AML 和ALL 患者骨髓DNMT1，SFRP1 基因甲基化及其与临床病理特征和预后相关性研究

目的 检测急性髓系白血病(acute myeloid leukemia，AML)和急性淋巴细胞白血病(acute lymphoblastic leukemia，ALL)患者骨髓中DNA 甲基化转移酶1(DNA methyltransferase 1，DNMT1)、分泌型卷曲相关蛋白1(secretedfrizzled related protein 1, SFRP1)基因甲基化及 mRNA 表达水平，探讨其与临床病理特征和预后的关系。方法 根据FBA (French-American-British classification systems；法- 美- 英分型系统) 标准选取2019 年11 月～ 2020 年11 月于邯郸市中心医院新诊断的急性白血病患者70 例，其中AML 50 例和ALL 20 例；另选取65 例非恶性血液病患者作为正常对照组。用甲基化特异性聚合酶链式反应(methylation-specific PCR，MSP) 检测所有研究对象骨髓标本中DNMT1 和SFRP1 基因甲基化状态，并分析两基因甲基化与AML 和ALL 患者临床参数之间的关系；用实时定量 PCR 检测所有研究对象化疗前和化疗缓解后骨髓标本中 DNMT1，SFRP1 和β-catenin mRNA 表达；Pearson 相关分析明确DNMT1，SFRP1 和β-catenin mRNA 水平之间的相关性；对所有患者进行随访，比较DNMT1 和SFRP1 基因甲基化与预后的关系。结果 AML 和ALL 患者中DNMT1 基因甲基化发生率分别为26.0% 和25.0%，较对照组(73.8%)显著下降(χ2=47.683，P ＜ 0.001)；SFRP1 基因甲基化发生率分别为78.0% 和85.0%，较正常对照组(18.5%)显著增加(χ2=55.265，P ＜ 0.001)，差异均有统计学意义。AML 组DNMT1 基因甲基化与WBC 水平、遗传学预后分组有明显相关性(χ2=6.524，5.732，均P ＜ 0.001)；SFRP1 基因甲基化与WBC 水平、BM 水平和遗传学预后分组有明显相关性(χ2=8.115, 5.395, 5.060，均P＜ 0.05)。ALL 组DNMT1 基因甲基化只与遗传学预后分组有关(χ2=4.802，P ＜ 0.05)；SFRP1 基因甲基化与WBC 水平、遗传学预后分组有明显相关性(χ2=4.920, 5.115，均P ＜ 0.05)。与对照组相比，AML 和ALL 患者化疗前DNMT1 和β-catenin mRNA 表达均显著升高(t=4.807 ～ 10.456，均P＜ 0.05)，SFRP1 表达显著下降(t=24.791，12.069，均P＜ 0.05)。与化疗前相比，AML 和ALL 患者化疗后DNMT1 和β-catenin mRNA 表达显著下降(t=3.461 ～ 6.374，均P ＜ 0.05)，SFRP1 表达显著上升(t=17.076，7.454，P ＜ 0.05)。两组患者DNMT1 与SFRP1 表达呈明显负相关(r=-0.328，-0.315，均P ＜ 0.05)；SFRP1 与β-catenin 表达呈明显正相关(r=0.682，0.728，均P ＜ 0.05)；两组患者DNMT1与β-catenin 表达均无明显相关性。两组患者DNMT1 基因甲基化生存率高于其未甲基化生存率(χ2=3.862，3.679，均P ＜ 0.05)；SFRP1 基因甲基化生存率低于其未甲基化生存率(χ2=2.927，3.155，均P ＜ 0.05)。结论 DNMT1 和SFRP1 基因甲基化与恶性血液病患者临床病理及预后相关，究其原因可能与DNMT1 和SFRP1 基因甲基化异常激活Wnt/β-catenin 信号通路有关。

Methylation of DNMT1 and SFRP1 Gene in Bone Marrow and Its Correlation with Clinicopathological Features and Prognosis in Patients with AML and ALL

Objective To detect the levels of DNA methyltransferase 1 (DNMT1) secretory frizzled related protein 1 (SFRP1) gene methylation and mRNA expression in bone marrow of patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), and explore their relationship with clinicopathological characteristics and prognosis. Methods From November 2019 to November 2020, 70 patients with newly diagnosed acute leukemia in Handan Central Hospital were selected according to FBA (French-American-British classification systems)criteria, including 50 patients with AML and 20 patients with ALL. Another 65 patients with non-malignant hematologic diseases were selected as normal control group. Methylation-specific PCR (MSP) was used to detect the methylation status of DNMT1 and SFRP1 genes in bone marrow samples of ALL subjects, and the relationship between methylation of DNMT1 and SFRP1 genes and clinical parameters of AML and ALL patients was analyzed. Real time quantitative PCR was used to detect DNMT1, SFRP1 and β- catenin mRNA expression in all subjects before chemotherapy and after chemotherapy. Pearson correlation analysis identifies correlations between DNMT1, SFRP1 and β-catenin mRNA levels. All patients were followed up to compare the relationship between DNMT1 and SFRP1 methylation and prognosis. Results The incidence of DNMT1 gene methylation in AML and ALL patients were 26.0% and 25.0%, respectively, which was significantly lower than that in the control group(73.8%) (χ2=47.683, P ＜ 0.001). The methylation rates of SFRP1 gene were 78.0% and 85.0%, respectively, which were significantly higher than those of normal control group (18.5%)(χ2=55.265, P ＜ 0.001), and the differences were statistically significant，respectively. There was significant correlation between DNMT1 methylation and WBC level in AML group and genetic prognosis group (χ2=6.524，5.732, all P ＜ 0.001). The methylation of SFRP1 was significantly correlated with WBC level, BM level and genetic prognosis group (χ2= 8.115, 5.395, 5.060, all P ＜ 0.05). DNMT1 gene methylation was only associated with genetic prognostic grouping in ALL group (χ2=4.802, P ＜ 0.05). The methylation of SFRP1 gene was significantly correlated with WBC level and genetic prognosis (χ2=4.920，5.115, all P ＜ 0.05).Compared with the control group, the mRNA expressions of DNMT1 and β-catenin in AML and ALL patients were significantly increased before chemotherapy (t=4.807 ～ 10.456, all P ＜ 0.05), while the expression of SFRP1 was significantly decreased (t=24.791, 12.069, all P ＜ 0.05). Compared with before chemotherapy, the mRNA expressions of DNMT1 and β-catenin in AML and ALL patients after chemotherapy were significantly decreased (t=3.461 ～ 6.374, all P ＜ 0.05), and the expression of SFRP1 was significantly increased (t=17.076, 7.454, all P ＜ 0.05). There was a significant negative correlation between DNMT1 and SFRP1 expression in two groups (r=-0.328，-0.315, all P ＜ 0.05). SFRP1 was positively correlated with β-catenin expression (r=0.682，0.728, all P ＜ 0.05).There was no significant correlation between DNMT1 and β-catenin expression in two groups. The methylated survival rate of DNMT1 in the two groups was higher than that in the non-methylated survival rate (χ2=3.862, 3.679, all P ＜ 0.05).The methylated survival rate of SFRP1 was lower than that of its unmethylated survival rate (χ2=2.927, 3.155, all P ＜ 0.05). Conclusion The methylation of DNMT1 and SFRP1 was related to the clinicopathology and prognosis of patients with hematological malignancies, and the reason may be related to the abnormal activation of the Wnt/β-catenin signaling pathway by DNMT1 and SFRP1 gene methylation.