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EPIGENETIC
BIOMARKERS
AND DIAGNOSTICS
Edited by
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
AMSTERDAM • BOSTON • HEIDELBERG • LONDON

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List of Contributors
Carolina Abril-Tormo IBSP-CV Biobank, FISABIO, Abdelhalim Boukaba Drug Discovery Pipeline,
Valencia, Spain Guangzhou Institutes of Biomedicine and Health,
Chinese Academy of Science, Guangzhou, People’s
Sahar Al-Mahdawi Department of Life Sciences,
Republic of China
College of Health & Life Sciences, Brunel Uni-
versity London, Uxbridge, UK; Synthetic Biology Eoin Brennan Diabetic Complications Division,
Theme, Institute of Environment, Health & Societ- Baker IDI Heart and Diabetes Institute, Melbourne,
ies, Brunel University London, Uxbridge, UK VIC, Australia
Diogo Almeida-Rios Cancer Biology and Epi- Enrique J. Busó Unidad Central de Investigación,
genetics Group – Research Center, Portuguese On- University of Valencia, Valencia, Spain
cology Institute, Porto, Portugal; Department of F. Javier Carmona Sanz Human Oncology &
Pathology, Portuguese Oncology Institute, Porto, Pathogenesis Program (HOPP), Memorial Sloan-
Portugal Kettering Cancer Center (MSKCC), New York, NY,
Sara Anjomani Virmouni Department of Life Sci- USA; Cancer Epigenetics and Biology Program
ences, College of Health & Life Sciences, Brunel (PEBC), Bellvitge Institute for Biomedical Research
University London, Uxbridge, UK; Synthetic Biol- (IDIBELL), Barcelona, Spain
ogy Theme, Institute of Environment, Health & Raimundo Cervera Hematology and Oncology
Societies, Brunel University London, Uxbridge, Unit, Biomedical Research Institute INCLIVA, Va-
UK lencia, Spain
Juan Ausio Department of Biochemistry and Mi- Alfredo Ciccodicola Institute of Genetics and Bio-
crobiology, University of Victoria, Victoria, BC, physics “Adriano Buzzati-Traverso”, CNR, Naples,
Canada Italy; Department of Science and Technology, Uni-
Bharati Bapat Lunenfeld Tanenbaum Research versity Parthenope of Naples, Italy
Institute, Mount Sinai Hospital, Toronto, ON, Joan Climent Hematology and Oncology Unit,
Canada; Department of Laboratory Medicine and Biomedical Research Institute INCLIVA, Valencia,
Pathobiology, University of Toronto, Toronto, ON, Spain
Canada; Department of Pathology, University Valerio Costa Institute of Genetics and Biophysics
Health Network, Toronto, ON, Canada “Adriano Buzzati-Traverso”, CNR, Naples, Italy
Charlotte L. Bevan Department of Surgery & Ana B. Crujeiras Laboratory of Molecular and Cel-
Cancer, Imperial Centre for Translational & Experi- lular Endocrinology, Instituto de Investigación San-
mental Medicine, Imperial College London, Ham- itaria (IDIS), Complejo Hospitalario Universitario
mersmith Hospital Campus, London, UK de Santiago (CHUS) and Santiago de Compostela
Jenefer M. Blackwell Telethon Kids Institute, The University (USC), Santiago de Compostela, Spain;
University of Western Australia, Subiaco, WA, Aus- CIBER Fisiopatología de la Obesidad y la Nutrición
tralia (CIBERobn), Madrid, Spain
Tiziana Bonaldi Department of Experimental On- Alessandro Cuomo Department of Experimental
cology, European Institute of Oncology, Milano, Oncology, European Institute of Oncology, Milano,
Italy Italy
xi
xii List of Contributors
Avery DeVries Department of Cellular and Molecu- Rosalba Giugno Department of Clinical and Experi-
lar Medicine, Arizona Respiratory Center and Ari- mental Medicine, University of Catania, Catania, Italy
zona Center for the Biology of Complex Diseases, Catherine Godson Conway Institute, Diabetes
University of Arizona, Tucson, AZ, USA Complications Research Centre, University College
Angel Diaz-Lagares Cancer Epigenetics and Biol- Dublin, Dublin, Ireland
ogy Program (PEBC), Bellvitge Biomedical Research Inês Graça Cancer Biology and Epigenetics Group
Institute (IDIBELL), Barcelona, Spain – Research Center, Portuguese Oncology Institute,
Roberta Esposito Institute of Genetics and Biophys- Porto, Portugal; School of Allied Health Sciences
ics “Adriano Buzzati-Traverso”, CNR, Naples, Italy (ESTSP), Polytechnic of Porto, Porto, Portugal
Alessandro Fatica Department of Biology and Bio- Kirsten Grønbæk Department of Hematology, Rig-
technology Charles Darwin, Sapienza University shospitalet, Copenhagen, Denmark
of Rome, Rome, Italy Shinji Hagiwara Diabetic Complications Division,
Alfredo Ferro Department of Clinical and Experi- Baker IDI Heart and Diabetes Institute, Melbourne,
mental Medicine, University of Catania, Catania, VIC, Australia
Italy Rui Henrique Cancer Biology & Epigenetics Group,
Claire E. Fletcher Department of Surgery & Cancer, IPO-Porto Research Center (CI-IPOP), Portuguese
Imperial Centre for Translational & Experimental Oncology Institute, Porto, Portugal; Department of
Medicine, Imperial College London, Hammer- Pathology, Portuguese Oncology Institute, Porto,
smith Hospital Campus, London, UK Portugal; Department of Pathology and Molecular
Ana-Maria Florea Department of Neuropathology, Immunology, Institute of Biomedical Sciences Abel
Heinrich-Heine-University Düsseldorf, Dussel- Salazar – University of Porto (ICBAS-UP), Porto,
dorf, Germany Portugal
Ernest Fraenkel Department of Biological Engi- José Santiago Ibañez Cabellos Center for Biomedi-
neering, Massachusetts Institute of Technology, cal Network Research on Rare Diseases, Medicine
Cambridge, MA, USA and Dentistry School, University of Valencia, Va-
lencia, Spain; Biomedical Research Institute INC-
Yu Fujita Division of Molecular and Cellular Medi-
LIVA, Valencia, Spain
cine, National Cancer Center Research Institute,
Tokyo, Japan Marisa Iborra Gastroenterology Department, Hos-
pital Universitari i Politècnic La Fe, Valencia, Spain
Tomohiro Fujiwara Department of Orthopaedic
Surgery, Okayama University Graduate School of Toyotaka Ishibashi Division of Life Science, Hong
Medicine, Dentistry, and Pharmaceutical Sciences, Kong University of Science and Technology, Kow-
Okayama, Japan; Center for Innovative Clinical loon, Hong Kong, HKSAR; Department of Biomed-
Medicine, Okayama University Hospital, Okaya- ical Engineer, Hong Kong University of Science
ma, Japan; Division of Molecular and Cellular and Technology, Kowloon, Hong Kong, HKSAR
Medicine, National Cancer Center Research Insti- Sarra E. Jamieson Telethon Kids Institute, The Uni-
tute, Tokyo, Japan versity of Western Australia, Subiaco, WA, Australia
Miriam Gagliardi Institute of Genetics and Bio- Carmen Jerónimo Cancer Biology & Epigenetics
physics “Adriano Buzzati-Traverso”, CNR, Naples, Group, IPO-Porto Research Center (CI-IPOP),
Italy Portuguese Oncology Institute, Porto, Portugal;
José Luis García-Giménez Center for Biomedical Department of Pathology and Molecular Immunol-
Network Research on Rare Diseases (CIBERER), ogy, Institute of Biomedical Sciences Abel Salazar
National Institute of Health Carlos IIII, Spain; – University of Porto (ICBAS-UP), Porto, Portugal
Department of Physiology, Medicine and Dentistry Sadhana Joshi Department of Nutritional Medi-
School, University of Valencia, Valencia, Spain; Bio- cine, Interactive Research School for Health Affairs,
medical Research Institute INCLIVA, University of Bharati Vidyapeeth University, Pune, Maharashtra,
Valencia, Valencia, Spain India
List of Contributors xiii
Phillip Kantharidis Diabetic Complications Divi- Takahiro Ochiya Division of Molecular and Cel-
sion, Baker IDI Heart and Diabetes Institute, Mel- lular Medicine, National Cancer Center Research
bourne, VIC, Australia Institute, Tokyo, Japan
Akira Kawai Department of Musculoskeletal On- Toshifumi Ozaki Department of Orthopaedic Sur-
cology, National Cancer Center Hospital, Tokyo, gery, Okayama University Graduate School of
Japan Medicine, Dentistry, and Pharmaceutical Sciences,
Vinita Khot Department of Nutritional Medicine, Okayama, Japan
Interactive Research School for Health Affairs, Federico V. Pallardó Center for Biomedical Net-
Bharati Vidyapeeth University, Pune, Maharashtra, work Research on Rare Diseases, Medicine and
India Dentistry School, University of Valencia, Valencia,
Lasse Sommer Kristensen Department of Hematol- Spain; Biomedical Research Institute INCLIVA, Va-
ogy, Rigshospitalet, Copenhagen, Denmark lencia, Spain
Ana Lluch Hematology and Oncology Unit, Bio- Lorena Peiró-Chova INCLIVA Biobank, INCLIVA
medical Research Institute INCLIVA, Valencia, Biomedical Research Institute, Valencia, Spain
Spain Marco Pellegrini Laboratory of Integrative Sys-
José Antonio López-Guerrero Laboratory of Mo- tems Medicine (LISM), Institute of Informatics and
lecular Biology and Biobank, Fundacion Instituto Telematics (IIT) and Institute of Clinical Physiology
Valenciano de Oncologia, Valencia, Spain (IFC), National Research Council (CNR), Pisa, Italy
Paula Lopez-Serra Epigenetic and Cancer Biology Tandy L.D. Petrov Department of Biology, The Uni-
Program (PEBC), Bellvitge Biomedical Research In- versity of Alabama at Birmingham, Birmingham,
stitute (IDIBELL), Barcelona, Spain AL, USA
Annita Louloupi Division of Molecular Pathology, Olga Bahamonde Ponce INCLIVA Biobank, INCLI-
The Netherlands Cancer Institute, Amsterdam, The VA Biomedical Research Institute, Valencia, Spain
Netherlands Mark A. Pook Department of Life Sciences, College
Luca Magnani Department of Surgery and Cancer of Health & Life Sciences, Brunel University Lon-
Imperial Centre for Translational and Experimen- don, Uxbridge, UK; Synthetic Biology Theme, In-
tal Medicine, Imperial College Hammersmith, Lon- stitute of Environment, Health & Societies, Brunel
don, UK University London, Uxbridge, UK
Jacobo Martínez-Santamaría IBSP-CV Biobank, Alfredo Pulvirenti Department of Clinical and Ex-
FISABIO, Valencia, Spain; Valencian Biobank Net- perimental Medicine, University of Catania, Cata-
work, FISABIO, Valencia, Spain nia, Italy
Maria R. Matarazzo Institute of Genetics and Bio- João Ramalho-Carvalho Cancer Biology & Epi-
physics “Adriano Buzzati-Traverso”, CNR, Naples, genetics Group, IPO-Porto Research Center
Italy (CI-IPOP), Portuguese Oncology Institute, Porto,
Aaron McClelland Diabetic Complications Divi- Portugal
sion, Baker IDI Heart and Diabetes Institute, Mel- Alberto Ramos Hematology and Oncology Unit,
bourne, VIC, Australia Biomedical Research Institute INCLIVA, Valencia,
Pamela Milani Department of Biological Engineer- Spain
ing, Massachusetts Institute of Technology, Cam- George Rasti Chromatin Biology Laboratory, Cancer
bridge, MA, USA Epigenetics and Biology Program (PEBC), Bellvitge
Yutaka Nezu Division of Molecular and Cellular Biomedical Research Institute (IDIBELL), Barcelona,
Medicine, National Cancer Center Research Insti- Spain
tute, Tokyo, Japan Nicole C. Riddle Department of Biology, The Uni-
Roberta Noberini Center of Genomic Science, Isti- versity of Alabama at Birmingham, Birmingham,
tuto Italiano di Tecnologia, Milano, Italy AL, USA
xiv List of Contributors
Peter H.J. Riegman Department of Pathology, Deepali Sundrani Department of Nutritional Medi-
Erasmus Medical Center, Rotterdam, The Neth- cine, Interactive Research School for Health Affairs,
erlands Bharati Vidyapeeth University, Pune, Maharashtra,
Carlos Romá Mateo Center for Biomedical Net- India
work Research on Rare Diseases, Medicine and Genevieve Syn Telethon Kids Institute, The Univer-
Dentistry School, University of Valencia, Valencia, sity of Western Australia, Subiaco, WA, Australia
Spain; Biomedical Research Institute INCLIVA, Va- Trygve O. Tollefsbol Comprehensive Cancer Cen-
lencia, Spain ter, Center for Aging, Comprehensive Diabetes
Francesco Russo Laboratory of Integrative Sys- Center, Nutrition Obesity Research Center, Cell Se-
tems Medicine (LISM), Institute of Informatics and nescence Culture Facility, University of Alabama at
Telematics (IIT) and Institute of Clinical Physiol- Birmingham, Birmingham, AL, USA
ogy (IFC), National Research Council (CNR), Pisa, Eneda Toska Human Oncology & Pathogenesis
Italy; Department of Computer Science, University Program (HOPP), Memorial Sloan-Kettering Can-
of Pisa, Pisa, Italy cer Center (MSKCC), New York, NY, USA
Fabian Sanchis-Gomar Department of Physiology, Marianne B. Treppendahl Department of Hematol-
Medicine and Dentistry School, University of Va- ogy, Rigshospitalet, Copenhagen, Denmark
lencia, Valencia, Spain; Biomedical Research Insti-
Toshikazu Ushijima Chief of Division of Epig-
tute INCLIVA, University of Valencia, Valencia,
enomics, National Cancer Center Research Insti-
Spain
tute, Tokyo, Japan
Juan Sandoval Epigenetic and Cancer Biology Pro-
Alejandro Vaquero Chromatin Biology Laboratory,
gram (PEBC), Bellvitge Biomedical Research Insti-
Cancer Epigenetics and Biology Program (PEBC),
tute (IDIBELL), Barcelona, Spain
Bellvitge Biomedical Research Institute (IDIBELL),
Flavia Scoyni Department of Biology and Biotech- Barcelona, Spain
nology Charles Darwin, Sapienza University of
Donata Vercelli Department of Cellular and Mo-
Rome, Rome, Italy
lecular Medicine, Arizona Respiratory Center and
Marta Seco Cervera Center for Biomedical Network Arizona Center for the Biology of Complex Diseas-
Research on Rare Diseases, Medicine and Dentistry es, University of Arizona, Tucson, AZ, USA
School, University of Valencia, Valencia, Spain;
Filipa Quintela Vieira Cancer Biology and Epi-
Biomedical Research Institute INCLIVA, Valencia,
genetics Group – Research Center, Portuguese
Spain
Oncology Institute, Porto, Portugal; School of Al-
Akifumi Shibakawa Department of Surgery & lied Health Sciences (ESTSP), Polytechnic of Porto,
Cancer, Imperial Centre for Translational & Experi- Porto, Portugal
mental Medicine, Imperial College London, Ham-
Yinan Zhang Division of Life Science, Hong Kong
mersmith Hospital Campus, London, UK
University of Science and Technology, Kowloon,
Nicolas G. Simonet Chromatin Biology Laboratory, Hong Kong, HKSAR
Cancer Epigenetics and Biology Program (PEBC),
Fang Zhao Lunenfeld Tanenbaum Research Insti-
Bellvitge Biomedical Research Institute (IDIBELL),
tute, Mount Sinai Hospital, Toronto, ON, Canada;
Barcelona, Spain
Department of Laboratory Medicine and Pathobi-
Ailsa Sita-Lumsden Department of Surgery & Can- ology, University of Toronto, Toronto, ON, Canada
cer, Imperial Centre for Translational & Experimen-
Wilbert Zwart Division of Molecular Pathology,
tal Medicine, Imperial College London, Hammer-
The Netherlands Cancer Institute, Amsterdam, The
smith Hospital Campus, London, UK
Netherlands
Olafur Andri Stefansson Cancer Research Labo-
ratory, Faculty of Medicine, University of Iceland,
Reykjavik, Iceland
Preface
Epigenetics is an emerging frontier of biology, as well as advanced technologies and tools

and its definition is continuously being adapted for their analysis. In this regard, epigenetic
based on new scientific findings. In fact, the biomarkers provide clinicians valuable infor-
NIH Roadmap Epigenomics Project has recently mation about the presence or absence of a dis-
defined epigenetics as “the heritable changes in ease (diagnostic value), the patient prognosis
gene activity and expression (in the progeny of (prognostic value), the response to a spe-
cells or of individuals) and also stable, long-term cific treatment (predictive value), the effects
alterations in the transcriptional potential of a of ongoing treatment ( therapy-monitoring
cell that are not necessarily heritable.” In this biomarkers), and the future risk of disease
regard, epigenetics includes DNA methylation, development (risk prediction). Furthermore,
noncoding RNAs, and histone posttranslational several advantages may arise from the use
modifications. This integrative definition of epi- of epigenetic biomarkers versus gene expres-
genetics reflects the potential of the discipline to sion in clinical practice, such as higher stabil-
expand beyond the control of a particular gene ity, for example, in biofluids. They can also fill
expression program for each cell type, defining clinical gaps by bridging genetic information,
the cellular and developmental identity and mRNA transcription, and protein translation.
function of cells, and, finally, translating this In consequence, the associations between
potential to health and disease conditions in epigenome alterations and diseases become
human beings. clearer, providing a way to act directly on
Due to the rapid progress in the field of gene expression by developing specific drugs
epigenetics, new and promising m ethodologies or even by adopting healthy lifestyles.
to advance biomedical research are being Many methodologies, including classical
developed. Epigenetic research and epigenetic methods and next-generation-sequencing-based
pharmaceutical drug development are now con- technologies, are available to clinicians and
sidered areas of great interest and promise in researchers to identify new epigenetic biomark-
the biomedical scene. The advantage of human ers and analyze them from several biological
epigenetics compared with human genetics and sources. In this context, genome-wide meth-
genomics is that it provides vital information ylation analysis, chromatin immunoprecipita-
about gene function in individual cell types, tion coupled with high-throughput platforms,
while incorporating information from the envi- and noncoding RNA sequencing are described
ronment and lifestyle, and unlike most genetic in this volume. Furthermore, some techniques
defects causative of human disease, epigenetic for DNA methylation analysis are more likely
alterations are modulable and reversible. The to be rapidly adopted in clinical laboratories,
volume Epigenetic Biomarkers and Diagnostics is such as EpiTYPER MassARRAY, methyl specific
intended to describe both epigenetic biomark- PCR (MSP), and pyrosequencing. On the other
ers that can be adopted into clinical routine hand, immunoassays are well established in
xv
xvi Preface
clinical laboratories for the analysis of histones corresponding fields for a broad target audience
(i.e., inflammatory and autoimmune diseases). such as advanced students, basic scientists, bio-
However, it is expected that the incorporation medical and biotechnological companies, as well
of mass spectrometry technologies into labo- as clinical researchers, clinicians (i.e., patholo-
ratories for clinical diagnostics (replacing rou- gists, immunologists, oncologists, endocrinolo-
tine immunoassays) will be the tendency in the gists, etc.) and analysts from clinical laboratories
coming years, as will be the analysis of histone who can adopt these potential biomarkers into
posttranslational modifications associated with clinical practice.
pathological states. In the coming years, epigenetics will continue
Although it is not possible to cover all epigenetic to provide an exciting future in biomedicine
markers, this volume includes chapters describ- and clinical practice. The chapters covered in
ing the most promising biomarkers for cancer (i.e., Epigenetic Biomarkers and Diagnostics highlight
breast, lung, colon, etc.), metabolic disorders (i.e., the unprecedented impact of epigenetics in
diabetes and obesity), autoimmune diseases, infer- clinical diagnostics and will contribute to the
tility, allergy, infectious diseases, and neurological discovery and development of new epigenetic
disorders; and, where possible, we will focus our biomarkers in the future.
attention on those which are feasible to be adopted
for clinical use. José Luis García-Giménez
This book was written in a comprehensive Valencia, Spain
manner by outstanding experts in their
C H A P T E R
1
Epigenetic Biomarkers: New Findings,
Perspectives, and Future Directions
in Diagnostics
José Luis García-Giménez1, Toshikazu Ushijima2,
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
O U T L I N E
1. Introduction 2 5. Epigenetic Biomarkers: An Overview

of Recent Advances 11
2. Epigenetic Mechanisms 3
2.1 DNA Methylation 3 6. Epigenetics: Perspective of Implantation
2.2 Histone PTMs and Histone Variants 5 in Clinical Laboratories 12
2.3 Noncoding RNAs 7
7. Perspectives of Epigenetics in Diagnostics 14
3. Epigenetic Biomarkers and In Vitro
List of Abbreviations 15
Diagnostics8
References15
4. Epigenetic Biomarkers and the Clinical
Laboratory10
Epigenetic Biomarkers and Diagnostics

http://dx.doi.org/10.1016/B978-0-12-801899-6.00001-2 1 Copyright © 2016 Elsevier Inc. All rights reserved.
2 1. EPIGENETIC BIOMARKERS AND DIAGNOSTICS
1. INTRODUCTION a chromosome without alterations in the DNA

sequence” [6]. It is evident that epigenetics is
The literal meaning of the term epigenetic is an emerging frontier of science, so its defini-
“above or on top of genetics,” although it has tion is continuously being adapted based on
had many different definitions over the years. new scientific findings. In fact, the NIH Road-
Conrad Hal Waddington was the first to define map Epigenomics Project defines epigenetics
epigenetics, in 1942, as “the branch of biology as “the heritable changes in gene activity and
which studies the causal interaction between expression (in the progeny of cells or of indi-
genes and their products, which bring the phe- viduals) and also stable, long-term alterations
notype into being” [1]. In 1990, Holiday defined in the transcriptional potential of a cell that are
epigenetics as “the study of the mechanisms not necessarily heritable” [7] (www.roadma-
of temporal and spatial control of gene func- pepigenomics.org). In this regard, epigenetics
tion during the development of organisms” [2]. includes DNA methylation, noncoding RNAs
A few years later, epigenetics was defined in a (ncRNAs), and histone posttranslational modifi-
narrower manner, as the different epigenetic cations (PTMs). This last integrative definition of
modifications or mechanisms that produce heri- epigenetics reflects the potential of epigenetics
table changes affecting gene expression without to go beyond the control of a particular gene
affecting the DNA sequence. In 2001, Jenuwein expression to produce a unique gene expression
and Allis proposed the histone code as an epi- program of each cell type, defining the cellular
genetic mechanism which is a critical feature of a and developmental identity [8], as well as poten-
genome-wide mechanism of information storage tial health and disease outcomes [9].
and retrieval and that considerably expands the Our epigenome is characterized by its abil-
information potential of the genetic code. There- ity to dynamically respond to intra- and extra-
fore, they pointed out that epigenetics imparts cellular stimuli, so that epigenetic changes are
a fundamental regulatory system beyond the reversible and in consequence are potential con-
sequence information of our genetic code [3]. By tributors to health and disease. Recent progress
2007, the definition of epigenetics had changed in the field of epigenetics opens promising ways
yet again, when Bird defined epigenetics as “the to advance biomedical research. Epigenetic
structural adaptation of chromosomal regions so research and epigenetic pharmaceutical drug
as to register, signal or perpetuate altered activ- development are now considered a bright spot
ity states” [4]. The same year, Goldberg, Allis, in the biomedical research field. This research
and Bernstein defined epigenetics as “the study will contribute to biomarker discovery, new
of any potentially stable and, ideally, heritable therapy development, computational and bioin-
change in gene expression or cellular phenotype formatics training, and the development of new
that occurs without changes in Watson-Crick next-generation sequencing (NGS) technologies
base pairing of DNA” [5]. It is evident that both and applications. The advantage of epigenetics
definitions proposed by Bird and Goldberg et al. compared to genetics is that it provides vital
were inclusive of transient chemical modifica- information about gene function in individual
tions of DNA and histones; modifications that cell types and incorporates information from
have not always been universally accepted the environment. Furthermore, several advan-
and that are still a subject of debate. Therefore, tages may arise from the use of epigenetic bio-
in 2008, a consensus definition was made at a markers versus gene expression (by measuring
Cold Spring Harbor meeting, giving a more mRNAs). Epigenetic biomarkers have shown
integrative definition for epigenetics as a “stably higher stability in fluids and formalin-fixed par-
heritable phenotype resulting from changes in affin-embedded (FFPE) biospecimens compared
to mRNAs. In addition, epigenetic biomarkers 5′ position of the cytosine base (5-methylcytosine
can benefit biomedical research and fill clinical (5mC)), which protrudes into the major groove
research gaps by bridging genetic information of DNA, representing a potential recognition
and mRNA expression. In consequence, this site for protein binding without changing the
makes the association between epigenome alter- Watson–Crick base pairing. The methyl group at
ations and diseases clearer, and also provides a the 5′ position of cytosine is donated by S-adeno-
way to act directly on them by developing spe- sylmethionine (SAM) via Dnmt1, Dnmt3A, and
cific drugs or adopting healthy lifestyles. One of Dnmt3B (DNA(cytosine-5-)methyltransferases
the most important issues is that the associations (DNMTs)). Importantly, CpG sites are under-
that have been found between epigenetics and represented and unevenly distributed across
certain diseases will have a synergistic effect on the human genome, giving rise to vast low-
the development of personalized medicine. In density CpG regions interspersed with CpG
recent years, epigenetics has aroused the inter- clusters located mainly in CpG islands [10].
est of different biomedical and scientific fields, Generally speaking, 5mCs play essential roles
and it also has impacted society. In 2010, TIME in maintaining cellular function and genome
Magazine published “Why Your DNA Isn’t Your stability.
Destiny” opening the public’s eyes to the latest In cancers, global DNA hypomethylation and
research and changing the general view about regional hypermethylation are almost always
DNA and its direct role in our lives. observed and have been associated with genome
instability, altered chromatin conformation, and
chromosome fragility [11–13]. At the same time,
2. EPIGENETIC MECHANISMS hypermethylation of CpG islands [14] and their
flanking regions, called CpG shores [15], is also
Three major events are mainly involved in observed in specific CpG islands, including those
epigenetic regulation and chromatin structure in promoters of tumor suppressor genes (driver
control: DNA methylation, histone PTMs, and methylation) and other genes methylated in
ncRNAs (i.e., microRNAs (miRNAs), long non- association with cancer development (passenger
coding RNAs (lncRNAs)). Disruption of one or methylation). Importantly, DNA methylation
more of these epigenetic mechanisms can lead can be detected by a wide range of sensitive and
to inappropriate expression of genes, resulting cost-effective techniques [16,17], as described
in an alterated state of cell homeostasis or dis- in Chapters 6–9 of Section II in this volume.
ease. In this regard, epigenetic-based biomark- Moreover, DNA methylation is a stable chemi-
ers are an important new research area. With the cal mark that is not easily altered, which makes
potent technologies now available, diagnostic it a feasible biomarker for diagnostics, prog-
tools can be created to analyze these biomarkers nostics, and treatment monitoring. The field of
and therefore contribute to the study of human DNA methylation has expanded with the iden-
diseases. Here, we summarize the three most tification of multiple cytosine variants thanks to
relevant mechanisms and discuss the technolo- the design of new methodologies. Ten-eleven
gies available to analyze them. translocation (Tet) family of cytosine oxygenase
enzymes (TETs) are responsible for oxidizing
5mCs into 5-hydroxymethylcytosine (5hmC),
2.1 DNA Methylation 5-formylcytosine (5fC), and 5-carboxylcytosine
DNA methylation is the most-studied DNA (5caC) by means of an α-ketoglutarate- and
modification since its discovery in the late 1940s. O2-Fe(II)-dependent reactions (Figure 1). Recent
It consists of the addition of a methyl group at the evidence reveals that these DNA demethylation
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-adeno-
sylmethionine 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 mod-
ifications 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.
intermediates are dynamic and participate in procedures in biomedicine and clinical research.
key regulatory functions in distal gene regula- In this regard, high-throughput methods for ana-
tory elements in mammalian genomes and also lyzing cytosine variants have appeared in recent
in biological processes, such as demethylation in years. Bisulfite sequencing (BS-seq), Infinium
zygote formation and in germ cell lineage (for a HumanMethylation 450K BeadChip (Illumina),
review see Ref. [18]). and immunoprecipitation of methylated DNA
Novel experimental designs consisting of followed by sequencing (MeDIP-seq) have been
chemical oxidation or chemical reduction of the developed to analyze 5mC. On the other hand,
modified cytosines allow the identification of Tet-assisted bisulfite sequencing (TAB-seq), 5hmC
5mC, 5hmC, 5fC, and 5caC at single-base reso- immunoprecipitation coupled to sequencing,
lution [19–21] and, when implemented in DNA oxidative bisulfite sequencing (oxBS-seq), and
sequencing protocols, will allow the analysis of oxidative bisulfite hybridization in the Infinium
whole genomes and the exploitation of these 450K BeadChip (oxBS-450K) have been designed
to map 5mC and 5hmC residues at single-base The hypothesis of the histone code proposes that
resolution on a genome-wide scale [22]. Using the control of the chromatin state is exerted by
named methylation-assisted bisulfite sequencing the PTMs [30]. Evidence accumulated in recent
(MAB-seq), the quantitation of the 5fC and 5caC years supports the idea that the PTM signature
residues at single-base resolution is possible [23], is altered in a wide range of diseases, such as
and using chemical modification-assisted bisulfite cancer [31,32], neurological syndromes [33,34],
sequencing (CAB-seq) the specific whole-genome and rare diseases such as Rubinstein–Taybi syn-
analysis of 5caC is possible [24]. By using reduced drome [35] and Cofin–Lowry syndrome [36].
bisulfite sequencing (redBS-seq), it is possible to In addition, clinicians have become increas-
identify 5fC [25]. ingly interested in histone PTMs in recent years
As shown in Figure 1, these different directed because it is possible to analyze them in specific
assays have been developed to study the cyto- regulatory domains of genes to obtain valuable
sine variants which participate in the regulation information for the diagnostics of disease [17].
of DNA methylation. Therefore, these methodol- Another additional level in the supramolecu-
ogies will contribute to explaining the intriguing lar structural organization of chromatin involves
epigenetic regulation underlying human pathol- the participation of histone variants (Figure 2).
ogies [26]. It is obvious that the direct application Histone variants differ in their primary sequence
of these high-throughput procedures in clinical of amino acids [37]. Variants for all of the histone
routine seems to be so far from reality. However, protein families have been described and have
these techniques will contribute to the identifi- emerged as important elements involved in
cation of passenger methylation underlying sev- chromatin dynamics and organization, and have
eral human diseases and also to develop clinical also served as scaffolds for other proteins that
epigenetics. create silenced regions in chromosomes [38,39].
Therefore, the incorporation of histone variants
in specific domains is very important, and their
2.2 Histone PTMs and Histone Variants regulation plays a crucial role in cellular pro-
Chromatin is composed of repeating arrays of cesses such as differentiation, proliferation, and
nucleosomes which comprise the essential units nuclear reprogramming. For this reason, muta-
of organization in eukaryotic chromosomes. tions in specific histone variants are involved
Each nucleosome is formed by 145 bp of DNA in human disease [40] like cancer, as recently
wrapped around a histone octamer. Each histone reviewed by Vardabasso et al. [41]. Due to the
octamer consists of two copies each of canonical importance of histone variants in cell differen-
histone H4, H3, H2A, and H2B or their variants. tiation and reprogramming it is not surprising
Histone can be chemically modified and when that they are closely related to male infertility, as
it is, chromatin state and gene expression are described later in this volume by Ausió, Zhang,
considered to be modified as well. The histone and Ishibashi in Chapter 24.
PTMs produced (i.e., acetylation, methylation, Interestingly, histones can also be found in
phosphorylation, butyrylation, hydroxybutyry- biological fluids such as blood, serum, and
lation, crotonylation, citrullination, formylation, plasma. Therefore, they have been proposed as
glycosylation, O-GlcNAcylation [27], carbon- clinical biomarkers since their presence in body
ylation, parsylation [28], and glutathionylation fluids suggests tissue damage, inflammation,
[29]) mainly occur on amino acids in the N-terminal and cellular apoptosis [42]. Recent evidence
tail domains (Figure 2). These chemical modi- supports the idea that extracellular histones con-
fications change the nucleosome structure tribute to human disease. Circulating nucleo-
and spread to different regions of the genome. somes were recently associated with disease
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 his-
tone 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).
progression in patients with thrombotic micro- consensus on the levels of circulating histones
angiopathies [43]. Ekaney et al. detected higher and nucleosomes that discriminate patients
levels of histone H4 in septic patients compared from healthy subjects, due in part to the diver-
to patients with multiple organ failure without sity of procedures used in their determination.
infection or to patients with minor trauma [44]. Currently, different commercial kits are
Zeerleder et al. found increased nucleosome lev- available for the identification of circulating
els in systemic inflammation and septic shock nucleosomes and histones in blood samples of
[45,46] and studied how these levels correlated autoimmune disease patients, mainly in drug-
with the severity of the inflammatory response induced systemic lupus erythomatosus [47].
and mortality in children affected by meningo- Many of them are based on enzyme immunoas-
coccal sepsis [46]. Unfortunately, there exists no says [48]. Furthermore, immunohistochemistry
and immunofluorescence-based procedures can characteristics of miRNAs [17,55,56]. This could
be used to analyze histone PTMs. However, a be due to their short length, particular biogen-
trend toward the use of mass spectrometry (MS)- esis, and strong association with proteins, or
based methodologies is gaining interest in clini- membrane-bound vesicles, such as exosomes,
cal diagnostic laboratories. MS methodologies microvesicles, or apoptotic bodies, etc. Impor-
can be employed in the analysis of histone PTMs tantly, miRNA expression is frequently altered in
and histone variants. In this volume, Noberini cancer and has shown promise as a tissue-based,
et al. discuss the potential of MS-based technolo- circulating biomarker for cancer classification
gies in biomarker discovery and their applica- and prognostics [56]. An interesting pioneer
tions in clinical epigenetics, focusing on novel study on this was performed by Volinia et al.
techniques to dissect the histone code in clini- They identified many overexpressed miRNAs
cal samples (Chapter 10). Importantly, MS holds in several solid tumors by performing a large-
great promise for clinical epigenetics because its scale miRNome analysis and found specific
potential allows histone PTM combinations to miRNA signatures for each tumor type [57]. Of
be dissected. great clinical importance is that unique miRNA
expression patterns can distinguish tumors from
different anatomical locations, and also differ-
2.3 Noncoding RNAs entiate various tumor subtypes at a single ana-
Only a small percentage of the transcribed tomic locus in kidney cancer [58], breast cancer
genes encode proteins, so these genome regions [59], and papillary renal carcinoma [60], among
were described as “dark matter RNAs” [49]. others.
ncRNAs are considered active participants in lncRNAs are generally defined as transcripts
controlling a wide range of biological processes, longer than 200 nucleotides that can be pro-
such as the regulation transcription of single cessed like mRNA, i.e., spliced and polyadenyl-
genes, as well as entire transcriptional programs ated [61]. These kinds of ncRNAs have been
[50]. Many thousands of regulatory nonpro- associated with neurological disorders [62], can-
tein-coding RNAs were demonstrated to be cer [63], and complex metabolic disorders [64].
transcribed in genome-wide studies, including There are currently a number of studies
miRNAs, small RNAs, PIWI-interacting RNAs, showing the interconnection between distinct
and various classes of lncRNAs [51]. Most clini- epigenetic events. For example, a subgroup
cal applications are being found for miRNAs of ncRNAs are additionally classified as epi-
and lncRNAs, so we will briefly describe these miRNAs because they can directly or indirectly
ncRNAs. Later chapters of this volume are regulate the expression of several components
focused on the ncRNA species and their poten- of the epigenetic machinery, such as DNA meth-
tial as clinical biomarkers. yltransferases or histone deacetylases, creating
miRNAs consist of a large family of short a very well-orchestrated mechanism [65,66] in
ncRNAs (17–25 nucleotides). These ncRNAs are such a way that the joint activities of different
involved in many biological processes [52] (i.e., epigenetic modifications result in a common
cellular development, differentiation, apoptosis, outcome. An altered balance of these processes
proliferation, tumor growth, metastatic dissemi- leads to pathological conditions, so it is impor-
nation, and resistance to therapy, among others) tant to evaluate the levels of ncRNAs (miRNAs
[53,54] as a consequence of their ability to con- and lncRNAs) in human biospecimens. High-
trol the expression (downregulation or upregu- throughput sequencing technologies, computa-
lation) of numerous genes. High stability and tional pipelines, and bioinformatics algorithms
low susceptibility to degradation are important allow us to identify the profile of dysregulated
ncRNAs in human diseases. In Chapter 11, diseases, infertility, and metabolic and neurode-
Costa et al. describe some of these approaches generative disorders. Some examples are shown
and tools for ncRNA analysis. In addition, Russo in section III of Epigenetic Biomarkers and Diag-
et al. (Chapter 12) discuss recent discoveries and nostics. Therefore, the identification of these
controversial topics on circulating ncRNAs and epigenetic changes serves to define healthy or
describe several public resources recently devel- disease states in humans and to set the basis for
oped for ncRNA analysis. In Table 1 the most the identification of epigenetic biomarkers.
relevant properties and information of miRNAs
and lncRNAs are summarized.
Epigenetic regulation does not always pro- 3. EPIGENETIC BIOMARKERS
duce gene regulation in a binary system that is AND IN VITRO DIAGNOSTICS
either ON or OFF. Epigenetic mechanisms can
be propagated over multiple cell divisions in A biomarker is a characteristic that is objec-
somatic cells. In addition, epigenetic information tively measured and evaluated as an indicator of
is modified during cellular differentiation and normal biologic processes, pathogenic processes,
partially erased in the germ line and the early or pharmacologic responses to a therapeutic
embryo [67]. We also know that lifestyle, environ- intervention. The United Nations World Health
ment, especially chronic inflammation, and nutri- Organization (WHO) defines a biomarker as
tion induce epigenetic changes during our life any substance, structure, or process that can be
[68–73] and that aberrant placement of epigenetic measured in the body or its products and which
marks and epigenetic dysregulation (produced influences or predicts the incidence of outcome
by mutations in genes that codify for epigenetic of diseases (Biomarkers in Risk Assessment: Valid-
machinery) are involved in disease. Thus, DNA ity and Validation, Environmental Health Criteria
methylation, histone PTMs, and ncRNAs play a Series, No. 222, WHO). In this regard, the goal
critical role in diseases such as cancer, infectious of clinical biomarkers is to provide clinicians
TABLE 1 General Information for Noncoding RNAs

Properties miRNAs lncRNAs
Length (nt) 17–25 >200
Poly-A tail No Yes
No. of sequences described in human 1881a 111685b
Found in Cell, tissues, body fluids/exosomes Cell, tissues, body fluids/exosomes
Stability in body fluids and FFPE High Low

biospecimens
Isolation RNA conserving miRNAs or miRNA RNA purification protocols

purification protocols
Procedures for the analysis RNA-seq, arrays, qRT-PCR RNA-seq, arrays, qRT-PCR
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).
miRNAs, microRNAs; lncRNAs, long noncoding RNAs; FFPE, formalin-fixed paraffin-embedded; qRT-PCR, quantitative real-time
polymerase chain reaction.
3. EPIGENETIC BIOMARKERS AND IN VITRO DIAGNOSTICS 9
with valuable information about the presence or changes based on disease evolution and intra- or
absence of a disease (diagnostic biomarker), the extraenvironmental cellular conditions while
patient prognosis (prognostic biomarker), the the latter, in contrast, is a static biomarker since
response to a specific treatment (predictive bio- our gene sequence generally does not change.
marker), the effects of ongoing treatment (ther- The potential of epigenetic biomarkers in
apy monitoring biomarkers), or a future risk of clinical practice is currently being demon-
disease development (risk markers) [74]. Based strated. The in vitro diagnostics (IVD) market
on these descriptions, recommended proper- is seeking new diagnostic and prognostic bio-
ties for clinical biomarkers are (1) they should markers which contribute to personalized medi-
be specific, sensitive, and stable; (2) they should cine, and epigenetics is currently contributing
be validated by different institutions in a large to this [77,78]. Recent advances in NGS have
number of samples followed by approval from allowed epigenetics to be used more easily both
the (US Food and Drug Administration) FDA by researchers and clinicians [79–81].
and/or (European Medicines Agency) EMA; and The exponential growth of the IVD market is
(3) although there exist excellent single markers evident at the moment, since the identification of
that serve for diagnostics, the use of biomarker biomarkers and the design of IVD tests facilitate
signatures instead of only one biomarker is pref- diagnostic, prognostic, and treatment monitor-
erable because the combination of biomarkers ing. Another point to consider is that the USFDA
increases the sensitivity and specificity [75]. encourages the integration of biomarkers into
One of the most important properties of epigen- drug development and their appropriate use in
etic marks is that they are highly stable (methyl- clinical practice. In that way, the effective inte-
ated DNA and miRNA) in multiple biospecimens gration of biomarkers into clinical development
(i.e., urine, blood, plasma, FFPE tissues, etc.). This programs may facilitate new medical product
is the case of miRNAs, which are very stable mol- development and promote personalized medi-
ecules in blood [56], urine [76], and also in FFPE cine [82,83]. In recent years, many epigenetic
[55], different from mRNA and proteins, as dis- drugs have been discovered, so the develop-
cussed by Peiró-Chova et al. in Chapter 2. ment of new, predictive, and sensitive biomark-
Based on these precedents, an epigenetic bio- ers for clinical practice is expected to reduce the
marker can be defined as “any epigenetic mark time and cost of drug development and also
or altered epigenetic mechanism (1) that can be contribute to overcoming problems during the
measured in the body fluids or tissues and (2) evaluation of new therapies during clinical trials
that defines a disease (detection), predicts the [84]. Therefore, the use of epigenetic biomarkers
outcome of diseases (prognostic) or response has tremendous potential to affect the success
to a therapy (predictive), monitors treatment rate of clinical trials and drug discovery. Proof
responses (therapy monitoring), or predicts risk of this is the growth observed in the number of
of future disease development (risk).” clinical trials using epigenetic drugs during last
In general, epigenetic biomarkers may repre- decade (Figure 3). In fact, epigenetic therapy
sent the effect of the environment and natural will be improved considerably after identifica-
history on the particular evolution of disease in tion of good pretreatment biomarkers predicting
each patient. Therefore, one of the most prom- response. Many large clinical trials in combina-
ising properties of epigenetic biomarkers is tion with novel high-throughput screening meth-
that they will contribute to the improvement of ods have contributed to these improvements, as
precision medicine. The advantage offered by described by Treppendahl et al. in Chapter 5.
epigenetic biomarkers versus genetic biomark- Specifically, epigenetic biomarkers are
ers is that the former is a dynamic one which coevolving and have reached a critical point
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 methyla-
tion analysis and chromatin immunoprecipita-
tion with high-throughput platforms, including
DNA microarrays and genome sequencing, have
permitted the performance of comprehensive
FIGURE 3 Number of clinical trials using epigenetic studies regarding the epigenetic bases of several
drugs and biomarkers reported by ClinicalTrials.gov from diseases. These technologies are well described
2000 to 2014. The figure presents the growth rate for clinical
trials in different statuses (i.e., not yet recruiting, recruiting,
by Toska and Carmona-Sanz in Chapter 6. NGS
enrolling by invitation, active but not recruiting, completed). has also revolutionized the analysis of miRNAs
The shape of the curve indicates a research area that is reach- and lncRNAs by allowing the application of
ing amount of interest in clinical practice. NGS-based methods for clinical epigenetics, as
described in Chapter 11 by Costa et al. A recent
wherein enough signatures have been identi- example of this application was published by
fied across various disease classes, most nota- Keller et al. In this study, the authors analyzed
bly in cancer, that some of these signatures and 863 miRNA signatures from 454 human blood
expression patterns can be validated and used biospecimens. The samples were obtained from
in the clinic [85,86]. The potential of epigenetic patients suffering from 14 different diseases,
biomarkers in clinical practice has directed them including lung cancer, pancreatic ductal ade-
to their use in IVD. In fact, the global epigenetics nocarcinoma, prostate cancer, ovarian cancer,
market was valued at an estimated $413.24 Wilms tumor, multiple sclerosis, periodontitis,
million in 2014 and it is expected to reach $783.17 sarcoidosis, myocardial infarction, and chronic
million in 2019. The IVD market is expected to obstructive pulmonary disease [87]. By utiliz-
experience an enormous expansion in the coming the data of dysregulated miRNAs in the
ing years as well, and epigenetics will clearly blood and developing mathematical algorithms,
contribute to that. Keller et al. were able to accurately predict dis-
ease in more than two-thirds of individuals
[87]. It is obvious that more ncRNAs are being
4. EPIGENETIC BIOMARKERS identified as research advances, so data inte-
AND THE CLINICAL LABORATORY gration in public databases such as the human
miRNA-associated disease database (HMDD)
Many methods, including classical methods [88] (www.cuilab.cn/hmdd) or miRandola for
and NGS-based methods, are available to clini- extracellular circulating miRNAs [89] serve as
cians and researchers to identify new epigenetic resources for researchers screening miRNA and
biomarkers, as reviewed by Petrove and Riddle in lncRNA profiles for a wide range of diseases.
Chapter 4. In recent years, technological improve- These databases are therefore fundamental tools
ments have allowed researchers to examine the for biomedical research as described by Russo
epigenome on a genome-wide fashion. et al. in Chapter 12.
5. Epigenetic Biomarkers: An Overview of Recent Advances 11
Since DNA methylation was the first epi- As we described in the previous section, PTMs
genetic mark to be widely studied, further have also led to a revolution in clinical diagnos-
progress has been made in the field of clinical tics. As an alternative to traditional antibody-
diagnostics. Some techniques for DNA meth- based methods in order to increase sensitivity
ylation analysis have the potential to be rap- and reproducibility, MS methods have emerged
idly well established in clinical laboratories. In as a powerful analytical tool to identify PTMs.
this regard, Buso and Iborra describe the use In Section II, Bonaldi and her team describe dif-
of Sequenom MassARRAY technology in the ferent approaches to analyze PTMs by MS, offer-
analysis of DNA methylation in Chapter 7. This ing a perspective on how this technology and its
MS-based method provides differences in DNA methods can be integrated into clinical practice
methylation at the individual CpG level. The (Chapter 10).
EpiTYPER MassARRAY system of Sequenom
is a candidate to be adopted in clinical labo-
ratories, although there exist some inconve- 5. EPIGENETIC BIOMARKERS: AN
niences to this technology, such as the fact that OVERVIEW OF RECENT ADVANCES
it requires expensive equipment and trained
technicians. Furthermore, other procedures Recent technical advances have contributed
that can be more rapidly adopted in clinical by making epigenome mapping increasingly
laboratories are described by Zhao and Bapat cost-efficient and less unmanageable. Therefore,
in Chapter 8. They describe MethyLight and research on disease-specific biomarkers will
multiplex MethyLight assays using TaqMan® continue over the following years. Furthermore,
fluorescent probes. These easy-to-use proce- the development of bioinformatic tools, which
dures allow the analysis of the specific meth- will increase the efficiency of the analysis of
ylated gene/s in a single or multiplex fashion. data generated in epigenetic research and epig-
These procedures are based on quantitative enome-wide association studies (EWAS), will
real-time (qRT) MSP (methylation-specific help to accomplish epigenetic biomarker devel-
polymerase chain reaction (PCR)) which is able opment projects [90,91]. Exciting advances are
to differentiate methylated from unmethylated rapidly occurring in this field, providing new
cytosines in DNA by a bisulfite treatment and biomarkers for diagnostics, prognostics, and
allele-specific primers. Because qRT-PCR tech- clinical monitoring for several diseases.
nologies are well adopted in clinical diagnos- In the field of DNA methylation, diverse
tics laboratories, MSP-based procedures and methylation-derived chemical modifications
their required equipment are very reliable tech- have recently been discovered, such as 5hmC,
nologies to be integrated into clinical labora- 5fC in mouse stem cells (ES) and brain cortex,
tories. In addition, pyrosequencing is another and 5caC in mice ES, [19]. The discovery of these
available methodology to be adopted in clinical kinds of new chemical modifications reveals the
laboratories. As described in Chapter 9, pyro- necessity for further investigations to elucidate
sequencing is a quantitative sequence-based their role in cell physiology and gene regulation,
detection technology which is applicable, for so as to provide new epigenetic biomarkers.
example, in toxicity tests to investigate the Emerging innovative methylation and hydroxy-
modification of DNA methylation levels upon methylation detection strategies are focused on
exposure to chemicals. In this regard, a number addressing the validation of DNA methylation-
of assays for CpG island analysis are available based biomarkers in order to provide potential
that have proven to be highly reproducible and applications for these kinds of biomarkers in
highly sensitive in clinical practice. diverse clinical settings [92].
Histone modifications as well as the enzymes Section III of this volume presents a number
that catalyze them have been explored for their of epigenetic biomarkers of cancer (i.e., lung
potential to be used as biomarkers and also as cancer, prostate cancer, and breast cancer), meta-
therapeutic targets and biomarkers in cancer [93] bolic syndrome, infertility, pregnancy compli-
and other diseases. However, some questions cations, allergy and respiratory diseases, and
regarding histone PTMs should be raised by also neurodegenerative disorders. This section
researchers, as pointed out by Boukaba A. et al. is focused on the description of DNA methyla-
in Chapter 3. The first question is why so many tion, histone PTMs, and ncRNA-based biomark-
PTMs? Are they truly PTMs or just histone ers, thus offering the most reliable biomarkers
chemical moieties of adducts that are catalyzed by to be used in clinical practice and serve as a use-
distinct enzymatic reactions? Although not real ful manual for clinicians and biomedical scien-
epigenetic PTMs introduced by histone modifiers, tists. In some chapters, a number of epigenetic
some of them could set the basis for the discovery biomarkers with clinical value and that are used
of new PTMs involved in pathological processes, in clinical diagnostics are mentioned. In other
thus serving as biomarkers. In the advent of new cases, although some of the epigenetic biomark-
MS procedures and recent achievements of MS- ers discussed are not used in clinical practice
based proteomics for qualitative and quantitative yet, they show promising diagnostic value and
characterization of histone PTMs and histone are candidates to be adopted promptly into the
variants, MS has been converted into a well- clinical setting.
established method in epigenetics research [94].
On the other hand, it has been shown that
miRNAs of cancer cells modulate the microen- 6. EPIGENETICS: PERSPECTIVE
vironment via noncell-autonomous mechanisms OF IMPLANTATION IN CLINICAL
such as angiogenesis, tumor immune invasion, LABORATORIES
and tumor–stromal interaction, favoring the
acquisition of hallmark cancer traits in neighbor- The use of epigenetic biomarkers in molecu-
ing cells to initiate cancer progression [95]. The lar diagnostics needs to reach important mile-
understanding of these molecular processes may stones. The knowledge of new methodologies
yield novel solutions to treat cancer tissues and and bioinformatic tools and the consideration of
design better therapeutic responses through the financial, regulatory, and bioethical aspects are
identification of new miRNA-based targets or important issues that must be taken into account
their use in treatment monitoring. New technolo- before the incorporation of epigenetic biomark-
gies have already contributed to our understand- ers into clinical practice [17]. For biomarker
ing of miRNA-based mechanisms not only in validation, it is necessary to perform clinical
cancer but also in the central nervous system and validation by enrolling eligible and clinically rel-
brain processing, including learning, memory, evant participants in a research study. For exam-
and cognition, setting the basis for the study of ple, a “detection” epigenetic biomarker should
incurable neurological disorders [96]. Moreover, have the power to differentiate between liver
miRNAs are also known as major constituents of cancer patients and liver cirrhosis patients, not
exosomes, extracellular vesicles that are proposed between liver cancer patients and age-matched
to transmit signals from cell to cell. In this regard, healthy volunteers.
knowing the function of specific miRNAs and in While there are a number of published
which particles can they be found in biological reports identifying strategies to recruit partici-
fluids can contribute to improving the knowl- pants for genetic research, there is scarce infor-
edge of ncRNAs for better biomarker design. mation regarding the recruitment strategy for
6. Epigenetics: Perspective of Implantation in Clinical Laboratories 13
epigenetic research [97]. In this regard, the ethical of new biomarkers by the medical community
conduct of epigenetic research in clinical trials is will improve success in diagnostic and also
extremely important. As we know, some epigen- therapeutic interventions by minimizing the
etic changes have an intergenerational influence secondary events produced by nonpersonal-
[98]. Indeed, it is of notable interest to reflect ized therapy. To introduce this concept to health
on ethics not only in epigenetic diagnostics but professionals we need specialists to educate
also in epigenetic clinical trials. In light of this, the health community about novel diagnostic
Jallo et al. discusses the strategy to be developed approaches based on epigenetics. (2) Increase
and implemented to enroll subjects for epigen- knowledge about new epigenetic-based biomarkers.
etic studies [97]. Following the performance of Direct communication with health profession-
epigenetic studies and once the epigenetic bio- als and medical societies will help change the
marker has been validated, biomedical research paradigm and adopt these epigenetic-based
is able to deliver a reliable and useful clinical biomarkers into clinical routine. Epigenetics is
biomarker. However, it is important to take into a relative young biomedical discipline. So many
account that clinical laboratories need easy-to- health professionals do not know the potential
use and low-cost technologies to analyze these of epigenetics and its clinical utility. So, meet-
biomarkers and prepare clinical reports [17], so ings, scientific journals, and clinical reviews and
it is evident that providing reliable epigenetic new edited volumes such as Epigenetic Biomark-
biomarkers is difficult and several obstacles may ers and Diagnostics may contribute to this task.
have to be solved along the way. Regarding the use of technologies required
To overcome the most important barriers for for epigenetic studies it is important to (3) intro-
the implementation of epigenetic biomarkers in duce new technologies into clinical laboratories. It
diagnostic laboratories, some issues regarding would be very useful to introduce NGS and MS
the clinical utility and clinical validity of epigen- spectrometry technologies into clinical laborato-
etic biomarkers and regulatory issues should be ries. NGS has transformed genomic medicine,
taken into account. In fact, to become a clinically because it has reduced the cost of large-scale
epigenetic biomarker approved by regulatory sequencing. By using NGS, it is possible to
agencies, the biomarker should be confirmed analyze an individual’s near-complete exome,
and validated, and it should be reproducible, genome, methylome, miRNome and also to ana-
specific, and sensitive. Therefore, it is of high lyze complete histone posttranslational maps to
importance to completely characterize the epi- assist in the diagnosis of a wide array of clini-
genetic biomarkers, first by analytical validation cal scenarios. For NGS, one of the most prom-
which ensures the consistency of the method, ising applications is based on the advances in
test, or technology used to measure the epigen- Nano-ChIP-Seq, which will allow the analysis
etic biomarker, and second to establish the clini- of specific chromatin regions from far fewer
cal validity which relates to the consistency and cells for embryology and development studies.
accuracy of the biomarker for diagnostics, pre- (4) Replace classical methods for other cost-effective
dicting the clinical outcome or monitoring the and time-effective technologies. For example, the
effect of a treatment. potential of MS-based assays instead of those
In addition, other issues require solving based on immunoassays for the analysis of his-
before using epigenetic biomarkers in clinical tone PTMs shows several advantages, including
routine, including changing the paradigm of quantification, high sensitivity, specificity and
clinical diagnostics based exclusively on genet- low cost, among others [99]. However, there is
ics. In this regard it is necessary to (1) change an associated problem consisting of the high cost
conventional diagnostic approaches. The adoption of this kind of technology and the requirement
of specialists in MS [99]. Importantly, it has been to align patient and clinician demands with the
estimated that the incorporation of MS in labora- decision-making of administrators. It is obvious
tories for clinical diagnostics (replacing routine that epigenetics has revolutionized biomedi-
immunoassays) would save about $250,000 per cine, and its potential applications in diagnostics
year in each tertiary hospital laboratory [100]. and disease treatment make it easy to be recog-
So, if the future tendency of clinical laborato- nized by public health administrators as a use-
ries is to replace immunoassays with MS-based ful tool in clinical practice. (8) Ethical issues. It is
procedures, it would be better that efforts were important to address the possible ethical issues
focused on the improvement of MS-based pro- regarding the adoption of epigenetics into clini-
cedures for the identification of histone PTMs. cal routine. Rothstein et al. have identified a
(5) Implement new applications for conventional number of issues in which epigenetics differs
methodologies. It is also possible to adapt con- from genetics. Therefore, epigenetic research
ventional technologies used in laboratories for which leads to new ethical issues addressing a
clinical diagnostics to epigenetic applications. In number of potential legal and ethical implica-
this regard, well-established qPCR can be used tions, including legal and moral responsibility,
to analyze DNA methylation by MSP assays in intergenerational considerations, and issues
clinical diagnostic. regarding access to health care [101,102].
Finally, other strategic considerations need to
be taken into account for the adoption of epigen-
etic biomarkers in clinical diagnostics, including 7. PERSPECTIVES OF EPIGENETICS
(6) government regulations. It would be very rele- IN DIAGNOSTICS
vant to work with different stakeholders to over-
come regulatory hurdles in different countries Since the first human methylome was pub-
around the world to achieve the implementa- lished in 2009 [103], technologies and biomedi-
tion of epigenetic diagnostics in clinical routine. cal research have offered a wide range of clinical
It is possible to use “in-house developed” tests applications for epigenetics. No doubt in the
in a Clinical Laboratory Improvement Amend- coming years epigenetics will overcome the
ments (CLIA)-certified reference laboratory to limitations of traditional genetics and genom-
perform different epigenetic tests. It is also pos- ics by providing new tools based on epigenetic
sible to apply for a less rigorous 510(k) process biomarkers and new epigenetic drugs able to
or for a more extensive premarket approval control the function of our genome. Epigenetics
application until full FDA or EMA review is will help address some unresolved questions in
performed. The “in-house developed” tests in our understanding of personalized medicine.
CLIA-certified laboratories may contribute to Epigenetic biomarkers (DNA methylation, PTM
adopt epigenetic assays in tertiary hospitals and in histones, and ncRNAs) serve in the dynamic
increase the knowledge of epigenetics in clini- study of physiopathological conditions, and
cal practice. (7) Third party. The adoption of new therefore, epigenetic biomarkers will serve to
technologies or epigenetic biomarkers by phy- predict the evolution of disease and to monitor
sicians and clinicians (prescribers) is subjected the effect of treatments on diseases. Further-
to decisions made by national/regional public more, the development of single-cell epigenetic
health administrators and insurance companies, assays or technologies such as Nano-ChIP-Seq,
which are ultimately responsible for the adop- which analyzes just a small number of cells, will
tion of new technologies. Therefore, it is very have a profound impact on clinical epigenetics,
important to perform clinical trials to validate because they solve the problem of limited tis-
the potential of epigenetic biomarkers and also sue availability and they also discern between
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C H A P T E R
2
The Importance of Biobanks in
Epigenetic Studies
Lorena Peiró-Chova1, Olga Bahamonde Ponce1, Carolina
Abril-Tormo2, Jacobo Martínez-Santamaría2,3, José Antonio
López-Guerrero4, Peter H.J. Riegman5
1INCLIVA Biobank, INCLIVA Biomedical Research Institute, Valencia, Spain; 2IBSP-CV Biobank,
FISABIO, Valencia, Spain; 3Valencian Biobank Network, FISABIO, Valencia, Spain; 4Laboratory
of Molecular Biology and Biobank, Fundacion Instituto Valenciano de Oncologia, Valencia, Spain;
5Department of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands
O U T L I N E
1. Biobanks 20 3. Epigenetics and Biobanking 27

1.1 Definition and Types of Biobanks 20 3.1 Epigenetic Biomarkers: Stability in
1.2 General Processes in Biobanks 21 Different Sample Sources 27
1.3 Ethical Issues in Biobanking 22 3.2 How Can Biobanks Contribute to
Epigenetic and Epigenomic Studies? 30
2. Biobanks in Biomedical Research 24
2.1 Biobanks as Research Infrastructures 24 4. Conclusions 31
2.1.1 IC and Governance 25
2.1.2 Associated Data 25
2.1.3 Sustainability 25 Acknowledgments32
2.2 Biobanks as Centers for Enhancing
References32
Security and Quality of Samples and Data 26

20 2. BIOBANKS AND EPIGENETICS
1. BIOBANKS like genomics and personalized medicine, and

may be classified according to different crite-
ria. When sorted by purpose, two large groups
1.1 Definition and Types of Biobanks
of biobanks can be discerned: population-based
Although there is no generally accepted defi- biobanks (PBs) and hospital-integrated biobanks
nition for “biobank,” and current definitions (HIBs). PBs developed first. They collect samples
often have slightly different meanings, the fol- from a large number of individual volunteers
lowing definition encompasses its essence: “A representing different population cohorts. This
biobank is a type of biorepository that stores bio- type of biobank stores both biomaterials and the
logical samples and data for use in research” [1]. associated information from these individuals,
In 2001, the Organization for Economic Co- mainly lifestyle habits, disease history, and envi-
operation and Development (OECD) defined ronmental data obtained for the most part from
biobanks as Biological Resources Centres (BRCs) participant questionnaires. Research performed
“that consist of service providers and reposito- on these biomaterials is usually devoted to the
ries of the living cells, genomes of organisms, epidemiological, exposition, and environmental
and information relating to heredity and func- risk factors for certain types of diseases in rela-
tions of biological systems…that must meet tion to the genotype of the participant [5,6]. HIBs
the high standards of quality and expertise are usually located at hospitals in which sam-
demanded by the international community of ples representing a variety of diseases are col-
scientist and industry for the delivery of bio- lected. Research performed with these samples
logical information and materials,…that must focuses mainly on biomarkers associated with
provide access to biological resources on which the progression of the disease or the treatment
R&D in the life sciences and the advancement of response [7]. In Spain, the development of a new
biotechnology depends” [2]. multidisciplinary model of biobanking has been
According to the International Society for Bio- promoted in which all human samples collected
logical and Environmental Repositories (ISBER), for biomedical research purposes from different
a biobank is “an entity that receives, stores, hospital departments are managed by a single
processes and/or disseminates specimens, as networked infrastructure created as a service
needed. It encompasses the physical location for supporting and enhancing cooperative bio-
as well as the full range of activities associated medical research. Nowadays many intermediate
with its operation” [3]. forms of biobanks can be found, making the dis-
In these two last definitions, the term biobank tinction between PBs and HIBs less pronounced.
refers to any type of collection of biological sam- In addition, biobanks can host two main
ples (plant, animal, microbe, human, etc.), but in types of collections: project-driven and general or
medical sciences the term is reserved for human archival collections [8]. A project-driven collection
specimens. In this sense, increasingly accepted means that specimens are collected and distrib-
are definitions like the one by the Public Popula- uted to answer specific research questions. The
tion Project in Genomics and Society (P3G): “an advantage this kind of collection has is that the
organized collection of human biological mate- investigator receives exactly what is requested.
rial and associated information stored for one or Nevertheless, its disadvantage is that it may
more research purposes” [4]. This review will take a long period of time (years/decades) to
focus on this kind of biobank. collect enough samples, especially if follow-up
Since the late 1990s, biobanks have become an data are required [8]. To the contrary, archival
important resource for biomedical research, sup- or general collections are devoted to establish-
porting many types of contemporary research ing reference collections. They are not meant to
1. Biobanks 21
meet particular research goals, but to be avail- relevant associated information. An operating
able in order to respond to multiple requests procedure is defined as each activity or set of
for an assortment of research uses. Hence, a activities using resources. It is managed, so the
large number of samples can be provided to elements of the input process can be transformed
researchers immediately, although the specifi- into results at the output level (ISO 9000:2005—
cally required data may not always be available. Quality Management Systems—Fundamentals
In archival collections, usually the data collected and Vocabulary). In turn, procedures are inter-
are less detailed than in project-driven collections related in a more or less sequential and multidi-
in which objectives of specific research projects mensional way. Thus, the application of a system
are previously defined [8]. In addition, in archi- based on processes within the organization of a
val collections from hospitals, the sample quality biobank, and the identification and definition of
needs only to be fit for diagnostic testing pur- the interactions between them, provides a holis-
poses. This certainly does not imply that these tic view of the operation of a biobank called the
samples would be useful in any technique avail- “process approach” (ISO 9001:2008—Quality
able for biomedical research. Management System—Requirements) (Figure 1).
These processes are mainly defined in the
quality management system adopted by a bio-
1.2 General Processes in Biobanks bank [9]. They are organized into three main
Biobanks, understood as technical units, levels: strategic processes, related to management
define their workflows into general operating efficiency; support processes, necessary to ensure
procedures for obtaining, handling, and dis- control over the available resources; and key
tributing human specimens, along with their processes. The last are defined in terms of the
FIGURE 1 A typical process model that could be implemented in biobanks according to a process approach in response to
the requirements of ISO 9001:2008.
nature of the biobank and the types of collections explicit purpose than SPREC: BRISQ (Biospecimen
it hosts. Key processes essentially refer to four Reporting for Improved Study Quality) [14] and
sequential common or fundamental processes MIABIS (Minimum Information About BIobank
for any type of biobank. These common key data Sharing) [15]. These initiatives have a greater
processes are, first, the reception of the biological capacity to cover the different variables that may
material, followed by its processing and preserva- be related to biobanking material and its associ-
tion, and finally, the distribution of that biological ated clinical and epidemiological information. In
material and associated data to researchers. the case of BRISQ, these variables are organized
Despite the fact that there are so many dif- into different levels, and they aim to character-
ferent types of biobanks, the preparation of ize part of the SOPs of the four key processes of a
biological material remains a key process. Such biobank. The purpose of BRISQ is to publish the
key processes should be described in the stan- conditions in which the used samples were pre-
dard operating procedures (SOPs). These SOPs served in order to enhance the reproducibility of
describe how to manipulate, for example, the results obtained. This can also have a normal-
peripheral blood to obtain blood components izing effect and decrease the variability associated
(serum, plasma, PBMCs, DNA, RNA, micro with the biomaterial and increase the feasibility
RNAs (miRNAs), histones, etc.) or other types of of the results obtained, for example, in large-scale
samples obtained without invasive procedures, epigenomic studies.
such as saliva, urine, feces, and tears. To improve Overall, the main objective of biobanks is to
the exchangeability of the samples needed to be be guarantors of the traceability and reproduc-
shared in multicenter research projects, these ibility of everything concerning the essence of
SOPs need to be standardized. Within an environ- biobanks. It is to provide the scientific commu-
ment where the biobanks are efficiently designed nity with biological material of high quality and
to answer different research questions, the han- appropriately characterized in order to enhance
dling of samples often differs. The variety may excellence in biomedical research.
be of importance when exchanging samples, and
this needs to be considered before the materials
1.3 Ethical Issues in Biobanking
are used. Moreover, there are biobanks with very
specific materials arising from surgical or clini- Biobanks have provoked questions on research
cal procedures, such as tumor and peritumoral and medical ethics, and have opened wide-
tissues, fresh, fixed, or postmortem tissues, cere- spread discussion. Since Chapter 3 describes the
brospinal fluid, and bone marrow. ethical concerns regarding epigenetic studies,
In order to harmonize the variations that may attention here is focused on the ethical issues
arise from the different SOPs used by different related to biobanking. The collection, storage,
biobanks, several initiatives have emerged across distribution, and use of biological materials and
best practice guidelines [10,2,11]. One of these is associated data should be conducted in a way
the SPREC (Standard PREanalytical Code) [12,13], that respects individuals, ensuring their privacy
which is understood as a tool for describing, using and confidentiality. Therefore, the following are
a seven-position code, the possible preanalyti- the fundamental ethical issues for biobanks:
cal variables associated with the preparation and the informed consent (IC) of the donors with the
processing of the biological samples (centrifuga- right to revoke their permission (including the
tion, precipitation, phase separation, etc.) with- informed opt-out systems when using residual
out the intervention of chemical agents. This tool materials); data protection; and anonymization or
is complemented by other initiatives that have a pseudoanonymization procedures of the biological
global character, but with a more general and less material for research.
1. Biobanks 23
Protecting autonomy through the process ensure the availability of donor ICs, current con-
of obtaining consent is important as it shows sent forms used in biobanking must be reviewed
respect for the individual. The principle of IC to ensure all these issues are carefully addressed
is largely recognized and considered a pillar in and explained by establishing practical and fea-
the practice of bioethics. Although it does not sible procedures. Otherwise, not only the act of
in itself protect a person, IC allows individu- consenting but also the right to revoke may be
als to exercise their fundamental right to decide only concepts with no real significance [1].
whether and how their biomaterials and associ- Still, protecting the donor against research
ated data will be used in research [1]. risks is a key responsibility for biobanks. In the
In recent years, biobank ethical debate has case of biobanking and the associated noninter-
focused especially on the validity of general or ventional research, the main risk for patients/
broad consent. Some would argue that the more donors is that identifiable personal data fall into
general the consent is, the less informed it becomes. the wrong hands. Hence, safeguarding confi-
Others would say that if the information provided dentiality is a paramount aspect of the protec-
covers all aspects relevant to the person’s choice, then tion of individuals and groups participating in
that person’s consent is appropriately informed. For biobanks [17].
those biobanks following the general collection The existence of many different levels of
model, a trend toward broad consent for future identifiability, coupled with their many differ-
research is now regarded as appropriate [16,17]. ent interpretations, has posed a major problem
Another discussion reflects the risks of the for discussions on confidentiality issues. Now, a
experiment and the conditions that need to be nomenclature proposed by the European Medi-
adhered to by stakeholders using the bioma- cines Agency (EMA) has been adopted by the
terials for medical research. A trial in which a International Conference on Harmonization of
patient is subjected to a potentially life-altering Technical Requirements (ICH), which brings
intervention is completely different from the sit- together the regulatory authorities in Europe,
uation where residual material and anonymized Japan, and the United States. Briefly, the nomen-
clinical data are used for medical research pur- clature is as follows. Identified data and samples
poses. In the latter case, an (informed) opt-out are labeled with personal identifiers such as
system is very capable of both safeguarding the name or identification numbers (e.g., social
unwanted use of biomaterials, and providing security or national insurance number). Coded
for the later revocation of the permission when data and samples are labeled with at least one
the only risk concerns privacy. This method is specific code and do not carry any personal
especially valuable when using archival materi- identifiers. Anonymized data and samples are
als for medical research and saves a lot of scarce initially single or double coded, but there is a
medical research resources, especially when the link with the subjects through the coding key(s).
patient contact with the pathologist involved is Anonymous data and samples are never labeled
not included in the normal routine [18]. with personal identifiers when originally col-
The right to revoke is mentioned in all consent lected, neither is a coding key generated. There-
forms for biomedical research. That notwith- fore, there is no potential to trace clinical data
standing, how it can be implemented in practice and samples to individual subjects [17].
is a challenge because biomaterial is exchanged, It is also recognized that the degree of data
data are distributed in complex databases, and protection is closely linked to questions of with-
sometimes samples undergo transformation drawal from research, dissemination of results to
into cell lines that can be exchanged and dupli- participants (generally or individually), follow-
cated. Therefore, although biobanks should up of participants, and third-party access to
research data. There are a variety of situations • t he need of large quantities of representative
ranging from identifying personal data (for biological samples with their associated
which there is significant protection, very lim- clinical information from different scenarios
ited access, and the possibility of withdrawal) to where the “-omics” technologies can be
anonymized data (for which there is free access applied, and
for research purposes, with no possibility of • the “globalization of biomedical research”
recontacting participants and no possibility of through an increasingly abundant and close
returning the results or of withdrawing from the collaboration among different research
project) [19]. With the realization that absolute groups around the world that implies the
data safety is an illusion, the idea of open consent interchange of biological samples and data.

has been proposed. This type of consent refrains
Hence, biobanks constitute a research infra-
from any promises of anonymity, privacy, or
structure committed to providing facilities,
confidentiality [20].
resources, and services to research groups in an
In genetic studies, including epigenetics,
open, transparent, and generous manner that
working with nonanonymized or even anony-
benefits not only science but also participants
mous samples and data is of special importance
(patients, healthy donors, etc.).
since the analysis of methylomes and miR-
Nomes, for example, could lead to incidental
findings affecting future generations, making it
2.1 Biobanks as Research Infrastructures
necessary to recontact participants.
Biobanks constitute an exclusive research
infrastructure that requires different governance
2. BIOBANKS IN BIOMEDICAL mechanisms than do project-based mechanisms.
RESEARCH This infrastructure necessitates a specialized level
of organization focused not only on ensuring the
The value of biobanks to translational medi-
quality of the biological samples and their asso-
cal science is widely recognized [21,22]. Promot-
ciated information but also on the access rules
ing translational research and the application of
and confidence for both end users: researchers,
innovative technology requires easy access sup-
as well as participants. Because of this operat-
port infrastructure to facilitate agile experimen-
ing philosophy, biobanks need to be dynamic
tal demonstrations of a hypothesis or simulated
and efficient in meeting the requirements of
models. In this scenario, biobanks constitute one
researchers, while preserving the rights and the
of the most attractive alternatives contributing
confidentiality of the donors. Overcoming this
to building bridges between all the biomedical
challenge requires the involvement of many
research specialties.
actors (clinicians, data managers, technicians,
Among the situations that have occurred in
pathologists, etc.) and institutional support.
the last decade of biomedical research and that
Trust between the biobank and the investigators
have resulted in the establishment of biobanks,
using the biobank is of major importance. The
the following deserve highlighting:
biomaterials and data must be available for their
• t he vertiginous development of the “-omics” projects. In addition, the trust between the bio-
disciplines (metabolomics, proteomics, bank and the participants is crucial. Within this
transcriptomics, genomics, epigenomics, force field, the biobank manager needs to find
etc.) in obtaining large amounts of data from the solution that best fits each situation. In order
high-quality samples [23], to guarantee a minimum of functionality within
2. Biobanks in Biomedical Research 25
the framework described above, at least the privacy will evolve over the life of the biobank.
following parameters should be considered. Hence, participants are key stakeholders in
these issues and CABs can serve as a represen-
2.1.1 IC and Governance tative voice of the community [30].
Rather than providing IC for a specific proj-
ect, participants in most biobanks offer an open 2.1.2 Associated Data
consent for multiple future biomedical projects, Most prevalent diseases are considered to be
the details of which cannot be provided at the caused by a large number of genetic and envi-
time of enrollment. For this reason, the gover- ronmental effects or modest gene–environment
nance mechanism must balance the needs of the interactions [31]. These interactions may also
scientific community and the participants with affect the response to treatments and patient
an emphasis on the recognition of participants, follow-up after clinical interventions. In these
trustworthiness, and adaptive management scenarios, the scientific value of biobanks is
[24,25]. Hence, formal governance structures greatly enhanced when they also have lifestyle,
are a common and necessary component of bio- risk factor data, and other clinical information
banks. Although the institutional review board available [5]. Clinically useful data commonly
(IRB) is an essential component for oversight include demographic information, general
and safety, most biobanks utilize a formal access health and functioning, personal and family
or oversight committee to approve the use of medical history, health behaviors (e.g., diet,
samples and data [26,27,28]. These committees physical activity, and smoking), medication use
may serve to review the scientific content of the (both prescription and over-the-counter medi-
research proposals as well as provide manage- cations), diagnosis (type of disease, biomark-
ment for finite biological samples. Members of ers, etc.), type of treatment, and follow-up. The
these committees should be skilled in scientific, systematic collection of family history data can
ethical, and clinical domains to provide an addi- also be highly useful, as it is generally stored as
tional level of safety and rigor to projects using unstructured text in the electronic health record
biobanks. (EHR), making it difficult to cost-effectively
In many countries, as well as in some col- retrieve it for research studies [32].
laborative research consortiums, the role of
participants is increasingly taken into account. 2.1.3 Sustainability
In some contexts, informal governance struc- Biobanks are often launched without a long-
tures, such as community advisory boards term plan of sustainability [5,17]. While a large
(CABs), can be an important component of component of the cost is the upfront collection
biobank governance [25]. CABs may provide and processing of samples, there are signifi-
advice about the efficacy of the IC process and cant costs to maintaining samples, data, and
the implementation of research protocols [29], access to a biobank. Cost recovery models vary
and they are representative of the community from institutional support to complete support
participating in the research being reviewed through user fees, although the latter are hard to
(for instance, cancer patient coalitions, rare set, given large initial costs and the life cycle of
disease advocacy groups, special and minority a biobank over decades [5]. More recently, it has
groups). Input from the community can add been suggested that biobanking in a clinical con-
insight into the perspectives of participants text might be incorporated into the cost of busi-
when questions arise. Issues with return of ness and embedded into the fee and insurance
results, academic/industry partnership, and reimbursement structure [33].
2.2 Biobanks as Centers for several challenges in using EHRs for research,

Enhancing Security and Quality of including data that are often incomplete, inac-
curate, conflicting, highly complex, and poten-
Samples and Data
tially biased [25].
To be successful, biobanks must pay close Another data source for biobanks are the
attention to collecting high-quality specimens data observations from completed studies con-
and relevant associated information. An impor- ducted using the biobank’s samples. The return
tant goal is to ensure a stable and comparable of the research data to the biobank at the com-
preanalytical phase, therefore SOPs and adher- pletion of the study should become a normal
ence to the SOPs should be implemented with procedure. This enables the secondary use of
sufficient sample handling information so that existing research data which can provide many
the history of each sample is completely retrace- opportunities for new discoveries beyond the
able [5]. This is especially the case when the scope of the original study, leading to the opti-
results show unexpected deviations. Quality mization of the resources. For example, infra-
metrics are key indicators of the usefulness of structures like the Mayo Clinic Biobank require
biobank specimens. For instance, DNA is funda- all the data generated using their materials
mental to epigenetic studies; numerous methods to be deposited into a secure central database
to estimate quality have been developed, includ- for future use [27]. In this context, the biobank
ing total DNA yield and DNA amplification by builds again on trust and acts as an honest broker
polymerase chain reaction (PCR) [34]. safeguarding privacy and confidentiality; hence,
The value of biobank samples is enhanced any study using returned data would need
by the presence of a high-quality information approval from the Access Committee and a
management system (IMS) to track overtime separate IRB approval [38]. Popular data types
data concerning enrollment and consent; sam- for reuse are genome-wide association and
ple acquisition, processing, storage, and dis- whole-exome sequencing data, which can be
tribution; quality assurance/quality control; used to reduce genotyping costs for subse-
collection and/or linkage to subject data (such quent studies and improve the characterization
as clinical data); data security and access; and of genetic variants that are clinically relevant
reporting functions. The IMS plays a critical and actionable. Another model would be that
role in providing sample and data accountabil- the data become part of the hospital data,
ity and in tracking a sample from collection to which also harbor the biobank and clinical
processing, storage, use, and final disposal. Use data. This way, the research is brought close to
of barcodes to enhance the tracking is strongly both the patient and to the biobank for reuse.
encouraged [35]. A robust IMS will be able to Clinicians are able to look into the research data
integrate large volumes of data from multiple and can contact the researchers about clinical
sources, including both clinical and research implications.
data. Use of recognized standards (like SPREC) Thus, biobanks have the capacity to gener-
[13,36] enhances the ability to harmonize with ate a large amount of genetic and epigenetic
other biobanks for the pooling of projects. data, some of which may have health implica-
Biobanks with an IMS that can be linked to tions for the participants and their relatives.
EHRs have an especially rich resource from This raises the need to address the return of
which to draw a wealth of data. Because of both primary research results and incidental
this, the development of methods to rapidly findings. In countries like Spain, this type of
extract phenotype data from the EHR is an information is considered a right of the partic-
active area of investigation [37]. Still, there are ipants of the study and is guaranteed by law.
3. Epigenetics and Biobanking 27
It is in the IC form where participants express will help ensure the adequate collection, pro-
their desire to be informed of the research cessing, and preservation of comparable, high-
results. Others recommend the results be quality samples.
returned to the biobank participants if certain
criteria are met [39,40], although the problem 3.1 Epigenetic Biomarkers: Stability
of what to do with genes with pleiotrophic
in Different Sample Sources
effects is yet to be resolved [41]. The process
of returning results to participants incurs sig- More and more studies are focused on the
nificant costs to biobanks and must be taken identification of biomarkers that may be of clini-
into account when planning for result disclo- cal utility. To deliver reliable clinical biological
sure [42]. Input from a CAB can also provide markers, researchers will have to look for those
important insights when developing return- characterized by high stability, reproducibility,
of-results policies and procedures. Further- sensitivity, and specificity. Epigenetic biomark-
more, it is necessary that biobank participants ers, which are based on methylation of specific
receive appropriate clinical follow-up with genes, miRNA signatures, and specific PTMs in
a geneticist and/or genetic counselor after histones, reflect all of these properties. Conse-
receiving research results, which may incur quently, they are promising candidates for deliv-
costs to participants [39]. ering potential clinical biomarkers.
When searching for clinical markers, research-
ers need to focus on the study of stable mol-
3. EPIGENETICS AND ecules. Therefore, it is necessary to assess the
BIOBANKING stability of epigenetic markers under various,
clinically relevant conditions. The character-
Epigenetics has recently emerged as a new ization of the degree of stability of epigenetic
and promising field within the genetics disci- biomarkers and the identification of determi-
pline. Lifestyle, nutrition, stress, diseases, and nants that may affect their integrity can provide
pharmacological interventions have a great critical information for the correct design and
impact on the epigenetic code of the cells by conduction of clinical trials and validation of
altering DNA methylation, miRNA expression, biomarkers for clinical practice. Therefore, well-
and histone posttranslational modifications designed studies evaluating the stability of epi-
(PTMs). These changes are inherently more genetic biomarkers under the different sample
plastic and dynamic than genetic mutations handling processes (collection, preparation of
and they contribute to the pathology of several samples, extraction procedures, preservation,
diseases. Moreover, there is an increasing need etc.) are required. In this regard, biobanks can
to find new biomarkers for diagnosis, progno- implement subprocesses with SOPs that ensure
sis, and therapy monitoring, hence epigenomic the stability of the preanalytical phase of the bio-
maps of cell types and tissues in specific dis- logical samples they host.
eases or physiological situations will provide Interestingly, intrinsic properties characterize
a valuable resource for the identification of the biomolecules that can be used for epigenetic
promising biomarkers and will also facilitate studies, making them suitable substrates for these
the characterization of targets for epigenetic purposes. Methylated DNA has been shown to be
drug development [43]. more stable in vivo and in vitro than unmethyl-
In this scientific context, the recruitment of ated DNA [44]. Furthermore, it has been demon-
biological samples must be entrusted to dedi- strated that methylated DNA circulates in blood
cated biobanks with dedicated personnel. This longer than unmethylated DNA does. Thus,
cell-free circulating methylated DNA can be a isolation of the exosomes from various body flu-
good candidate for diagnostic biomarkers [44]. ids, including plasma, malignant ascites, urine,
In fact, methylated DNA has been determined amniotic fluid, breast milk, and saliva [76,77].
in serum and plasma [45–49], urine and semen The stability of exosomes and exosomal miRNA
[50–52], bronchoalveolar lavage fluid [53], saliva in body fluids and their accurate detection by
[54], sputum [55,56], ductal lavage fluid [57], and quantitative PCR further support their potential
fine-needle aspirates [58]; a fact which gives an as noninvasive, or at least minimally invasive,
idea of the high stability of methylated DNA. biomarkers for disease [78]. In this regard, it is
miRNAs are a large family of short noncod- important to point out that procedures for iso-
ing RNAs (17–25 nucleotides) [59], which are lating exosomes and miRNAs, along with meth-
involved in many biological processes and can- odologies for quantifying miRNA species from
cer [60,61]. The miRNAs have been described biological material, are relatively recent devel-
as highly stable molecules, and some authors opments. Thus, SOPs for the collection, process-
have attributed their stability to nuclease resis- ing, and preservation of biological specimens
tance due to smaller nucleic acid size [62] and/or from which exosomes and miRNAs can be iso-
microvesicular containment [63]. Therefore, the lated must be carefully evaluated before these
study of these stable small RNA molecules molecules are used for further research as can-
involved in a broad spectrum of biological pro- didate biomarkers. In this regard, biobanks can
cesses may lead to the discovery of promising play a crucial role, fine-tuning these procedures
new biomarkers with a diagnostic, prognostic, and ensuring the quality of samples.
or therapeutic value [43,64]. The miRNAs can be readily isolated not only
Mitchell et al. [65] and Chen et al. [66] were from body fluids but also from fresh or fixed
the first to report the stability of miRNAs in tissues. The stability of these small molecules
serum and their potential use as biomarkers for in tissue samples of compromised quality, such
disease. Since then, many other investigations as formalin-fixed paraffin-embedded (FFPE)
have analyzed the stability of miRNAs over samples, has also been evaluated [79]. Forma-
time, taking into account preanalytical condi- lin fixation and the paraffin-embedding process
tions in body fluids, such as serum and plasma cause enzymatic degradation and even a chemi-
[67] and urine [68]. cal modification of RNA, giving rise to cross-
Additionally, specific miRNA signatures links with proteins and making RNA extraction
with clinical value have been identified in the difficult [80,81,82]. However, small RNAs are
body fluids of cancer patients, making cell-free easily extracted after proteinase K digestion.
miRNAs good candidates for minimally inva- Therefore, miRNAs from FFPE samples are sim-
sive biomarkers [69]. The miRNAs and miRNA ilarly recovered than miRNAs from optimally
signatures from serum, urine, saliva, cerebrospi- preserved frozen samples, thus supporting the
nal fluid [70,71], and exosomes [72] have been high degree of stability of miRNAs [79]. Thus,
described as promising diagnostic biomarkers. tissue samples, mainly found in HIBs, could be
As has been pointed out before, some authors the starting material for early biomarker stud-
have attributed the unusual stability of miRNA ies. Retrospective studies using tissue samples
in several biofluids, as well as in tissues, to their for the identification of miRNA signatures in
location within exosomes [73], since these vesi- diseases may be the first step in the discovery
cles act as a barrier to nucleases. of candidate biomarkers. Consequently, once
Exosomes containing miRNAs have been potential biomarkers are identified, methods
described as playing a role in cell-to-cell commu- can be optimized in specific SOPs so as to detect
nication [74,75]. Recent studies have described the and validate these specific miRNAs in body
3. Epigenetics and Biobanking 29
fluids in order to obtain useful minimally inva- nonfixed samples are further recommended for
sive epigenetic biomarkers to be used in every- MS studies because protein extraction from FFPE
day clinical practice. tissues faces challenges due to deparaffinization
Histones are other biomolecules that can be and cross-link reversion. Despite the advances
used as epigenetic biomarkers, and both the in proteomics, factors affecting the stability and
presence of circulating histones and altered utility of FFPE biomaterials will require detailed
PTMs in histones in specific tissue or cell lines evaluation and validation, and PTM enrich-
are indicative of pathological conditions. ment procedures would be required. In conclu-
Extracellular histones have been demon- sion, the PTM-directed analysis in FFPE would
strated to function as endogenous danger sig- be complicated by dynamic changes during the
nals. Levels of circulating histones as well as fixation process and due to complications of
nucleosomes are increased in patients with can- affinity enrichment procedures [90]. Since the
cer, inflammation, and infection [83,84]. Hence, optimal starting material for MS studies, such
detecting the levels of histones and nucleosomes as fresh-frozen tissues, are not always readily
in biological fluids could set the basis for the available, the optimization of methods for the
design of new biomarkers. EDTA (10 mmol/L) extraction of proteins from different sources of
avoids further digestion of nucleosomes through biological material to carry out these studies is
the Ca+2- and Mg+2-dependent endonucleases also needed [91].
present in blood. These processed samples have Other important experimental procedures
shown good stability for at least 6–12 months at used in epigenomic studies are chromatin immu-
−80 and −20 °C [85]. In addition, a 5-year stor- noprecipitation (ChIP) experiments followed by
age at −80 °C revealed a decrease of about 32% real-time PCR or sequencing analysis [92]. ChIP
of the nucleosome values, suggesting that long- experiments consist of the immunoprecipita-
term stability of these kinds of proteins in blood tion of the chromatin using specific antibod-
derivatives can be compromised during the ies that recognize proteins bound to chromatin
storage time [86]. Clarifying what are the opti- and specific PTMs in histones. Although FFPE
mal processes for preserving the integrity of tissue samples comprise a potentially valuable
these proteins in body fluids, thus avoiding the resource for retrospective biomarker discovery
degradation by endogenous proteases, is a chal- studies, these biological samples have draw-
lenge in which biobanks can contribute actively backs, as described earlier. These samples often
by providing SOPs that ensure the integrity of display degradation and loss of antigenicity
these nuclear proteins. due to harsh fixation conditions and prolonged
Histone PTMs play an important role in the storage. These factors can affect the ease of
epigenetic regulation, and irregular patterns of chromatin preparation for experiments of ChIP-
histone global acetylation and methylation have seq and require optimized protocols for good
frequently been observed in various diseases results. Some commercial kits partially solve
[87,88]. As a consequence, quantitative analysis this problem. For example, Active Motif, Inc. has
of the PTMs can be very useful in identifying developed a ChIP-IT® FFPE Chromatin Prepa-
new biomarkers, looking for therapeutic targets, ration Kit that contains specifically formulated
or monitoring treatment. Mass spectrometric reagents and an improved protocol to extract
(MS) approaches, such as selected reaction mon- high-quality chromatin from histological slides
itoring, can quantify histone PTMs directly from or tissue sections. Moreover, there also exists lit-
core histone samples [89]. Therefore, it is very erature where optimized ChIP-seq procedures
important to characterize the source from which to obtain good-quality chromatin from FFPE
histones have been purified. In this context, samples are described [93].
Overall, the stability of candidate biomarkers Furthermore, biobanks can contribute to the
is a prerequisite for the adequate identification systematization, protocolization, standardiza-
and validation of epigenetic biomarkers which tion, and innovation of technical procedures that
may be used in clinical practice to improve the could facilitate researchers with optimal starting
accuracy of diagnosis, predict prognosis, and material for their investigations. The implemen-
monitor disease progression and response to tation and standardization of technical proce-
therapy. The nature and essence of biobanks dures in biobanks may contribute to decrease the
make them a feasible source for providing amount of resources needed in order to acquire
researchers this appreciated biomaterial and the biomaterial required for epigenetic studies
associated data of high quality. [43]. Moreover, as previously mentioned, the
return of research data to biobanks at the comple-
tion of a study could lead to an optimization of
3.2 How Can Biobanks Contribute to
the resources, avoiding the need for researchers
Epigenetic and Epigenomic Studies? to reanalyze the same parameters while reducing
Large-scale epigenomic studies have revo- the cost and time required for subsequent studies.
lutionized three major areas in science: basic Large-scale epigenomic projects and stud-
gene regulatory processes, cellular differentia- ies focusing on the identification of epigenetic
tion and reprogramming, and the role of epi- biomarkers require a large number of biological
genetic regulation in disease [94]. In a similar samples with their associated clinical data. Nev-
way, epigenetics has accelerated biomedical ertheless, a single biobank may not always be
research in three major areas: the identifica- able to provide the number of samples needed to
tion of pathogenic pathways and targets par- provide statistical power in this kind of studies.
ticipating in disease, the identification of new Thus, there is a need for biobanks to network. The
epigenetic biomarkers, and the development standardization of procedures between biobanks
of epigenetic drugs for treating diseases. The plays a key role in these networks [95]. The par-
optimal conditions for the collection, process- ticipation of several biobanks in multicenter proj-
ing, and long-term preservation of biological ects requires all of them to adopt common SOPs
samples need to be defined before perform- [15]. Hence, networking between biobanks also
ing biomarker discovery studies. Furthermore, requires a multidirectional flow of information.
taking into account the preanalytical variables Consequently, the proper management and trace-
associated with the above-mentioned processes ability of preanalytical variables associated with
is very important for the success of epigenetic samples are crucial to ensuring effective inter-
and epigenomic studies. Additionally, there is a connection and interoperability among biobanks
need for adequate isolation and detection proce- [96]. Thus, the coordination and integration of
dures for the specific biomolecules that are to be biobanks in a way that allows them to share SOPs
analyzed as candidate biomarkers, as described and information about the samples they hold
in the previous sections. Moreover, an adequate may facilitate large-scale collaborative projects.
selection of cell types, tissues, and body fluids For the shared information, it is crucial to look
of high quality to carry out biomedical research carefully at the annotations used and translate
should be ensured. Consequently, biobanks them to a common format wherever necessary.
play a crucial role in ensuring the availability to Finally, genes, epigenetic regulation, and envi-
researchers of high-quality biological samples ronment are important contributors in the devel-
that have been collected, processed, and pre- opment of human diseases [97]. Individuals have
served the best way, following standardized different genomes and environmental exposure
operating procedures. histories, the latter also defined as exposomes [98],
4. Conclusions 31
affecting their epigenomic signature. Epigenetics on the implications of epigenetic studies. Bio-
and exposomes are tightly associated, and it banks would act as providers of high-quality
is surprising how environment can affect the biological samples with well-annotated data to
human epigenome. Regarding the impact of the scientific community, facilitating the deep
environment on epigenetics, biobanks interested research collaborations needed for large-scale
in human biomonitoring that host samples from epigenomic projects (Figure 2). Therefore, dedi-
humans exposed to different environmental con- cated biobanks are a key step in the development
ditions (exposure to chemicals, for example) have of personalized/precision medicine [5,99,100].
appeared recently in great quantity. The number
of this type of biobank is exploding. Environmental
specimen biobanks can be integrated into PBs.
4. CONCLUSIONS
These biobanks may make possible the develop-
ment of interesting epigenomic projects that shed
To conclude, the following are some of the
light on how environment affects our health.
most important aspects that have been dis-
Overall, the identification and analysis of
cussed in this chapter.
candidate biomarkers to be translated to clinics
require a proper research ecosystem that can be • D uring the last few years, biobanks
mediated by biobanks. Before performing bio- have become an important resource for
marker discovery studies, optimal material with biomedical research, constituting an
the associated data needs to be used, and ade- exclusive infrastructure that requires specific
quate isolation and detection procedures need governance mechanisms.
to be defined. To a large degree, biobanks can • Biobanks must pay close attention to the
cover the area of regulatory and ethical issues, collection of high-quality specimens and
as well as that of social engagement and focus their relevant associated information. They
must conduct their activities in a way that
respects individuals by ensuring their
privacy and confidentiality. Biobanks also
need to be dynamic and efficient in order to
meet the requirements of researchers.
• Epigenetic biomarkers, which are based on
the methylation of specific genes, miRNA
signatures, and specific PTMs in histones, are
promising clinical biomarkers because they
are highly stable under different conditions.
• Biobanks provide critical research and
infrastructure support for biomedical
research in the field of personalized
medicine, facilitating the discovery and
validation of epi- and genetic associations
with exposome interactions. These
interactions will shed light onto disease
FIGURE 2 A schematic representation of how biobanks pathogenesis which can ultimately
contribute to epigenetic and epigenomic studies, provid-
ing high-quality samples, data, and services to the scien-
be translated into risk assessment/
tific community in an ethical framework, facilitating deep stratification schemes, new diagnostic and
research collaborations through biobanking networks. pharmacogenomic tools, and new drugs.
• A
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C H A P T E R
3
Epigenetic Mechanisms as Key
Regulators in Disease: Clinical
Implications
Abdelhalim Boukaba1, Fabian Sanchis-Gomar2,3, José Luis
García-Giménez2,3,4
1Drug Discovery Pipeline, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Science,
Guangzhou, People’s Republic of China; 2Department of Physiology, Medicine and Dentistry School,
University of Valencia, Valencia, Spain; 3Biomedical Research Institute INCLIVA, University of Valencia,
Valencia, Spain; 4Center for Biomedical Network Research on Rare Diseases (CIBERER), Spain
O U T L I N E
1. Introduction 38 2.2 The Tricarboxylic Acid Cycle and

1.1 The Cross talk between Metabolism Epigenetics44
and Epigenetics 38
3. Lifestyle, Nutrition, and Physical
1.2 Epigenetic Mechanisms in Gene
Activity in Epigenetic Regulation 45
Regulation38
1.2.1 General Overview 38 4. Epigenetic Mechanisms Underlying
1.2.2 DNA Methylation 39 Complex Diseases 47
1.2.3 Histone Modifications 40 4.1 MetS and Diabetes 47
1.2.4 MicroRNAs and Long Noncoding 4.2 Alzheimer’s Disease 49
RNAs41
5. Epigenetics in Rare Diseases—Clinical
2. Epigenetics and Metabolism Intersection 42 Implications50
2.1 Relevant Genes in the Methionine Cycle 5.1 ICF Syndrome 50
Contribute to Epigenetic Regulation 42 5.2 Rett Syndrome 53

38 3. EPIGENETICS AND DISEASE
5.3 RSTS1 Syndrome 54 7. Conclusions 58

5.4 Weaver Syndrome 2 55
5.5 Friedreich’s Ataxia 56
Acknowledgments59
6. Epigenetic Biomarkers and Clinical
Laboratory57 References59
1. INTRODUCTION chromatin translates into a defined outcome on

its transcriptional status. That is, it examines the
1.1 The Cross talk between Metabolism question, how do the combinatorial epigenetic
and Epigenetics signatures that portray any gene unit drive its
expression pattern? The set of instructions that
Modulation of gene expression is of para- chromatin receives locally could be regarded as
mount importance for the survival of organisms a set of signal inputs which are converted into
and cells in an internal milieu that undergoes effector signal outputs through complex molec-
continuous changes. Cell growth, cell differen- ular interactions engaging multicomponent pro-
tiation, migration, transport regulation of amino tein complexes. The nucleosomal nature of the
acids, regulation of enzymatic activity (i.e., telom- chromatin imposes a barrier to transcription but
erase, proteasome, etc.), DNA synthesis, and regu- the nucleosome is emerging as a signaling unit
lation of the different cell death mechanisms are on which complex biological transactions take
processes that control not only the fate of cells place [3]. These transactions are difficult to dis-
but also the organismal proneness to pathologi- sect individually, but they integrate into the very
cal situations like cancer, neurodegeneration, and nature of any chromatin-templated process.
impairment of immunity. Numerous metabolites Chromatin is composed of repeating arrays
fuel the enzymatic activities of different epigene- of nucleosomes, which are the building block of
tic machineries. Hence, epigenetic gene regulation eukaryotic chromosomes [4]. Each nucleosome
and metabolism participate in a perfect intricate is characterized by 147 bp of DNA wrapped
coordination in which the deregulation of any around a histone octamer. A histone octamer
component could have detrimental consequences consists of two copies each of canonical histone
for the cell and ultimately for the organism. H4, H3, H2A, and H2B or their variants that are
Originally it was proposed that epigenetics is organized in a special configuration to accom-
the study of heritable changes in gene expres- modate ∼1.6 superhelical turns of DNA [5].
sion that are not due to modifications in the Both the tails and the globular domains of the
DNA sequence [1]. Nowadays, epigenetics is histones and their variants are subjected to over
the study of all regulatory aspects of chromatin- 100 posttranslational modifications (PTMs).
associated processes no matter if they are herita- Conceptually, a modification can either disrupt
ble or not [2], as described in detail in Chapter 1. the association of histones with the DNA, hence
dictate the topology of the chromatin, define
1.2 Epigenetic Mechanisms chromosome territories, or act as scaffolds to
in Gene Regulation influence the recruitment of transcription factors
and other chromatin-associated proteins.
1.2.1 General Overview Epigenetic alterations can produce the same
Epigenetic gene regulation refers to how a phenotypic defect as a null mutant when they
specific structural and chemical configuration of induce the improper silencing of a gene. Or, the
1. Introduction 39
contrary could result in overexpression of one in some chapters dedicated to it in the present
particular gene and its protein product. How- book. Hence, not surprisingly it is implicated
ever, the existence of multilevel gene control can in embryonic development, cell differentiation,
act at the mRNA or protein level just to add to parental gene imprinting, gene transcriptional
the complexity of this scenario. In consequence, regulation, and genome stability, as well as
one expects that the interpretation of these mul- chromatin structure, only to mention classical
tilevel gene controls mediated by epigenetic aspects of its functionalities. Some aspects are
regulation can be used for the comprehension only starting to emerge such as the control of
of pathological phenotypes which in turn, could the rate of elongation or the modulation of splic-
translate into clinical applications. Hence, it is ing [9,10], which dictates the frequency and the
important for clinicians to use specific epigene- prevalence of protein variants. Another aspect is
tic signature alterations for diagnosis, prognosis, the deregulation of splicing, which is associated
personalized therapy, and treatment monitor- with a huge number of human diseases [11].
ing. It is crucial for translational epigenetics to Initially, DNA methylation was regarded as
understand how alterations in the epigenetic a static modification whose pattern is stably
signatures of a gene can and do occur, and inherited, qualifying it for the status of a true
result in transcriptional defect(s). Intermediary epigenetic mark. This dogma has been reversed
metabolites, by-products of the metabolism, by the discovery that ten-eleven translocases
diet, environmental insults, lifestyle, etc., have (TETs) proteins are able to oxidize the 5-meth-
the potential to alter the epigenetic context with ylcytosine (5mC) to 5-hydroxymethylcytosine
immediate outcome on our health status. (5hmC), and to 5-formylcytosine (5fC), and
The genetic information is not always 5-carboxylcytosine (5caC), as reviewed by
expressed in a binary system being either OFF Wu and Zhang [12]. DNA methylation is cata-
or ON (0 or 1). Rather, its expression displays lyzed by the maintenance methylase, DNMT1
varying degrees of intensities even within the (DNA(cytosine-5-)-methyltransferase 1), and by
same homogeneous population of cells as dem- the de novo methylases, DNMT3A and 3B (DNA
onstrated by single cell transcriptome analysis. (cytosine-5-)-methyltransferase 3 alpha/beta).
Transcriptional activation within a population These enzymes are essential for embryonic
of identical cells obeys a stochastic model due development (reviewed in Ref. [13]) and also
to intrinsic and extrinsic parameters inherent to for genomic stability as we describe for ICF syn-
a multistep process which requires the hierar- drome (immunodeficiency, centromeric region
chical coordination of multicomponent protein instability, and facial anomalies syndrome) in
machineries [6]. In addition, the processing of the following sections.
nucleosomal DNA, itself, may cause stalling of Two ways by which DNA methylation affects
the polymerase at nicks, for example, caused by gene regulation have been postulated. It can
the elongation process, or by the oxidative activ- alter the binding of transcription factor(s) to
ities of some chromatin modifiers which uses their cognate sequence(s), especially for those
H2O2 as a cofactor [7]. bearing the CpG dinucleotide. Alternatively,
CpG methylation is recognized by specific pro-
1.2.2 DNA Methylation tein factors (readers), which recruit chromatin-
DNA methylation is the best studied epi- modifying complexes to target sequences. CpG
genetic modification since its discovery [8]. It methylation is recognized by MeCP2 (methyl
affects the 5′ carbon position of cytosine mostly CpG-binding protein 2) and MBD2 (methyl
in the context of CpG dinucleotide. DNA meth- CpG-binding domain protein 2) (reviewed in
ylation plays crucial roles in all chromatin- Ref. [13]). Interestingly, mutations in MeCP2
templated processes and diseases as illustrated produce one of the most frequent syndromes
related to intellectual disability in women, crotonylation, citrulination, formylation, glyco-

called Rett syndrome (RTT), as we describe next. sylation, O-GlcNAcylation [19], carbonylation,
Unmethylated CpGs are also recognized by spe- PARsylation, and glutathionylation [20,21].
cific modules containing proteins such as CXXC What are the functional roles of such modifi-
zinc finger. Readers of 5hmC have also been cations? It is clear that epigenetic regulation is
identified in recent screenings, highlighting that inherently linked to metabolism and it is impor-
this modification is an epigenetic mark and not tant not only for basic science to uncover the
an intermediary product during the demethyl- functionalities of such modifications but also for
ation of cytosines [14]. translational epigenetics, as it provides new bio-
Mapping DNA methylation signatures is rela- markers to diagnose or identify druggable tar-
tively easy compared with histone PTMs profil- gets to treat diseases.
ing. Hence clinically, they harbor great potential The chromatin is inherently defined by its
value as diagnostic and prognostic biomarkers. epigenetic state. Epigenetic state refers to the
The possibility to screen blood-circulating DNA pattern of histone modifications, DNA meth-
for alterations in DNA methylation also adds to ylation, and nucleosome positioning including
its value. Alterations in DNA methylation pat- histone variants. Locally chromatin domains
terns have been linked to a growing number of are defined by epigenetic signatures that dis-
human pathologies. These issues are discussed tinguish them structurally and functionally
in detail in this chapter. from other domains. Namely, the pattern of
histone modifications has served to redefine,
1.2.3 Histone Modifications biochemically, the subdivision of chromatin
The repertoire of histone modifications as domains and extended its classical cytological
well as the enzymes implicated in the modifi- partition in heterochromatin and euchromatin.
cation process exceeds largely the scope of any Thus, at any given moment, any human cell
review article, not only because of their number has a defined epigenome that is portrayed by
but also mainly because of the diversity and the epigenetic modifications of each one of the
the complexity of the processes in which they ∼30 million nucleosomes that make up human
are involved. Not surprisingly, a few chapters chromosomes, and by the epigenetic modifica-
have been devoted to this topic in the present tions of cytosine of the underlying DNA, mainly
book. Readers are referred to the histone modi- in the context of CpG dinucleotide. The epig-
fication database [15], or to snapshot of histone enome is highly dynamic and it is subjected
modifications [16]. For the scope of this chapter, to continuous fluctuations as a result of the
histone modifications as well as the enzymes action of chromatin-modifying and demodify-
that catalyze them have been explored for their ing enzymes, which make a steady state virtu-
potential as therapeutic targets and biomarkers ally nonexistent. The acetyl group turnover on
in cancer [17]. The first question is why so many histones, for example, has been shown to last
PTMs in histones? Next, are they truly PTMs or for less than 30 min in yeast. Knowing that most
just transient histone-chemical moiety adducts histone modifications could be removed by coun-
that are catalyzed by illicit enzymatic reactions? termodifying enzymes or erasers, it is logical to
Indeed, some HATs (histone acetyltransferases) postulate that they are subjected to either fast or
may use acetyl-CoA or propionyl-CoA to either slow turnover. Thus, even single-cell epigenom-
acetylate or propionylate histones. The enzymes ics is only a snapshot of the epigenomic landscape
that remove acetyl groups are also able to of that cell at that moment. Even though the num-
remove propionyl moieties [18]. Other additions ber of histone PTMs exceeds astronomically the
include butyrylation, hydroxybutyrylation, number of total histones that can exist in a unique
1. Introduction 41
configuration, the exploitation of the unimagi- chromatin proteins are normally part of mac-
nable number of possible epigenomes a single romolecular complexes. To fulfill the combi-
cell can adopt may still need quantic computing natorial readout requirement of the epigenetic
power that is yet to come. code, only a few subunits have to bear at least
In 2000, Brian Turner [22] early on posited one module reader. Chromatin-targeting com-
that histone acetylation on different residues plexes with such characteristics are known to be
may configure an epigenetic code that is recog- widespread.
nized by specific protein readers. In the same In summary, the dynamic nature of all
year, this hypothesis was further extended by chromatin-associated processes requires the
Strahl and Allis under the name of the histone coordinated interplay between writers, eras-
code hypothesis [23], stating that a histone mod- ers, and readers to drive forward the process.
ification can either enhance or impede another While drugs targeting some epigenetic erasers
giving rise to specific combinatorial configura- have been approved by the US Food and Drug
tions to histone tails which are recognized by Administration (FDA) to be used in antitumor
specific module-containing proteins. However, treatment, and others are in clinical trial, a mile-
the finding that different reader modules can stone has been achieved by targeting a chro-
recognize the same histone PTM or the same matin reader to treat cancer [29,30], namely,
reader module can recognize a PTM regardless the BET protein family (bromodomain and
of its sequence context casted a serious shadow extraterminal: BRD2, BRD3, BRD4, and BRDT
on its validity [24,25]. Moreover, readers have (bromodomain 2/3/4/testis-specific, respec-
very low affinity to their target(s) when singu- tively)), which shares a common structural
larly considered in vitro, which contrasts with configuration featuring tandem bromodomains
their anticipated specificity in a chromatin con- at their N-terminus. The BET proteins play a
text [26]. This hypothesis has been conceptually fundamental role in transcriptional elongation
reformulated to accommodate further players and cell-cycle progression by regulating the
and to gain wider perspective [27]. The ratio- transcription of key oncogenes. Recurrent trans-
nale is that different modules combine to create locations involving BRD3/4 are associated with
diverse interfaces of recognition, which confer the aggressive form of nuclear protein in testis
increasing specificity. The thermodynamics of (NUT)-midline carcinoma (NMC) [31]. NMC is
this process is favored because it engages more primarily a chromosomal rearrangement involv-
interactions. Thus, the readout of histone PTMs ing the fusion of most of the coding region of
is combinatorial and multivalent, and explains NUT protein located on chromosome 15q14 with
per se the logics underpinning the spatial and BRD4 (75% of cases) located on chromosome
temporal coordination observed in vivo on all 19p13.1 or BRD3 [32]. This rearrangement results
chromatin-templated processes. in aggressive forms of carcinomas. Targeting the
The findings of protein domains, which inter- BET bromodomains has proven to be promising
act specifically with definite histone modifica- against NMC, as well as in other hematological
tions and with methylated cytosine (readers), malignancies (reviewed in Ref. [29]).
support the existence of an epigenetic code.
Moreover, an early study provided a mechanis- 1.2.4 MicroRNAs and Long
tic workflow of how the epigenetic code dictates Noncoding RNAs
the directionality to the chain of events during Epigenetic regulation is also mediated by
transcriptional activation [28]. Chromatin pro- noncoding RNAs (ncRNAs) which are func-
teins bearing distinct combinations of epigenetic tional, but not protein-coding RNAs. In gen-
module readers have been described. Moreover, eral, ncRNAs can regulate gene expression at
the transcriptional and posttranslational level. genome, thereby modifying chromatin states
Those ncRNAs include microRNAs (miRNAs) and influencing gene expression [43].
and long noncoding RNAs (lncRNAs), among
others. miRNAs are a large family of short RNA
molecules, with an average size of 17–25 nucleo- 2. EPIGENETICS AND
tides [33]. lncRNA transcripts are longer than METABOLISM INTERSECTION
200 nucleotides and poorly conserved. lncRNAs
are transcribed from all over the genome, includ- 2.1 Relevant Genes in the Methionine
ing intergenic regions, domains overlapping Cycle Contribute to Epigenetic
one or more exons of another transcript on the Regulation
same strand (sense) or on the opposite strand
(antisense), or intronic regions of protein-coding The methionine cycle and transsulfuration
genes [34]. pathway have special relevance in the con-
ncRNAs contribute to the dynamics of the trol of S-adenosylmethionine (SAM), which
epigenome, so they are key players in the is the most important methyl donor for DNA
regulation of gene expression and cellular methyltransferases (DNMTs) and histone
activity, and both miRNAs and lncRNAs play methyltransferases (HMTs) (Figure 1). In the
a role in heterochromatin formation, histone methionine cycle, the methylenetetrahydro-
PTMs, DNA methylation, and gene silencing. folate reductase gene (MTHFR) is the enzyme
miRNAs are involved in several biological that reduces 5,10-methylenetetrahydrofolate
processes, such as cellular development, dif- to 5-methyltetrahydrofolate, necessary for the
ferentiation, apoptosis, cell proliferation, and transference of a methyl group to the methio-
cancer [35,36]. These small RNA molecules nine. Mutations in the MTHFR gene produce
regulate gene expression mainly by base- low levels of SAM [44]. Genetic polymorphisms
pairing to the 3′-untranslated region (3′-UTR) in the MTHFR gene results in high homocyste-
of target mRNAs, although evidence suggests ine (HCys) levels and low methionine produc-
that miRNAs can also bind to coding regions or tion, which in turn may lead to accumulation of
to the 5′-untranslated region (5′-UTR) of some S-adenosylhomocysteine (SAH) and low levels
mRNAs [37,38]. miRNAs can downregulate of SAM in human population [45]. The low lev-
gene expression by inhibiting mRNA transla- els of SAM and accumulation of SAH alter the
tion or by degradation of mRNA molecules, as SAM/SAH methylation ratio inducing global
well as increasing mRNA translation of some hypomethylation of DNA and histones [45],
targets [39]. which contribute to genetic instability and
The role of lncRNAs in epigenetic regula- chromosome aberrations [46]. Vitamin B12
tion is dual because they can serve as scaf- and SAM/SAH are essential in regulating the
folds for chromatin-modifying complexes and activity of the methionine synthase (MS) and
they can guide other epigenetic machinery to it has been found that a deficit in MS produces
the target site of the chromatin [40,41]. It has alterations in genome-wide methylation [47],
also been reported that some lncRNAs show so affecting the epigenetic program.
enhancer-like activity, and they regulate alterna- The transsulfuration pathway is a very impor-
tive splicing and other posttranscriptional RNA tant pathway for the production of cysteine
modifications [42] that determine the activity (Cys)-containing amino acids and the synthesis
of our genome. Many lncRNAs act by form- of glutathione, one of the most important mam-
ing complexes with chromatin-modifying pro- malian nonenzymatic antioxidants (Figure 1).
teins and recruiting them to specific sites in the The transsulfuration pathway involves the
2. Epigenetics and Metabolism Intersection 43
FIGURE 1 Diagram showing the methionine cycle, transsulfuration pathway, and glutathione synthesis (GSH) path-
way and its connection to epigenetic metabolites. Abbreviations in the diagram: B6 and B12, vitamins B6 and B12, respec-
tively; BHMT, betaine homocysteine methyltransferase; CBS, cystathionine β-synthase; CTH, cystathionase; DNMTs,
DNA methyltransferases; GCLC and GCLM, catalytic and modulatory subunits of γ-glutamylcysteine synthetase; GGT,
γ-glutamyltranspeptidase; GLS, glutaminase; GNMT, glycine N-methyltransferase; GSS, glutathione synthetase; HMT, his-
tone methyltransferases; MAT, methionine adenosyltransferase; MS, methionine synthase; SAHH, S-adenosylhomocysteine
hydrolase; and THF, tetrahydrofolate.
interconversion of Cys and HCys, through the disease called cystathionine β-synthase defi-
intermediate cystathionine. The production of ciency (ICD-10 E72.1; ORPHA394) that produces
homocysteine through transsulfuration allows an accumulation of HCys. Hyperhomocystein-
the conversion of HCys to methionine, which as emia induces oxidative stress, which has been
described above is catalyzed by MS [48]. Follow- postulated as a mechanism that produces hypo-
ing the steps of the transsulfuration pathway, methylation of several substrates [49,50] includ-
HCys is transformed into cystathionine by CBS ing DNA [51]. In patients with cystathionine
(cystathionine β-synthase) (Figure 1). Mutations β-synthase deficiency, the levels of HCys, SAM,
in the CBS gene are found in patients with a rare and SAH were significantly elevated compared
Another random document with
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La Sicilia era così venuta tutta omai in potere de' Romani e,
compiuta la conquista, si attese a darle uno stabile assetto. M.
Valerio Levino che avea compiuto l'impresa di guerra, attese
anch'esso ad esaurir questo compito.
Già, da quando alla fine della prima guerra punica la Sicilia era stata
costituita in provincia, avea dovuto esserle dato un particolare
ordinamento, ma è ignoto a noi quale precisamente si fosse. Che vi
venisse mandato uno speciale annuo governatore sin dal principio, lo
dice Appiano [196], ma quale speciale grado egli avesse, non par
chiaro. Un pretore, no di certo, quantunque tale apparirebbe dalla
parola usata dallo storico greco (στρατηγός); giacchè è noto che
solo tra il 525 ed il 529, verosimilmente nel 527 [197], si portò a
quattro il numero de' pretori, per destinarne uno al governo della
Sardegna ed un altro a quello della Sicilia; ed il primo, di cui si sa
che andò in tale qualità a reggere la Sicilia, fu C. Flaminio [198]. Sino
a quel tempo fu dunque uno dei due pretori in carica ad
amministrarla direttamente, o l'amministrò una persona eletta da un
d'essi? o eletta dal popolo [199]? e fu un questore [200], o un
praefectus iuri dicundo [201]? Ecco tante domande, le cui risposte
possono poggiare soltanto sopra più o meno fondate induzioni.
Del resto le condizioni anormali e lo stato di guerra in cui la Sicilia si
trovò ancora avvolta dopo il 527, fecero sì che consoli vi prendessero
la direzione della guerra, ed allora i pretori, destinati al suo governo,
ebbero limitata ad una zona più ristretta la loro attività e dovettero
subordinare il loro imperio a quello del magistrato superiore [202]. In
virtù di questo suo imperio M. Claudio Marcello [203], espugnata
Syracusae, nel ricevere le legazioni di altre città, ne determinò in
varia maniera la condizione, prendendo consiglio dall'attitudine da
esse tenuta. Ma ordinata definitivamente non avea potuto essere la
Sicilia, già neppur tutta riconquistata e sgombra de' Cartaginesi; e,
poichè li ebbe scacciati anche da Agrigentum [204] ed ebbe
ricondotto l'ordine in tutta l'isola, di nuovo in gran parte turbata e
fatta ribelle dopo la partenza di Marcello [205]; fu M. Levino (210 av.
C.) che dette alla provincia il suo stabile assetto, la sua forma; e, per
chi volesse accettare l'ingegnosa interpretazione fatta da un
autore [206] di un noto passo di Livio [207], quell'assetto rimase poi
immutato sino a' tempi di Verre e di Cicerone. Tuttavia noi
dell'ordinamento di Levino abbiamo così poche notizie e tanto
generiche, che non ce ne consentono una conoscenza ampia e
sicura; e poichè, per giunta, qui si parla dell'amministrazione della
Sicilia solo in via d'introduzione al processo di Verre e per quel tanto
che giovi a meglio intendere e chiarire quel fatto storico, non è
possibile, e non importa neanche molto, trattar qui alcune quistioni
d'ordine assai speciale, che esigono una trattazione indipendente.
Tra il governo di Levino e quello di Verre nuovamente fu sconvolta la
Sicilia, non più da guerre esterne, ma da un moto intestino, dalle
due guerre servili di Euno e di Atenione; e, chiusa che fu la prima di
esse, secondo la saggia politica romana che non si preoccupava
soltanto di vincere, ma ancor più di assicurare i frutti della vittoria,
non s'affidava soltanto all'opera breve e violenta delle armi, ma
ancora a quella più lenta e durevole di proficui ordinamenti; dieci
legati furon dal senato mandati in Sicilia perchè insieme al Console
Rupilio, vincitore della ribellione, componessero le cose di Sicilia; ed
è la Sicilia, quale uscì riordinata dalle mani di questi suoi legislatori,
che noi conosciamo relativamente un po' meglio, grazie a Cicerone;
ed è quello che anche a noi importa meglio sapere per il nostro
argomento.
A che cosa precisamente si limitasse o si estendesse l'opera di
Rupilio e de' dieci commissari, non possiamo in maniera certa trarre
dalle fonti che ce ne danno notizia [208]. Per quanto è possibile
dedurne, l'opera loro dovette principalmente rivolgersi a regolare la
vita giuridica ed economica, sopratutto nell'intento di allontanare
quelle cause, che aveano prodotta la prima guerra servile e, non
molti anni di poi, benchè per periodo più breve, fecero riardere la
seconda.
Secondo un'ipotesi già accennata, potè ben darsi che ne' privilegî
delle varie città, nella loro condizione rispetto a Roma non venissero
introdotte mutazioni, od almeno mutazioni notevoli.
Non si dovea trattare, infatti, per la natura della guerra, di punire o
premiare città, che avessero parteggiato a favore o contro di Roma,
ma piuttosto di riordinare i rapporti interni, ed eliminare sopratutto
ogni forte ragione di malcontento, e temperare i danni dell'economia
servile e scongiurare o frenare i moti minacciosi che ne derivavano.
Ma, che che sia di ciò, come ultime venute nel tempo, sotto il
periodo della Repubblica, per quel che poterono innovare e per
quello che confermarono, per quello che largirono e per quel che
riconobbero, le leges Rupiliae (Lex Rupilia, leges Rupiliae, decretum)
valsero come la carta fondamentale della vita pubblica siciliana.
Le città di Sicilia.
Le città di Sicilia possono calcolarsi, anche nel periodo della

Repubblica, a sessantotto [209].
Il numero di sessantasette, mentovato da Diodoro [210], sale a
sessantotto se vi si aggiunge, come bisogna aggiungervi, Syracusae,
ed allo stesso computo si giunge con Livio [211], considerando, come
sembra riesca evidente, che nel numero di sessantasei, cui accenna,
non ha potuto comprendere Syracusae ed Agrigentum, della cui
espugnazione ha subito prima dato conto. Similmente in niente
contrasta a tutto questo la menzione fatta da Cicerone [212] di
centotrenta censori, eletti, in numero di due per ciascuna, da
sessantacinque città; giacchè niente toglie (ed anzi la relazione di
que' tratti col contesto lo suggerisce) che siano state lasciate fuori le
tre città federate.
Di queste sessantotto città, tre -- Messana [213], Tauromenium [214] e
Netum [215] -- erano città federate; cinque -- Centoripae, Halaesa,
Segesta, Halicyae e Panhormus -- benchè senza trattato, erano
libere ed immuni da tributi (liberae et immunes); trentaquattro
comuni -- quelli degli Agyrinenses (Cic. in Verr. A. S. III, 27, 67),
Aetnenses (id. 44, 104), Acestenses (id. 36, 83), Agrigentini (id. 43,
103) Amestratini (39, 88), Apollonienses (43, 103), Assorini (l. c.),
Catinenses (l. c.), Calactini (43, 101), Coephaloeditani (43, 103),
Citarini (l. c.), Capitini (l. c.), Entellini (l. c.), Enguini (l. c.), Gelenses
(l. c.), Hennenses (42, 100), Heraclienses (43, 103), Haluntini (l. c.),
Helorini (l. c.), Herbitenses (32, 75), Hyblenses (43, 102),
Imacharenses (43, 100), Inenses (43, 103), Ietini (l. c.), Leontini
(44, 104), Mutycenses (43, 101), Menaeni (43, 102), Murgetini (48,
103), Petrini (39, 90) Soluntini (43, 103), Scherini (43, 103),
Thermitani (42, 99) Tissenses (38, 86), Tyndaritani (43, 103) --
erano città decumanae: le rimanenti città erano censoriae. Il numero
preciso ed il nome di queste ultime non ci sono stati tramandati; ma,
per via indiretta [216], è sembrato poterne fissare il numero a
ventisei, anche per argomento di una notizia già citata di Livio [217];
e, quando si ritenessero prive di valore le critiche fatte all'indice
pliniano, riescirebbe molto ovvio averne anche il nome, detraendo da
quell'elenco le quarantadue città di altra categoria già menzionate.
In tal caso le ventisei città censorie dovrebbero riconoscersi in quelle
di Megara, Syracusa, Camerina, Lilyboeum, Mytistratum, Acrae,
Bidis, Drepanum, Ergetia, Echetla, Eryx, Herbessus, Hadranum, e
quelle degli Iponenses, Macellini, Naxii, Noini, Parapini, Phintienses,
Semelitani, Selinuntini, Symaetii, Talarienses, Triocalini, Tyracinenses,
Zanclaei [218].
La condizione delle città.
Questa varia classificazione delle città siciliane era stata determinata

da criterî di governo, tendenti a rompere ogni unità di consenso ed
ogni formazione di un maggior senso di solidarietà nella regione, da
considerazioni di opportunità rispondenti alla maggiore o minore
importanza militare ed economica delle varie città, e, come i fonti,
anche frammentarî ed incompleti, sono in grado molte volte di
dimostrare specificatamente [219], avea avuto la sua causa
immediata e la sua base nel diverso atteggiamento tenuto dalla città
durante le due guerre puniche, e nel modo, come erano venute
all'amicizia od in potere de' Romani, e ne' diversi servizî che
potevano ancora rendere [220].
Città federate ed indipendenti.
Le tre città foederatae, congiunte a Roma da un foedus, e
materialmente comprese ne' termini della provincia, legalmente ne
erano fuori, e teoricamente formavano come tre distinti stati
rimpetto a Roma, con completa autonomia politica e legislativa e
proprietà esclusiva del suolo del loro territorio rimpetto a tutti.
Messana nondimeno avea l'obbligo di fornire una nave oneraria al
popolo romano, la qual cosa, agli occhi di Cicerone, era quasi come
un'impronta di servitù (quasi quaedam nota servitutis) [221]. Che
quest'obbligo si estendesse anche alle altre due città foederatae ed
alle liberae sine foedere, è sembrato a qualcuno [222], ma in realtà
nelle orazioni di Cicerone, e negli stessi tratti citati a documento (Cic.
in Verr. AS. IV 9, 19; 67, 150; V, 17, 42; 20, 51; 24, 60), si fa
riferimento sempre a Messana. Altrove (Cic. in Verr. AS V, 19, 50) si
dice espressamente che Tauromenium era stata esentata da
quest'obbligo; e quindi, anche a voler ritenere inerente al foedus
l'obbligo di contribuire la nave, non si sa se l'altra città ne fosse
esentata; e in ogni modo nulla se ne può dedurre riguardo alle città
liberae et immunes sine foedere.
Le città libere ed esenti da tributo, benchè non in virtù di trattato
(liberae et immunes sine foedere), materialmente erano nella stessa
condizione delle alleate, e la differenza vera tra le une e le altre era,
anzichè nella loro posizione, nel fondamento di queste, che, non
risultando da contratto ma da concessione unilaterale, era
necessariamente precario.
L'immunità poi loro concessa, se questa condizione non era limitata
alla sola Halicya, non rifletteva tutto il territorio, ma il territorio
coltivato da cittadini de' comuni immuni; sicchè gl'incolae pagavano
il tributo, se coltivavano parte di quel suolo (AS. III, 40, 91). Ciò
spiega e dimostra alla sua volta la natura d'imposta insieme
personale e fondiaria della decima in Sicilia (Voigt, op. cit. II, p. 403,
n. 475).
A queste, come alle città alleate, era stato imposto, dal 73 a. G. C.
con la lex Cassia Terentia, come obbligo straordinario, la provvisione
del frumento commesso da Roma e pagato ad un prezzo dallo stesso
committente fissato a tre sesterzî il modio per la seconda decima che
davano le città decumane, ed a tre sesterzî e mezzo per l'altro che
veniva indistintamente prelevato da tutte le città [223].
Città decumane.
Dalle trentaquattro città decumane il Governo di Roma prelevava la

decima di tutti i prodotti agricoli, frumento, vino ecc., e da questo
sistema d'imposta veniva ad esse la definizione del loro stato legale.
Il suolo, o meglio il possesso di esso [224] era stato loro lasciato, o
reso, come si amò meglio dire in seguito, riassumendo
nell'elaborazione di un concetto giuridico riflesso questo stato di
cose [225]; e il tributo imposto era come il segno dell'alto dominio di
Roma e ne alimentava l'erario. Queste decime, non esatte
direttamente dallo Stato, venivano locate a' pubblicani, od anche alle
città che avevano parimenti facoltà di concorrere. Tutto il frumento
così esatto o comprato poteva ammontare per la prima decima a tre
milioni di modii, ad altrettanto per la seconda e per l'imperatum ad
ottocentomila modî; in tutto 6,800,000.
Città censorie.
Restava il reddito delle città censorie. Quanto alle città censorie il

loro suolo era più propriamente comparabile all'ager vectigalis. Di
questo suolo delle città censorie dice Cicerone [226] che, essendo
divenuto suolo pubblico del popolo romano, fu nondimeno loro
restituito (illis est redditus), e soggiunge: questo territorio suole
esser locato da' censori. E ad evitare ogni contraddizione tra l'una e
l'altra cosa, occorre bene ammettere che la locazione avesse per
oggetto il tributo (vectigal) imposto a' detentori di campi, non già i
campi stessi [227]. Il tributo del suolo censorio si locava a Roma,
mentre quello decumano si locava presso i questori di Lilybaeum e di
Syracusae; ma sarebbe un errore volere limitare a questo la
differenza reale delle due categorie.
L'attribuzione a due diverse competenze indicava meglio la
distinzione di suolo semplicemente provinciale e suolo più
propriamente e direttamente pubblico; ma la distinzione maggiore
doveva consistere nell'entità del tributo che, mentre per l'agro
decumano era proporzionale e limitato ad un decimo del prodotto
secondo la lex Hieronica, per l'agro censorio invece era qualche cosa
di determinato [228], senza che noi siamo in grado di dire a quanto
ammontasse.
La differente sede e la diversa competenza della locazione delle
decumae e del tributo dell'agro censorio, non fu talvolta con rigidità
osservata. Infatti non essendovi stati censori dal 673 al 683 di Roma,
la locazione venne fatta da' consoli e nel 679 il Senato [229] permise
a' consoli di locare anche le decime de' vini, olii e de' cereali di
minore importanza, che regolarmente avrebbero dovuto essere
locate da' questori in Sicilia. Che poi, durante l'amministrazione di
Verre in Sicilia (681-3), a' questori di Sicilia ricadesse anche la
locazione dell'agro censorio, è un errore in cui, mi pare, il Voigt [230]
sia caduto, considerando come censorio il territorio di Leontini,
ch'era invece decumano [231].
Altri tributi.
Si ha inoltre notizia da Cicerone [232] di un tributo, che sarebbe stato

pagato da tutti i Siciliani, in proporzione dell'avere, a norma de' censi
fatti da due censori nominati per ogni città. Ma la menzione di
questo tributo, dato in maniera indiretta, trattando della creazione
dei censori, è la più incompleta ed oscura. Che fosse un tributo
dovuto all'erario romano, sembra da escludere, tenuto anche conto
che delle accuse fatte a Verre e che riguardano ogni lato del suo
governo, alcuna non ve n'è che abbia relazione a questo. Ed inoltre il
fatto che l'elezione di questi censori era lasciata alle città (Cic. in
Verr. AS III, 53, 131), l'osservazione che il censo fatto da questi
censori creati da Verre fu tale che la cosa pubblica di nessuna città
poteva amministrarsi con esso (Cic. l. c. 55, 138), e la
considerazione che due censori per città, nel numero di centotrenta,
importano l'inclusione delle civitates liberae et immunes; tutto induce
a ritenere che questo tributo avesse effetto ed impiego tutto locale.
Infatti, un'amministrazione comunale ivi sussistente, servizî pubblici,
polizia, non possono intendersi senza una finanza comunale.
I redditi de' pascoli, se questi rientravano nel suolo censorio, si
ottenevano per mezzo di pubblicani [233]; se erano compresi nel
territorio decumano, ignoriamo qual sistema si tenesse. Oltre a
questi tributi diretti, vi era il tributo indiretto, che gravava in genere
su tutto quanto fosse esportato dalla Sicilia, senza distinzione della
persona cui appartenesse, e consisteva nella vicesima, il cinque per
cento del valore della merce, che sotto nome di portorio si esigeva
come dazio di esportazione [234].
Altri redditi dovevano pure venire da altri cespiti ed imposte
indirette, in tutto sei [235], secondo una lettura del testo di Cicerone,
ed in cui erano forse compresi diritti sulla pesca, sul sale e
l'estrazione de' metalli [236].
Ordinamento locale.
Ma, mentre da una ad un'altra di queste città v'erano così notevoli

differenze nella posizione verso Roma e nella misura de' tributi; tutte
in genere avevano una loro organizzazione comunale, con magistrati,
senati, censori, funzionarî locali.
Si ha menzione del senatus non solo di comunità privilegiate e
conosciute come foederatae, liberae immunes, quali Messana,
Segesta, Halaesa, Centoripae, Panhormus [237], ma anche di
decumanae [238] e perfino di censoriae [239], e di oppida miserrima et
desertissima [240], con regolari manifestazioni sotto forma di
legationes, mandata, postulata, litterae, testimonia [241]. E col senato
naturalmente vi erano del pari magistrati di ordine vario, cui erano
affidate le attribuzioni esecutive.
Si hanno così i proagori [242], i censori [243], di cui abbiamo fatto
cenno, nominati due per città, edili e questori [244], e poi molteplici
altri funzionarî di vario ordine e di varie attribuzioni come
τριακάδαρχοι, σιτοφύλαχοι [245] ginnasiarchi [246] oltre ad altri uffici
subordinati, come quelli di ὐπογραφεῖς, ὑπηρέτας, κάρυξ [247].
Accanto a questi uffizî civili vi erano poi i sacerdozî [248], i quali
alcune volte, come a Gela, ad Agrigentum, a Melitta, a Segesta
(ἱεράπολος o ἱέροθύτης) [249] compariscono come eponimi, ed a
Syracusae (άμφίπολος Διὸς Ὀλυμπίου) [250] veniva adoperato il nome
del sommo sacerdote a controsegnare gli atti pubblici e gli anni.
Ricorre anche la menzione de' decemprimi [251] quinqueprimi [252],
che, indicati così distintamente e forse in contrapposizione a'
magistrati, denotano probabilmente, più che altro, un grado di
anzianità e di maggiore importanza di alcuni senatori.
Sistemi d'elezione.
La nomina a questi diversi uffici aveva luogo in modo diverso.

La loro creazione era regolata da norme spesso diverse da una ad
un'altra città, norme spesso date da magistrati romani, anche per
facoltà avuta dal senato; come quelle date da C. Claudio Pulcro e da
C. Marcello (95 a. C.) ad Halaesa, che spontaneamente ne faceva
richiesta [253], da Scipione (207 av. C.) e da P. Rupilio (132 a. C.) nel
dedurre coloni ed Agrigentum ed Heraclea [254].
Per tali regolamenti ad Halaesa era stabilita a trenta anni l'età per
entrare in Senato ed erano dati altri provvedimenti circa la capacità,
il censo ed altre cose [255]: ad Agrigentum e ad Heraclea era
provveduto a che vecchi cittadini e nuovi coloni avessero una uguale
rappresentanza nel senato locale [256].
Niente conosciamo del numero de' senatori: nella discussione non vi
era ordine rigoroso, ma per consuetudine parlava prima il più
vecchio, e, tacendo tutti, si traeva a sorte il nome di qualcuno, e
quegli era chiamato a parlare [257]. Un indirizzo democratico non era
in genere favorito de' Romani, ma in città predilette, come
Centoripae (Cic. in Verr. AS II, 68, 163), vi è accenno a deliberazioni
popolari dirette.
I senatori venivano creati, sembra, per cooptazione [258]; i censori
invece per mandato diretto del popolo [259]; e per voti ed in comizî
sappiamo anche più chiaramente ch'erano eletti i sacerdoti [260]. In
Syracusae la scelta era limitata a tre, non tribù, come qualcuno
vuole [261], ma schiatte (ex tribus generibus) [262].
Tutte queste elezioni, riferendosi ad amministrazioni di ordine
interno, avrebbero dovuto restar indipendenti dall'azione del
governatore della provincia; ma il diritto di veto, d'intercessio ch'egli
avea, e che, naturalmente, trovava la sua ragione d'essere nella
tutela della sovranità romana, lo metteva anche in grado di esercitar
tali azioni, tali abusi da mettergli in mano come la facoltà stessa
della nomina [263].
Le leges Rupiliae.
Il decretum di Rupilio poi, quello che si potrebbe dire la Charta

largita alla Sicilia, avea inteso soprattutto a regolare la vita giuridica
e giurisdizionale della Sicilia. Ciò ne costituiva, sembra, la parte
precipua, od almeno quella di cui, relativamente, noi abbiamo
maggiore notizia.
Quanto alle norme giuridiche imperanti in Sicilia, noi non abbiamo
conoscenza del loro contenuto e del modo, onde ciascun rapporto
era regolato.
Le notizie che abbiamo intorno alle riforme legislative di Diocle nel
339 a. u. c.=416 a. G. C., di Cephalo nel 412=342 sotto Timoleone,
e di Polydoro sulla fine del V secolo sotto Jerone [264], sono notizie
giunte a noi in forma molto indeterminata: di Diocle legislatore è
dubbia fino l'identificazione [265].
Quello invece che sappiamo, è che in Sicilia la vita giuridica
funzionava sotto l'impero dello statuto personale.
I Siciliani aveano le loro leggi [266], che erano speciali di ciascuna
città e rimasero intatte a regolare i loro rapporti reciproci. Vigeva poi,
d'altra parte, riguardo a' cittadini romani o per quella parte che fosse
stato imposto, il diritto romano [267], ed accanto all'uno e l'altro
diritto particolare vi è accenno a' iura communia [268], pe' quali
sembra doversi intendere: iura gentium, quel diritto delle genti, che
serviva di complemento all'uno ed all'altro.
Vi erano insomma tutti quegli elementi dalla cui fusione si
apprestava ad uscire, come poi uscì, il diritto romano quale noi lo
conosciamo.
La lex Rupilia, o meglio decretum Rupilii, per quel tanto che noi ne
sappiamo, avea sopra tutto atteso a riconoscere direttamente od
indirettamente la validità e l'applicabilità e regolare la coesistenza e
l'applicazione di questi varî principî o statuti, specialmente
determinando la competenza e la costituzione dell'autorità
giudicante.
Il diritto giudiziario in Sicilia veniva così a riposare su questi
principî [269]. Il giudizio di un Siciliano con un Siciliano avea luogo
nella comune loro città, ed era deciso secondo le leggi, cui entrambi
obbedivano; nel giudizio tra due Siciliani di città diverse, il pretore
dava esso i giudici secondo le norme del decreto di Rupilio; nella
causa di un privato con una città era costituito a giudice il senato di
una città estranea a' due litiganti. Nelle cause tra Siciliani e cittadini
romani, veniva dato un giudice della nazionalità del convenuto e non
dell'attore: romano quindi se la causa era promossa da un Siciliano;
siciliano se la causa avea luogo ad istanza di un Romano.
Per le restanti cose dice Cicerone, erano scelti giudici dal
conventus [270] de' cittadini Romani; espressione che, usata
dapprima per indicare i cittadini romani che convenivano in un luogo
per l'esercizio delle loro azioni giudiziarie, acquistò un significato più
specifico, secondo alcuni di universitates civium Romanorum,
residenti (consistentium) in città le quali non godevano della
cittadinanza romana [271] e secondo altri di diocesi giuridiche [272].
Che cosa si dovesse intendere per ceterae res, non è ben chiaro:
parrebbe dovesse intendersi di cause non promosse da un privato;
ma deve voler avere un significato più comprensivo, nell'intento di
abbracciare ogni altro caso non preveduto.
La lex Hieronica.
Un'altra menzione vi era finalmente ed era che i giudizî tra decumani

ed agricoltori aveano luogo secondo la lex frumentaria chiamata
Hieronica [273].
La lex Hieronica ha la massima importanza nelle Verrine e la sua
compiuta conoscenza sarebbe capitale per istudiare tutto quanto
concerne Verre.
Ma quanto è grande il bisogno che noi abbiamo di un'estesa ed
esatta conoscenza di quella legge, altrettanto è grande la scarsezza
di dati per tutto quanto ad essa si riferisce. Noi non sappiamo
nemmeno con precisione a chi riferirla, se a Jerone I od al II; ed
anzi lo stesso nome può per qualcuno [274] non valere come un
indizio sicuro dell'autore della legge, tenuto conto che i riformatori
delle leggi di Diocle non furono nè Jerone, nè Timoleone, ma
Cephalo e Polydoro.
Il dritto di prelevare una decima parte de' prodotti del suolo può farsi
risalire ben alto nella storia de' tiranni siracusani: anche in Atene
Pisistrato prelevava una decima [275]. Ma come va spiegato questo
suo particolare appellativo e questa sua estensione a trentaquattro
città di ogni parte della Sicilia; e si deve dire a tutta la Sicilia,
considerando che quella delle città censorie fu una condizione resa
deteriore del loro particolare atteggiamento verso i Romani, durante
la guerra?
Pur facendo risalire il sistema d'imposizione allo stesso Gelone, fu
forse Jerone I, che lo estese a buona parte della Sicilia e gli dette il
nome con l'estendere il suo potere ed il suo credito?
Ovvero, in un ultimo rimaneggiamento avvenuto sotto Jerone II,
prese il nome da costui, e la sua legge tributaria venne indi da'
Romani assunta a base del loro sistema d'imposizione nella fusione
delle due circoscrizioni prima distinte e contrapposte di Lilybeo e di
Syracusae e nella costituzione della provincia di Sicilia allargata?
Senza documenti, senza dati, senza possibilità d'induzioni sicure, son
semplici ipotesi queste, che hanno un valore tutto relativo.
Ma la lex Hieronica, come la troviamo menzionata nelle Verrine [276],
non era più semplicemente una legge tributaria, bensì anche una
legge giudiziaria. Com'è cosa tutt'altro che infrequente nell'antichità,
la legge che stabiliva un rapporto giuridico, essa stessa, come ad
integrarlo, determinava i mezzi di esecuzione, la sanzione della sua
infrazione e tutta la procedura degli annessi giudizi.
Se non che nella lex Hieronica, quale Cicerone ce la mostra,
ricorrono i recuperatores, il vadimonium, il conventus, e se queste
parole, come pare, debbono essere prese nel loro significato tecnico
e non come espressione approssimativa di istituti giuridici stranieri,
ne viene che la lex Hieronica, nella forma approssimativamente a noi
nota, più che un'impronta greca, quale conformemente al suo nome,
dovrebbe avere, ha una fisonomia prevalentemente romana. E tutto
ciò trova la sua spiegazione, quando si consideri che la lex Hieronica,
accolta nel suo nome e nel suo fondamento di legge tributaria,
venne sotto il dominio romano, trasformata ed ampliata in una legge
giudiziaria [277]. Quale fosse tutto l'ordinamento giudiziario voluto
dalla legge, è dubbio, oltre che nelle particolarità, anche ne' suoi
tratti generali, tanto che è stato possibile ad alcuni scrittori
considerare la lex Hieronica semplicemente come tributaria [278],
mentre altri ne limitano l'efficacia alla semplice costituzione
dell'autorità giudicante [279] ed altri le mette altre arbitrarie
limitazioni, circoscrivendo le sue disposizioni procedurali soltanto alle
controversie riflettenti le denunzie delle coltivazioni (professiones) ed
il pagamento della decima (pactiones) [280].
Il contenuto delle regole di vita giudiziaria contenuto nella lex
Hieronica è difficile a sapere, perchè non se ne hanno notizie dirette,
ma invece tutto quello che se ne può sapere, bisogna dedurlo dalle
violazioni che Verre, secondo Cicerone, ne avrebbe fatte durante la
sua amministrazione in Sicilia.
Sembra [281] dunque che a dirimere le controversie tra decumani ed
aratores, sotto il quale appellativo vengono compresi possessori del
suolo e coltivatori diretti, la lex Hieronica avesse disposto la
costituzione di un collegio giudicante, a comporre il quale erano
chiamati aratores e negotiatores, non si sa se per giudicare sotto
forma di tribunale misto, o alternandosi con vicenda a noi ignota.
Probabilmente delle persone abilitate all'esercizio di questo potere
giurisdizionale era redatto un albo, che peraltro aveva piuttosto lo
scopo di agevolare il compito del pretore e non mai quello di limitare
con criteri esclusivi la libertà d'elezione. La natura delle controversie
tra decumani ed aratores, sommarie per se stesse e tali da dovere
essere sbrigate con sollecitudine, esigeva che le persone chiamate a
risolverle vi funzionassero come recuperatores; ed infatti son questi
che ordinariamente ricorrono ne' giudizî riferiti di Cicerone. Tuttavia
si deduce dal tratto relativo alla lex Hieronica [282] come questa avea
norme anche su la scelta di iudices. La distinzione netta tra la
competenza de' recuperatores e quella del judex, che è ancora
oscura nella sua classificazione generale [283], è anche qui nel caso
particolare non chiara.
Suppone qualcuno [284] che l'impiego del judex piuttosto che de'
recuperatores potesse dipendere da un accordo mutuo delle due
parti, che del resto non ricorreva se non di rado.
Un'altra delle condizioni favorevoli che la lex Hieronica, in
concorrenza con altre leggi, garentiva all'arator, era che la
competenza fosse determinata dal luogo dell'esazione [285]. La
controversia circa il pagamento della decima sorgeva come un
incidente dell'esecuzione, ed a ciò principalmente, alla connessione
ch'era tra l'una cosa e l'altra, si deve la competenza eccezionale, la
competenza del luogo di esazione; e, poichè il luogo di esazione era
d'ordinario nelle vicinanze del campo, la competenza veniva a
convertirsi in una condizione di favore per l'arator.
Del modo ora come questo giudizio si svolgeva, Cicerone
direttamente proprio si limita a dire che la legge era quale un tiranno
ed un Siciliano avevano potuto scrivere, acuta e severa e diligente,
quale si conveniva a chi non avea altri tributi da esigere [286].
Tale legge, continua Cicerone (l. c.) era fatta in modo che per essa
l'arator era assoggettato al decumano con tutte le cure convenienti
all'esazione ed in modo che diveniva impossibile all'arator frodare il
decumano con l'asportare, o rimuovere o celare il frumento.
I decumani, in fondo, come assuntori dell'esazione de' tributi,
venivano ad essere guardati con occhio benevolo dal Governo
romano, che in maniera più o meno diretta, avea interessi non
difformi da' loro. Onde, in tutto il procedimento dell'esazione de'
tributi, si era sempre più venuto insinuando un elemento, che teneva
del carattere pubblico più che del privato e che improntava di tale
fisonomia la procedura.
Le facoltà intese ad impedire l'amozione e l'asportazione del
frumento e poi la stessa esecuzione dovevano risolversi facilmente in
un diritto di pegno.
II diritto di pegno, assicurato secondo il diritto ellenico al pubblicano,
cominciò a sperimentarsi con l'assistenza del magistrato, ch'era di
guarentigia allo stesso arator. La denunzia delle colture (professio),
fatta prima dell'appalto, quando il pubblicano non aveva ancora
acquistati i suoi diritti, faceva sì che la sua violazione valesse come
una violazione di una disposizione d'ordine pubblico. Il pubblicano
aveva tre vie innanzi a sè nello esigere le decime e poteva, come
spesso accadeva [287], cedere il suo diritto d'esazione alle singole
città, che pensavano ad effettuare dal canto loro la riscossione;
poteva mettersi, procedendo direttamente all'esazione, d'accordo
con l'arator, ed allora il suo diritto veniva a poggiare su di una base
contrattuale, alla cui esecuzione era assicurata la protezione del
governatore provinciale [288]; poteva finalmente esservi disaccordo
tra decumano ed arator, ed allora avea origine una controversia,
intorno al cui giudizio si può dire che fosse un giudizio sommario,
che stesse di mezzo tra il diritto pubblico ed il privato, e, vertendo
tra persone di diversa categoria, alla imparzialità sua doveva vegliare
il governatore sopra tutto con la scelta de' recuperatores [289].
La lesione de' diritti de' pubblicani assumeva un carattere pubblico e
di qui le multe e l'ampiezza de' mezzi esecutivi.
I poteri del governatore.
Tale era l'ordinamento effettivo, su cui riposava il governo della

Sicilia; ma resta ad indicare quale fosse il valore legale di esso. Tutto
questo ordinamento, in altri termini, avea l'autorità e la forza di una
legge? Domanda interessante questa, la cui risposta ci aiuta meglio
a considerare ed a comprendere l'atteggiamento del governatore
della provincia rimpetto ad essa.
È sicuro che Cicerone tutte le volte che parla dell'ordinamento dato
sotto Rupilio alla provincia, tiene a distinguer bene che non si
trattava di una legge; e tale era chiamata solo impropriamente da'
provinciali [290].
Il Senato con l'estensione, che veniva sempre più dando alle sue
attribuzioni ed a' suoi poteri, era venuto sviluppando questa sua
facoltà e questa ingerenza nell'ordinamento provinciale, che avea
stretto rapporto con l'amministrazione stessa della provincia e con la
sorveglianza su' suoi governatori. E l'ordinamento, infatti, si veniva
per molta parte compiendo dal Senato sia sotto forma di delegazione
che sotto forma di ratifica di disposizioni adottate dagli stessi
governatori [291].
Perciò non si saprebbe rigorosamente determinare quanto tali
ordinamenti emanati dal Senato potessero ritenersi efficaci ed
obbligatori per i governatori di provincia [292], e non è perfettamente
giusto [293] sostenere che avessero interamente il valore di una
legge. Bisogna ammettere non solo che doveano cedere il luogo ad
una legge, ove questa vi era, ma ancora che «formalmente» [294] il
governatore poteva non ritenersi assolutamente stretto da quegli
ordinamenti.
A ciò si aggiunga che il potere, nella realtà legislativo, inerente al jus
edicendi [295] avea limiti tanto poco precisi, che nella sua
esplicazione, specialmente nel campo del dritto privato, si convertiva
in una vera fonte legislativa. Perciò è la cosa più ardua il voler
decidere in quanto secondo le fondamentali regole costituzionali e in
quanto anche, secondo i partiti e le necessità della pratica, poteva
ritenersi legato da quegli ordinamenti un governatore, sopra tutto in
provincia, dove la preoccupazione di raffermare od estendere il
dominio romano e quello di fondere ed armonizzare insieme i diversi
statuti personali ed i cozzanti interessi di così varî elementi dovea
imporsi al governatore e far sì che, in provincia, forse ben più che a
Roma, il suo editto fosse uno strumento di continua evoluzione del
diritto.
Le condizioni economiche della Sicilia.
E in Sicilia, anche più che altrove, codesto compito era interessante

e difficile.
La sua posizione ne avea fatto l'oggetto di contesa ed il luogo di
convegno di tutti i popoli che si erano disputato l'imperio del
Mediterraneo; e, pure decisa la lotta a favore di Roma, restavano
ancora tutte queste diverse razze, diverse consuetudini, diverse
norme giuridiche a fondere insieme, ed intanto occorreva renderne
possibile la coesistenza.
Inoltre, ruinato il ceto agricolo romano ed italico, e fattosi vorticoso il
crescere del proletariato e dato allo Stato, dopo i Gracchi,
quell'indirizzo, che metteva tra i suoi compiti l'alimentazione del
proletariato cittadino; la Sicilia, sino alla conquista dell'Egitto uno de'
maggiori granai d'Italia, avea per la vita politica ed economica, per la
sussistenza stessa di Roma un'importanza capitale. Il particolare
sistema di amministrazione della Sicilia (Cicerone non si stanca mai
di ripeterlo e di farlo intendere) era in teoria tale che l'interesse suo
e quello dello Stato dominante erano intimamente connessi. La
prosperità crescente della Sicilia voleva dire una decima più
abbondante, il frumento imperatum comperato a miglior prezzo e,
con l'aumento dell'importazione e dell'esportazione, una quota più
alta di tributi indiretti.
Ma l'ordinamento provinciale della Sicilia, per quanto Roma avesse
cercato favorirlo, per necessità di cose e di tempi, non avea punto
evitati tanti di quegl'inconvenienti, che ne impedivano il completo
sviluppo economico; e, tanto più potea essere naturale l'opera
pratica, adattata a' singoli casi, del governatore, che rendeva meno
sensibili alcune asprezze, che attenuava o s'ingegnava di eliminare
alcuni mali.
Il sistema d'impedire alle diverse parti della provincia di trovare in se
stessa un centro, a cui fortemente riconnettersi, e quello di creare
barriere tra parti e parti della provincia, per far sì che propositi di
autonomia non sorgessero, o non venissero messi in atto; erano
cose a cui i Romani non aveano saputo, nè potuto rinunziare
interamente nell'amministrazione della Sicilia. Di qui tanti di
quegl'inceppi, che non poteva fare a meno di comprimere lo
svolgimento di tutte le sue attività.
Benchè ci avvenga d'incontrare un commune Siciliae, è affatto da
escludere ogni organizzazione regionale. Tutte le città vivevano
ciascuna a sè, trovando il proprio centro, fuori dell'isola, in Roma, e,
nell'isola, in colui che ne era il rappresentante, nel governatore.
V'era un più stretto legame tra alcune città, ma erano le diciassette,
che s'erano mantenute sempre fedeli a Roma; ed i rapporti più
stretti, che le univano, aveano semplicemente carattere e scopo
religioso.
A ciò si aggiunga che la classe dominante romana avea vivo
l'interesse, chiaro il disegno di attrarre a sè, come più poteva, tutte
le fonti di ricchezza e, come meglio le riusciva, di monopolizzarle.
Di tutta l'azione di questi diversi interessi pubblici e privati, la Sicilia
sentì vivamente gli effetti e con danno della sua economia.
Se il commercium interdetto ad ogni altro sul territorio di
Segesta [296] dipendeva non da altro che dalla sua condizione di città
libera, che la facea considerare come uno stato indipendente anche
rimpetto a' Romani; quello che lo stesso Cicerone ci dice di
Agrigentum [297], mi pare possa ben dar luogo a supporre che tra
città e città si erano elevate barriere, ed era impedito o circoscritto il
commercio ai cittadini di una in un'altra città. E, pur ritenendo che la
natura del suolo provinciale, sia di città censorie che di decumane,
non ammetteva ne' detentori un diritto di dominio, non è detto che
anche il semplice trasferimento del possesso non potesse essere più
o meno limitato dal divieto o dalla mancanza d'ogni riconoscimento
giuridico. Ne nasceva allora che, posti tali inceppi, i cittadini romani,
che, come tali, aveano il commercium nelle città soggette di Sicilia,
poteano vittoriosamente fare agli altri la concorrenza.
È sicuro in ogni modo che campi ed aziende agricole siciliane
vennero in buona parte, nelle proporzioni e sotto forma di latifondi,
in mano di cittadini Romani, i quali, menandone innanzi la coltura, si
avvalevano del lavoro servile, e davano luogo ad una industria
agricola, che, diretta ad esaurire gli uomini impiegati ed il suolo,
ruinava l'agricoltura e fomentava quel malessere che divampò due
volte nelle guerre servili.
La produzione nel primo terzo del primo secolo si è fatta
ascendere [298] in tutta la Sicilia ad otto milioni di medimni (ogni
medimno siciliano è di litri 52,392) [299] di frumento e quattro milioni
di medimni di altri cereali; ma, considerando che la sola decima del
frumento delle città decumane ascendeva a cinquecentomila
medimni, potrebbe fors'anche il computo portarsi più alto [300].
Intanto, secondo calcoli che naturalmente possono accettarsi solo
come approssimativi [301], la popolazione che, tra la fine del quinto e
il principio del quarto secolo, ascendeva a circa 800000 abitanti e
appresso avea avuto un incremento e sotto Agatocle avea potuto
salire ad un milione; colla morte del tiranno avea cominciato a
decadere, e se, ristabilita la pace dopo ottanta anni di guerre, la
regione riavea sotto i Romani un periodo di relativa risurrezione
economica, era il numero degli schiavi che cresceva in proporzione
assai notevole, assai più che innanzi non fosse stato, sino a
pareggiare il numero de' liberi.
A lenire alcuni inconvenienti, a stornare alcuni mali, ad esercitare in
tutto un'azione benefica, potea giovar molto l'opera del governatore,
fornito, così com'era, di largo potere; e, del pari, se mosso da
impulsi diversi, potea recar molto male.
Di settantasei de' governatori della Sicilia, che esercitarono l'ufficio
loro prima di Verre, noi abbiamo notizia [302]; ma dell'opera ivi
spiegata da loro, specialmente quando non si riferisce ad imprese di
guerra, poco sappiamo. Di M. Valerio Levino, primo tra i governatori
dell'intera Sicilia, sappiamo bensì che cercò di svilupparne la
produzione anche artificialmente, incitando personalmente [303], alla
coltura del suolo.
In questa provincia fu mandato come governatore,
settantesimosettimo di quelli a noi noti, C. Verre.
V.
HOMO AMENS AC PERDITUS?
Chi era C. Verre?

Le Verrine.
Lo scrittore, il poeta, l'oratore, l'artista sono anche tanti giustizieri. In

questo arruffìo quotidiano della vita, mentre essi, gravati
dell'incommodo bagaglio delle loro idealità, passano assai spesso
punzecchiati, derisi, angosciati; gli uomini piccoli, che per la loro
stessa natura non perdono mai di vista la terra, s'insinuano in tutti i
meandri del multiforme inganno umano, tendono reti e stanno in
agguato, ghignando, sugl'improvvidi che vi cadono, scavano ad altri
la terra sotto i piedi; e da questo lavorio di tutti i giorni assurgono
pieni di oro e di fango, carichi di uffizî e di onori, e, tra l'incenso, che
loro sale alle nari e le maledizioni che si perdono inascoltate nell'aria,
arrivano freschi, riposati, sereni al termine della loro giornata
terrena.
Ma se essi inciampano il poeta, lo scrittore, l'artista, in un'ora della
vita in cui il loro cuore è colmo d'infinita amarezza, il poeta, lo
scrittore, l'artista, che sanno anche essere giustizieri, li acciuffano
con la mano febbrilmente potente, e sulla fronte, insolitamente
rivolta verso il cielo, imprimono come un suggello il marchio della
condanna.
Si spengono allora gli echi dolenti delle angoscie che essi
suscitarono; scompare fin la traccia delle ville sontuose e de' palagi
superbi, onde guardarono con piglio insolente sul mondo e sulla vita;
le statue, ch'essi si eressero, vanno in pezzi; ma vive ancora nel
verso, nell'orazione, nel libro il povero clandestino epigramma, che
anonimamente ronzava intorno a loro; e, raccomandati all'opera di
chi li fece eterni, passano alla posterità cinti di un nimbo d'infamia,
monumenti vivi dello stridente contrasto tra la gente che impera e
quella che langue, esempî di tanti altri, che vissero della loro vita e
godettero della loro fortuna.
Tale è la sorte di Verre. Non son molti davvero i personaggi
dell'antichità, specialmente di ordine secondario, delle cui gesta
possiamo dire di sapere tanto come di Verre. Ma tutto quello che
sappiamo di lui, sgorga da una fonte sola, e questa non è una fonte
spassionata ed impersonale; anzi è un atto di accusa, dove la
narrazione si trova incastrata tra invettive, sarcasmi, dileggi.
E, quasi che ciò non bastasse, l'accusa non è soltanto la reazione di
un'anima offesa dallo spettacolo dell'ingiustizia, ma, che che
Cicerone faccia per attenuarne l'effetto, è l'assalto di un avversario
ed un atto coordinato a tutto un piano di condotta politica e
destinato ad avere un'azione immediata sugli avvenimenti politici del
tempo.
Pure, benchè la fonte sia così unilaterale e ci manchi generalmente
la possibilità del controllo, convenientemente usata ed interpretata,
ci dà modo di formarci un concetto non incompiuto dell'uomo e del
tempo.
Chi ha qualche volta osservato l'indole e le fasi dello svolgimento de'
drammi giudiziari, come usa chiamarli, ha veduto come spesso, nelle
mani di chi accusa o difende, i fatti vengano abilmente svisati ed
affermati o smentiti secondo un preconcetto disegno. Ma tal'altra
volta i fatti son tali, o così provati, che si contende piuttosto della
loro importanza e della interpretazione a cui possono andare
soggetti, ed allora anche un'accusa od una difesa può esser un
documento storico di notevole valore. E questo sembra sia il caso
delle «Verrine». In esse per giunta troviamo riflettuto, sia pure per
farne strazio, tutto il sistema di difesa di Verre, e di tratto in tratto ci
è dato gettare uno sguardo sull'ambiente esteriore, e conoscere ciò
che si operava dietro la scena e da quali impressioni era dominata e
come si moveva la folla, che, assistendovi, partecipava anch'essa a
questo così importante episodio della vita romana.
Accanto a qualche esagerazione ed a qualche interpretazione
forzata, da alcuni particolari stessi che non hanno diretto rapporto
con l'accusa, balza fuori in qualche modo la figura dell'accusato. Da
tutto l'insieme di dati piccoli e grandi, da quella sfida cinica alla
giustizia ed all'opinione [304], da quella furberia talvolta sino
infantile [305] e da quella cieca e sventata imprevidenza, da quella
passione delle opere di arte morbosamente irresistibile [306], da
quella sua irritabilità [307] che, eccitata reagisce feroce, da quella sua
rapacità che diventa crudeltà, da quel suo furioso entusiasmo della
libidine e dell'orgia [308], e da quella sua incontinenza che non
conosce confine, si ricompone -- se tutti que' dati son veri -- una
figura di delinquente quale la scienza moderna [309] lo concepisce:
un tipo di degenerato morale, che da un lato si ricongiunge alla follia
e dall'altro presenta come un ritorno atavico dell'uomo primitivo, del
selvaggio. Tra la civiltà greca, in mezzo a cui vive, egli appare
veramente il tardo nepote de' favoleggiati compagni di Romolo,
assertori del diritto della forza, rapitori di donne, banditi; o come
un'anticipazione di quello che sarà tra un secolo Nerone, amatore
dell'arte cieco ed impotente, che vuol trasportare nella vita un suo
grande sogno di lusso e di piacere e, dominato da invincibili impulsi,
piega e spezza tutto quanto si para come un ostacolo al suo egoismo
armato del potere.
E insieme alla figura di Verre viene a galla tutto un altro gruppo di
figure, quale intraveduta appena, quale sbozzata e pur chiara; e
tutta una serie di concetti morali dominanti la vita pubblica e la
privata, e i buoni e i cattivi istinti della folla e l'ambiente stesso della
vita giornaliera de' dominatori e dei soggetti; tutto un momento
insomma della vita romana, che il processo di Verre rievoca e
ricompone intorno a Verre, fatto centro di tanti interessi, ire e
passioni, che, compenetrandosi con la sua sorte e la sua persona, le
danno una particolare importanza storica.
C. Verre e la sua famiglia.
Chi egli fosse e da quale famiglia uscisse, non parve sempre, nè a
tutti ben chiaro. Il nome romano, che ha la particolarità di
determinare così bene un uomo ne' suoi rapporti pubblici e privati,
nelle sue relazioni famigliari e di schiatta, appare in C. Verre,
piuttosto che monco, suscettibile di ambigue interpretazioni. Fu
creduto anche che Verre fosse solo il cognome, e il nome gentilizio,
per tutto taciuto, fosse Cornelio; ma è invece il contrario ch'emerge
dal testo stesso di Cicerone [310], e si può affermare con sicurezza
che quello di Verre fu un nome gentilizio [311], e, cosa del resto non
rara, egli non ebbe cognome. Nè il nome stesso ha il carattere
eccezionale, che da principio si sarebbe inchini ad attribuirgli, se può
ritenersi soltanto come una forma più antica di Verrius [312], della cui
schiatta la famiglia di Verre potrebbe così essere stata un ramo,
distratto sempre più dal tronco comune.
Vi sarebbe qualche motivo per credere che non potesse già vantare
parentele illustri e potenti [313]; in ogni modo era nobile [314]. Suo
padre, C. Verre anch'esso [315], dovea aver nome d'insigne
manipolatore di elezioni e vivea in una cerchia d'incettatori di
voti [316], incettatore egli stesso, addestrando nell'arte, perchè non si
perdesse, altri membri di sua famiglia, un Q. Verre [317] per esempio,
della tribù Romilia.
Malgrado ciò, o forse per ciò, non gli mancò un posto nel senato.
Sua madre avea comune la schiatta ed il nome con Q. Tadio [318].
Questi i congiunti che avea per parte de' suoi genitori. Il suo
matrimonio appresso lo legò con vincoli di affinità a Vezio, cavaliere
romano [319]: un legame non sappiamo di qual genere e tra chi, lo
rese anche affine de' Metelli, ma solo alla vigilia del suo giudizio [320].
Di qual famiglia fosse il genero non sappiamo. L'anno della sua
nascita ci è del pari ignoto, ma sapendo della sua morte avvenuta, e
non per causa naturale, nel 43 av. C. e che, pretore in Sicilia era
padre di un figlio presso ad uscire dall'adolescenza e di una figliola
già fatta sposa [321]; possiamo da tutto ciò argomentare che
all'epoca della sua pretura probabilmente egli doveva avere un'età
non maggiore o di poco a quella richiesta per covrire l'ufficio.
Egli dunque dovè fare con rapidità e con fortuna la sua carriera, a
cui si preparò non con pazienza di studî e nobile esercizio di vita;
ma, piuttosto, sulla scorta dell'esempio paterno e, conforme
all'indole dei tempi, emergendo nel lieto pandemonio delle gazzarre
cittadine.
Non si logorò proprio negli studî; e se Cicerone non lo calunnia
anche in questo [322], non trovò nemmeno il tempo di acquistare una
conveniente cultura, neppure quello di apprendere il greco, cosa
relativamente comune per gli uomini del suo stato. Ma Cicerone
certamente esagerò anche questa volta.
In ogni modo da adolescente si sarebbe annunziato, quale sarebbe
poi stato maturo. Cicerone fa le viste di non volersi occupare della
sua vita di adolescente; ma ciò non è che un artifizio retorico;
ricorrendo alla figura della preterizione, trova benissimo il modo di
rievocare le orgie notturne e la gioventù spesa tra lenoni, biscazzieri,
mezzani, e i buchi fatti al patrimonio paterno; quella sua prima
milizia non ricca che d'ignominie, e in cui profuse oro insieme ed
onori; tutta insomma l'impura adolescenza che ora si rinnovava nel
figliuolo [323].
La questura di Verre.
Questo stesso suo modo di vita non doveva essergli d'impedimento a

salire nelle nuove condizioni della vita romana e specialmente
nell'infuriare delle dissensioni civili; e lo vediamo infatti entrare nella
vita pubblica come questore [324] del console Cn. Papirio Carbone
nella lotta della parte mariana e sillana. Cicerone gli fa tener questa
carica quattordici anni prima della causa e, benchè, secondo quella
che ad alcuni può parere la più usuale maniera di contare, si sarebbe
aspettato che avesse detto quindici [325], riesce evidente che
intendeva riferirsi all'anno 84 a. C. [326].
Il fatto che Cicerone lo fa restare questore ancora sino al sacco di
Rimini [327], il quale non potè accadere che nell'82, e ad anno
abbastanza avanzato, ha dato origine ad una viva controversia
sull'epoca vera della questura di Verre; onde, mentre qualcuno [328]
l'assegna all'anno 84, altri [329] l'attribuisce all'anno 83, ed altri [330]
ancora all'anno 82. Che Cicerone assegnasse l'entrata di Verre in
carica all'anno 84 è fuor di dubbio e non pare lo facesse per una
svista, giacchè egli si ferma molto alle particolarità del fatto, rileva il
nome di L. Scipione, siccome quello del console succeduto a Cn.
Papirio Carbone e mostra di ben conoscere il seguito de' fatti di
quegli anni, che del resto si erano svolti sotto i suoi occhi.
D'altra parte per l'allusione al sacco di Rimini, per la menzione de'
conti resi a P. Lentulo e L. Triario [331], questori nell'anno 81, si deve
pure ritenere, che nell'anno 82, C. Verre occupava l'ufficio di
questore.
Di fronte a questi fatti restano prive di valore le opinioni unilaterali di
quelli che vorrebbero far valere la data di un anno o quella di un
altro, e pare si debba pensare ad una prorogazione dell'ufficio o ad
una rielezione di Verre all'ufficio di questore; cosa tanto più probabile
in quanto avrebbe fatto riscontro appunto alla reiterazione del
consolato di Cn. Papirio Carbone [332].
Cronologicamente dunque la notizia di Cicerone non offre materia ad
appunti: merita bensì di essere accolto con ogni cautela il giudizio
che dà del modo onde l'ufficio fu gerito e degli incidenti che
l'accompagnarono. Verre infatti nell'anno 82 si staccò dalla parte
mariana per aderire alla sillana, e Cicerone non ha parole sufficienti
per bollare più e più volte questa sua defezione [333]. A sentire il suo
accusatore, prima di tutto C. Verre non fece questa apostasia per
considerazioni politiche, ma semplicemente a scopo di rapina. Infatti
da' conti resi della sua questura appariva com'egli dovesse avere
ancora presso di sè seicentomila sesterzi, quanti costituivano la
differenza tra il denaro ricevuto e lo speso, e, intanto, a' suoi
successori non seppe darne altra giustificazione se non d'averli
lasciati a Rimini, dove a cagione del sacco doveano essere andati
perduti [334]. Ora come accadessero le cose, noi non siamo in grado
di dire; ma si può ben dire che la giustificazione di Verre non
menava univocamente a quell'interpretazione che Cicerone le dava.
E tanto più apparisce parziale, quanto più passionato ed esagerato è
il biasimo che Cicerone getta su Verre, indipendentemente da ogni
ragione di furto, pel semplice abbandono del suo console.
Per averne un chiaro intendimento occorre mettere in relazione quel
fatto con altri avvenimenti del tempo stesso. L. Cornelio Silla era da
poco sbarcato in Italia e già dalla parte avversa molti cominciavano
ad inclinare verso di lui. L'esercito di L. Scipione abbandonava il suo
comandante e si rendeva a Silla [335], e colle vittorie de' suoi legati e
l'affermarsi della sua fortuna, la demoralizzazione e la sfiducia
entravano nel campo nemico; e le diserzioni spesseggiavano tanto
più, quanto l'uomo, a' suoi benigno ed a' nemici crudo, sapeva con
tutte le arti del fascino personale e con tutte le minacce della più
inesorabile vendetta attrarre a sè gli uomini [336]. Se qualche cosa è
notevole, è precisamente questo: che Verre potè ancor tanto tempo
dopo la venuta di Silla in Italia restare fedele alla sua parte, e non
passò all'avversario, se non quando la sua parte si andava ormai
dissolvendo senza rimedio e i suoi interessi di Romano e di nobile lo
gettavano in braccio a Silla; e probabilmente non si poteva dire
ch'egli abbandonava il suo console, ma che il suo console
abbandonava lui e l'esercito e la sua parte, cercando in Africa uno
scampo [337]. Il peggio che in questo può dirsi di Verre è che, al pari
di tanti altri, in questa e nelle posteriori guerre civili, anch'egli finì
per obbedire a quel senso dell'opportunità, che, se oggi è perfino il
programma e la bandiera di un partito, fu sempre come la bussola
de' moltissimi che, nella vita civile e nella politica, in ogni tempo
ebbero a supremo scopo all'esistenza il salvataggio della borsa e
della pelle.
Verre potè dire d'aver assicurato l'una cosa e l'altra. Silla lo mandò
nel Beneventano tra i suoi amici, per diffidenza come vuole
Cicerone [338], per ragione di uffizio, se come è lecito dedurre dalla
resa di conti fatta a' questori dell'anno successivo, egli tenne la
carica sino alla fine dell'anno [339]. Silla in ogni modo, com'era natura
dell'uomo, lo beneficò e lo protesse, e Verre potè andare innanzi
nella sua carriera. A Roma dopo questa sua questura, rimase appena
tre giorni [340], occupato del resto, come sembra, in missioni ed
incarichi, probabilmente nel Beneventano.
La legazione e la proquestura di Verre.
Due anni dopo, nell'anno 80, Cn. Dolabella di parte aristocratica,

stato l'anno innanzi pretore, va come propretore in Cilicia ed, a
proprio legato, non sa eleggersi miglior compagno di Verre, e dopo
la morte del suo questore C. Malleolo gli affida anche la gestione
della questura in sua vece [341]. Che legazione e che proquestura!
Qui Cicerone aggrava la mano e fa Verre responsabile non solo di
quello ch'egli potè commettere, ma di quello altresì che commisero il
suo capo ed i suoi dipendenti. Che cosa in verità fece di suo
proposito e per suo conto, che cosa come ministro del propretore?
Noi non lo possiamo sapere completamente. Dolabella era ornai
tramontato dall'orizzonte e a Cicerone fa commodo addebitare tutto
a Verre, anche quello di cui Dolabella era stato riconosciuto
colpevole in giudizio [342], ricorrendo ad induzioni ed illazioni,
invocando la testimonianza di gente danneggiata e stizzita, o
disposta ad accusare anche per propria difesa [343]. In ogni modo, se
anche alcuni fatti non ebbero origine in un preconcetto disegno, ma
furono l'incidente di una notte di crapula [344]; se in alcuni atti egli fu
il braccio allungato di Dolabella, non bisogna durar molta fatica per
ammettere che il suo passaggio, più che quello di un legato del
popolo romano, dovè sembrare talvolta quello di qualche
infortunio [345]. Soltanto, di questi infortunî in pellegrinaggio si era
destinati a vederne più d'uno.
I Dolabella erano di razza rapaci e fraudolenti [346] e C. Malleolo, il
questore, era tutt'altro che un puritano [347]. Un uomo dal senso
morale molto mediocre, come era Verre, non poteva proporsi altro
che di accomodarsi alle leggi dell'ambiente e farne suo pro, e così
fece. A quella scuola egli apprese e ritenne per poi, se mai, emulare
o superare i maestri. Era, pare, la sua prima scorsa su' poderi del
popolo romano ed egli vi faceva, come è dire, il suo noviziato; per
non perdere tempo anzi incominciò dal viaggio. La sua casa era
ancora poco adorna di statue greche, e la selvetta che la circondava
sarebbe stata lieta di porgere le sue ombre alle divinità
elleniche [348]; i provinciali aveano ancora molta lana da potersi
tosare e, se anche un colpo di forbici arrivava alla pelle, ciò li
avrebbe resi più umili; e, finalmente, a lui che usciva dalla guerra
sillana, gli ozî di Marte, dio infido e terribile, doveano parere utili a
propiziarsi Afrodite, e tutte le avventure galanti della provincia
poteano fornire un diletto, lieto a godere oggi e bello a narrare
domani.
Il viaggio.
All'opera dunque! A Sicione fa al magistrato cittadino richiesta di

danaro. Non si arrende? L'avarizia può più del nome romano e della
paura? Ebbene, lo si caccia in uno stambugio e, acceso un buon
fuoco di legna verdi, lo si lascia a considerare se ha minor pregio
l'oro, o dà maggior molestia il fumo [349]; citra sanguinis effusionem!
Di oro intanto sarà meno avara in Atene Minerva, e al magistrato
romano che la vide e non ne partì a mani vuote, ne rimarrà due
volte grato il ricordo [350]. Sono ancora troppo ricchi, troppo ornati
questi templi di Grecia e d'Oriente, pur dopo che vi passò, da tanti
anni, il conquistatore romano: ha troppe statue Apollo nel suo
tempio, a Delo ove nacque, e nella notte è bene che ne sparisca
qualcuna. Ma Apollo è qualche volta un dio iroso e capace di
difendere la sua proprietà, come un qualunque avaro mortale, anche
invocando da Poseidone un uragano: la tempesta infatti viene e la
nave, che porta la sacrilega preda, si sfascia ed il turbato flutto del
mare riporta alla riva le statue sacre di Apollo; sicchè -- fu
superstizione o necessità di resipiscenza? -- Dolabella deve farle
rimettere là donde furono prese e donde il popolo, tacito, ne
deplorava la mancanza, pur non avendo il coraggio di
reclamarle [351].
Ma non tutti gli dèi sanno o vogliono difendersi come Apollo: a Samo
il tempio di Giunone resta a dirittura spogliato [352]; a Tenedo lo
stesso dio indigete Tene non riesce a restar fermo al suo posto [353];
Diana di Perga vede depredato non solo il suo tempio, ma sè stessa
degli ornamenti muliebri che porta [354]. E invano tentano gli uomini
di sopperire alla neghittosità degli iddii; a' legati di Samo, che vanno
a muoverne lamento, C. Nerone risponde che vadano a Roma, e
Roma è lontana, e, quando essi vi arrivino, potranno avere la
sorpresa di vedere che, in prova d'animo generoso, il loro
depredatore si è offerto ad ornare per un giorno delle opere d'arte
rubate i fòri e le vie [355].
Intanto bellissime statue sono portate via di forza da Chio, da
Erythrae, da Alicarnasso [356]; da Aspendo, in cospetto di tutti, su
carri tirati da buoi s'asporta tutto quanto stava ad ornamento di
edifizî pubblici e di templi e, tra l'altro, quel citarista, che per la
perfezione sua era passato in proverbio e si dicea capace di cantare
ogni cosa [357].
Non perdona agli dèi il nuovo padrone e tanto meno perdona agli
uomini; ma, in cambio, come ama piacere, ad ogni costo, alle donne!
Il suo amore egli lo insinua, lo offre per tutto, più spesso lo impone a
dirittura.
L'avventura di Lampsaco.
Vi è una missione per i re Sadala e Nicomede, e Verre fa sì che Cn.

Dolabella gliela commetta; una felice occasione a queste spedizioni
feconde e senza pericoli. Sulla sua via è Lampsaco, ed egli vi fa
sosta con tutto il suo seguito. Dolce e generoso è il vino di Lampsaco
e belle ne sono le donne; ma bella più di tutte è la figliuola di
Filodamo, bella e pudica. Verre è ospite di Janitore, di cui non gli
riesce potere abbandonare la casa, e a Filodamo impone, ospite non
desiderato, non accetto, Rubrio, un suo bracco, lo stesso appunto
che avrebbe scovata la bella fanciulla. Filodamo tuttavia vuol fare
ammenda dell'ospitalità mal concessa, e la casa del ricco anfitrione
s'apre per accogliere, convitati da Rubrio, Verre e il seguito suo. Fu
intanto pensiero preconcetto, come vuole Cicerone, o fu caso? Ma,
libando e crescendo l'eccitazione del convito, Rubrio si rivolge a
Filodamo per suggerirgli di chiamar lì la sua bella figliuola e, al
timido schermirsi di Filodamo, altre voci si levano più pertinaci

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For information on all Academic Press publications

Publisher: Mica Haley

Epigenetics is an emerging frontier of biology, as well as advanced technologies and tools

1. Introduction 2 5. Epigenetic Biomarkers: An Overview

Epigenetic Biomarkers and Diagnostics

1. INTRODUCTION a chromosome without alterations in the DNA

FIGURE 1 Dynamic DNA methylation and demethylation catalyzed by DNA(cytosine-5-)methyltransferases (DNMTs)

TABLE 1 General Information for Noncoding RNAs

Length (nt) 17–25 >200

Poly-A tail No Yes

No. of sequences described in human 1881a 111685b

Found in Cell, tissues, body fluids/exosomes Cell, tissues, body fluids/exosomes

Stability in body fluids and FFPE High Low

Isolation RNA conserving miRNAs or miRNA RNA purification protocols

In Section II of this volume we also provide

1. Biobanks 20 3. Epigenetics and Biobanking 27

Epigenetic Biomarkers and Diagnostics

1. BIOBANKS like genomics and personalized medicine, and

2.2 Biobanks as Centers for several challenges in using EHRs for research,

1. Introduction 38 2.2 The Tricarboxylic Acid Cycle and

Epigenetic Biomarkers and Diagnostics

5.3 RSTS1 Syndrome 54 7. Conclusions 58

1. INTRODUCTION chromatin translates into a defined outcome on

related to intellectual disability in women, crotonylation, citrulination, formylation, glyco-

Le città di Sicilia possono calcolarsi, anche nel periodo della

Questa varia classificazione delle città siciliane era stata determinata

Dalle trentaquattro città decumane il Governo di Roma prelevava la

Restava il reddito delle città censorie. Quanto alle città censorie il

Si ha inoltre notizia da Cicerone [232] di un tributo, che sarebbe stato

Ma, mentre da una ad un'altra di queste città v'erano così notevoli

La nomina a questi diversi uffici aveva luogo in modo diverso.

Il decretum di Rupilio poi, quello che si potrebbe dire la Charta

Un'altra menzione vi era finalmente ed era che i giudizî tra decumani

Tale era l'ordinamento effettivo, su cui riposava il governo della

E in Sicilia, anche più che altrove, codesto compito era interessante

Chi era C. Verre?

Lo scrittore, il poeta, l'oratore, l'artista sono anche tanti giustizieri. In

Questo stesso suo modo di vita non doveva essergli d'impedimento a

Due anni dopo, nell'anno 80, Cn. Dolabella di parte aristocratica,

All'opera dunque! A Sicione fa al magistrato cittadino richiesta di

Vi è una missione per i re Sadala e Nicomede, e Verre fa sì che Cn.

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1. Introduction 2 5. Epigenetic Biomarkers: An Overview

TABLE 1 General Information for Noncoding RNAs

1. Biobanks 20 3. Epigenetics and Biobanking 27

2.2 Biobanks as Centers for several challenges in using EHRs for research,

1. Introduction 38 2.2 The Tricarboxylic Acid Cycle and

5.3 RSTS1 Syndrome 54 7. Conclusions 58