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

人类雄激素受体基因（HUMARA）被用于x染色体失活（XCI）模式的分析，因为在与XCI相关的5'-启动子区域附近存在多态性短串联重复（STR）。我们介绍了一种新的基于PCR的HUMARA方法来分析MDS患者造血的克隆性，称为HUMARA甲基化特异性PCR （HUMARA- msp）测定，该方法通过亚硫酸酯修饰而不是甲基化敏感的限制性内切酶来分析HUMARA基因的甲基化状态。虽然原始的MSP方法显示是否存在甲基化带，但我们的HUMARA-MSP方法识别了甲基化带和非甲基化带的模式。由于该方法可以在每个PCR管中识别未甲基化或甲基化的等位基因，并且根据甲基化状态显示相反的条带模式，因此我们可以独立评估XCI模式两次。该方法可避免酶切不完全产生假结果，亚硫酸酯不完全修饰不影响结果。极少量的样品，如造血菌落，也可用于HUMARA-MSP测定。HUMAR-MSP检测方法简便、灵敏、适用性广，可为血液病发病机制的分析提供参考。

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.