Epigenetic Study of XIST Gene from Female and Male Cells by Pyrosequencing

Kim, Hwan Hee; Yun, Yeo Jin; Song, Min Ae; Lee, Suman

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.