Epigenetics in Human Disease

591 Pages·2012·20.002 MB·English

1
CHAPTER
Epigenetics of Human
Disease
Trygve O. Tollefsbol
University of Alabama at Birmingham, Birmingham, AL, USA
CHAPTER OUTLINE
1.1 Introduction 1 1.10 Cardiovascular Disease and
1.2 Epigenetic Variation Methods 2 Epigenetics 5
1.3 Cancer Epigenetics 2 1.11 Epigenetics of Human Infectious
1.4 Epigenetics of Neurological Diseases 5
Disease 3 1.12 Reproductive Disorders and
1.5 Autoimmunity and Epigenetic Aberrations 5
Epigenetics 3 1.13 Stem Cell Epigenetics in Human
1
1.6 Human Imprinting Disorders 4 Disease 5
1.7 Epigenetics of Obesity 4 1.14 Epigenetics of Aging and Age-
1.8 Diabetes: The Epigenetic Associated Diseases 6
Connection 4 1.15 Conclusion 6
1.9 Epigenetics and Allergic References 6
Disorders 4
1.1 INTRODUCTION
Epigeneticsdoesnot involve changesin DNA sequence but is nevertheless able toinfluence
heritablegene expression through anumberof processes such as DNA methylation, modifi-
cationsof chromatin and non-coding RNA. Aberrations in DNA methylation are common
contributorstodisease.Forexample,imprintingdiseasessuchastheAngelman,SilvereRussell,
PradereWilli and BeckwitheWiedemann syndromes are often associated withalterations in
DNA methylation [1].Human diseases attributableto DNA methylation-based imprinting
disorders,however,havenotbeenlimitedtothesegeneticdiseasesasdiabetes,schizophrenia,
autismandcancerhavealsobeenassociatedwithaberrationsinimprinting.Abnormalitiesof
theenzymesthatmediateDNAmethylationcanalsocontributetodiseaseasillustratedbythe
rareImmunodeficiencyeCentromereinstabilityeFacialanomalies(ICF)syndromecausedby
mutations inDNA methyltransferase3B (DNMT3B).Likewise, Rettsyndrome, related to
mutationsin themethyl-binding domain (MBD) protein,MeCP2,leads todysregulationsin
geneexpressionandneurodevelopmentaldisease[2].Perhapsmostcommonly,DNAmethy-
lationaberrations can often contribute tocancer eitherthrough DNA hypo-or hyper-
methylation.DNAhypomethylationleadstochromosomalinstabilityandcanalsocontribute
T.Tollefsbol(Ed):EpigeneticsinHumanDisease.DOI:10.1016/B978-0-12-388415-2.00001-9
Copyright(cid:1)2012ElsevierInc.Allrightsreserved.
Epigenetics in Human Disease
tooncogeneactivation,bothcommonprocessesinoncogenesis,andDNAhypermethylation
isoften associated withtumorsuppressor gene inactivationduring tumorigenesis.
Histone modifications frequentlycontribute to disease development and progressions and
histoneacetylationordeacetylationarethemostcommonhistonemodificationsinvolvedin
diseases.Aberrations in histone modifications can significantlydisrupt gene regulation,
acommon factor in disease, and could potentiallybe transmissible acrossgenerations [3].
Histone modifications haveinfact been associated with anumberofdiseasessuch as cancer
and neurological disorders.Collaborations betweenDNA methylation and histone modifi-
cations can occurand either or both of these epigenetic processes maylead todisease devel-
opment [4].
Non-codingRNAs are an emerging areaof epigenetics and alternations intheseRNAs,espe-
ciallymicroRNAs(miRNAs),contributetonumerousdiseases.miRNAscaninhibittranslation
ofmRNA if the miRNA binds tothe mRNA,a process that leads toits degradation, or the
0
miRNAmaypartiallybindtothe3 endofthemRNAandprohibittheactionsoftransferRNA
[5].AlthoughmiRNAshavebeenassociatedwithanumberofdiseasessuchasCrohn’sdisease
[6],theirroleintumorigenesisisnowestablishedandisconsideredtobeafrequentepigenetic
aberrationin cancer.
Collectively,epigenetic processes are now generallyaccepted to playakeyrole in human
diseases.Asthe knowledgeofepigenetic mechanisms inhumandiseases expands,it is
expectedthatapproachestodiseasepreventionandtherapyusingepigeneticinterventionswill
also continue todevelop and mayeventuallybecome mainstays in disease management.
1.2 EPIGENETIC VARIATION METHODS
Technologicaladvancesoftenserveasamajorstimulusforknowledgedevelopmentandthe
2 field of epigenetics is no exception in this regard. Recent advances in epigenetic-based
methodshaveservedasmajordrivingforcesinthefascinatingandever-expandingepigenetic
phenomenathathavebeenrevealedespeciallyoverthepastdecade.Althoughgenome-wide
maps have been developed, there is still a need for maps of the human methylome and
histonemodificationsinhealthyanddiseasedtissues,asdiscussedinChapter2.Epigenetic
variation is especially prominent in human diseases and established techniques such as
bisulfite genomic methylation sequencing and chromatin immunoprecipitation (ChIP)
analyses are revealing numerous epigenetic aberrations involved in disease processes.
However, cutting-edge advances in comparative genomic hybridization (CGH) and micro-
arrayanalysesaswellasquantitativeanalysisofmethylatedalleles(QAMA)andmanyother
developing technologies are now facilitating the elucidation of epigenetic alterations in
diseasethatwerepreviouslyunimagined.Combinationsofepigenetictechnologiesarealso
emergingthatshowpromiseinleadingtonewadvancesinunderstandingtheepigeneticsof
disease.
1.3 CANCER EPIGENETICS
Asmentioned above, DNA methylation is often animportantfactorincancerdevelopment
andprogression.DNAmethylationchangescannowbereadilyassessedfrombodyfluidsand
applied tocancerdiagnosis as wellas the prognosis ofcancer (Chapter3). Epigenome refer-
encemapswilllikelyhaveanimpactonourunderstandingofmanydifferentdiseasesandmay
lead the way tobreakthroughs in the diagnosis, prevention and therapyof human cancers.
Histonemodificationsarefrequentlyalteredinmanyhumancancersandthedevelopmentof
ahistone modification signaturemaybe developed that will aid in the prognosis and treat-
mentofcancers(Chapter4).Thesehistonemapsmayalsohavepotentialinguidingtherapyof
human cancers.MicroRNAs (miRNAs) are central tomanycellular functions and theyare
frequentlydysregulated during oncogenesis (Chapter 5).In fact, miRNAexpression profiles
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Epigenetics of Human Disease
maybemoreusefulthangeneexpressionprofilesforclinicalapplicationssincetherearefewer
mRNA regulatorymolecules.These miRNA profilesmaybe applicable toidentifying various
cancersortostratifytumorsinadditiontoservingprognosticortherapeuticroles.Epigenetic
therapyfor canceris perhaps oneof the most excitingand rapidlydeveloping areasof
epigenetics.AsdiscussedinChapter6,approachesareavailablefortargetingenzymessuchas
theDNMTs,histoneacetyltransferases(HATs),histonedeacetylases(HDACs),histonemethyl-
transferases(HMTs) and histone demethylases (HDMTs). The development of drug-based
inhibitors of these epigenetic-modifying enzymes could be furtherimproved throughdrug
combinationsorevennaturalplant-basedproducts,manyofwhichhavebeenfoundtoharbor
propertiesthatcanmimictheoftenmoretoxicandperhapslessbioavailableepigeneticdrugs
that are currently inuse.
1.4 EPIGENETICS OF NEUROLOGICAL DISEASE
One of the newer areas of epigenetics that has been rapidlyexpanding is its role in neuro-
logicaldisordersordiseases.Thesedisordersarenotlimitedtothebrainasthediseasetarget,
butalsoofteninvolvenutritionalandmetabolicfactorsthatcontributeaswelltoconditions
such as neurobehavioral diseases (Chapter 7). At this point, however, the number of
neurodevelopmental disorders that have been associated with epigenetic aberrations is not
veryextensive. A possible explanation for this is that the pervasive nature of epigenetic
processes could serve as a negative selective force against more localized disease such as
neurodevelopmentaldisorders(Chapter8).Infact,manyneurodevelopmentaldisordersare
due to partial loss-of-function mutations or are X-chromosomal mosaics with recessive
X-linked mutations. Neurodegenerative diseases such as Alzheimer’s disease have been
increasinglyassociatedwithalternationsinepigeneticprocesses.Environmentalfactorssuch
as diet and exposure to heavy metals may lead to the epigenetic changes often involved in
Alzheimer’s disease eventuallycontributing to increased amyloid b peptide (Chapter 9). 3
Thesefactorsmaybeginearlyinlifeandmanifestaslate-onsetformsofAlzheimer’sdisease.
Fortunately, as reviewed in Chapter 10, a number of new approaches are currently being
developed that could have translational potential in preventing or treating manyof the
epigenetic changes that are being revealed as an important component of neurobiological
disorders.
1.5 AUTOIMMUNITY AND EPIGENETICS
There is astrong association betweenenvironmental factors,age and the development of
autoimmune disorders.Epigenetic processes are central toaging and are also animportant
mediator between the environment and disease and it is thoughtthat these factors maybe
importantin the development and progression ofnumerous autoimmune diseases.For
example,systemiclupuserythematosus(SLE)andrheumatoidarthritis(RA)areautoimmune
disorders that have frequentlybeenassociated with aberrations in epigenetic mechanisms
(Chapter11).Oftentheepigenomicandsequence-specificDNAmethylationchangesfoundin
SLE and RA affectkey genes inimmune function. Two challenges are toincrease the use of
high-throughputapproachestothesediseasestomineforadditionalgeneaberrationsandto
translatetheseepigeneticchangestotheclinicthroughthedevelopmentofnovelapproaches
for preventing or treating SLE and RA.Fortunately, there is hope for epigenetic therapyof
autoimmune disorders as reviewedin Chapter12. Much ofthe current researchfor drug
developmentrelevanttoautoimmunedysfunctionisfocusedoncorrectingalterationsinDNA
methylation and histone acetylation. However,recent excitingadvances suggest promising
avenues for drug development as applied tomiRNAs.Forinstance, miRNAsor inhibitors of
miRNAtoimpactDNAmethylationmayhaveutilityinaffectinggenetranscriptioninimmune
cells that often lead to the development of SLE.
Epigenetics in Human Disease
1.6 HUMAN IMPRINTING DISORDERS
BothDNAmethylationandhistonemodificationscanimpactimprintingcentersthatcontrol
parent-of-origin-specificexpressionandleadtohumanimprintingdisorders.Thesedisorders,
such as Angelman,PradereWilli, SilvereRusselland BeckwitheWiedemann syndromes
frequentlyinvolve epigenetic changesthat contribute tothesedisorders and theyoften
manifest at averyyoungage (Chapter13). However,both epigenetic and genetic factors are
often important in humanimprintingdisorders and the development of epigenetic therapy
approachesin this particular arearepresentsaconsiderablechallenge.Advances are being
made in understanding the epigenetic basisof human imprintingdisorders which may
providebreakthroughs in treating these tragic diseases.
1.7 EPIGENETICS OF OBESITY
Rare obesity-associated imprintingdisorders have been described and dietary modulation
effortshavesuggestedanepigeneticcomponentmayexistinthesedisorders.Infact,themajor
roleofenvironmentalfactorsinobesitystronglysuggestsaroleofepigeneticchangessuchas
those involving DNA methylation in obesity (Chapter 14).Early-life environmental factors
could be especially important incontrolling epigenetic aberrations thatmaycontribute to
obesityas reviewedinChapter 15. Itis likelythatincreased identificationofobesity
biomarkersandtheirassociatedepigeneticfactorsmayleadtonewadvancesincontrollingthe
extant epidemic in childhood obesity inmanydeveloped countries.It is highly likely that
nutritionalor lifestyleinterventions eitherduring pregnancyor early inlife could impact
processes such as DNA methylationand histone modifications that are highly responsiveto
environmental stimuli and lead tomeansto control obesityat veryearlyages.
1.8 DIABETES: THE EPIGENETIC CONNECTION
4
Similartoobesity,environmentalfactorsarealsooftenimportantinthedevelopmentoftype
2diabetes.Non-geneticriskfactorssuchasagingandasedentarylifestylehavebeenassociated
withepigeneticaberrationscharacteristicoftype2diabetes(Chapter16).Sincemarkerssuch
asDNAmethylationhavebeenshowntovaryindiabeticversusnon-diabeticindividuals,itis
very possible thatepigenetic manifestations mayhave akeyrolein the pathogenesis oftype
2diabetes.However,multisystem studies are currently needed tofurther substantiate this
conceptandadditionalstudiesonthepredictionandpreventionoftype2diabetesaresorely
needed. Histonemodifications havealso been strongly implicatedindiabetesasreviewedin
Chapter17.Infact,HDACinhibitorsmayhavepotentialintreatingdiabetesintheshortterm.
Nutritionalcompounds thatlead to HDAC inhibition mayhave potentialintreating type
2diabetes as wellas the development of miRNA-based therapeutics that wouldhave greater
targeting potential.
1.9 EPIGENETICS AND ALLERGIC DISORDERS
Consistent with manyotherepigenetic diseases,earlyenvironmental factors appear to be
acriticalcomponenttothedevelopmentofnumerousallergicdisorders.Forexample,expos-
ureto specificfactors in utero maybe associated with epigeneticaberrations that affect gene
expression,immuneprogrammingandthedevelopmentofallergicmaladiesintheoffspring
(Chapter18).Additionally,thistransgenerationalcomponentmayallowforthetransmittance
ofepigeneticchangestofuturegenerationsbeyondtheoffspringleadingtoallergicdisorders.
Novelearlyinterventions into epigenetic-modifyingfactorssuch as maternal diet may
contributetoaneventualdeclineinallergy-baseddisorders.Asthmaisacommondisorderof
thisnatureandthereissomeevidencethatcorticosteroidsexerttheiranti-inflammatoryeffects
inpart byinducing acetylation of anti-inflammatorygenes (Chapter19).The potential
recruitmentof HDAC2 to activatedinflammatory genesbycorticosteroids maybe akey
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Epigenetics of Human Disease
mechanismforepigenetic-basedtherapyofallergicdisorderssuchasasthma.Futureeffortsare
nowbeingdirectedtowardmodifiersofotherepigeneticprocessesinallergicdisorderssuchas
histone phosphorylation and ubiquitination.
1.10 CARDIOVASCULAR DISEASE AND EPIGENETICS
Atherosclerosis is amajorprecipitating factor in cardiovascular diseasesand the functions of
smoothmuscle cells (SMCs) and endothelialcells(ECs)are central tothe development of
atherosclerosis.Mounting evidence hasindicated that epigenetic processes such as DNA
methylation and histone acetylation havecriticalfunctions in modulating SMC and EC
homeostasis.TheSMCandECproliferation,migration,apoptosisanddifferentiationnotonly
contribute toatherosclerosis,but also cardiomyocytehypertrophyand heart failure, as
reviewedin Chapter 20. The role of HDACs incardiovasculardisease such as arteriosclerosis
hasbeenshowingpromise,althoughconcernssurroundthetissue-specificityoftheseagents.
Giventhisconcern,thedevelopmentofhighlyselectiveandcelltype-specificHDACinhibitors
mayhave potential inepigenetic-basedtherapies for cardiovascular diseases of varied types.
1.11 EPIGENETICS OF HUMAN INFECTIOUS DISEASES
Acommon theme is the environmental impacton theepigenome and its role inepigenetic
disease processes. Consistent with this concept, bacterial and viralinfections often cause
epigenetic changes in host cells thatleadtopathologyas reviewedinChapter 21. The
consequencesoftheseepigenome-modifyinginfectionsarenotlimitedtoneoplasia.Thereare,
infact,manyotherdiseasesthathaveanepigeneticbasisinducedbyinfectiousagentssuchas
diseases ofthe oral cavity.Evenorganisms like protozoa can contributeto host epigenetic
dysregulation. Knowledgeaccumulated regardingepigenetic “invaders”of the genome and
their pathological consequenceswill undoubtedly lead tothe development of more sophis-
5
ticatedandnovelapproachestocontrollingandtreatingepigenetic-basedinfectiousdiseases.
1.12 REPRODUCTIVE DISORDERS AND EPIGENETIC
ABERRATIONS
Endometriosis,or the presence of functional endometrial-like tissues outsideof the uterine
cavity, is often secondary to hormonal and immunological aberrations.Most excitinginthe
context of epigenetics, however, is thatmanyrecent studies have indicated that
endometriosis mayhave animportant epigenetic component that contributes to its patho-
logicalprogression(Chapter22).AnumberofinvestigationshaveindicatedHDACinhibitors
maybe effectivein treating endometriosis.There is also potential for the development of
epigenetic biomarkers for endometriosis such as changesin DNA methylation aswellas
miRNA-based biomarkers. Epigenetic processes are also gainingincreasingimportance in
endometrialcancer (Chapter23). Damage tothe mismatch repairsystem appears toplay
asignificantroleinthedevelopmentofendometrialcancerthroughthemechanismofhMLH1
hypermethylation.Thesefindingsmayhaveimportantepigenetictherapeuticimplicationsfor
endometrialcancer and could also have potential for the prevention,diagnosis and risk
assessment ofendometrialcancer.
1.13 STEM CELL EPIGENETICS IN HUMAN DISEASE
Stem cell-based therapeutic approaches could lead to powerfulmeans of treating human
diseasesandepigeneticregulatorysignalsplayanimportantroleinthemaintenanceofstem
cell potency (Chapter24).Chromatinmodifications and dynamics appear to have an
importantroleinconservationofpluripotencyandthedifferentiationofembryonicstemcells
whichare central factors instem cell-based therapeutics.In fact, severalepigenetic disorders
havebeenmodeledinvitrothroughtheuseofinducedpluripotentstemcells(iPSCs)fromthe
Epigenetics in Human Disease
cells ofpatients.Understanding the basic epigenetic changes central to these processes may
haveconsiderablepotentialinthetreatmentofhumanepigeneticdiseases.Non-codingRNAs
alsoparticipateinstemcellrenewalanddifferentiation(Chapter25).Theroleofepigenetics
and non-coding RNAs mayprovidemanyuseful tools for manipulatingstem cell program-
mingas applied to therapyof epigenetic-based diseases.
1.14 EPIGENETICS OF AGING AND AGE-ASSOCIATED DISEASES
Fewprocessesareaspervasiveasagingwhichimpactsnotonlytheentirephysiologicalfitness
ofan organism, but also its predisposition to developing age-related diseaseswhich is
comprisedofanever-growinglistofdiseases.Itisnowapparentthatepigeneticprocessesare
major components of aging, which opens manyavenuesto humandiseases(Chapter26).
Althoughaging isnot considered adisease in and of itself, it is perhapsthe most frequent
contributor tohumandisease. Therefore, delayingthe epigenetic aberrations associated with
aging through epigenetic intervention and treating epigenetic-basedage-associated diseases
couldhaveatremendousimpactontheroleofepigeneticsinhumandisease.Althoughthey
areonoppositesidesofthelifespanspectrum,earlydevelopmentalprocessesarelikelylinked
tolaterlife aging and age-associated diseases (Chapter27). The role of nutrition, hormones
andmetabolicenvironmentearlyinlifecanhaveeffectsthroughoutlife,influenceepigenetic
pathwaysand markers and manifest inthe formof aging and age-related diseases.Consid-
erableinterestisnowfocusedontheimpactofearlylifeepigeneticimpactsandtheoutcomeof
theseeffectsonthemyriadofage-associateddiseaseswhichcomprisemuchofthepathology
thatforms the basis of human disease.
1.15 CONCLUSION
Epigeneticprocessesnotonlytakemanyforms,buttheyalsocanreadilybecomeexpressedas
6 humandiseases.Thesediseases,thatcanbelooselygroupedundertheheadingof“epigenetic
diseases”, are vast and the list of diseases thatfit into this description is rapidly growing.
Elucidationof the epigenetic aberrations inhumandiseases not only hasimplications for
epigenetic-based therapy, but also forrisk assessment, prevention, progression analysis,
prognosisand biomarkerdevelopment. Acommon theme of manyepigenetic-based human
diseases is the role ofthe environment. This maytake varied forms,ranging from maternal
nutrition to infectiousagents.Exciting advances are rapidlydeveloping that are contributing
significantlytowardthemanagementofhumandiseasesthroughepigeneticintervention.Itis
anticipated that epigenetic-based preventive and therapeutic strategieswillcontinue to
developat arapid pace and mayassumearole at the forefront ofmedicine inthe not too
distant future.
References
[1] FallsJG,PulfordDJ,WylieAA,JirtleRL.Genomicimprinting:implicationsforhumandisease.AmJPathol
1999;154:635e47.
[2] YasuiDH,PeddadaS,BiedaMC,ValleroRO,HogartA,NagarajanRP,etal.Integratedepigenomicanalysesof
neuronalMeCP2revealaroleforlong-rangeinteractionwithactivegenes.ProcNatlAcadSciUSA
2007;104:19416e21.
[3] CavalliG,ParoR.TheDrosophilaFab-7chromosomalelementconveysepigeneticinheritanceduringmitosis
andmeiosis.Cell1998;93:505e18.
[4] EstellerM.Cancerepigenomics:DNAmethylomesandhistone-modificationmaps.NatRevGenet
2007;8:286e98.
[5] GibneyER,NolanCM.Epigeneticsandgeneexpression.Heredity(Edinb)2010;105:4e13.
[6] WuF,ZhangS,DassopoulosT,HarrisML,BaylessTM,MeltzerSJ,etal.IdentificationofmicroRNAsassociated
withilealandcolonicCrohn’sdisease.InflammBowelDis2010;16:1729e38.
2
CHAPTER
Methods and Strategies
to Determine Epigenetic
Variation in Human
Disease
Yoshihisa Watanabe, Masato Maekawa
Hamamatsu University School of Medicine, Hamamatsu, Japan
CHAPTER OUTLINE
2.1 Introduction 8 2.2.10DNAMethylationAnalysisby
2.2 DNA Methylation Analysis 8 Pyrosequencing 10 7
2.2.1Methylation-Sensitive 2.2.11 Matrix-AssistedLaser
Restriction Enzymes 9 DesorptionIonization Time-
2.2.2Bisulfite Conversion of of-Flight Mass
Unmethylated Cytosines, Spectrometry 10
PCRand Sequencing 9 2.2.12 NewTechnologies 11
2.2.3Comparative Genomic 2.2.13 Computational Tools 11
Hybridization (CGH)and 2.3 Histone Modification
Microarray Analysis 9 Analysis 11
2.2.4Bisulfite Treatment andPCR 2.4 Non-Coding RNA Analysis:
Single-Strand Conformation MicroRNA 12
Polymorphism (SSCP) 2.5 Analysis of Genome DNA
(BiPS) 9 Replication Program Based on
2.2.5Methylation-Sensitive Single- DNA Replication Timing 14
Nucleotide Primer 2.6 Strategy for Epigenomic
Extension 9 Investigation Based on
2.2.6Combined Bisulfite and Chromosomal Band
Restriction Analysis 10 Structures 15
2.2.7Quantitative Bisulfite 2.7 Overview of Recent Epigenetic
Sequencing using genome-Wide or Bioinformatic
Pyrosequencing Studies and Strategies 19
Technology 10 2.8 General Overview and Future
2.2.8MethyLight Technology 10 Perspective 22
2.2.9Quantitative Analysis of References 22
Methylated Alleles
(QAMA) 10
T.Tollefsbol(Ed):EpigeneticsinHumanDisease.DOI:10.1016/B978-0-12-388415-2.00002-0
Copyright(cid:1)2012ElsevierInc.Allrightsreserved.
Epigenetics in Human Disease
2.1 INTRODUCTION
Epigeneticsis not onlyoneof the most rapidly expanding fields of study in biomedical
research but is also one ofthe most excitingand promisingin terms of increasing our
understanding ofdisease etiologies and of developing new treatmentstrategies.Among the
recentlandmarkeventsinthisfieldarethecharacterizationofthehumanDNAmethylomeat
single nucleotide resolution, the discoveryof CpG island shores,the identification of new
histone variantsand modifications,and development ofgenome-widemapsof nucleosome
positions. Much of our increasedunderstanding is the result of technological breakthroughs
thathavemade it feasible toundertake large-scale epigenomicstudies.Thesenew method-
ologies have enabled everfiner mappingof the epigenetic marks,such as DNA methylation,
histone modifications and nucleosomepositioning, thatare criticalfor regulating the
expressionofbothgenesandnoncodingRNAs[1].Inturn,wehaveagrowingunderstanding
ofthe consequences ofaberrant patternsof epigenetic marksand ofmutations inthe
epigenetic machinery in the etiologyof disease.
However,thereareseveralaspectsofthemethodsusedtoanalyzeepigeneticvariationassoci-
atedwithdiseasethatpresentpotentialproblems.First,thetissueusedtoobtaintheDNA.This
dependstosomeextentonthenatureofthedisease,andcaninfluencetheanalyticalmethods
thatareemployed.Forexample,theDNAofsometissuesmayhavealowincidenceofmoieties
withthediagnosticpatternofmethylation,whichwouldlimitthechoiceofanalyticmeth-
odologiestothosewithhighsensitivityforthesemolecularsignatures.Second,different
diseasesmayrequireanalysisofeitherregionalorgenome-wideepigeneticvariation,withthe
choicedependingonthepredictedvariationinthespecificdisease.Thecontinuingincreasein
thenumberof“epigenetic”diseasesmeansthatthelistofmethodsthatarepracticalforthe
differentdiseasesisalsoincreasing.Third,epigeneticvariationcanbeaconsequenceoracause
ofthedisease.Therefore,useofstrategiesthatcandifferentiatetherole,orotherwise,of
8 epigeneticvariationinthecausalityofadiseaseisfundamental.Itmight,forexample,allow
determinationofwhetherepigeneticvariationisamarkerofdiseaseprogression,apotential
therapeutictarget,orausefulmarkerforassessingtheefficiencyofatherapy.
Althoughthenewtechnologieshaveprovidedconsiderableinsightsintoepigeneticaspectsof
disease,thereisstillconsiderablymoreworkthatneedstobecarriedout.Inparticular,thereis
agreat need for detailed descriptions of human DNA methylomes and for maps of histone
modifications and nucleosome positions in healthyand diseased tissues.A number of
international projects and initiatives have been established to meet this need: the NIH
Roadmap Epigenomics Program, the ENCODE Project, the AHEAD Project, and the
EpigenomicsNCBIbrowser,amongothers[2,3].Theavailabilityofdetailedepigeneticmaps
will be of enormous value to basic and applied research and will enable pharmacological
research to focus on the most promising epigenetictargets.
Thischaptersummarizes some ofthe contemporarymethods used to studyepigenetics and
highlights new methodsand strategies that have considerablepotential for future epigenetic
and epigenomic studies.
2.2 DNA METHYLATION ANALYSIS
MethylationofcytosinebasesinDNAisnotonlyanimportantepigeneticmodificationofthe
genome but is also crucial to the regulation of manycellular processes. DNAmethylation is
important inmanyeukaryotesfor both normalbiologyand disease etiology[1].Therefore,
identifyingwhich genomic sites DNAare methylated and determining howthis epigenetic
mark ismaintainedor lost is vital to our understanding ofepigenetics.Inrecent years,the
technologyusedfor DNA methylation analysishasprogressed substantially: previously,
analyseswereessentiallylimitedtospecificloci,butnow,theycanbeperformedonagenome-
wide scale tocharacterize the entire "methylome"with single-base-pairresolution [4].
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Methods and Strategies to Determine Epigenetic Variation in Human Disease
Thenewwealthofprofilingtechniquesraisesthechallengeofwhichisthemostappropriateto
select for agivenexperimental purpose. Here, welist differentmethodologiesavailable for
analyzing DNA methylation and brieflycompare their relativestrengths and limitations [5].
We also discuss important considerations for data analysis.
2.2.1 Methylation-Sensitive Restriction Enzymes
The identification of DNA methylation sites using methylation-sensitive restriction enzymes
requireshigh-molecular-weightDNA and is limitedbythe target sequence of the chosen
enzyme.The use ofrestriction enzymesthat are sensitive toCpG methylationwithintheir
cleavagerecognitionsites[6]isarelativelylow-resolutionmethod,butitcanbeusefulwhen
combined with genomic microarrays[7,8].
2.2.2 Bisulfite Conversion of Unmethylated Cytosines, PCR and
Sequencing
Conversion ofunmethylatedsequences withbisulfite followed byPCRamplification and
sequencing analysesprovidesan unbiased and sensitivealternativeto the use of restriction
enzymes.Thisapproachisthereforegenerallyregardedasthe“gold-standardtechnology”for
detection of 5-methyl cytosine as it enables mapping ofmethylated sites at single-base-pair
resolution[9].ThebisulfitemethodrequiresaprolongedincubationoftheDNAsamplewith
sodium bisulfite;duringthis period, unmethylatedcytosines in the single-stranded DNA are
deaminated touracil.However, the modified nucleoside 5-methylcytosine is immune to
transformation and, therefore,anycytosines that remain followingbisulfite treatmentmust
havebeenmethylated.Thismethod is currentlyone of the most popularapproachesto
methylationanalysisandyieldsreliable,high-qualitydata[9,10].Thedrawbacktothemethod
is that it is labor-intensiveand is not suitable for screening large numbers ofsamples.
9
2.2.3 Comparative Genomic Hybridization (CGH) and Microarray
Analysis
AcombinationofCGHandmicroarrayanalysiscanovercomethelimitationsofthebisulfite
method. This combination can enable high-throughput methylation analyses.The various
advantagesanddisadvantagesofthisapproachhavebeenreviewedpreviously[11e13].Recent
high-throughput studieshaveusedprotein affinity to enrich for methylated sequencesand
thenexploitedthesesequencesasprobesingenomicmicroarrays.MethylatedDNAfragments
can be affinity-purified either with ananti-5-methyl cytosine antibodyor byusing the DNA-
binding domain of amethyl-CpG-bindingprotein [14,15].
2.2.4 Bisulfite Treatment and PCR Single-Strand Conformation
Polymorphism (SSCP) (BiPS)
The combination of bisulfite treatment with PCR-basedsingle-strandDNAconformation
polymorphism(SSCP) analysisoffers apotentiallyquantitativeassayfor methylation [16].
Thiscombinationapproach,sometimesreferredtoasBiPSanalysis,canbeusedfortherapid
identificationof the methylation status of multiplesamples, for the quantification of
methylationdifferences,andforthedetectionofmethylationheterogeneityinamplifiedDNA
fragments.This techniquehas been successfully used toinvestigate the methylation status of
the promoter regionofthe hMLH1, p16, and HIC1 genes in several cancer cell lines and
colorectal cancertissues [17].
2.2.5 Methylation-Sensitive Single-Nucleotide Primer Extension
Methylation-sensitivesingle-nucleotideprimerextension(MS-SNuPE)isatechniquethatcan
be used for rapid quantitation of methylationat individualCpGsites [18,19]. Treatment of
genomicDNAwithsodiumbisulfiteisusedtoconvertunmethylatedcytosinetouracilwhile
Epigenetics in Human Disease
leaving 5-methylcytosine unaltered. Strand-specific PCR isperformed togenerate aDNA
templateforquantitativemethylationanalysisusingMS-SNuPE.Thisprotocolcanbecarried
usingmultiplexreactions,thusenablingthesimultaneousquantificationofmultipleCpGsites
ineach assay.
2.2.6 Combined Bisulfite and Restriction Analysis
The combined bisulfite and restriction analysis (COBRA) approachinvolves combiningthe
bisulfiteandrestrictionanalysisprotocols[20].Itisrelativelysimpletousewhilestillretaining
quantitativeaccuracy. Althoughboth COBRA and MS-SNuPE are quantitative, they havethe
restrictionsthattheformercanonlyanalyzeaspecificsequencebecauseitutilizesrestriction
enzymes and thelatteris somewhat laborious.MS-SnuPE hasalso beencombined with
microarrayanalysisto allowparallel detection of DNA methylation incancer cells [19].
2.2.7 Quantitative Bisulfite Sequencing using Pyrosequencing
Technology
Quantitaivebisulfitesequencingusingpyrosequencingtechnology(QBSUPT)isbasedonthe
luminometric detection ofpyrophosphate releasefollowing nucleotide incorporation [21].
The advantage ofQBSUPT is thatquantitativeDNA methylation data are obtained directly
fromPCR products,without the need for cloningand sequencingalargenumberof clones.
However, QBSUPTcannot be used to analyze haplotype-specificDNA methylation patterns.
Thus,while verysensitive, this assaymaybe more suited tolaboratorydiagnosis.
2.2.8 MethyLight Technology
MethyLight technology provides atool forthequantitativeanalysisof methylatedDNA
sequences viafluorescencedetection in PCR-amplified samples [22].This method hastwo
10 particular advantages: first, the fluorescentprobe can be designed todetect specific DNA
methylationpatterns,not simply to discriminate methylatedfromunmethylatedsequences;
second, ithasthe potentialability torapidlyscreen hundredsoreventhousandsofsamples.
2.2.9 Quantitative Analysis of Methylated Alleles (QAMA)
QAMA is aquantitativevariation ofMethyLight thatusesTaqMan probesbased on minor
groovebinder(MGB)technology[23].QAMAhasthemainadvantageofbeingsimpletoset
up, makingit suitablefor high-throughput methylation analyses.
2.2.10 DNA Methylation Analysis by Pyrosequencing
Pyrosequencingis areplication-based sequencing method inwhichaddition ofthe correct
nucleotidetoimmobilizedtemplateDNAissignaledbyaphotometricallydetectablereaction.
Thismethodhasbeenadapted toquantify methylationof CpG sites.The templateDNAis
treatedwithbisulfiteandPCRisusedforsequencing;theratioofTandCresiduesisthenused
toquantifymethylation. Pyrosequencing offers ahigh-resolutionand quantitativelyaccurate
measurement of methylation ofclosely positioned CpGs [24].
2.2.11 Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass
Spectrometry
Tostetal. [25]described amethodusing matrix-assistedlaserdesorption ionization time-of-
flight (MALDI-TOF) for analysisand quantificationof methylation at CpGs.Although the
methodrequiresgene-specificamplification,andshouldthereforebeconsideredacandidate
gene method, it is amenable toautomation as it can make use of the EpiTYPER platform
developed bySequenom. EpiTYPER canbe used todetermine methylation status following
gene-specific amplification of bisulfite-treated DNA followed byin vitro transcription, base-
specific RNAcleavage and MALDI-TOFanalysis [26]. Althoughit is not a genome-wide