Analysis of the efficiency of the separation of normal hematopoietic stem cells of myelodysplastic syndrome for auto stem cell transplantation

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The human androgen-receptor gene (HUMARA) has been used for analysis of X-chromosome inactivation (XCI) patter because of a polymorphic short tandem repeat (STR) near the 5'-promoter region correlated with XCI.We introduced a novel PCR based HUMARA method to analyze the clonality of the hematopoiesis of MDS patients named HUMARA methylation-specific PCR (HUMARA-MSP) assay, which analyzed methylation status of the HUMARA gene by bisulfite modification instead of a methylation-sensitive restriction enzyme. Although the original MSP method shows whether there is a methylated band, or not, our HUMARA-MSP method identifies the pattems of methylated and unmethylated bands. Because this method identifies either unmethylated or methylated alleles in each PCR tube and shows opposite band patterns dependent on methylation status, we can assess the XCI pattern independently twice. This method can avoid false results by incomplete enzyme digestion and incomplete bisulfite modification will not affect the results. Extremely small quantities of samples, such as hematopoietic colonies, were also available for HUMARA-MSP assay. The HUMAR-MSP assay may facilitate the analysis for pathogenesis of hematological disorders because of its simplicity, sensitivity and wide applicability.