Quantification of Unmethylated Alu (QUAlu): a tool to assess global hypomethylation in routine clinical samples

// Raquel Buj 1,2 , Izaskun Mallona 1,2 , Anna Diez-Villanueva 1,2 , Victor Barrera 1 , Didac Mauricio 2,3,4 , Manel Puig-Domingo 2,3,4 , Jordi L. Reverter 2,3 , Xavier Matias-Guiu 5 , Daniel Azuara 6 , Jose L. Ramirez 2,7 , Sergio Alonso 1,2 , Rafael Rosell 2,7 , Gabriel Capella 6 , Manuel Perucho 1,2,8 , Mercedes Robledo 9,10 , Miguel A. Peinado 1,2 and Mireia Jorda 1,2 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 Correspondence to: Mireia Jorda, 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 Abstract 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 ( Q uantification of U nmethylated Alu ), a new technique to estimate the Percentage of U n M ethylated 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.