A new assay for the analysis of X-chromosome inactivation based on methylation-specific PCR

The pattern of X-chromosome inactivation in females is currently evaluated by assays of differential methylation in the genes between the active and t he inactive X chromosomes, with methylation-sensitive enzymes. We report a new assay in the human androgen receptor (HUMARA) locus involving a methyla tion-specific polymerase chain reaction (M-PCR) technique, independent of t he use of restriction enzymes. The assay involves the chemical modification of DNA with sodium bisulfite and subsequent PCR. By using the assay with s pecific primers for the methylated allele, we obtained an X-inactivation pa ttern based on the ratio of the maternal inactive X to the paternal inactiv e X. These patterns were consistent with those obtained by conventional PCR assay at the same locus in 48 female cases. We also obtained another X-ina ctivation pattern based on the ratio of the maternal active X to the patern al active X by using specific primers for the unmethylated allele. The latt er pattern was complementary to the former pattern, and a combination of th ese patterns produced a reliable X-inactivation pattern. The assay revealed that 12 (11%) of the 105 normal females had nonrandom inactivation pattern s (>80:20 or <20.80). Four patients with an X; autosome translocation showe d extremely non-random patterns, and these results were consistent with tho se obtained by previous molecular/cytogenetic studies. We conclude that M-P CR provides an accurate assay for X-inactivation and that it can be perform ed on various DNA samples unsuitable for restriction digestion.