Buj Raquel, Mallona Izaskun, Díez-Villanueva Anna, Barrera Víctor, Mauricio Dídac, Puig-Domingo Manel, Reverter Jordi L, Matias-Guiu Xavier, Azuara Daniel, Ramírez Jose L, Alonso Sergio, Rosell Rafael, Capellà Gabriel, Perucho Manuel, Robledo Mercedes, Peinado Miguel A, Jordà Mireia
Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Barcelona, Spain.
Germans Trias i Pujol Health Sciences Research Institute (IGTP), Badalona, Barcelona, Spain.
Oncotarget. 2016 Mar 1;7(9):10536-46. doi: 10.18632/oncotarget.7233.
Hypomethylation of DNA is a hallmark of cancer and its analysis as tumor biomarker has been proposed, but its determination in clinical settings is hampered by lack of standardized methodologies. Here, we present QUAlu (Quantification of Unmethylated Alu), a new technique to estimate the Percentage of UnMethylated Alu (PUMA) as a surrogate for global hypomethylation. QUAlu consists in the measurement by qPCR of Alu repeats after digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation. QUAlu performance has been evaluated for reproducibility, trueness and specificity, and validated by deep sequencing. As a proof of use, QUAlu has been applied to a broad variety of pathological examination specimens covering five cancer types. Major findings of the preliminary application of QUAlu to clinical samples include: (1) all normal tissues displayed similar PUMA; (2) tumors showed variable PUMA with the highest levels in lung and colon and the lowest in thyroid cancer; (3) stools from colon cancer patients presented higher PUMA than those from control individuals; (4) lung squamous cell carcinomas showed higher PUMA than lung adenocarcinomas, and an increasing hypomethylation trend associated with smoking habits. In conclusion, QUAlu is a simple and robust method to determine Alu hypomethylation in human biospecimens and may be easily implemented in research and clinical settings.
DNA 低甲基化是癌症的一个标志，有人提出将其作为肿瘤生物标志物进行分析，但由于缺乏标准化方法，在临床环境中对其进行测定受到阻碍。在此，我们介绍了QUAlu（未甲基化Alu的定量分析），这是一种估计未甲基化Alu百分比（PUMA）以替代整体低甲基化的新技术。QUAlu包括在用对DNA甲基化具有不同敏感性的同裂酶消化基因组DNA后，通过qPCR测量Alu重复序列。已对QUAlu的性能进行了可重复性、真实性和特异性评估，并通过深度测序进行了验证。作为应用实例，QUAlu已应用于涵盖五种癌症类型的多种病理检查标本。QUAlu在临床样本中的初步应用的主要发现包括：（1）所有正常组织的PUMA相似；（2）肿瘤的PUMA各不相同，其中肺癌和结肠癌中的水平最高，甲状腺癌中的水平最低；（3）结肠癌患者的粪便PUMA高于对照个体；（4）肺鳞状细胞癌的PUMA高于肺腺癌，且与吸烟习惯相关的低甲基化趋势增加。总之，QUAlu是一种简单且可靠的方法，用于测定人类生物样本中的Alu低甲基化，并且可以在研究和临床环境中轻松实施。
