2000 Fiscal Year Final Research Report Summary
Establishment of carrier detection method of X-linked diseases by methylation-specific PCR
| Project/Area Number |
11670752
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pediatrics
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| Research Institution | National Center of Neurology and Psychiatry (2000)
Shinshu University (1999) |
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Principal Investigator |
KUBOTA Takeo National Center of Neurology and Psychiatry, Head, 疾病研究第2部, 室長 (70293511)
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| Co-Investigator(Kenkyū-buntansha) |
AGEMATSU Kazunaga Shinshu University School of Medicine, Assistant Professor, 大学院・医学研究科・移植免疫感染症, 講師 (60262721)
FUKUSHIMA Yoshimitsu Shinshu University School of Medicine, Professor, 医学部・衛生学, 教授 (70273084)
GOTO Yu-ichi National Center of Neurology and Psychiatry, Director, 疾病研究第2部, 部長 (20225668)
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| Project Period (FY) |
1999 – 2000
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| Keywords | X-chromosome / inactivation / methylation / carrier detection / methylation-specific PCR / androgen receptor gene / HUMARA gene / nonrandom pattern |
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| Research Abstract |
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.
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