Epigenetic Biomarkers and Diagnostics

Epigenetic Biomarkers and Diagnostics

Editor

José Luis García-Giménez

Center for Biomedical Network Research on Rare Diseases (CIBERER), Madrid, Spain

Medicine and Dentistry School

Biomedical Research Institute INCLIVA, University of Valencia, Spain

Table of Contents

Cover image

Title page

Copyright

List of Contributors

Preface

Chapter 1. Epigenetic Biomarkers: New Findings, Perspectives, and Future Directions in Diagnostics

1. Introduction

2. Epigenetic Mechanisms

3. Epigenetic Biomarkers and In Vitro Diagnostics

4. Epigenetic Biomarkers and the Clinical Laboratory

5. Epigenetic Biomarkers: An Overview of Recent Advances

6. Epigenetics: Perspective of Implantation in Clinical Laboratories

7. Perspectives of Epigenetics in Diagnostics

List of Abbreviations

Chapter 2. The Importance of Biobanks in Epigenetic Studies

1. Biobanks

2. Biobanks in Biomedical Research

3. Epigenetics and Biobanking

4. Conclusions

List of Abbreviations

Chapter 3. Epigenetic Mechanisms as Key Regulators in Disease: Clinical Implications

1. Introduction

2. Epigenetics and Metabolism Intersection

3. Lifestyle, Nutrition, and Physical Activity in Epigenetic Regulation

4. Epigenetic Mechanisms Underlying Complex Diseases

5. Epigenetics in Rare Diseases—Clinical Implications

6. Epigenetic Biomarkers and Clinical Laboratory

7. Conclusions

List of Abbreviations

Chapter 4. The Evolution of New Technologies and Methods in Clinical Epigenetics Research

1. Introduction

2. DNA Methylation

3. Histone Modifications

4. Chromatin Structure

5. Noncoding RNAs

6. Conclusions and Outlook

List of Abbreviations

Chapter 5. Biomarkers and Methodologies for Monitoring Epigenetic Drug Effects in Cancer

1. Introduction

2. Predicting Response to DNMTi Treatment

3. Monitoring Single-Locus DNA Methylation Biomarkers in Clinical Samples

4. Monitoring Global DNA Methylation in Clinical Samples

5. Conclusions

List of Abbreviations

Chapter 6. Genome-Wide Techniques for the Study of Clinical Epigenetic Biomarkers

1. Introduction

2. First Approaches to Analyze 5-Methylcytosine Levels at a Genomic Scale

3. DNA Methylation Arrays

4. Quantification of 5mC Oxidative Derivatives

5. Affinity Enrichment of Methylated DNA

6. Reading the DNA Methylome: Genome Sequencing Applications

7. Conclusions

List of Abbreviations

Chapter 7. Sequenom MassARRAY Technology for the Analysis of DNA Methylation: Clinical Applications

1. Introduction

2. DNA Methylation

3. Methods for DNA Methylation Analysis

4. DNA Methylation Studies by Sequenom

5. Conclusions

Glossary

List of Abbreviations

Chapter 8. The Role of Methylation-Specific PCR and Associated Techniques in Clinical Diagnostics

1. Overview of DNA Methylation as an Epigenetic Marker

2. DNA Methylation in Diagnostics

3. Restriction Enzyme Digestion for Analyzing DNA Methylation

4. Methylation-Specific PCR and Associated Techniques

5. Sodium Bisulfite Conversion

6. Methylation-Specific PCR

7. Methylation-Sensitive Dot Blot Assay

8. MethyLight

9. Technical Specifications for MethyLight

10. Applications of MethyLight Technique

11. Multiplex MethyLight

12. Methylation-Sensitive Multiplex Ligation-Dependent Probe Amplification

13. Pyrosequencing

14.. High-Resolution Melting Analysis

15. Emerging Applications

16. Future of DNA Methylation in Clinical Applications

List of Abbreviations

Chapter 9. Pyrosequencing and Its Application in Epigenetic Clinical Diagnostics

1. Introduction

2. Detection of Epigenetic Changes By Pyrosequencing: DNA Methylation

3. Pyrosequencing and the Assessment to miRNAs

4. Conclusions

Glossary

List of Abbreviations

Chapter 10. Mass Spectrometry for the Identification of Posttranslational Modifications in Histones and Its Application in Clinical Epigenetics

1. Introduction

2. MS Analysis of hPTMs

3. Toward Clinical Applications of MS Analysis of hPTMs

4. Conclusions and Perspectives

List of Abbreviations

Chapter 11. High-Throughput Analysis of Noncoding RNAs: Implications in Clinical Epigenetics

1. Introduction

2. ncRNAs: Definitions and Innovative Technologies for Their Analysis

3. How the Epigenome Controls ncRNAs and How ncRNAs Control the Epigenome

4. ncRNAs and Epigenetics: Clinical Relevance

5. Conclusions and Future Perspectives

Glossary

List of Abbreviations

Chapter 12. Circulating Noncoding RNAs as Clinical Biomarkers

1. Introduction

2. MicroRNAs

3. Long Noncoding RNAs

4. Circular RNAs

5. Resources for Circulating RNAs

6. Conclusion and Future Perspective

List of Abbreviations

Chapter 13. DNA Methylation Biomarkers in Lung Cancer

1. Introduction

2. Methylation

3. Epigenetic-Associated Regulatory Elements

4. DNA Methylation in Cancer

5. DNA Methylation in Lung Cancer

6. Conclusions

List of Abbreviations

Chapter 14. DNA Methylation Alterations as Biomarkers for Prostate Cancer

1. Introduction

2. The Aberrant DNA Methylation Landscape of PCa

3. Promoter Hypermethylation in PCa

4. Hypomethylation in PCa

5. DNA Methylation-Based Markers for PCa Detection, Management, and Risk Estimation

6. Cancer Detection and Diagnosis

7. Prognosis and Prediction of Response to Therapy

8. Conclusions and Perspectives

Glossary

List of Abbreviations

Chapter 15. DNA Methylation in Breast Cancer

1. Introduction

2. Major Milestones in Breast Cancer and DNA Methylation Research

3. DNA Methylation

4. DNA Methylation as a Therapeutic Target

5. Concluding Remarks and Future Prospects

List of Abbreviations

Chapter 16. DNA Methylation in Obesity and Associated Diseases

1. Introduction

2. An Overview of Epigenetics: Focus on DNA Methylation

3. Environmental and Lifestyle Factors Influencing DNA Methylation

4. DNA Methylation Pattern and Obesity Susceptibility

5. Epigenetics as a Link between Obesity and Its Codiseases

6. DNA Methylation as Biomarker of Disease

7. Epigenetic Drugs and Functional Foods for Overcoming Metabolic Diseases

8. Conclusion

List of Abbreviations

Chapter 17. DNA Methylation Biomarkers in Asthma and Allergy

1. Introduction

2. Allergic Diseases: Phenotypes and Genes

3. Why Has Epigenetics Become a Buzz Word in Allergic Disease?

4. What Have We learned So Far from Allergic Disease Epigenetics?

5. Conclusion: Where Is the Field Going?

List of Abbreviations

Chapter 18. DNA Methylation for Prediction of Adverse Pregnancy Outcomes

1. Introduction

2. Epigenetics

3. Epigenetic Mechanisms

4. Regulation of Gene Expression by Epigenetic Mechanisms

5. Epigenetic Changes during the Mammalian Life Cycle

6. DNA Methylation during Embryonic Development

7. Maternal Nutrition, DOHaD, and Epigenetics

8. Epigenetic Regulation in the Placenta and Adverse Pregnancy Complications

9. Conclusion and Future Prospects

Glossary

List of Abbreviations

Chapter 19. Epigenetics in Infectious Diseases

1. Introduction

2. miRNA Profiles

3. DNA Methylation Patterns

4. Histone marks

5. Concluding remarks

Glossary

List of Acronyms and Abbreviations

Chapter 20. DNA Methylation in Neurodegenerative Diseases

1. Introduction

2. DNA Methylation and the CNS

3. Alzheimer’s Disease

4. Parkinson’s Disease

5. Amyotrophic Lateral Sclerosis

6. Huntington’s Disease

7. Fragile X-Associated Tremor/Ataxia Syndrome

8. Friedreich Ataxia

9. Spinocerebellar Ataxia 7

10. Conclusion

List of Abbreviations

Chapter 21. The Histone Code and Disease: Posttranslational Modifications as Potential Prognostic Factors for Clinical Diagnosis

1. Introduction

2. Histone Modifications and Disease

3. Conclusions

Glossary

List of Abbreviations

Chapter 22. Histone Posttranslational Modifications and Chromatin Remodelers in Prostate Cancer

1. Introduction

2. Epigenetic-Based Markers for PCa Detection, Management, and Risk Estimation

3. Epigenetic Silencing as a Therapeutic Target in PCa

4. Conclusion

Glossary

List of Abbreviations

Chapter 23. Histone Posttranslational Modifications in Breast Cancer and Their Use in Clinical Diagnosis and Prognosis

1. Histone Marks and the Epigenetic Landscape

2. Breast Cancer Subtypes, Hormonal Dependence, and Epigenetic Regulation

3. Epigenetic Factors in Mammary Gland Development and Cancer

4. Epigenetic Complexes as Mutational Targets in Luminal Breast Cancer

5. Epigenetic Modifications as Biomarkers for Treatment Sensitivity and Outcome

6. Epigenetic Modifiers as Potential Drug Targets in Cancer

7. Conclusive remarks

Glossary

List of Abbreviations

Chapter 24. Histone Variants and Posttranslational Modifications in Spermatogenesis and Infertility

1. Introduction

2. Histone Variants and their Role in Infertility

3. The Effects of Histone PTMs

4. Brief Overview of the Role of Spermiogenesis-Specific Nuclear Basic Proteins: TNPs and Protamines in Infertility

5. Can Histone Variants be Used as Biomarkers for Infertility?

6. Closing Remarks

Glossary

List of Abbreviations

Chapter 25. Circulating Histones and Nucleosomes as Biomarkers in Sepsis and Septic Shock

1. Introduction

2. Epidemiology and Clinical Features of Sepsis, Severe Sepsis, and Septic Shock

3. Genetics, Epigenetics, and Molecular Pathways Underlying Sepsis

4. Extracellular Nucleosomes and Histones and Their Role in the Physiopathology of Sepsis and Septic Shock

5. Circulating Histones as Biomarkers of Prognosis and Treatment Monitoring

6. Therapeutic Approaches to Antagonize Histone-Mediated Toxicity

7. Conclusions

Glossary

List of Abbreviations

Chapter 26. Chromatin Landscape and Epigenetic Signatures in Neurological Disorders: Emerging Perspectives for Biological and Clinical Research

1. Introduction

2. The Involvement of Epigenetic Modifications in HD

3. The Involvement of Epigenetic Modifications in ALS

4. Conclusions and Future Perspectives

Glossary

List of Abbreviations

Chapter 27. MicroRNA Deregulation in Lung Cancer and Their Use as Clinical Tools

1. Introduction to Noncoding RNA in Human Cell Physiology

2. miRNA Biology and Mechanism of Action

3. miRNA Alteration in Lung Cancer Progression

4. miRNA-Based Biomarkers for Lung Cancer Diagnosis, Prognosis, and Treatment

5. Interplay between Genetics and Epigenetics in miRNA Regulation and Lung Cancer

6. miRNA-Based Anticancer Therapy

7. Concluding Remarks

List of Abbreviations

Chapter 28. Circulating Nucleic Acids as Prostate Cancer Biomarkers

1. Introduction—The Urgent Requirement for New Prostate Cancer Biomarkers

2. Circulating miRNAs as Biomarkers

3. Circulating mRNAs as Biomarkers

4. Cell-Free DNAs as Biomarkers

5. Conclusions

List of Abbreviations

Chapter 29. MicroRNAs in Breast Cancer and Their Value as Biomarkers

1. Introduction

2. Global Deregulation of miRNAs in Cancer

3. The Biological Significance of miRNAs in Breast Cancer Development

4. Genetic Variants in miRNAs and Their Binding Sites

5. Acquired Genomic and Epigenomic Changes Affecting miRNAs

6. miRNAs as Regulators of the DNA Damage Response

7. DNA Repair Genes as Targets of miRNAs: A Bridge toward Personalized Treatment?

8. miRNAs as Prognostic Biomarkers

9. The Use of miRNAs in Screening for Early Breast Cancer Development

10. Conclusions

Glossary

List of Abbreviations

Chapter 30. MicroRNAs in Bone and Soft Tissue Sarcomas and Their Value as Biomarkers

1. Introduction

2. miRNAs in Bone Sarcomas

3. miRNAs in Soft Tissue Sarcomas

4. Circulating miRNAs in Bone and Soft Tissue Sarcomas

5. Conclusions

list of Abbreviations

Chapter 31. miRNAs in the Pathophysiology of Diabetes and Their Value as Biomarkers

1. Introduction

2. miRNAs Regulating Pancreatic β-Cell Function and Insulin Secretion

3. miRNAs in Insulin Target Tissues: Liver, Skeletal Muscle, and Adipose Tissue

4. miRNA Responses to Antidiabetic Therapies

5. Pharmacological Targeting of miRNAs in Diabetes

6. Circulating miRNAs as Promising Clinical Biomarkers for Diabetes

7. Conclusion

List of Abbreviations

Index

Copyright

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List of Contributors

Carolina Abril-Tormo,     IBSP-CV Biobank, FISABIO, Valencia, Spain

Sahar Al-Mahdawi

Department of Life Sciences, College of Health & Life Sciences, Brunel University London, Uxbridge, UK

Synthetic Biology Theme, Institute of Environment, Health & Societies, Brunel University London, Uxbridge, UK

Diogo Almeida-Rios

Cancer Biology and Epigenetics Group – Research Center, Portuguese Oncology Institute, Porto, Portugal

Department of Pathology, Portuguese Oncology Institute, Porto, Portugal

Sara Anjomani Virmouni

Department of Life Sciences, College of Health & Life Sciences, Brunel University London, Uxbridge, UK

Synthetic Biology Theme, Institute of Environment, Health & Societies, Brunel University London, Uxbridge, UK

Juan Ausio,     Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada

Bharati Bapat

Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada

Department of Pathology, University Health Network, Toronto, ON, Canada

Charlotte L. Bevan,     Department of Surgery & Cancer, Imperial Centre for Translational & Experimental Medicine, Imperial College London, Hammersmith Hospital Campus, London, UK

Jenefer M. Blackwell,     Telethon Kids Institute, The University of Western Australia, Subiaco, WA, Australia

Tiziana Bonaldi,     Department of Experimental Oncology, European Institute of Oncology, Milano, Italy

Abdelhalim Boukaba,     Drug Discovery Pipeline, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Science, Guangzhou, People’s Republic of China

Eoin Brennan,     Diabetic Complications Division, Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia

Enrique J. Busó,     Unidad Central de Investigación, University of Valencia, Valencia, Spain

F. Javier Carmona Sanz

Human Oncology & Pathogenesis Program (HOPP), Memorial Sloan-Kettering Cancer Center (MSKCC), New York, NY, USA

Cancer Epigenetics and Biology Program (PEBC), Bellvitge Institute for Biomedical Research (IDIBELL), Barcelona, Spain

Raimundo Cervera,     Hematology and Oncology Unit, Biomedical Research Institute INCLIVA, Valencia, Spain

Alfredo Ciccodicola

Institute of Genetics and Biophysics Adriano Buzzati-Traverso, CNR, Naples, Italy

Department of Science and Technology, University Parthenope of Naples, Italy

Joan Climent,     Hematology and Oncology Unit, Biomedical Research Institute INCLIVA, Valencia, Spain

Valerio Costa,     Institute of Genetics and Biophysics Adriano Buzzati-Traverso, CNR, Naples, Italy

Ana B. Crujeiras

Laboratory of Molecular and Cellular Endocrinology, Instituto de Investigación Sanitaria (IDIS), Complejo Hospitalario Universitario de Santiago (CHUS) and Santiago de Compostela University (USC), Santiago de Compostela, Spain

CIBER Fisiopatología de la Obesidad y la Nutrición (CIBERobn), Madrid, Spain

Alessandro Cuomo,     Department of Experimental Oncology, European Institute of Oncology, Milano, Italy

Avery DeVries,     Department of Cellular and Molecular Medicine, Arizona Respiratory Center and Arizona Center for the Biology of Complex Diseases, University of Arizona, Tucson, AZ, USA

Angel Diaz-Lagares,     Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain

Roberta Esposito,     Institute of Genetics and Biophysics Adriano Buzzati-Traverso, CNR, Naples, Italy

Alessandro Fatica,     Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy

Alfredo Ferro,     Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy

Claire E. Fletcher,     Department of Surgery & Cancer, Imperial Centre for Translational & Experimental Medicine, Imperial College London, Hammersmith Hospital Campus, London, UK

Ana-Maria Florea,     Department of Neuropathology, Heinrich-Heine-University Düsseldorf, Dusseldorf, Germany

Ernest Fraenkel,     Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

Yu Fujita,     Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan

Tomohiro Fujiwara

Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan

Center for Innovative Clinical Medicine, Okayama University Hospital, Okayama, Japan

Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan

Miriam Gagliardi,     Institute of Genetics and Biophysics Adriano Buzzati-Traverso, CNR, Naples, Italy

José Luis García-Giménez

Center for Biomedical Network Research on Rare Diseases (CIBERER), National Institute of Health Carlos IIII, Spain

Department of Physiology, Medicine and Dentistry School, University of Valencia, Valencia, Spain

Biomedical Research Institute INCLIVA, University of Valencia, Valencia, Spain

Rosalba Giugno,     Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy

Catherine Godson,     Conway Institute, Diabetes Complications Research Centre, University College Dublin, Dublin, Ireland

Inês Graça

Cancer Biology and Epigenetics Group – Research Center, Portuguese Oncology Institute, Porto, Portugal

School of Allied Health Sciences (ESTSP), Polytechnic of Porto, Porto, Portugal

Kirsten Grønbæk,     Department of Hematology, Rigshospitalet, Copenhagen, Denmark

Shinji Hagiwara,     Diabetic Complications Division, Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia

Rui Henrique

Cancer Biology & Epigenetics Group, IPO-Porto Research Center (CI-IPOP), Portuguese Oncology Institute, Porto, Portugal

Department of Pathology, Portuguese Oncology Institute, Porto, Portugal

Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar – University of Porto (ICBAS-UP), Porto, Portugal

José Santiago Ibañez Cabellos

Center for Biomedical Network Research on Rare Diseases, Medicine and Dentistry School, University of Valencia, Valencia, Spain

Biomedical Research Institute INCLIVA, Valencia, Spain

Marisa Iborra,     Gastroenterology Department, Hospital Universitari i Politècnic La Fe, Valencia, Spain

Toyotaka Ishibashi

Division of Life Science, Hong Kong University of Science and Technology, Kowloon, Hong Kong, HKSAR

Department of Biomedical Engineer, Hong Kong University of Science and Technology, Kowloon, Hong Kong, HKSAR

Sarra E. Jamieson,     Telethon Kids Institute, The University of Western Australia, Subiaco, WA, Australia

Carmen Jerónimo

Cancer Biology & Epigenetics Group, IPO-Porto Research Center (CI-IPOP), Portuguese Oncology Institute, Porto, Portugal

Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar – University of Porto (ICBAS-UP), Porto, Portugal

Sadhana Joshi,     Department of Nutritional Medicine, Interactive Research School for Health Affairs, Bharati Vidyapeeth University, Pune, Maharashtra, India

Phillip Kantharidis,     Diabetic Complications Division, Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia

Akira Kawai,     Department of Musculoskeletal Oncology, National Cancer Center Hospital, Tokyo, Japan

Vinita Khot,     Department of Nutritional Medicine, Interactive Research School for Health Affairs, Bharati Vidyapeeth University, Pune, Maharashtra, India

Lasse Sommer Kristensen,     Department of Hematology, Rigshospitalet, Copenhagen, Denmark

Ana Lluch,     Hematology and Oncology Unit, Biomedical Research Institute INCLIVA, Valencia, Spain

José Antonio López-Guerrero,     Laboratory of Molecular Biology and Biobank, Fundacion Instituto Valenciano de Oncologia, Valencia, Spain

Paula Lopez-Serra,     Epigenetic and Cancer Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain

Annita Louloupi,     Division of Molecular Pathology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

Luca Magnani,     Department of Surgery and Cancer Imperial Centre for Translational and Experimental Medicine, Imperial College Hammersmith, London, UK

Jacobo Martínez-Santamaría

IBSP-CV Biobank, FISABIO, Valencia, Spain

Valencian Biobank Network, FISABIO, Valencia, Spain

Maria R. Matarazzo,     Institute of Genetics and Biophysics Adriano Buzzati-Traverso, CNR, Naples, Italy

Aaron McClelland,     Diabetic Complications Division, Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia

Pamela Milani,     Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

Yutaka Nezu,     Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan

Roberta Noberini,     Center of Genomic Science, Istituto Italiano di Tecnologia, Milano, Italy

Takahiro Ochiya,     Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan

Toshifumi Ozaki,     Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan

Federico V. Pallardó

Center for Biomedical Network Research on Rare Diseases, Medicine and Dentistry School, University of Valencia, Valencia, Spain

Biomedical Research Institute INCLIVA, Valencia, Spain

Lorena Peiró-Chova,     INCLIVA Biobank, INCLIVA Biomedical Research Institute, Valencia, Spain

Marco Pellegrini,     Laboratory of Integrative Systems Medicine (LISM), Institute of Informatics and Telematics (IIT) and Institute of Clinical Physiology (IFC), National Research Council (CNR), Pisa, Italy

Tandy L.D. Petrov,     Department of Biology, The University of Alabama at Birmingham, Birmingham, AL, USA

Olga Bahamonde Ponce,     INCLIVA Biobank, INCLIVA Biomedical Research Institute, Valencia, Spain

Mark A. Pook

Department of Life Sciences, College of Health & Life Sciences, Brunel University London, Uxbridge, UK

Synthetic Biology Theme, Institute of Environment, Health & Societies, Brunel University London, Uxbridge, UK

Alfredo Pulvirenti,     Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy

João Ramalho-Carvalho,     Cancer Biology & Epigenetics Group, IPO-Porto Research Center (CI-IPOP), Portuguese Oncology Institute, Porto, Portugal

Alberto Ramos,     Hematology and Oncology Unit, Biomedical Research Institute INCLIVA, Valencia, Spain

George Rasti,     Chromatin Biology Laboratory, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain

Nicole C. Riddle,     Department of Biology, The University of Alabama at Birmingham, Birmingham, AL, USA

Peter H.J. Riegman,     Department of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands

Carlos Romá Mateo

Center for Biomedical Network Research on Rare Diseases, Medicine and Dentistry School, University of Valencia, Valencia, Spain

Biomedical Research Institute INCLIVA, Valencia, Spain

Francesco Russo

Laboratory of Integrative Systems Medicine (LISM), Institute of Informatics and Telematics (IIT) and Institute of Clinical Physiology (IFC), National Research Council (CNR), Pisa, Italy

Department of Computer Science, University of Pisa, Pisa, Italy

Fabian Sanchis-Gomar

Department of Physiology, Medicine and Dentistry School, University of Valencia, Valencia, Spain

Biomedical Research Institute INCLIVA, University of Valencia, Valencia, Spain

Juan Sandoval,     Epigenetic and Cancer Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain

Flavia Scoyni,     Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy

Marta Seco Cervera

Center for Biomedical Network Research on Rare Diseases, Medicine and Dentistry School, University of Valencia, Valencia, Spain

Biomedical Research Institute INCLIVA, Valencia, Spain

Akifumi Shibakawa,     Department of Surgery & Cancer, Imperial Centre for Translational & Experimental Medicine, Imperial College London, Hammersmith Hospital Campus, London, UK

Nicolas G. Simonet,     Chromatin Biology Laboratory, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain

Ailsa Sita-Lumsden,     Department of Surgery & Cancer, Imperial Centre for Translational & Experimental Medicine, Imperial College London, Hammersmith Hospital Campus, London, UK

Olafur Andri Stefansson,     Cancer Research Laboratory, Faculty of Medicine, University of Iceland, Reykjavik, Iceland

Deepali Sundrani,     Department of Nutritional Medicine, Interactive Research School for Health Affairs, Bharati Vidyapeeth University, Pune, Maharashtra, India

Genevieve Syn,     Telethon Kids Institute, The University of Western Australia, Subiaco, WA, Australia

Trygve O. Tollefsbol,     Comprehensive Cancer Center, Center for Aging, Comprehensive Diabetes Center, Nutrition Obesity Research Center, Cell Senescence Culture Facility, University of Alabama at Birmingham, Birmingham, AL, USA

Eneda Toska,     Human Oncology & Pathogenesis Program (HOPP), Memorial Sloan-Kettering Cancer Center (MSKCC), New York, NY, USA

Marianne B. Treppendahl,     Department of Hematology, Rigshospitalet, Copenhagen, Denmark

Toshikazu Ushijima,     Chief of Division of Epigenomics, National Cancer Center Research Institute, Tokyo, Japan

Alejandro Vaquero,     Chromatin Biology Laboratory, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain

Donata Vercelli,     Department of Cellular and Molecular Medicine, Arizona Respiratory Center and Arizona Center for the Biology of Complex Diseases, University of Arizona, Tucson, AZ, USA

Filipa Quintela Vieira

Cancer Biology and Epigenetics Group – Research Center, Portuguese Oncology Institute, Porto, Portugal

School of Allied Health Sciences (ESTSP), Polytechnic of Porto, Porto, Portugal

Yinan Zhang,     Division of Life Science, Hong Kong University of Science and Technology, Kowloon, Hong Kong, HKSAR

Fang Zhao

Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada

Wilbert Zwart,     Division of Molecular Pathology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

Preface

Epigenetics is an emerging frontier of biology, and its definition is continuously being adapted based on new scientific findings. In fact, the NIH Roadmap Epigenomics Project has recently defined epigenetics as the heritable changes in gene activity and expression (in the progeny of cells or of individuals) and also stable, long-term alterations in the transcriptional potential of a cell that are not necessarily heritable. In this regard, epigenetics includes DNA methylation, noncoding RNAs, and histone posttranslational modifications. This integrative definition of epigenetics reflects the potential of the discipline to expand beyond the control of a particular gene expression program for each cell type, defining the cellular and developmental identity and function of cells, and, finally, translating this potential to health and disease conditions in human beings.

Due to the rapid progress in the field of epigenetics, new and promising methodologies to advance biomedical research are being developed. Epigenetic research and epigenetic pharmaceutical drug development are now considered areas of great interest and promise in the biomedical scene. The advantage of human epigenetics compared with human genetics and genomics is that it provides vital information about gene function in individual cell types, while incorporating information from the environment and lifestyle, and unlike most genetic defects causative of human disease, epigenetic alterations are modulable and reversible. The volume Epigenetic Biomarkers and Diagnostics is intended to describe both epigenetic biomarkers that can be adopted into clinical routine as well as advanced technologies and tools for their analysis. In this regard, epigenetic biomarkers provide clinicians valuable information about the presence or absence of a disease (diagnostic value), the patient prognosis (prognostic value), the response to a specific treatment (predictive value), the effects of ongoing treatment (therapy-monitoring biomarkers), and the future risk of disease development (risk prediction). Furthermore, several advantages may arise from the use of epigenetic biomarkers versus gene expression in clinical practice, such as higher stability, for example, in biofluids. They can also fill clinical gaps by bridging genetic information, mRNA transcription, and protein translation. In consequence, the associations between epigenome alterations and diseases become clearer, providing a way to act directly on gene expression by developing specific drugs or even by adopting healthy lifestyles.

Many methodologies, including classical methods and next-generation-sequencing-based technologies, are available to clinicians and researchers to identify new epigenetic biomarkers and analyze them from several biological sources. In this context, genome-wide methylation analysis, chromatin immunoprecipitation coupled with high-throughput platforms, and noncoding RNA sequencing are described in this volume. Furthermore, some techniques for DNA methylation analysis are more likely to be rapidly adopted in clinical laboratories, such as EpiTYPER MassARRAY, methyl specific PCR (MSP), and pyrosequencing. On the other hand, immunoassays are well established in clinical laboratories for the analysis of histones (i.e., inflammatory and autoimmune diseases). However, it is expected that the incorporation of mass spectrometry technologies into laboratories for clinical diagnostics (replacing routine immunoassays) will be the tendency in the coming years, as will be the analysis of histone posttranslational modifications associated with pathological states.

Although it is not possible to cover all epigenetic markers, this volume includes chapters describing the most promising biomarkers for cancer (i.e., breast, lung, colon, etc.), metabolic disorders (i.e., diabetes and obesity), autoimmune diseases, infertility, allergy, infectious diseases, and neurological disorders; and, where possible, we will focus our attention on those which are feasible to be adopted for clinical use.

This book was written in a comprehensive manner by outstanding experts in their corresponding fields for a broad target audience such as advanced students, basic scientists, biomedical and biotechnological companies, as well as clinical researchers, clinicians (i.e., pathologists, immunologists, oncologists, endocrinologists, etc.) and analysts from clinical laboratories who can adopt these potential biomarkers into clinical practice.

In the coming years, epigenetics will continue to provide an exciting future in biomedicine and clinical practice. The chapters covered in Epigenetic Biomarkers and Diagnostics highlight the unprecedented impact of epigenetics in clinical diagnostics and will contribute to the discovery and development of new epigenetic biomarkers in the future.

José Luis García-Giménez,     Valencia, Spain

Chapter 1

Epigenetic Biomarkers

New Findings, Perspectives, and Future Directions in Diagnostics

José Luis García-Giménez¹, Toshikazu Ushijima²,  and Trygve O. Tollefsbol³     ¹Department Physiology, Center for Biomedical Network Research on Rare Diseases, National Institute of Health Carlos IIII, Institute of Health Research INCLIVA, Medicine and Dentistry School, University of Valencia, Valencia, Spain     ²Chief of Division of Epigenomics, National Cancer Center Research Institute, Tokyo, Japan     ³Comprehensive Cancer Center, Center for Aging, Comprehensive Diabetes Center, Nutrition Obesity Research Center, Cell Senescence Culture Facility, University of Alabama at Birmingham, Birmingham, AL, USA

Abstract

Our epigenome is characterized by its ability to dynamically respond to intra- and extracellular stimuli. These epigenetic changes are reversible and consequently are potential contributors to health and disease. Epigenetic biomarkers are emerging as tools for the screening and early detection of various diseases, for prognostic and treatment monitoring, and for predicting future risk of disease development. In clinical practice, this will contribute to the development of in vitro diagnostic and prognostic tools, which in turn may also lead to advances in personalized medicine. In this chapter we propose a definition for epigenetic biomarkers and describe different technologies used for their discovery and analysis. Furthermore, we focus our attention on issues and obstacles that need to be solved before adopting them into clinical routine. Finally, we present a perspective on the implantation of epigenetic biomarkers in clinical laboratories and their use in clinical diagnostics.

Keywords

Diagnostic laboratory; Epigenetic biomarkers; Epigenetics; In vitro diagnostics; Market

Outline

1. Introduction 2

2. Epigenetic Mechanisms 3

2.1 DNA Methylation 3

2.2 Histone PTMs and Histone Variants 5

2.3 Noncoding RNAs 7

3. Epigenetic Biomarkers and In Vitro Diagnostics 8

4. Epigenetic Biomarkers and the Clinical Laboratory 10

5. Epigenetic Biomarkers: An Overview of Recent Advances 11

6. Epigenetics: Perspective of Implantation in Clinical Laboratories 12

7. Perspectives of Epigenetics in Diagnostics 14

List of Abbreviations 15

References 15

1. Introduction

The literal meaning of the term epigenetic is above or on top of genetics, although it has had many different definitions over the years. Conrad Hal Waddington was the first to define epigenetics, in 1942, as the branch of biology which studies the causal interaction between genes and their products, which bring the phenotype into being [1]. In 1990, Holiday defined epigenetics as the study of the mechanisms of temporal and spatial control of gene function during the development of organisms [2]. A few years later, epigenetics was defined in a narrower manner, as the different epigenetic modifications or mechanisms that produce heritable changes affecting gene expression without affecting the DNA sequence. In 2001, Jenuwein and Allis proposed the histone code as an epigenetic mechanism which is a critical feature of a genome-wide mechanism of information storage and retrieval and that considerably expands the information potential of the genetic code. Therefore, they pointed out that epigenetics imparts a fundamental regulatory system beyond the sequence information of our genetic code [3]. By 2007, the definition of epigenetics had changed yet again, when Bird defined epigenetics as the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states [4]. The same year, Goldberg, Allis, and Bernstein defined epigenetics as the study of any potentially stable and, ideally, heritable change in gene expression or cellular phenotype that occurs without changes in Watson-Crick base pairing of DNA [5]. It is evident that both definitions proposed by Bird and Goldberg et al. were inclusive of transient chemical modifications of DNA and histones; modifications that have not always been universally accepted and that are still a subject of debate. Therefore, in 2008, a consensus definition was made at a Cold Spring Harbor meeting, giving a more integrative definition for epigenetics as a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence [6]. It is evident that epigenetics is an emerging frontier of science, so its definition is continuously being adapted based on new scientific findings. In fact, the NIH Roadmap Epigenomics Project defines epigenetics as the heritable changes in gene activity and expression (in the progeny of cells or of individuals) and also stable, long-term alterations in the transcriptional potential of a cell that are not necessarily heritable [7] (www.roadmapepigenomics.org). In this regard, epigenetics includes DNA methylation, noncoding RNAs (ncRNAs), and histone posttranslational modifications (PTMs). This last integrative definition of epigenetics reflects the potential of epigenetics to go beyond the control of a particular gene expression to produce a unique gene expression program of each cell type, defining the cellular and developmental identity [8], as well as potential health and disease outcomes [9].

Our epigenome is characterized by its ability to dynamically respond to intra- and extracellular stimuli, so that epigenetic changes are reversible and in consequence are potential contributors to health and disease. Recent progress in the field of epigenetics opens promising ways to advance biomedical research. Epigenetic research and epigenetic pharmaceutical drug development are now considered a bright spot in the biomedical research field. This research will contribute to biomarker discovery, new therapy development, computational and bioinformatics training, and the development of new next-generation sequencing (NGS) technologies and applications. The advantage of epigenetics compared to genetics is that it provides vital information about gene function in individual cell types and incorporates information from the environment. Furthermore, several advantages may arise from the use of epigenetic biomarkers versus gene expression (by measuring mRNAs). Epigenetic biomarkers have shown higher stability in fluids and formalin-fixed paraffin-embedded (FFPE) biospecimens compared to mRNAs. In addition, epigenetic biomarkers can benefit biomedical research and fill clinical research gaps by bridging genetic information and mRNA expression. In consequence, this makes the association between epigenome alterations and diseases clearer, and also provides a way to act directly on them by developing specific drugs or adopting healthy lifestyles. One of the most important issues is that the associations that have been found between epigenetics and certain diseases will have a synergistic effect on the development of personalized medicine. In recent years, epigenetics has aroused the interest of different biomedical and scientific fields, and it also has impacted society. In 2010, TIME Magazine published Why Your DNA Isn’t Your Destiny opening the public’s eyes to the latest research and changing the general view about DNA and its direct role in our lives.

2. Epigenetic Mechanisms

Three major events are mainly involved in epigenetic regulation and chromatin structure control: DNA methylation, histone PTMs, and ncRNAs (i.e., microRNAs (miRNAs), long noncoding RNAs (lncRNAs)). Disruption of one or more of these epigenetic mechanisms can lead to inappropriate expression of genes, resulting in an alterated state of cell homeostasis or disease. In this regard, epigenetic-based biomarkers are an important new research area. With the potent technologies now available, diagnostic tools can be created to analyze these biomarkers and therefore contribute to the study of human diseases. Here, we summarize the three most relevant mechanisms and discuss the technologies available to analyze them.

2.1. DNA Methylation

DNA methylation is the most-studied DNA modification since its discovery in the late 1940s. It consists of the addition of a methyl group at the 5′ position of the cytosine base (5-methylcytosine (5mC)), which protrudes into the major groove of DNA, representing a potential recognition site for protein binding without changing the Watson–Crick base pairing. The methyl group at the 5′ position of cytosine is donated by S-adenosylmethionine (SAM) via Dnmt1, Dnmt3A, and Dnmt3B (DNA(cytosine-5-)methyltransferases (DNMTs)). Importantly, CpG sites are underrepresented and unevenly distributed across the human genome, giving rise to vast low-density CpG regions interspersed with CpG clusters located mainly in CpG islands [10]. Generally speaking, 5mCs play essential roles in maintaining cellular function and genome stability.

In cancers, global DNA hypomethylation and regional hypermethylation are almost always observed and have been associated with genome instability, altered chromatin conformation, and chromosome fragility [11–13]. At the same time, hypermethylation of CpG islands [14] and their flanking regions, called CpG shores [15], is also observed in specific CpG islands, including those in promoters of tumor suppressor genes (driver methylation) and other genes methylated in association with cancer development (passenger methylation). Importantly, DNA methylation can be detected by a wide range of sensitive and cost-effective techniques [16,17], as described in Chapters 6–9 of Section II in this volume. Moreover, DNA methylation is a stable chemical mark that is not easily altered, which makes it a feasible biomarker for diagnostics, prognostics, and treatment monitoring. The field of DNA methylation has expanded with the identification of multiple cytosine variants thanks to the design of new methodologies. Ten-eleven translocation (Tet) family of cytosine oxygenase enzymes (TETs) are responsible for oxidizing 5mCs into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by means of an α-ketoglutarate- and O2-Fe(II)-dependent reactions (Figure 1). Recent evidence reveals that these DNA demethylation intermediates are dynamic and participate in key regulatory functions in distal gene regulatory elements in mammalian genomes and also in biological processes, such as demethylation in zygote formation and in germ cell lineage (for a review see Ref. [18]).

Figure 1  Dynamic DNA methylation and demethylation catalyzed by DNA(cytosine-5-)methyltransferases (DNMTs) and ten-eleven translocase (TET) proteins. 5-methylcytosine (5mC) is produced by the activity of DNMTs using S-adenosylmethionine as methyl donor. 5mC can be converted to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by iron-dependent dioxygenases TET proteins. Some methods exist to identify the cytosine modifications at single-base resolution in whole-genome studies. Bisulfite genomic sequencing (BS-seq), Infinium 450K DNA methylation (Illumina), and direct immunoprecipitation of methylated DNA (MeDIP-seq) are used for 5mC studies. DNA pull-down using 5hmC antibodies in combination with genome-wide sequencing (hMeDIP-seq), Tet-assisted (bisulfite) reduced representation bisulfite sequencing (TAB-RRBS), and oxidative bisulfite coupled to 450K BeadChip (oxBS-450K) to study 5hmC. The method methylation-assisted bisulfite sequencing (MAB-seq) serves for mapping genome-wide 5fC and 5caC. However, using the reduced bisulfite sequencing (redBS-seq) or chemical modification-assisted bisulfite sequencing it is possible to identify 5fC or 5caC, respectively.

Novel experimental designs consisting of chemical oxidation or chemical reduction of the modified cytosines allow the identification of 5mC, 5hmC, 5fC, and 5caC at single-base resolution [19–21] and, when implemented in DNA sequencing protocols, will allow the analysis of whole genomes and the exploitation of these procedures in biomedicine and clinical research. In this regard, high-throughput methods for analyzing cytosine variants have appeared in recent years. Bisulfite sequencing (BS-seq), Infinium HumanMethylation 450K BeadChip (Illumina), and immunoprecipitation of methylated DNA followed by sequencing (MeDIP-seq) have been developed to analyze 5mC. On the other hand, Tet-assisted bisulfite sequencing (TAB-seq), 5hmC immunoprecipitation coupled to sequencing, oxidative bisulfite sequencing (oxBS-seq), and oxidative bisulfite hybridization in the Infinium 450K BeadChip (oxBS-450K) have been designed to map 5mC and 5hmC residues at single-base resolution on a genome-wide scale [22]. Using named methylation-assisted bisulfite sequencing (MAB-seq), the quantitation of the 5fC and 5caC residues at single-base resolution is possible [23], and using chemical modification-assisted bisulfite sequencing (CAB-seq) the specific whole-genome analysis of 5caC is possible [24]. By using reduced bisulfite sequencing (redBS-seq), it is possible to identify 5fC [25].

As shown in Figure 1, these different directed assays have been developed to study the cytosine variants which participate in the regulation of DNA methylation. Therefore, these methodologies will contribute to explaining the intriguing epigenetic regulation underlying human pathologies [26]. It is obvious that the direct application of these high-throughput procedures in clinical routine seems to be so far from reality. However, these techniques will contribute to the identification of passenger methylation underlying several human diseases and also to develop clinical epigenetics.

2.2. Histone PTMs and Histone Variants

Chromatin is composed of repeating arrays of nucleosomes which comprise the essential units of organization in eukaryotic chromosomes. Each nucleosome is formed by 145  bp of DNA wrapped around a histone octamer. Each histone octamer consists of two copies each of canonical histone H4, H3, H2A, and H2B or their variants. Histone can be chemically modified and when it is, chromatin state and gene expression are considered to be modified as well. The histone PTMs produced (i.e., acetylation, methylation, phosphorylation, butyrylation, hydroxybutyrylation, crotonylation, citrullination, formylation, glycosylation, O-GlcNAcylation [27], carbonylation, parsylation [28], and glutathionylation [29]) mainly occur on amino acids in the N-terminal tail domains (Figure 2). These chemical modifications change the nucleosome structure and spread to different regions of the genome. The hypothesis of the histone code proposes that the control of the chromatin state is exerted by the PTMs [30]. Evidence accumulated in recent years supports the idea that the PTM signature is altered in a wide range of diseases, such as cancer [31,32], neurological syndromes [33,34], and rare diseases such as Rubinstein–Taybi syndrome [35] and Cofin–Lowry syndrome [36]. In addition, clinicians have become increasingly interested in histone PTMs in recent years because it is possible to analyze them in specific regulatory domains of genes to obtain valuable information for the diagnostics of disease [17].

Another additional level in the supramolecular structural organization of chromatin involves the participation of histone variants (Figure 2). Histone variants differ in their primary sequence of amino acids [37]. Variants for all of the histone protein families have been described and have emerged as important elements involved in chromatin dynamics and organization, and have also served as scaffolds for other proteins that create silenced regions in chromosomes [38,39]. Therefore, the incorporation of histone variants in specific domains is very important, and their regulation plays a crucial role in cellular processes such as differentiation, proliferation, and nuclear reprogramming. For this reason, mutations in specific histone variants are involved in human disease [40] like cancer, as recently reviewed by Vardabasso et al. [41]. Due to the importance of histone variants in cell differentiation and reprogramming it is not surprising that they are closely related to male infertility, as described later in this volume by Ausió, Zhang, and Ishibashi in Chapter 24.

Interestingly, histones can also be found in biological fluids such as blood, serum, and plasma. Therefore, they have been proposed as clinical biomarkers since their presence in body fluids suggests tissue damage, inflammation, and cellular apoptosis [42]. Recent evidence supports the idea that extracellular histones contribute to human disease. Circulating nucleosomes were recently associated with disease progression in patients with thrombotic microangiopathies [43]. Ekaney et al. detected higher levels of histone H4 in septic patients compared to patients with multiple organ failure without infection or to patients with minor trauma [44]. Zeerleder et al. found increased nucleosome levels in systemic inflammation and septic shock [45,46] and studied how these levels correlated with the severity of the inflammatory response and mortality in children affected by meningococcal sepsis [46]. Unfortunately, there exists no consensus on the levels of circulating histones and nucleosomes that discriminate patients from healthy subjects, due in part to the diversity of procedures used in their determination.

Figure 2  Graphical representation of nucleosomes showing histone octamer and histone tails for histone H3.3.

On the top of the figure is shown partially the primary sequence of histone H3.3 in which main posttranslational modifications (PTMs) in amino acids (acetylation, ac; monomethylation, me1; dimethylation, me2; trimethylation, me3; biotinylation, yellow triangle; citrullination, red square; phosphorylation, green square; and glutathionylation, blue oval) are represented. H3 variants are showed into the box at the bottom of the figure, in which is compared the alignment of human noncentromeric histone H3 variants. Differences in amino acid sequence among H3.3, H3.2, H3.1, H3.1t (testicular variant), and H3.Y are shown in italics (in online version they are shown colored in red).

Currently, different commercial kits are available for the identification of circulating nucleosomes and histones in blood samples of autoimmune disease patients, mainly in drug-induced systemic lupus erythomatosus [47]. Many of them are based on enzyme immunoassays [48]. Furthermore, immunohistochemistry and immunofluorescence-based procedures can be used to analyze histone PTMs. However, a trend toward the use of mass spectrometry (MS)-based methodologies is gaining interest in clinical diagnostic laboratories. MS methodologies can be employed in the analysis of histone PTMs and histone variants. In this volume, Noberini et al. discuss the potential of MS-based technologies in biomarker discovery and their applications in clinical epigenetics, focusing on novel techniques to dissect the histone code in clinical samples (Chapter 10). Importantly, MS holds great promise for clinical epigenetics because its potential allows histone PTM combinations to be dissected.

2.3. Noncoding RNAs

Only a small percentage of the transcribed genes encode proteins, so these genome regions were described as dark matter RNAs [49]. ncRNAs are considered active participants in controlling a wide range of biological processes, such as the regulation transcription of single genes, as well as entire transcriptional programs [50]. Many thousands of regulatory nonprotein-coding RNAs were demonstrated to be transcribed in genome-wide studies, including miRNAs, small RNAs, PIWI-interacting RNAs, and various classes of lncRNAs [51]. Most clinical applications are being found for miRNAs and lncRNAs, so we will briefly describe these ncRNAs. Later chapters of this volume are focused on the ncRNA species and their potential as clinical biomarkers.

miRNAs consist of a large family of short ncRNAs (17–25 nucleotides). These ncRNAs are involved in many biological processes [52] (i.e., cellular development, differentiation, apoptosis, proliferation, tumor growth, metastatic dissemination, and resistance to therapy, among others) [53,54] as a consequence of their ability to control the expression (downregulation or upregulation) of numerous genes. High stability and low susceptibility to degradation are important characteristics of miRNAs [17,55,56]. This could be due to their short length, particular biogenesis, and strong association with proteins, or membrane-bound vesicles, such as exosomes, microvesicles, or apoptotic bodies, etc. Importantly, miRNA expression is frequently altered in cancer and has shown promise as a tissue-based, circulating biomarker for cancer classification and prognostics [56]. An interesting pioneer study on this was performed by Volinia et al. They identified many overexpressed miRNAs in several solid tumors by performing a large-scale miRNome analysis and found specific miRNA signatures for each tumor type [57]. Of great clinical importance is that unique miRNA expression patterns can distinguish tumors from different anatomical locations, and also differentiate various tumor subtypes at a single anatomic locus in kidney cancer [58], breast cancer [59], and papillary renal carcinoma [60], among others.

lncRNAs are generally defined as transcripts longer than 200 nucleotides that can be processed like mRNA, i.e., spliced and polyadenylated [61]. These kinds of ncRNAs have been associated with neurological disorders [62], cancer [63], and complex metabolic disorders [64].

There are currently a number of studies showing the interconnection between distinct epigenetic events. For example, a subgroup of ncRNAs are additionally classified as epi-miRNAs because they can directly or indirectly regulate the expression of several components of the epigenetic machinery, such as DNA methyltransferases or histone deacetylases, creating a very well-orchestrated mechanism [65,66] in such a way that the joint activities of different epigenetic modifications result in a common outcome. An altered balance of these processes leads to pathological conditions, so it is important to evaluate the levels of ncRNAs (miRNAs and lncRNAs) in human biospecimens. High-throughput sequencing technologies, computational pipelines, and bioinformatics algorithms allow us to identify the profile of dysregulated ncRNAs in human diseases. In Chapter 11, Costa et al. describe some of these approaches and tools for ncRNA analysis. In addition, Russo et al. (Chapter 12) discuss recent discoveries and controversial topics on circulating ncRNAs and describe several public resources recently developed for ncRNA analysis. In Table 1 the most relevant properties and information of miRNAs and lncRNAs are summarized.

Epigenetic regulation does not always produce gene regulation in a binary system that is either ON or OFF. Epigenetic mechanisms can be propagated over multiple cell divisions in somatic cells. In addition, epigenetic information is modified during cellular differentiation and partially erased in the germ line and the early embryo [67]. We also know that lifestyle, environment, especially chronic inflammation, and nutrition induce epigenetic changes during our life [68–73] and that aberrant placement of epigenetic marks and epigenetic dysregulation (produced by mutations in genes that codify for epigenetic machinery) are involved in disease. Thus, DNA methylation, histone PTMs, and ncRNAs play a critical role in diseases such as cancer, infectious diseases, infertility, and metabolic and neurodegenerative disorders. Some examples are shown in section III of Epigenetic Biomarkers and Diagnostics. Therefore, the identification of these epigenetic changes serves to define healthy or disease states in humans and to set the basis for the identification of epigenetic biomarkers.

3. Epigenetic Biomarkers and In Vitro Diagnostics

A biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. The United Nations World Health Organization (WHO) defines a biomarker as any substance, structure, or process that can be measured in the body or its products and which influences or predicts the incidence of outcome of diseases (Biomarkers in Risk Assessment: Validity and Validation, Environmental Health Criteria Series, No. 222, WHO). In this regard, the goal of clinical biomarkers is to provide clinicians with valuable information about the presence or absence of a disease (diagnostic biomarker), the patient prognosis (prognostic biomarker), the response to a specific treatment (predictive biomarker), the effects of ongoing treatment (therapy monitoring biomarkers), or a future risk of disease development (risk markers) [74]. Based on these descriptions, recommended properties for clinical biomarkers are (1) they should be specific, sensitive, and stable; (2) they should be validated by different institutions in a large number of samples followed by approval from the (US Food and Drug Administration) FDA and/or (European Medicines Agency) EMA; and (3) although there exist excellent single markers that serve for diagnostics, the use of biomarker signatures instead of only one biomarker is preferable because the combination of biomarkers increases the sensitivity and specificity [75].

Table 1

General Information for Noncoding RNAs

miRNAs, microRNAs; lncRNAs, long noncoding RNAs; FFPE, formalin-fixed paraffin-embedded; qRT-PCR, quantitative real-time polymerase chain reaction.

a miRbase v21 (Kozomara A, Griffiths-Jones S. miRBase: annotating high confidence microRNAs using deep sequencing data. Nucl Acids Res 2014 January; 42(D1): D68–D73).

b LNCipedia.org v3.1 (Volders P-J, Verheggen K, Menschaert G, Vandepoele K, Martens L, Vandesompele J and Mestdag P. An update on LNCipedia: a database for annotated human lncRNA sequences. Nucl Acids Res 2014 2015 January; 43(D1): D174-D180).

One of the most important properties of epigenetic marks is that they are highly stable (methylated DNA and miRNA) in multiple biospecimens (i.e., urine, blood, plasma, FFPE tissues, etc.). This is the case of miRNAs, which are very stable molecules in blood [56], urine [76], and also in FFPE [55], different from mRNA and proteins, as discussed by Peiró-Chova et al. in Chapter 2.

Based on these precedents, an epigenetic biomarker can be defined as any epigenetic mark or altered epigenetic mechanism (1) that can be measured in the body fluids or tissues and (2) that defines a disease (detection), predicts the outcome of diseases (prognostic) or response to a therapy (predictive), monitors treatment responses (therapy monitoring), or predicts risk of future disease development (risk).

In general, epigenetic biomarkers may represent the effect of the environment and natural history on the particular evolution of disease in each patient. Therefore, one of the most promising properties of epigenetic biomarkers is that they will contribute to the improvement of precision medicine. The advantage offered by epigenetic biomarkers versus genetic biomarkers is that the former is a dynamic one which changes based on disease evolution and intra- or extraenvironmental cellular conditions while the latter, in contrast, is a static biomarker since our gene sequence generally does not change.

The potential of epigenetic biomarkers in clinical practice is currently being demonstrated. The in vitro diagnostics (IVD) market is seeking new diagnostic and prognostic biomarkers which contribute to personalized medicine, and epigenetics is currently contributing to this [77,78]. Recent advances in NGS have allowed epigenetics to be used more easily both by researchers and clinicians [79–81].

The exponential growth of the IVD market is evident at the moment, since the identification of biomarkers and the design of IVD tests facilitate diagnostic, prognostic, and treatment monitoring. Another point to consider is that the USFDA encourages the integration of biomarkers into drug development and their appropriate use in clinical practice. In that way, the effective integration of biomarkers into clinical development programs may facilitate new medical product development and promote personalized medicine [82,83]. In recent years, many epigenetic drugs have been discovered, so the development of new, predictive, and sensitive biomarkers for clinical practice is expected to reduce the time and cost of drug development and also contribute to overcoming problems during the evaluation of new therapies during clinical trials [84]. Therefore, the use of epigenetic biomarkers has tremendous potential to affect the success rate of clinical trials and drug discovery. Proof of this is the growth observed in the number of clinical trials using epigenetic drugs during last decade (Figure 3). In fact, epigenetic therapy will be improved considerably after identification of good pretreatment biomarkers predicting response. Many large clinical trials in combination with novel high-throughput screening methods have contributed to these improvements, as described by Treppendahl et al. in Chapter 5.

Specifically, epigenetic biomarkers are coevolving and have reached a critical point wherein enough signatures have been identified across various disease classes, most notably in cancer, that some of these signatures and expression patterns can be validated and used in the clinic [85,86]. The potential of epigenetic biomarkers in clinical practice has directed them to their use in IVD. In fact, the global epigenetics market was valued at an estimated $413.24 million in 2014 and it is expected to reach $783.17 million in 2019. The IVD market is expected to experience an enormous expansion in the coming years as well, and epigenetics will clearly contribute to that.

Figure 3  Number of clinical trials using epigenetic drugs and biomarkers reported by ClinicalTrials.gov from 2000 to 2014.

The figure presents the growth rate for clinical trials in different statuses (i.e., not yet recruiting, recruiting, enrolling by invitation, active but not recruiting, completed). The shape of the curve indicates a research area that is reaching amount of interest in clinical practice.

4. Epigenetic Biomarkers and the Clinical Laboratory

Many methods, including classical methods and NGS-based methods, are available to clinicians and researchers to identify new epigenetic biomarkers, as reviewed by Petrove and Riddle in Chapter 4. In recent years, technological improvements have allowed researchers to examine the epigenome on a genome-wide fashion.

In Section II of this volume we also provide a description of the most relevant technologies that have contributed to epigenetic biomarker identification and analysis and outline which are feasible and useful technologies to be adopted in clinical laboratories.

These new technological improvements have set the basis for new biomarker identification and analysis. In this context, genome-wide methylation analysis and chromatin immunoprecipitation with high-throughput platforms, including DNA microarrays and genome sequencing, have permitted the performance of comprehensive studies regarding the epigenetic bases of several diseases. These technologies are well described by Toska and Carmona-Sanz in Chapter 6. NGS has also revolutionized the analysis of miRNAs and lncRNAs by allowing the application of NGS-based methods for clinical epigenetics, as described in Chapter 11 by Costa et al. A recent example of this application was published by Keller et al. In this study, the authors analyzed 863 miRNA signatures from 454 human blood biospecimens. The samples were obtained from patients suffering from 14 different diseases, including lung cancer, pancreatic ductal adenocarcinoma, prostate cancer, ovarian cancer, Wilms tumor, multiple sclerosis, periodontitis, sarcoidosis, myocardial infarction, and chronic obstructive pulmonary disease [87]. By utilizing the data of dysregulated miRNAs in the blood and developing mathematical algorithms, Keller et al. were able to accurately predict disease in more than two-thirds of individuals [87]. It is obvious that more ncRNAs are being identified as research advances, so data integration in public databases such as the human miRNA-associated disease database (HMDD) [88] (www.cuilab.cn/hmdd) or miRandola for extracellular circulating miRNAs [89] serve as resources for researchers screening miRNA and lncRNA profiles for a wide range of diseases. These databases are therefore fundamental tools for biomedical research as described by Russo et al. in Chapter 12.

Since DNA methylation was the first epigenetic mark to be widely studied, further progress has been made in the field of clinical diagnostics. Some techniques for DNA methylation analysis have the potential to be rapidly well established in clinical laboratories. In this regard, Buso and Iborra describe the use of Sequenom MassARRAY technology in the analysis of DNA methylation in Chapter 7. This MS-based method provides differences in DNA methylation at the individual CpG level. The EpiTYPER MassARRAY system of Sequenom is a candidate to be adopted in clinical laboratories, although there exist some inconveniences to this technology, such as the fact that it requires expensive equipment and trained technicians. Furthermore, other procedures that can be more rapidly adopted in clinical laboratories are described by Zhao and Bapat in Chapter 8. They describe MethyLight and multiplex MethyLight assays using TaqMan® fluorescent probes. These easy-to-use procedures allow the analysis of the specific methylated gene/s in a single or multiplex fashion. These procedures are based on quantitative real-time (qRT) MSP (methylation-specific polymerase chain reaction (PCR)) which is able to differentiate methylated from unmethylated cytosines in DNA by a bisulfite treatment and allele-specific primers. Because qRT-PCR technologies are well adopted in clinical diagnostics laboratories, MSP-based procedures and their required equipment are very reliable technologies to be integrated into clinical laboratories. In addition, pyrosequencing is another available methodology to be adopted in clinical laboratories. As described in Chapter 9, pyrosequencing is a quantitative sequence-based detection technology which is applicable, for example, in toxicity tests to investigate the modification of DNA methylation levels upon exposure to chemicals. In this regard, a number of assays for CpG island analysis are available that have proven to be highly reproducible and highly sensitive in clinical practice.

As we described in the previous section, PTMs have also led to a revolution in clinical diagnostics. As an alternative to traditional antibody-based methods in order to increase sensitivity and reproducibility, MS methods have emerged as a powerful analytical tool to identify PTMs. In Section II, Bonaldi and her team describe different approaches to analyze PTMs by MS, offering a perspective on how this technology and its methods can be integrated into clinical practice (Chapter 10).

5. Epigenetic Biomarkers: An Overview of Recent Advances

Recent technical advances have contributed by making epigenome mapping increasingly cost-efficient and less unmanageable. Therefore, research on disease-specific biomarkers will continue over the following years. Furthermore, the development of bioinformatic tools, which will increase the efficiency of the analysis of data generated in epigenetic research and epigenome-wide association studies (EWAS), will help to accomplish epigenetic biomarker development projects [90,91]. Exciting advances are rapidly occurring in this field, providing new biomarkers for diagnostics, prognostics, and clinical monitoring for several diseases.

In the field of DNA methylation, diverse methylation-derived chemical modifications have recently been discovered, such as 5hmC, 5fC in mouse stem cells (ES) and brain cortex, and 5caC in mice ES, [19]. The discovery of these kinds of new chemical modifications reveals the necessity for further investigations to elucidate their role in cell physiology and gene regulation, so as to provide new epigenetic biomarkers. Emerging innovative methylation and hydroxymethylation detection strategies are focused on addressing the validation of DNA methylation-based biomarkers in order to provide potential applications for these kinds of biomarkers in diverse clinical settings [92].

Histone modifications as well as the enzymes that catalyze them have been explored for their potential to be used as biomarkers and also as therapeutic targets and biomarkers in cancer [93] and other diseases. However, some questions regarding histone PTMs should be raised by researchers, as pointed out by Boukaba A. et al. in Chapter 3. The first question is why so many PTMs? Are they truly PTMs or just histone chemical moieties of adducts that are catalyzed by distinct enzymatic reactions? Although not real epigenetic PTMs introduced by histone modifiers, some of them could set the basis for the discovery of new PTMs involved in pathological processes, thus serving as biomarkers. In the advent of new MS procedures and recent achievements of MS-based proteomics for qualitative and quantitative characterization of histone PTMs and histone variants, MS has been converted into a well-established method in epigenetics research [94].

On the other hand, it has been shown that miRNAs of cancer cells modulate the microenvironment via noncell-autonomous mechanisms such as angiogenesis, tumor immune invasion, and tumor–stromal interaction, favoring the acquisition of hallmark cancer traits in neighboring cells to initiate cancer progression [95]. The understanding of these molecular processes may yield novel solutions to treat cancer tissues and design better therapeutic responses through the identification of new miRNA-based targets or their use in treatment monitoring. New technologies have already contributed to our understanding of miRNA-based mechanisms not only in cancer but also in the central nervous system and brain processing, including learning, memory, and cognition, setting the basis for the study of incurable neurological disorders [96]. Moreover, miRNAs are also known as major constituents of exosomes, extracellular vesicles that are proposed to transmit signals from cell to cell. In this regard, knowing the function of specific miRNAs and in which particles can they be found in biological fluids can contribute to improving the knowledge of ncRNAs for better biomarker design.

Section III of this volume presents a number of epigenetic biomarkers of cancer (i.e., lung cancer, prostate cancer, and breast cancer), metabolic syndrome, infertility, pregnancy complications, allergy and respiratory diseases, and also neurodegenerative disorders. This section is focused on the description of DNA methylation, histone PTMs, and ncRNA-based biomarkers, thus offering the most reliable biomarkers to be used in clinical practice and serve as a useful manual for clinicians and biomedical scientists. In some chapters, a number of epigenetic biomarkers with clinical value and that are used in clinical diagnostics are mentioned. In other cases, although some of the epigenetic biomarkers discussed are not used in clinical practice yet, they show promising diagnostic value and are candidates to be adopted promptly into the clinical setting.

6. Epigenetics: Perspective of Implantation in Clinical Laboratories

The use of epigenetic biomarkers in molecular diagnostics