半巢式甲基化特异性PCR在急性淋巴细胞白血病患者p15基因甲基化检测中的应用

Detection of p15 methylation and deletion status in acute lymphoblastic leukemia using hn-MSP

目的探讨半巢式甲基化特异性PCR（hn—MSP）的特性并了解p15基因甲基化和缺失在急性淋巴细胞白血病（ALL）发病中可能起到的作用。方法运用hn—MSP方法和基因组硫化修饰PCR（BSP）后直接测序方法，分析在25例初诊或复发不同阶段ALL患者以及部分恶性血液病细胞株中p15基因的甲基化和缺失状态，以10名健康者或非恶性血液病患者为对照组。以Molt-4细胞株为阳性对照，以对照组单个核细胞为阴性对照，分析hn-MSP方法的敏感性和特异性。结果hn-MSP产物克隆测序结果与BSP结果一致，hn—MSP检测p15基因甲基化敏感性可达到1×10-5。25例ALL患者p15基因甲基化发生率为68．0％（17例）；25例ALL患者中3例p15基因外显子1缺失。对照组p15基因无甲基化或缺失。结论p15基因甲基化状态在ALL患者中有较高的检出率，hn—MSP对分析p15基因甲基化状态具有较高的特异性和敏感性。

Objective To explore the characteristics of hemi-nested methylation specific polymerase chain reaction Oan-MSP） and to find out the possible relatiqnship between patterns of methylation or deletion and the developmet of adult acute lymphoblastie leukemia（ALL）. Methods hn-MSP and bisulfit-sequencing PCR （BSP） were designed and adopted to analyze p15 gene methylatian or deletion patterns in 25 adult ALL patients,malignant hematopathy cell lines and normal lymphocytes, hn-MSP and BSP products were cloned and sequenced.The sensitivity and specificity of hn-MSP were also analized. Results The sequencing results of hn-MSP and BSP products were consistent, and the sensitivity of detection of p15 methylation was up to 1.0×10-5. 17 adult ALL patients （68 %） were p15 gene hypermethylation and 3 patients were with deletion of p15 gene exon 1.There were no hypermethylation or deletion in the 10 controls.Conclusions The detection rate of p15 methylation in many tumors, especially in adult ALL, is frequent high. hn-MSP is highly sensitive and specific in analyzing p15 methylation.