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

女性X染色体失活的模式目前已经通过使用甲基化敏感酶在活性和非活性X染色体之间的基因中的差异甲基化的测定来评估。我们报告了一种新的测定在人类雄激素受体（HUMARA）基因座使用甲基化特异性PCR（M-PCR）技术，独立的限制性内切酶的使用。用亚硫酸氢钠对DNA进行化学修饰，然后用PCR方法检测甲基化等位基因，根据材料失活X与父源失活X的比值，获得了X失活模式，与48例女性患者的常规PCR检测结果一致。我们还获得了另一个X-失活模式的基础上的比率，母本的活性X，父本的活性X使用特异性引物的非甲基化等位基因。后一种模式与前一种模式是互补的，这些模式的组合产生了可靠的X-失活模式。105名正常女性中有12名（11%）表现出非随机失活模式（>80：20或<20：80）。四个病人有X光片;常染色体易位表现出极强的非随机性，这一结果与以往的分子/细胞遗传学研究结果一致。我们的结论是，M-PCR提供了一个准确的测定X-灭活，这可以进行各种DNA样品不适合限制性消化。

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.