected by factors other than X-inactivation, making analysis of clonality with the M27B probe impossible. These individuals should be clearly identified in studies of clonality.