采用甲基化特异性聚合酶链反应研究急性白血病p15基因CpG岛甲基化

目的　探讨ｐ１５ 基因ＣｐＧ岛甲基化作为各型急性白血病通用的基因标志物的可行性。方法　采用甲基化特异性聚合酶链反应（ＭＳＰ）法检测４０ 例初治或复发急性白血病、５ 例完全缓解期白血病及８ 例（ 份） 正常骨髓的ｐ１５ 基因ＣｐＧ岛甲基化，并对ＭＳＰ产物进行序列测定。结果　急性白血病患者ｐ１５ 基因ＣｐＧ岛甲基化阳性率为８０％ ，ＡＭＬ与ＡＬＬ的阳性率（分别为８３％ 和７３％） 差异无显著性；５ 例完全缓解期白血病患者中１ 例ｐ１５ ＣｐＧ岛甲基化阳性（该例患者不久白血病复发）；正常骨髓８ 例均为阴性，表明ｐ１５ ＣｐＧ岛甲基化为白血病细胞所特有；ＭＳＰ产物测序结果与理论结果相符合，ＭＳＰ敏感度可达１０ －３。结论　ｐ１５ ＣｐＧ岛甲基化在各型急性白血病中均有很高的发生率，可以作为各型急性白血病通用的微量残留病（ＭＲＤ） 指标；白血病完全缓解期ｐ１５ ＣｐＧ 岛甲基化仍为阳性可能预示着复发；ＭＳＰ法ＤＮＡ需要量极少，不需要有特异性酶切位点，无同位素污染，对标本要求不高，敏感度高，是甲基化研究的一个简便、敏感、特异的新手段。

Study on the methylation of p15 gene CpG islands in acute leukemia: using methylation specific PCR method

Objective To explore the feasibility of the methylation of p15 gene CpG islands as a common gene marker for all types of acute leukemias(AL). Methods The methylation of p15 gene CpG islands in bone marrow from 40 cases of newly diagnosed or relapsed AL, 5 cases of AL in CR and 8 of normal subjects was analyzed by using methylation specific PCR methods(MSP), and the product of MSP was sequenced. Results p15 gene CpG islands were methylated in 80% (32/40) of the newly diagnosed or relapsed AL, no difference between AML and ALL was observed. One positive result was found in the 5 AL patients in CR and this patient soon relapsed. The bone marrow cells from 8 normal subjects did not have p15 gene CpG islands methylation, which suggested that such methylation was peculiar to AL cells. DNA sequencing confirmed the right expected sequence. The sensitivity of MSP was 10 -3 . Conclusion The methylation of p15 gene CpG islands occurs very commonly in every types of AL. It can be used as a gene marker for ALs and for minimal residual disease in CR. MSP needs neither special methylation sensitive restricted sites, nor high volume of DNA. There is no radioactive pollution, and the sample need not be fresh. MSP is a very sensitive, simple and specific method for the detection of DNA methylation.