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

甲基化特异性PCR检测X连锁疾病携带者方法的建立

基本信息

批准号：
11670752
负责人：
KUBOTA Takeo
金额：
$ 1.86万
依托单位：
National Center of Neurology and Psychiatry (2000)Shinshu University (1999)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

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.

目前，通过使用甲基化敏感酶对活性和非活性 X 染色体之间基因的差异甲基化进行分析，评估了女性 X 染色体失活的模式。我们报告了一种使用甲基化特异性 PCR (M-PCR) 技术对人雄激素受体 (HUMARA) 基因座进行的新测定，该技术与限制性酶的使用无关。该测定涉及用亚硫酸氢钠对 DNA 进行化学修饰以及随后的 PCR。通过使用甲基化等位基因的特异性引物进行测定，我们根据材料失活 X 与父本失活 X 的比率获得了 X 失活模式。这些模式与 48 名女性病例中同一位点的常规 PCR 检测结果一致。我们还使用未甲基化等位基因的特异性引物，根据母源活性 X 与父源活性 X 的比率获得了另一种 X 失活模式。后一种模式与前一种模式互补，这些模式的组合产生了可靠的 X 失活模式。通过测定，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）。四名 X 患者；常染色体易位表现出极其非随机的模式，这些结果与之前的分子/细胞遗传学研究的结果一致。我们得出的结论是，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酶切的各种 DNA 样品进行检测。