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的酶在活性和非活性X染色体之间的基因中，通过测定法评估了女性中X染色体灭活的模式。我们报告了使用甲基化特异性PCR（M-PCR）技术在人类雄激素受体（HUMARA）基因座中进行的新测定，而与限制性酶的使用无关。该测定涉及用硫酸钠对DNA进行化学修饰和随后的PCR。通过具有甲基化等位基因的特异性引物的测定，我们获得了基于材料无效X与父亲无效X的比例的X灭活模式。这些模式与这些常规pcrcccrain ccrains she ny sheny8888888888。我们还使用未甲基化等位基因的特定引物，基于母体活性X与父亲活性X的比率获得了另一种X灭活模式。后一种模式与以前的模式互补，这些模式的组合产生了可靠的X灭活模式。通过测定，105名正常女性中有十二名（11％）表现出非随机失活模式（> 80：20或<20：80）。四名X患者；常染色体易位显示出极为非随机模式，这些结果与先前的分子/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X灭活的准确测定，可以在不适合限制消化的各种DNA样品上执行。