Orsini Paola, Impera Luciana, Parciante Elisa, Cumbo Cosimo, Minervini Crescenzio F, Minervini Angela, Zagaria Antonella, Anelli Luisa, Coccaro Nicoletta, Casieri Paola, Tota Giuseppina, Brunetti Claudia, Ricco Alessandra, Carluccio Paola, Specchia Giorgina, Albano Francesco
Department of Emergency and Organ Transplantation (D.E.T.O.), Hematology Section, University of Bari, P.zza G. Cesare, 11 70124, Bari, Italy.
Diagn Pathol. 2018 Dec 22;13(1):98. doi: 10.1186/s13000-018-0777-x.
Alu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. Alu sequences lie in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu methylation has been associated with tumor aggressiveness, and also previously discussed in hematological malignancies, by applying different approaches. Moreover, today different techniques designed to measure global DNA methylation are focused on the methylation level of specific repeat elements. In this work we propose a new method of investigating Alu differential methylation, based on droplet digital PCR (ddPCR) technology.
Forty-six patients with hematological neoplasms were included in the study: 30 patients affected by chronic lymphocytic leukemia, 7 patients with myelodysplastic syndromes at intermediate/high risk, according with the International Prognostic Scoring System, and 9 patients with myelomonocytic leukemia. Ten healthy donors were included as controls. Acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with decitabine (DEC) hypomethylating agent, was also analyzed. DNA samples were investigated for Alu methylation level by digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation, followed by ddPCR.
Using ddPCR, a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, was observed. Moreover, comparing the global Alu methylation levels at diagnosis and after azacytidine (AZA) treatment in MDS patients, a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis was evident. We also observed a significant decrease of the Alu methylation level in CLL patients compared to HD, and, finally, for CMML patients, a decrease of Alu sequences methylation was observed in patients harboring the SRSF2 hotspot gene mutation c.284C>D.
In our work, we propose a method to investigate Alu differential methylation based on ddPCR technology. This assay introduces ddPCR as a more sensitive and immediate technique for Alu methylation analysis. To date, this is the first application of ddPCR to study DNA repetitive elements. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose.
Alu重复序列属于短散在重复元件（SINEs）类别，在人类基因组中包含约25%的CpG位点。Alu序列位于基因丰富区域，因此其甲基化是一种重要的转录调控机制。Alu甲基化异常与肿瘤侵袭性相关，此前也通过不同方法在血液系统恶性肿瘤中进行过讨论。此外，如今设计用于测量整体DNA甲基化的不同技术都聚焦于特定重复元件的甲基化水平。在本研究中，我们提出了一种基于液滴数字PCR（ddPCR）技术研究Alu差异甲基化的新方法。
本研究纳入了46例血液系统肿瘤患者：30例慢性淋巴细胞白血病患者、7例根据国际预后评分系统处于中/高危的骨髓增生异常综合征患者以及9例粒单核细胞白血病患者。纳入10名健康供者作为对照。还分析了急性早幼粒细胞白血病来源的NB4细胞系，该细胞系未处理或用去甲基化药物地西他滨（DEC）处理。通过用对DNA甲基化具有不同敏感性的同裂酶消化基因组DNA，随后进行ddPCR，研究DNA样本的Alu甲基化水平。
使用ddPCR观察到，与未处理的细胞相比，用DEC处理的NB细胞提取的DNA中Alu整体甲基化水平显著降低。此外，比较骨髓增生异常综合征患者诊断时和阿扎胞苷（AZA）治疗后的Alu整体甲基化水平，治疗后Alu序列甲基化水平与诊断时相比有统计学意义的显著降低。我们还观察到慢性淋巴细胞白血病患者的Alu甲基化水平与健康对照相比显著降低，最后，对于粒单核细胞白血病患者，在携带SRSF2热点基因突变c.284C>D的患者中观察到Alu序列甲基化降低。
在我们的研究中，我们提出了一种基于ddPCR技术研究Alu差异甲基化的方法。该检测方法将ddPCR引入作为一种更敏感、更直接的Alu甲基化分析技术。迄今为止，这是ddPCR首次应用于研究DNA重复元件。这种方法可能有助于有助于有助于对血液系统恶性肿瘤患者进行诊断/预后分析有用。
