Corcoran Martin, Parker Anton, Orchard Jenny, Davis Zadie, Wirtz Michaela, Schmitz Oliver J, Oscier David
Department of Molecular Biology and Haematology, Royal Bournemouth Hospital, Bournemouth, Dorset, BH7 7DW, UK.
Haematologica. 2005 Aug;90(8):1078-88.
ZAP-70 expression is a recognized prognostic marker in chronic lymphocytic leukemia (CLL). The aim of this study was to analyze whether the methylation status of the ZAP-70 gene is associated with expression of the ZAP-70 protein.
Patients with CLL (n=87), acute lymphoblastic leukemia (n=13), mantle cell leukemia (n=13) and splenic marginal zone lymphoma (n=14) of known immunoglobulin gene mutation (IgVH) status were studied. The methylation status of the 5' region of ZAP-70 was analyzed by combined bisulphite restriction analysis (COBRA), southern blotting and bisulphite sequencing in 10 CLL patients and in normal T/NK and B cells. Further COBRA of a single CpG site located 334bp downstream of the ZAP-70 transcription start site (C-334) was then performed on all patients.
ZAP-70 expression status in CLL and normal peripheral blood lymphocytes is associated with the methylation status of the intron1-exon2 boundary region of ZAP-70 and methylation status of C-334 determined by COBRA is representative of methylation in this region. Of 87 CLL patients, 51/53 ZAP-70 negative patients had methylation at C-334 and 30/32 ZAP-70 positive patients did not have methylation (p<0.0001); a similar association was seen in all other diseases. Median survivals of methylated and unmethylated CLL patients were 211 and 85 months, respectively (p<0.0001).
Measuring ZAP-70 methylation status at C-334 is a simple and reproducible method for predicting prognosis in CLL, which is closely associated with ZAP-70 expression and IgVH gene mutational status. Methylation of a highly conserved intronic region of ZAP-70 may be responsible for regulation of expression in normal and neoplastic cells.
ZAP-70表达是慢性淋巴细胞白血病（CLL）中公认的预后标志物。本研究旨在分析ZAP-70基因的甲基化状态是否与ZAP-70蛋白的表达相关。
对已知免疫球蛋白基因突变（IgVH）状态的CLL患者（n = 87）、急性淋巴细胞白血病患者（n = 13）、套细胞白血病患者（n = 13）和脾边缘区淋巴瘤患者（n = 14）进行研究。通过联合亚硫酸氢盐限制性分析（COBRA）、Southern印迹法和亚硫酸氢盐测序法分析了10例CLL患者以及正常T/NK和B细胞中ZAP-70 5'区域的甲基化状态。然后对所有患者进行了位于ZAP-70转录起始位点下游334bp处的单个CpG位点（C-334）的进一步COBRA分析。
CLL和正常外周血淋巴细胞中ZAP-70的表达状态与ZAP-70内含子1-外显子2边界区域的甲基化状态相关，通过COBRA确定的C-334甲基化状态代表该区域的甲基化情况。在87例CLL患者中，51/53例ZAP-70阴性患者在C-334处发生甲基化，30/32例ZAP-70阳性患者未发生甲基化（p<0.0001）；在所有其他疾病中也观察到类似的关联。甲基化和未甲基化的CLL患者的中位生存期分别为211个月和85个月（p<0.0001）。
检测C-334处的ZAP-70甲基化状态是预测CLL预后的一种简单且可重复的方法，它与ZAP-70表达和IgVH基因突变状态密切相关。ZAP-70高度保守内含子区域的甲基化可能负责正常细胞和肿瘤细胞中表达的调控。
