Establishment of carrier detection method of X-linked diseases by methylation-specific PCR

目前，女性X染色体失活的模式是通过使用甲基化敏感酶对活跃的和非活跃的X染色体之间的基因进行差异甲基化来评估的。我们报道了一种新的分析人类雄激素受体(HUMARA)基因座的方法，该方法使用甲基化特异的聚合酶链式反应(M-PCR)技术，不需要使用限制性内切酶。该方法包括用亚硫酸氢钠对DNA进行化学修饰和随后的PCR。通过甲基化等位基因的特异性引物检测，我们得到了基于材料失活X和父亲失活X的比率的X失活模式。这些模式与48例女性同一基因座的常规PCR检测结果一致。我们还使用针对未甲基化等位基因的特异引物，根据母亲的活性X与父亲的活性X的比率，获得了另一种X失活模式。后一种模式是对前一种模式的补充，这些模式的组合产生了可靠的X失活模式。通过检测，105名正常女性中有12名(11%)表现出非随机的失活模式(&gt；80：20或&lt；20：80)。4例X；常染色体易位患者表现出非常非随机的模式，这些结果与先前的分子/细胞遗传学研究结果一致。我们的结论是，M-PCR提供了一种准确的X-失活检测方法，可以对不适合限制性内切酶消化的各种DNA样本进行检测。

The pattern of X-chromosome inactivation in females has currently been evaluated by the assays through diffential methylation in the genes between the active and the inactive X chromosomes using methylation-sensitive enzymes. We report a new assay in the human androgen receptor (HUMARA) locus using a methylation-specific PCR (M-PCR) technique, independent of the use of restriction enzymes. The assay involves the chemical modification of DNA with sodium bisulfite and the subsequent PCR.By the assay with specific primers for the methylated allele, we obtained an X-inactivation pattern based on the ratio of the material inactive X to the paternal inactive X.These patterns were consistent with those by the conventional PCR assay at the same locus in 48 female cases. We also obtained another X-inactivation pattern based on the ratio of the maternal active X to the paternal active X using specific primers for the unmethylated allele. The latter pattern were complementary to the former pattern, and combination of these patterns produced a reliable X-inactivation pattern. By the assay, twelve (11%) of the 105 normal females showed nonrandom inactivation patterns (>80 : 20 or <20 : 80). Four patients with a X ; autosome translocation showed extremely nonrandom patterns, and these results were consistent with those by the previous molecular/cytogenetic studies. We conclude that M-PCR provides a accurate assay for X-inactivation, which can be performed on various DNA samples unsuitable for restriction digestion.