| Epigenetic Study of XIST Gene from Female and Male Cells by Pyrosequencing. |
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Hwan Hee Kim, Yeo Jin Yun, Min Ae Song, Suman Lee |
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Functional Genomics Lab, CHA Stem Cell Research Institute, School of Medicine, CHA University, Bundang-Gu, Sungnam-Si, Kyunggi-Do, 463-836, Korea. |
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| Abstract |
OBJECTIVE
X inactivation is the silencing one of the two X chromosomes in female mammals for gene dosage on the X-chromosome between female and male. X inactivation is controlled by X inactive-specific transcript (XIST) gene, untranslated RNA. XIST is expressed only from the inactive X (Xi), not expressed from the active X (Xa). The Xist promoter is methylated on the silent Xist allele on the Xa in somatic cells, and less methylated on the Xist-expressing Xi. We investigated the difference of XIST methylation pattern of the promoter and 5'-region of XIST from male (XY) and female (XX) subjects. METHODS: The direct quantification of XIST methylation is required for clinical application of normal XX and XY blood. Methylation percentage of eight CpG sites (-1696, -1679, -1475, -1473, -1469, +947, +956, +971) of XIST gene were diagnosed by pyrosequencing. RESULTS: We directly quantitated the methylation percentage of the promoter and 5'-end of XIST by pyrosequencing. The average methylation percentages at CpG6-8 sites (+947, +956, +971) were 45.2% at CpG6, 49.9% at CpG7, and 44.2% at CpG8 from normal female and normal male were 90.6%, 96.7%, 87.8%, respectively. Nether CpG 1-5sites (-1696, -1679, -1475, -1473, -1469) had any effect on XX and XY. CONCLUSION: This method is sensitive for quantifying the small percentage change in the methylation status of XIST, and may be used for diagnosis. |
| Key Words:
XIST; CpG; DNA methylation; Pyrosequencing |
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