儿童非霍奇金淋巴瘤ZO-1基因甲基化状态分析及临床意义

ZO-1 gene methylation status and its clinical significance in children with non- Hodgkin lymphoma

摘要:

目的 探讨紧密连接蛋白（ZO-1）基因启动子区甲基化状态在儿童非霍奇金淋巴瘤（NHL）Ⅳ期检测中的临床意义，以进一步寻找病因及早期诊断方法。方法 将55 例确诊为NHL Ⅳ期（T 细胞型40 例，B 细胞型15 例）患儿的骨髓标本作为病例组，20 例非血液肿瘤患儿骨髓标本作为对照组。采用甲基化特异性PCR 法（MS-PCR）检测病例组与对照组骨髓中ZO-1 基因启动子区甲基化状态，并检测光密度积分值（IOD）。逆转录PCR 法（RT-PCR）检测病例组与对照组ZO-1 基因mRNA 的表达情况。结果 MS-PCR 检测结果显示病例组治疗前39 例出现ZO-1 基因甲基化条带，阳性率为70.9%（39/55），对照组20 例均呈非甲基化状态；治疗过程中，行动态观察NHL 患儿47 例，其中T 细胞型32 例，B 细胞型15 例，治疗前ZO-1 基因甲基化阳性率分别为72% 和67%，两者差异无统计学意义（P>0.05）。T 细胞型与B 细胞型NHL 患儿治疗前与化疗早期、中期比较差异均无统计学意义（P>0.05），而与化疗后期比较差异均有统计学意义（P<0.05）。RT-PCR 检测结果显示病例组中ZO-1 甲基化阳性病例均无mRNA 表达，对照组患儿均表达ZO-1 mRNA。外周血白细胞总数与ZO-1 基因IOD 值无直线相关关系（r=0.093，P=0.575）；骨髓中恶性肿瘤细胞数与ZO-1 基因IOD 值呈正相关关系（r=0.669，P<0.001）。结论 ZO-1 基因在儿童NHL 中呈特异性高甲基化状态，其程度与骨髓中恶性肿瘤细胞数呈正相关。ZO-1 基因可能成为新的分子标志，为临床早期诊断、疗效判断、预后评价、微小残留检测提供新的指标。

Abstract:

Objective To investigate the methylation status of zonula occludens-1 (ZO-1) gene promoter and its clinical significance in children with stage IV non-Hodgkin lymphoma (NHL) and to provide a basis for further etiological study and early diagnosis of this disease. Methods Fifty-five children with a confirmed diagnosis of stage IV NHL (40 cases of T-NHL and 15 cases of B-NHL) were selected as the case group, and 20 children with diseases other than hematologic malignancies were selected as the control group. Bone marrow samples were collected from these subjects. Methylation-specific PCR (MS-PCR) was applied to evaluate the methylation status of ZO-1 gene promoter, and the integrated optical density (IOD) was determined. RT-PCR was used to measure the mRNA expression of ZO-1. Results MS-PCR showed that the methylated bands of ZO-1 gene promoter were found in 39 (70.9%) of 55 patients in the case group before treatment, while no ZO-1 gene promoter methylation was detected in the control group. With close tracking of 47 cases in the study group, consisting of 32 cases of T-NHL and 15 cases of B-NHL, the rates of ZO-1 gene promoter methylation prior to treatment were 72% and 67%, respectively, (P>0.572). The cases of T-NHL and B-NHL showed no significant changes in methylation rate in the early and middle phases of chemotherapy (P>0.05), but they showed significant changes in methylation rate in the late phase of chemotherapy (P<0.05). RT-PCR showed that the NHL cases carrying methylated ZO-1 gene had no mRNA expression of ZO-1, while all children in the control group had mRNA expression of ZO-1. There was no linear relationship between the total number of peripheral blood leukocytes and ZO-1 gene IOD (r=0.093, P=0.575); a positive correlation was found between the number of malignant cells in bone marrow and ZO-1 gene IOD (r=0.669, P<0.001). Conclusions ZO-1 gene shows a hypermethylation status in children with NHL, and the methylation level is positively correlated with the number of malignant cells in bone marrow. ZO-1 may be used as a novel molecular marker in early diagnosis, outcome assessment, prognostic evaluation, and detection of minimal residual disease.