Quantification of Unmethylated Alu (QUAlu): a tool to assess global hypomethylation in routine clinical samples
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Raquel Buj1,2, Izaskun Mallona1,2, Anna Díez-Villanueva1,2, Víctor Barrera1, Dídac Mauricio2,3,4, Manel Puig-Domingo2,3,4, Jordi L. Reverter2,3, Xavier Matias-Guiu5, Daniel Azuara6, Jose L. Ramírez2,7, Sergio Alonso1,2, Rafael Rosell2,7, Gabriel Capellà6, Manuel Perucho1,2,8, Mercedes Robledo9,10, Miguel A. Peinado1,2 and Mireia Jordà1,2
1 Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Barcelona, Spain
2 Germans Trias i Pujol Health Sciences Research Institute (IGTP), Badalona, Barcelona, Spain
3 Department of Endocrinology and Nutrition, University Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain
4 ISCIII Center for Biomedical Research on Diabetes and Metabolic Associated Diseases (CIBERDEM), Madrid, Spain
5 Department of Pathology and Molecular Genetics, University Hospital Arnau de Vilanova and University of Lleida, Biomedical Research Institute of Lleida (IRBLLEIDA), Lleida, Spain
6 Catalan Institute of Oncology (ICO-IDIBELL), L’Hospitalet de Llobregat, Barcelona, Spain
7 Catalan Institute of Oncology (ICO), Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain
8 Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain
9 Hereditary Endocrine Cancer Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain
10 ISCIII Center for Biomedical Research on Rare Diseases (CIBERER), Madrid, Spain
Mireia Jordà, email:
Miguel A. Peinado, email:
Keywords: DNA hypomethylation, Alu repeats, human cancer, biomarker, routine clinical biospecimens
Received: November 23, 2015 Accepted: January 25, 2016 Published: February 07, 2016
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.
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