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Korean Journal of Reproductive Medicine 2010;37(1):25-31.
Published online March 1, 2010.
Epigenetic Study of XIST Gene from Female and Male Cells by Pyrosequencing.
Hwan Hee Kim, Yeo Jin Yun, Min Ae Song, Suman Lee
Functional Genomics Lab, CHA Stem Cell Research Institute, School of Medicine, CHA University, Bundang-Gu, Sungnam-Si, Kyunggi-Do, 463-836, Korea.
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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