Oxidative stress, genomic features and DNA repair in frail elderly: a systematic review

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AgeingResearchReviews37(2017)1–15

ContentslistsavailableatScienceDirect

Ageing

Research

Reviews

j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / a r r

Review

Oxidative

stress,

genomic

features

and

DNA

repair

in

frail

elderly:

A

systematic

review

María

Sánchez-Flores

a,b,1

,

Diego

Marcos-Pérez

a,b,1

,

Solange

Costa

c

_,

_João

_Paulo

_Teixeira

c

_,

Stefano

Bonassi

d,e

,

Eduardo

Pásaro

a

_,

_Blanca

_Laffon

a,∗,2

_,

_Vanessa

_Valdiglesias

a,c,2

a_Universidade_da_Coru˜_na,_DICOMOSA_Group,_Department_of_Psychology,_Area_of_{Psychobiology,}_Ediﬁcio_de_Servicios_Centrales_de_{Investigación,}_Campus

Elvi˜nas/n,15071,ACoru˜na,Spain

b_Universidade_da_Coru˜_na,_Department_of_Cell_and_Molecular_Biology,_A_Coru˜_na,_Spain

c_EPIUnit_Instituto_de_Saúde_Pública,_Universidade_do_Porto,_Rua_das_Taipas,_n◦_135,_4050-600_Porto,_Portugal d_Department_of_Human_Sciences_and_Quality_of_Life_Promotion,_San_Raffaele_University,_Rome,_Italy e_Unit_of_Clinical_and_Molecular_{Epidemiology,}_IRCCS_San_Raffaele_Pisana,_Rome,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received9April2017

Receivedinrevisedform28April2017 Accepted2May2017

Availableonline6May2017 Keywords: Cellularbiomarkers DNArepair Frailty Genomicinstability Elderly Oxidativestress

a

b

s

t

r

a

c

t

Frailtyisanemerginggeriatricsyndromecharacterizedbyhighervulnerabilitytostressors,withan

increasedriskofadversehealthoutcomessuchasmortality,morbidity,disability,hospitalization,and

institutionalization.Althoughitisgenerallyrecognizedtohaveabiologicalbasis,noparticularbiological

traithasbeenconsistentlyassociatedtofrailtystatussofar.Inthiswork,epidemiologicalstudies

eval-uatingassociationoffrailtystatuswithalterationsatcellularlevel−namelyoxidativestress,genomic

instabilityandDNAdamageandrepairbiomarkers−wererevisedandcompared.Atotalof25studies

fulﬁlledinclusion/exclusioncriteriaand,consequently,wereincludedinthereview.Variationsof

oxida-tivestressbiomarkerswereoftenassociatedtofrailtystatusinolderpeople.Onthecontrary,genomic

instabilityseemsnottobelinkedtofrailty.Theonlystudywhichaddressedthepossiblerelationship

betweenDNArepairmodulationsandfrailtystatusalsofailedinﬁndingassociation.Despitethelarge

numberofcellularalterationsknowntobeassociatedwithfrailty,studiesonthisissuearestillveryscarce

andlimitedtosomeofthepossiblecellulartargets.TheestablishedlinkbetweenDNArepair,genomic

instability,andageandage-relateddisorders,encouragedeeperinvestigationsonthisline.

Contents

1. Introduction...2

1.1. Biologicalbasisoffrailty...2

2. Materialandmethods...3

2.1. Bibliographicsearch...3

2.2. Selectioncriteria...3

3. Resultsanddiscussion...4

3.1. Oxidativestressbiomarkers...4

3.2. Genomicbiomarkers...11

3.2.1. Individualgeneticbackground...11

3.2.2. Genomicinstability...12

3.2.3. Epigenetics ... 12

3.3. DNArepairability...12

4. Conclusions...13

Acknowledgments...13

References...13

∗ Correspondingauthor.

E-mailaddress:blaffon@udc.es(B.Laffon).

1 _These_authors_contributed_equally_to_this_work.

2 _These_authors_contributed_equally_to_the_senior_authorship_of_this_manuscript.

http://dx.doi.org/10.1016/j.arr.2017.05.001

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2 M.Sánchez-Floresetal./AgeingResearchReviews37(2017)1–15

1. Introduction

Theconceptoffrailtyisgettingmoreandmoreattentionasa newandmoreaccuratewaytodefinebiologicalageaswellasto identifyvulnerabilityin elderly.Frailtyisanimportantgeriatric syndromethatrepresentsariskfactorforthoseover65yearsold, anditsprevalenceincreaseswithadvancingage(Topinková,2008). Identifyingfrailpeopleasearlyaspossibleissociallyand econom-icallycrucialsinceevidencefromdifferentstudiessuggeststhat frailtystatus,particularlyatitsveryearlieststages,mightpresent characteristicsofreversibility(Espinozaetal.,2012;Gilletal.,2006; Rolandetal.,2014).Thus,frailtycriteriashouldbeusedby geria-triciansandhealthcareprofessionalsinordertoprovideaproper andpersonalizedcaretotheolderindividual.Butcurrently,thereis noconsensusinaninternationalofficialdefinitionoffrailty,being consideredasacondition,syndromeorstatusdependingonthe authororpublication.

Currently,thereareseveraltoolsthatareusedfor screening frailtyinolderadults.Thetwomostwell-knowninstrumentsto identifyfrailtyarethephenotypicmodelproposedbyFriedetal. (2001),and thedeficitaccumulationmodeldevelopedby Rock-wood,Mitnitskiandcollaborators(Mitnitskietal.,2001;Rockwood etal.,2005).ThemodelproposedbyFriedetal.isbasedonfive cri-teriathatincludeshrinkingorunintentionalweightloss,muscular weakness,self-reportedexhaustion,slowwalkandlowphysical activitylevel.Thoseindividualswiththreeormoreofthose crite-riaareconsideredfrail,whilethosewithoneortwoareconsidered pre-frail,andthoseindividualswhodonotshowanyofthoseare considerednon-frailorrobust(Friedetal.,2001).Thecumulative modelproposedbyRockwoodandMitnitski(Mitnitskietal.,2001; Rockwoodetal.,2005),alsocalledfrailtyindex(FI),iscalculated astheratiobetweenthenumberofdeficitstheindividualpresents dividedbythetotalofdeficitsconsideredinthecomputation.The deficitsevaluated include physical criteria, neurological exami-nations,psychologicalsymptoms,andclinicallaboratoryvalues, amongothers.

Inadditiontothesemodels,therearemany otherscreening toolstoidentifyfrailty,includinganumberofvariantsofthe origi-nalcriteria.Forinstance,Montesantoscaleisapopulation-specific surveyconsistingofaclusteranalysisbasedonthreephenotypic parameters[MiniMentalStateExamination(MMSE)(Folsteinetal., 1975),hand gripstrength andGeriatric DepressionScale (GDS) (Sheikhand Yesavage,1986)], adjustedby physicaland clinical parameters(height,weight,knee-to-floorheightandwaistandhip circumferences,functionalactivityandhealthstatus)(Montesanto et al., 2010). Hospital admissionrisk profile (HARP) is another differentinstrumentfor stratifyingolderpatientsatthetime of hospitaladmission,accordingtotheirriskofdevelopingnew dis-abilitiesinactivitiesofdailyliving(ADL)followingacutemedical illnessandhospitalization(Sageretal.,1996).Thisindexisbased ondemographic information (age, gender,mental status, living arrangement,race),abilitytoperformsixADL(bathing,dressing, transferring,walking,toileting,andeating)andseveninstrumental activitiesofdaily living(IADL)(managingfinances,taking med-ications,telephoning, shopping,using transportation, preparing meals,and doing housework) two weeks beforeadmission. An abbreviatedMMSE(range0–21)isalsoobtainedduringthe admis-sioninterview.

Ina recent review theexistenceof more than260different versionsofthefrailtyphenotypepublishedintheliteraturewas reported.And even though allof them might potentially iden-tifyfrailty,themodificationsintroducedintheoriginalphenotype criteriahadimportantimpactonitsclassificationandpredictive ability(Theouetal.,2015).Thisobservationwaslaterconfirmedby Dentetal.(2016),whopublishedanothercompletereview collect-ingallthefrailtymeasurementsemployedtodatetoidentifythis

syndrome,andalsopointedoutthatmanyfrailtymeasurements weremodifiedsomewhatfromtheiroriginalvalidatedversion,and thatthiscouldeventuallyhaveastrikingimpactonfrailty classifi-cation.Afterreviewingatotalof422studiesonfrailtyandreporting 29differentfrailtyinstruments,Dentetal.(2016)concludedthat thereisnotasingleoptimalfrailtyassessmentcriteriaamongthose proposedsofar,andclaimedtheneedofunifyingcriteria,possibly throughthereleaseofguidelines,toestablishastandard measure-mentcriteriaforfrailty,whichwillmakeeasiertheidentificationof frailsubjectsinclinicalpractice,andwillmakethedifferent stud-iescomparable.Thiswould alsohelp toknowtheactualfrailty prevalenceworldwide.

Still,despitethehighnumberofavailableinstrumentsto mea-surefrailtyandtheirdifferentbasisandcriteria,itseemsthatthe predictivevalueoffrailtyfornegativehealthoutcomes,including falls,hospitalizations,disability,institutionalization,andmortality, isconsistentlyconﬁrmedformostassessmenttools,target popula-tions,andsettings(Cleggetal.,2013;Friedetal.,2009;Theouetal., 2013).

Despitealltheeffortsfocusedonestablishingacommon def-initionanda moreaccuratemeasurement offrailty,thecriteria proposedbyFriedandcollaboratorsisstillthetoolmostfrequently employed to identify frail individuals in clinical practice and research.Theworkingdeﬁnitionoffrailtyestablishedaccording toFried’scriteria,however,itisonlybasedonphysicalsymptoms andsigns.Itneglectsotherpotentiallyimportantcomponentsof thesyndromesuchasmood,cognition,sensoryimpairments,and socioeconomicaspectsofolderadults’lives(AbellanvanKanetal., 2008;Zaslavskyetal.,2013).Moreover,nobiologicalmodiﬁcations areconsidered.Numerousresearchershavearguedthatfrailtyisa multidimensionalandmultisystemprocessthatcannotbe compre-hensivelycapturedbyapplyingphysicalcriteriaonly(Dentetal., 2016;Zaslavskyetal.,2013).

Consequently,inthelastyears,moreandmoreauthors empha-sized thelimitations of classifyingfrail subjects onlyaccording toclinicalparameters,highlightingtheneedofothermarkersof frailtyatdifferentlevels,andevenconsideringseparatelyphysical andcognitivefrailty(DulacandAubertin-Leheudre,2016;Kelaiditi etal.,2013;Woodsetal.,2013).

1.1. Biologicalbasisoffrailty

Although,frailtywas initiallylinked tophysical decline and consideredassynonymousofdisabilityorcomorbidity,currently itisbecomingrecognizedasa distinctclinicalsyndromewitha biological basis (Langet al., 2009).Indeed, frailtyis commonly acceptedtohaveastrongbiologicalcomponentthatresultsfrom cumulativecellulardamageoverthelife-course(Dentetal.,2016). As people age, many systems and processes can be modiﬁed (reviewedinFielding,2015).Similarly,anumberofphysiological processes/functionshavebeendemonstratedtobealteredinfrail subjects(reviewedinWalston,2004).

However,thespeciﬁcpathophysiologicalchangesinvolvedin frailtyaetiologyremainundeﬁned.Thisismainlyduetothefact thatnosinglesystemimpairmentcharacterizesfrailty.Instead,it seemsthatanintertwinednetworkofbiologicalanomaliesat dif-ferentlevelsis likelytobepartof thepathophysiologicalchain ofeventsleadingtofrailty(Zaslavskyetal.,2013).In2009,Lang etal.alreadyhighlightedtheimportanceofimprovingour under-standingonthecomplexbiologicalfactorsleadingtoage-related muscleloss(sarcopenia, atypicalclinicalsignin frailsubjects), beyondthoseattributatoasimpledecreaseinphysicalactivityand todeleteriouschronicundernutrition.

Alltheprocessesorphysiologicalfunctionsknowntobealtered infrailpatientscanbegroupedintothreedifferentdimensions, accordingtotheorganismiclevel affected(Fig.1).Atthe

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cellu-M.Sánchez-Floresetal./AgeingResearchReviews37(2017)1–15 3

Fig.1.Biologicalbasisoffrailty.

larlevel,frailtystatushasbeenlinkedtodeﬁcienciesincellular repair abilityand consequentDNA damage accumulation(Dent etal., 2016).The biologicalconsequences of increasedlevelsof damagecanbewideranging,includingalteredgeneexpression, genomicinstability,mutations,lossofcelldivisionpotential,cell death,impairedintercellularcommunication,tissue disorganiza-tion,organdysfunctions,andincreasedvulnerabilitytostressand othersourcesofdisturbance(Rattan,2006).Atthesystematiclevel, moreandmoreevidencesuggeststhatfrailty-associated physio-logicaldysregulationinvolvesmulti-organsystems,includingthe musculoskeletal,immune,endocrine,hematologic,and cardiovas-cularsystems(Friedetal.,2009;Lietal.,2011).Finally,asmore systemsshowabnormalfunction,frailtyseverityincreases,andall thesedysregulations,althoughinitiallysilent,becomephysically evident,affectingthewholeorganism,showingupas the clini-calsignsoffrailty:musclemassloss,cognitiveimpairmentand sensorialloss,amongothers.

Recentresearcheffortshavehelpedtobetterdeﬁnethe clin-icalandphysiologicalcharacteristicsoffrailty(Langetal.,2009; Sieber,2017).Theuseofbiomarkerstoidentifyfrailsubjectsnot only wouldbe a moreprecise and objective methodfor frailty identiﬁcation, but also would allow to compare epidemiologi-cal studies drawing suitable conclusions from them. However, anddespitetheselastevidencesonthebiologicalbasisoffrailty, upto now nobiological featurehasbeen validated tobeused asa usefulbiomarkertoidentifyfrailtystatus.In thiswork,all clinical/epidemiological studies conducted in older frail adults, evaluatinganyalterationatthecellularlevel−including biomark-ersofoxidativestress,genomicalterationsandDNArepair−were reviewed,inordertoidentifyparametersthatcouldbeassociated withfrailtyconditionandconsequentlybeproposedasbiomarkers offrailty.

2. Materialandmethods

2.1. Bibliographicsearch

Theidentiﬁcation and selection ofstudies tobe includedin thereviewwascarriedoutthroughanextensiveliteraturesearch usingthePubMeddatabase(NationalLibraryofMedicine,National

InstitutesofHealth,Bethesda,MD,USA;http://www.ncbi.nih.gov/ PubMed),andwasupdatedtoMarch2016.

Thesearchstrategydevelopedcomprisedtwotermsthatwere intersectedusingtheBooleanterm“AND”.Theﬁrstoneincluded descriptorsrelatedtofrailty(‘frail’,‘frailty’or‘frailtyelderly’),and thesecondoneincludeddescriptorsrelatedtobiomarkersatthe cellularlevel (‘cellular damage’, ‘DNA’,‘genomic’, ‘oxidative’,or ‘DNArepair’).Allsearcheswerefocusedonintitleorabstract.

2.2. Selectioncriteria

Eligiblestudiestobeincludedin thereviewwereallstudies conductedinhumans,writteninEnglishorSpanish,andfocused onpopulationsofolderadults(meanage≥60yearsold).Study individualsmusthavebeenclassifiedaccordingtheirfrailtystatus followinganyofthecurrentlyvalidatedcriteriafor frailty iden-tification. Moreover,anycellularormolecularbiomarkersmust havebeenevaluatedusinganymethods.Inparticular,the stud-iesselectedforthisreviewcanbeclassifiedwithinthefollowing subgroups:

-Thoseevaluatingoxidativestressbiomarkersinfrailandnon-frail olderadults

-Thoseevaluatinggenomicbiomarkersinfrailandnon-frailolder adults

-ThoseevaluatingDNArepairabilityinfrailandnon-frailolder adults

Studiesnotconsideringfrailtybutageing,reviews,studies car-riedoutinanimals,andarticlesnotwritteninEnglishorSpanish wereexcluded.Finally,studiesusingcriterianotvalidatedto iden-tifyfrailindividuals,oremployingfrailtyasaconfounderinsteadof outcome,werealsonotincludedinthisreview.Wheneverthesame groupofauthorspublishedpapersonthesamegroupofpatients, onlythemostrecentormostcompletereportwasconsidered.

Twohundredandsixty-sixcitations(afterexcludingduplicates) were initiallyobtained and manuallyreviewed (Fig.2).Among them,75resultedeligibleafteraninitialrevisionofabstracts.The wholepublicationsofallthesestudieswerefullyreviewed,finding 21studiesthatfulfilledtheselectioncriteria.Otherfiveadditional

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Fig.2.Flowchartofsystematicreview.*Onepaper(Collertonetal.,2012)addressedthethreeaspectsconsideredinthissystematicreview.

publications were identiﬁed and included in the revision after reviewingthereferencessectionofpublishedarticles.

3. Resultsanddiscussion

Asaresultofthesearchingprocess,atotalof26studies pub-lishedfrom2008to2017suitedinclusion/exclusioncriteriaand, consequently,wereincludedinthisreview.Amongthem,8 eval-uated oxidative stress biomarkers and 17 genomic biomarkers. Besides,onestudy(Collertonetal.,2012)evaluatedoxidativestress andgenomicbiomarkers,plusDNArepairabilityonthesame pop-ulationofolderadults.Allstudiesbutone(Pereiraet al.,2016) werewrittenin English.Accordingtotheaffiliationof thefirst author,thesestudiesweremostlyconductedinEurope(54%),USA (27%),andAsia(15%).Thenumberofindividualsanalyzedperstudy rangedfrom15to5275,withameansamplesizeof499pergroup. Fourpapersonly(15.4%)includedlessthan100individuals,73% includedmorethan300,andineightoutof26reviewedstudies (30.8%)thesamplepopulationsizewaslargerthan1000subjects, confirmingtherobustnessofthedataevaluatedinthisreview.The meanageofthestudiedindividualsrangedfrom61.9to99years pergroup,withatotalmeanageof66.8years.Consideringall stud-iesinwhichpatientswereclassifiedbyfrailty,themeanageinfrail groupswas78.1years,inpre-frailgroups76.4years,andinnon-frail groups74.4years.

Regardingthecriteriaemployedtoidentifyfrailpeople,almost allstudies(24outof26)employedeitherFried’sorRockwood’s criteria(FI),orevenbothofthem.Amongthesestudies,thegreat majority(67%)employedFried’scriteriaforfrailtyidentification, andonlyintwostudiesamodifiedversionwasusedinsteadofthe originalone.Fiveoutof24studies(21%)usedFItoidentifyfrail subjects,andthreeoutof24studies(13%)employedbothofthem. However,thenumberofitemsanalyzedinstudiesemployingFI wasalwaysdifferentamongstudies,rangingfrom17to40.The tworemainingstudiesnotusingFried’sorRockwood’scriteriawere Bellizzietal.(2012)andPereiraetal.(2016),whichemployedthe Montesantoetal.(2010)method,andtheHospitalAdmissionRisk Profile(HARP)togetherwithFI,respectively.

All studies included in this review are presented in tables (Tables1and2)anddescribedinthefollowingsectionsaccording tothetypeofparameterevaluated.Althoughperforminga meta-analysisofthesestudieswasinitiallyconsidered,itwasnotpossible tocarryitoutduetothehighvariabilityintheoutcomesaddressed togetherwiththelimitednumberofstudiesassessingthesame outcome.

3.1. Oxidativestressbiomarkers

Reactiveoxygenspecies(ROS)arefreeradicalsformedduring thecellularmetabolism.Generally,theseROSarenotharmfulfor thecellsincetheyareneutralizedbycellularantioxidantsystems,

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M. Sánchez-Flores et al. / Ageing Research Reviews 37 (2017) 1–15 5 Table1

Studiesassessingoxidativestressbiomarkers.

Study Totalpopulation

(meanage±SD)

Casepopulation(mean age±SD)

Controlpopulation (meanage±SD)

Frailtycriteria Outcomes Assaymethod Results

Bleetal.(2006) n=827(73.6±6.4)446 females,381males

Frailn=54 Pre-frailn=313; non-frailn=460

Friedetal.(2001) PlasmaVit-Elevels Reverse-phaseHPLC LevelsofvitaminEdecreased graduallyfromthenon-frailto thefrailgroup

Collertonetal.(2012) n=552(85)332 females,220males Frailn=119(85)92 females,27males Pre-frailn=333(85) 193females,140 males; Non-frailn=100(85) 47females,53males Friedetal.(2001) FI(40deﬁcits) Plasmalipid peroxidation: isoprostanesiPF2 alpha-IIIandiPF2 alpha-VI

LC/MS/MS Noassociationlipid peroxidationwithfrailty

Inglesetal.(2014) n=742 Frailn=54(78.8±6.0) 36females,18males Pre-frailn=278 (73.8±4.7)160 females,118males; n=410non-frail (72.4±4.2)237 females,173males

Friedetal.(2001) Plasmalipid peroxidation:MDA

HPLC Westernblot

FrailpeoplehadhigherMDA levelsthannon-frailsubjects. Plasmaprotein

carbonylation

Frailpeoplehadhigherlevels ofproteincarbonylationthan non-frailsubjects

Liuetal.(2016) n=1919 Frailn=142(77±6)77 females,65males Pre-frailn=864 (72±7)495females, 369males; non-frailn=913 (69±6)463females, 450males

Friedetal.(2001) PlasmaLpPLA2activity andmass,serum ICAM-1andMCP-1, and urine8-epi-PGF␣isoprostanes

ELISA Frailtywasindividually associatedwithisoprostanes, LpPLA2mass,ICAM-1,and MCP-1.

Pre-frailtywasindividually associatedwithelevatedlevels ofLpPLA2activity,ICAM-1, andMCP-1

Inthoseindividuals≥70years old,associationsbetween isoprostanesandfrailty,and betweenpre-frailtyandMCP-1 werenolongersigniﬁcant

Namiokaetal.(2016) n=140 Frailn=34(82.3±6.1) 23females,11males Pre-frailn=62 (80.5±4.9)40females, 22males; Non-frailn=44 (78.2±6.0)19, females,25males

Friedetal.(2001) PlasmadROM Freeradicalanalyzer system

(spectrophotometry)

dROMlevels:frail> pre-frail>non-frail individuals PlasmaBAP Freeradicalanalyzer

system

(spectrophotometry)

BAPlevelsweresigniﬁcantly lowerinthefrailgroupthanin thenon-frailgroup

Endogenousplasma anti-oxidants:albumin, bilirubin,uricacid

Bromocresolpurple staining,vanadicacid oxidation,anduricase assay,respectively

Bilirubinlevelswere signiﬁcantlylowerinthefrail groupthaninthenon-frail group.Noassociationalbumin oruricacidwithfrailty Urinary8-OHdGand

8-epiPGF2␣

HPLCandenzyme immunoassay, respectively

Urinaryexcretionsof8-OHdG and8-epiPGF2␣were signiﬁcantlyhigherinthefrail andpre-frailgroupsthaninthe non-frailgroup

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6 M. Sánchez-Flores et al. / Ageing Research Reviews 37 (2017) 1–15 Table1(Continued)

(meanage±SD)

Pereiraetal.(2016) n=15(88±9.78) Highriskoffrailty (HARP)n=7 Intermediateorlow riskoffrailty (HARP)n=7 HARP FI Lymphocyte membranelipid peroxidation: conjugateddienesand trienes

UVspectrophotometry Elderlypatientswithahigher degreeoffrailtyhad signiﬁcantlyhigherlevelof dienesinbothfrailtyscales, andsigniﬁcantlyhigherlevelof trienesonlyinFI Intermediateorsevere frailty(FI)n=12 Non-frail/mild frailty(FI)n=3 Plasmaprotein oxidation:MDA proteinadducts

TBARStest Elderlypatientswithahigher degreeoffrailty(HARP) presentedsigniﬁcantlyhigher levelsofMDAthanthosewith milderlevelsoffrailty

Saumetal.(2015) n=2518 Frailn=210 (73.7±6.0)136 females,74males Pre-frailn=1463 (70.3±6.2)820 females,643males; non-frailn=845 (67.8±5.8)367 females,478males

Friedetal.(2001) PlasmadROM Spectrophotometry CorrelationbetweendROM levelsandfrailtywas statisticallysigniﬁcant,but attenuatedafteradjustment withmultiplecovariates.

PlasmaTTL Aninversestatistically

signiﬁcantassociationwith frailtywasobservedforTTL.

PlasmaBAP NoassociationBAPwithfrailty

Serviddioetal.(2009) n=62(76.7±5.1) 23females,39males

Frailn=43 n=19non-frail Friedetal.(2001) GSSG Alkalinehydrolysisof N-ethylmaleimide

Asigniﬁcantincreaseinthe GSSGwasobservedinfrail patientswhencomparedto non-frail

WholebloodtotalGSH level

Spectrophotometry NoassociationGSHlevelwith frailty

Frailpatientsexhibitedan increaseintheGSSG/GSHratio ascomparedtonon-frail PlasmaMDAandHNE

proteinsadducts

Spectroﬂuorimetry MDAandHNEadductswere signiﬁcantlyhigherinfrailtyas comparedtonon-frailpatients Plasmaoxidized

proteins

WesternBlot Anappreciabledecreasein oxidizedproteinswasdetected innon-frailsubjectswhen comparedtofrailpatients

Wuetal.(2009) n=90 Frailn=21(79.9±5.8) Pre-frailn=56 (76.8±5.8); non-frailn=13 (73.1±5.3)

Friedetal.(2001) Serum8-OHdG ELISA Frailsubjectshadhigherserum 8-OHdGlevelthanpre-frailand non-frailindividuals

8-OHdG,8-hidroxy-2_{-deoxyguanosine;}_{BAP,biological}_antioxidant_potential;_dROM,_derivatives_ofreactive_oxygen_metabolites;_ELISA,_{enzyme-linked}_{immunosorbent}_assay;_FI,frailty_index;_{GSH,glutathion;}_GSSG,_glutathione

disulﬁde,oxidizedglutathione;HARP,hospitaladmissionriskproﬁle;HNE,4-hidroxy-2,3-nonenal;HPLC,highperformanceliquidchromatography;ICAM-1,intracellularadhesionmolecule-1;LC,liquidchromatography; LpPLA-2,lipoproteinphospholipaseA2;MCP-1,monocytechemoattractantprotein-1;MDA,malondialdehyde;MS,massspectrometry;TBARS,thiobarbituricbindingacidreactivespecies;TTL,totalthiollevels;Vit-E,vitaminE.

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M. Sánchez-Flores et al. / Ageing Research Reviews 37 (2017) 1–15 7 Table2

Studiesassessinggenomicbiomarkers.

(meanage±SD)

Asharetal.(2015) n=4892;2774females, 2118males

na na Friedetal.(2001) mtDNAcopynumber qPCR LowermtDNAcopynumber wassigniﬁcantlyassociated withprevalentfrailtyonlyin whiteparticipants

Bellizzietal.(2012) Cross-sectionalstudy: n=318(S1+S2). S1:n=217(medianage 75years)123females, 94males,classiﬁedin non-frail,pre-frailand frail.

S2:n=101(medianage 99years)51females,50 males,classiﬁedinfrail andveryfrail

na na Montesantoetal.

(2010)

GlobalDNA methylation

CpGlobalassay GlobalDNAmethylationlevels werecorrelatedwithfrailtyin S1butnotinS2.

Longitudinalstudy7 yearslater: n=37,randomsample fromS1pre-frailand nonfrailgroups

LowerglobalDNAmethylation levelsinpre-frailsubjectsthat becamefrailafter7years

Braultetal.(2014) n=53(≥75yearsold) na na Friedetal.(2001) TLinleukocytesand aortictissue

NoassociationbetweenTLand frailty.

Associationbetweenlonger leukocyteandaorticT/Sratio andgreaternumberofclinical frailtycriteria.

Breitlingetal.(2016) Dataset1:n=969 (62.1±6.5)484 females,485males

na na _FI₍₃₄_deﬁcits) DNAmethylationage

acceleration (methylationage minuschronological age) Inﬁnium HumanMethylation450 BeadChip AssociationofDNA methylationageacceleration withFIincreasedwith increasingageacceleration Dataset2:n=851

(63±6.7)464females, 387males

TL qPCR InteractionbetweenTLand

epigeneticageaccelerationdid notimprovethepredictionof FI

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8 M. Sánchez-Flores et al. / Ageing Research Reviews 37 (2017) 1–15 Table2(Continued)

(meanage±SD)

Collertonetal.(2012) n=552(85)332 females,220males Frailn=119(85)92 females,27males Pre-frailn=333(85) 193females,140 males; non-frailn=100(85) 47females,53males Friedetal.(2001) FI(40deﬁcits) TL qPCR NoassociationofTLorionized radiation-inducedDNA damageandrepairwithfrailty Ionized

radiation-inducedDNA damageandrepair

Automated ﬂuorimetricalkaline DNAunwinding

Collertonetal.(2013) n=1173(85.5) Frailn=696(FI), n=477(Friedetal., 2001)

3ethnicallymatched populationcontroldata sets

Friedetal.(2001)

FI(40deﬁcits)

mtDNAhaplogroups SequenomMassARRAY Noassociationbetween commongeneticvariantsof mtDNAandfrailty

Collertonetal.(2014) n=321(85)184 females,137males na na Friedetal.(2001)(CHS modiﬁed) DNAmethylationin CpGislands Highlyquantitative pyrosequencing AssociationCpGisland methylationwithfrailty n=231(85)148

females,83males

LINE-1methylation levels(surrogatefor genome-wideDNA methylationlevels)

Noassociationgenome-wide methylation(LINE-1)with frailty

Gaoetal.(2017) Discoverysetn=978 (62.1±6.5)

Frailn=100 Pre-frailn=443; non-frailn=435

FI(34deﬁcits) DNAmethylation proﬁles

IlluminaHuman Methylation450 BeadChip

17smoking-relatedCpGsites wereassociatedwiththeFI. 9ofthosesiteswere designatedasfrailtyassociated loci.Sixofthem[cg02657160 (CPOX),cg05673882(POLK), cg07826859(MYO1G), cg19859270(GPR15), cg23667432(ALPP),and cg25189904(GNG12)]were mappedshowingmethylation intensityin

frail<pre-frail<non-frail SI,basedonthose9 smoking-relatedCpGsites, manifestedamonotonic dose-responserelationship withtheFI Validationsetn=531 (62.0±6.6) Frailn=48 Pre-frailn=220; non-frailn=263

Hoetal.(2011) n=348females(74.2) Frailn=152 Pre-frailn=165; non-frailn=32

Friedetal.(2001) SNPvariations(134 genes,1354SNP)

BeadArray(Illumina customGoldenGate 1536SNPpanel)

20SNP(from11genes) associatedwithfrailty(not signiﬁcantly)

Jylhavaetal.(2013) n=174 Frailn=144 (nonagerians)101 females,43males

n=30youngcontrols (range19–30years)21 females,9males

Friedetal.(2001) Totalcf-DNAinplasma Fluorimetry (Quant-iTTM_DNA

high-sensitivityassay kit)

Innonagenarians: Higherlevelsoftotaland unmethylatedcf-DNAwere associatedwithincreased frailty

Unmethylatedcf-DNA ELISA(DNA MethylationKit) Genomicequivalentsof

theRNaseP-coding cf-DNA

qPCR Noassociationwithfrailty

Alurepeatcf-DNA qPCR Noassociationwithfrailty mtDNAcopynumber qPCR mtDNAcopynumberwas

directlycorrelatedwith increasedfrailty

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M. Sánchez-Flores et al. / Ageing Research Reviews 37 (2017) 1–15 9

Kimetal.(2015) n=320.Parentsatleast 90yearsoldandtheir offspring(50–80years old)

na na FI(34deﬁcits) Genome-widelinkage

scanningfollowedby ﬁne-scaleassociation mapping Genotyping:BeadChip array(Illumina InﬁniumLinkage24 set); associationmapping:I lluminaGoldenGate assay

Theyfoundthreesites associatedwithhealthyageing

Marzettietal.(2014) n=142(74.9±6.5)84 females,58males Frailn=74 Pre-frail/non-frail: n=68 Friedetal.(2001) FI(30deﬁcits)

TL qPCR Noassociationwithfrailty

(FriedorFI)

Matteinietal.(2010) n=326females(74.1) na na Friedetal.(2001) 56SNPfromsix candidategenes involvedinVitamin B12metabolic pathway:MTHFR, MTR,MTRR,CBS,TCN1 andTCN2

IlluminaBeadArray SNPintheTCN2showed signiﬁcantassociationwith frailty.

TwoSNPinMTRRshowed2–4 timesgreateroddsofbeing frailcomparedtorobust

Mooreetal.(2010) Pilotstudy:n=315 Frailn=154(75±4.45) 112females,42males.

Non-frail:n=161 (81.35±3.16)111 females,50males

Friedetal.(2001) mtDNAvariations (SNP)

Oligonucleotide sequencingmicroarray

ThreemtDNASNPs(mt146, mt204,andmt228)were associatedwithfrailty Cross-sectionalstudy:

totaln=5275

Frailwhite:n=262 (77.36±6.36)174 females,88males;frail black:n=102 (75.44±6.76)76 females,26males Non-frailwhite: n=4223(72.43±5.37) 2377females,1846 males;non-frailblack: n=688(72.05±5.09) 425females,263males

Real-timePCRTaqMan assaysforindividual SNPselectedfor follow-up(mt146, mt204,andmt228)

mt204Callelewasassociated withgreaterlikelihoodof frailty

Saumetal.(2014) n=3537(61.9±6.6) 1963females, 1574males.

na na FI(34deﬁcits) RelativeTL(T/Sratio) qPCR NodifferenceoftheFIbetween

theT/Sratiotertileswas observed.

TLmeasurementsbytheT/S ratiowerehighlycorrelated withabsoluteTL Validationina subpopulationn=20 AbsoluteTLinbase pairs Southernblot Valdiglesiasetal. (2015) n=180 Frailn=93 (76.9±6.6)52females, 41males Non-frailn=87 (72.9±6.1)50females, 37males

Friedetal.(2001) MNfrequency CBMNwithautomated scoring

NoassociationbetweenMN frequencyandfrailty

Wooetal.(2008) n=2006.1030females (72.02±5.191),976 males(72.75±5.026)

na na FI(17deﬁcits) TL qPCR NocorrelationbetweenTLand

frailty Yuetal.(2015) n=2006(72.4±5.1) 1030females,976 males Frailn=127 Pre-frailn=967; non-frailn=912,

Friedetal.(2001) TL qPCR NoassociationbetweenTLand frailtyatbaseline,norin4year follow-up

CBMN,cytokinesis-blockedmicronucleustest;cf-DNA,cellfreeDNA;CHS,cardiovascularhealthstudy;LINE-1,longinterspersednuclearelements1;MN,micronucleus;mtDNA,mitochondrialDNA;na,notavailable;qPCR,quantitative PCR;SI,Smokingindex;SNP,singlenucleotidepolymorphism;T/Sratio,meantelomererepeatcopytosinglegenecopynumber;TL,telomerelength.

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but theirproduction can alsobe induced byexogenous agents thatincludeionizingradiation,airpollutionandawiderangeof chemicals(Halliwell,2007).Insuchcases,theimbalancebetween increasedROSproduction and antioxidant defensesleadstoan oxidativestressstatewhichishighlydestructiveforthecelland theorganism.Ithasbeendemonstratedthatoxidativestressplays animportantroleinneurodegenerativediseases,oftenassociated withageing(MiglioreandCoppede2002;Perryetal.,2002);indeed oxidativeDNAdamageisoneoftheeventsthatcanbedetected ear-lierinthepathogenesisofthesediseases.Furthermore,oxidative stressisconsideredariskfactorforageing(CoppedèandMigliore, 2009).

Besides, increases in oxidative stress with ageing may also contributetothedevelopmentofchronicinflammationand dis-ease(Woodsetal.,2012).Indeed,thereareavarietyofpotential mechanismslinkingoxidative stress toinflammation,including disturbances in the redox equilibrium, decrease of oxidation-sensitive biomolecules related to immune response, such as vitaminsor5,6,7,8-tetrahydrobiopterin(BH4),oralterationsin pat-ternrecognitionreceptorsoftheinnateimmunesystem,suchas toll-likereceptors(Gilletal.,2010;Strasseretal.,2017). Accord-ingly,anumberofpreviousstudieshaverelatedimmunesystem alterationstofrailty(reviewedinFulopetal.,2015).Still,thiskind ofimmunologicalalterations,althoughmaybeclassifiedascellular alterationsinsomecases,wereconsideredbiomarkersat system-aticleveland,consequently,notincludedinthepresentreview.

Therelationshipbetweenoxidativestressandfrailtyhasbeen studiedin several works. Table 1 shows all humanpopulation studiesevaluating thepossibleassociation betweenmarkers of oxidativestressandfrailtyinoldage.Atotalof9studieswerefound intheliteratureaddressingthisissue.Thetotalantioxidantcellular abilityorthelevelsofsomespeciﬁcantioxidantsinthefrailsubjects regardingthepre-frailandnon-frailindividualswasmeasuredin4 (44.4%)outof9reviewedstudies.In7studies,directeffectsofROS onlipids(71%)(Collertonetal.,2012;Inglesetal.,2014;Liuetal., 2016;Pereira etal.,2016;Serviddioetal.,2009),proteins(57%) (Inglésetal.,2014;Pereiraetal.,2016;Serviddioetal.,2009)and DNA(29%)(Namiokaetal.,2016;Wuetal.,2009)wereevaluated; evaluationofmorethanonetargetinthesamestudywascommon. Moreover,justin2cases(Namiokaetal.,2016;Saumetal.,2015), thelevelsofderivativesofreactiveoxygenmetabolites(d-ROM) weredetermined.

Resultsfromthesestudiesdonotclarifyifoxidativestressis consequenceoffrailty(orviceversa),orif−assuggestedbyLiuetal. (2016)−thereisabidirectionalrelationshipwherethepresence ofoneofthemincreasestheriskoftheother.

CellshavedifferentmechanismstolimitthelevelsofROSand thedamagetheyinduce.Thecellulardefensemechanismsagainst oxidativestressincludebothenzymatic(e.g.catalase,superoxide dismutase)andnon-enzymatic(e.g.vitaminE[vit-E],glutathione) antioxidants,whichplayacentralroleinmaintainingthecellular redoxbalanceessentialforcellsurvival(Birbenetal.,2012). Sev-eralalterationsindifferentantioxidantsinassociationwithfrailty havebeenobtainedinthestudiesreviewed.Forinstance,vit-E,or ␣-tocopherol,isthemajorlipophilicantioxidantinhumans;low levelsofvit-Eareconsideredanindirectbiomarkerofoxidative stressand,therefore,it hasbeenhypothesizedtobeassociated withanincreasedriskoffrailty.Accordingtothis,Bleetal.(2006) foundasigniﬁcantdecreaseofvit-Elevelsinfrailindividuals,after adjustmentformultipleconfounders,inapopulationof827older individualsclassiﬁedaccordingtoFried’scriteria.

Also,increasesofbothoxidizedglutathione(GSSG)andthe oxi-dized/reducedglutathioneratio(GSSG/GSH),butnormalreduced glutathione(GSH)levels,werefoundin43frailindividuals(Fried’s criteria)regarding19non-frailsubjects(Serviddioetal.,2009).

Investigating the association between oxidative stress and frailty,Saumetal.(2015)evaluatedthreebiomarkersofoxidative stressin2518individualsclassifiedaccordingtoFried’scriteria: totalthiollevels(TTL),d-ROM,andbiologicalantioxidantpotential (BAP).Whilstnostatisticallysignificantdifferencewasobserved inthelevelsofBAPinfrailindividualsascomparedwiththe pre-frailandnon-frailsubjects,thefrailgroupshowedhigherd-ROM butlowerTLLlevels.Also,significantpositivecorrelationsbetween d-ROMand BAP,and BAPand TTLwereobserved, aswellas a weakinverseassociationbetweend-ROMand TTL.Theauthors concludedthatTTLwasthebiomarkermostconsistentlyassociated withfrailtyinalltheregressionmodels.

Namiokaetal.(2016)studiedoxidativestressinasampleof 140olderadultswithmild-to-moderateAlzheimerDisease, clas-sifiedaccordingtotheirfrailtystatususingtheFried’scriteria.To thatend,levelsofseveraloxidativestressbiomarkerswere evalu-atedincludingd-ROM,BAP,andendogenousplasmaantioxidants (namelyalbumin,bilirubin,anduricacid).Consistentlywiththe observationsbySaumetal.(2015),resultsshowedsignificantly increasinglevelsofdROMinnon-frail<pre-frail<frailgroups.On theotherhand,BAPandplasmabilirubinlevelsweresignificantly lowerinthefrailgroup.Plasmaalbuminanduricacidlevelsshowed nosignificantdifferencesamongthethreegroups.

ROS attack all biological molecules including DNA, proteins andlipids.Lipidsandlipoproteinsareparticularlysusceptibleto ROSattackbecausehydrogenabstraction bya radicalcan initi-ateadevastatingchainreaction:lipidperoxidation(Andersonand Phillips,1999).Severalstudiesevaluatedlipidperoxidationand oxidizedproteinlevelsinrelationtofrailty.Serviddioetal.(2009) analyzedplasmalevelsofmalonaldehyde(MDA)and 4-hydroxy-2,3-nonenal (HNE) protein adducts (both of them recognized biomarkersoflipidperoxidation),and plasmaoxidizedproteins (asexpressionofoxidativeproteindamage)inapopulationof62 oldindividuals,classiﬁedasfrailandnon-frailaccordinglytothe Fried’scriteria.HigherlevelsofMDAandHNE adductsand oxi-dizedproteinswereobservedinfrailindividualsascomparedwith non-frailsubjects.

Liuetal.(2016)studiedtheassociationoffrailty(Fried’scriteria) withseveraloxidativestressbiomarkersrelatedtocardiovascular disease,namelyisoprostanes(accuratemarkersoflipid peroxida-tion)andthelipoproteinphospholipaseA2(LpPLA2)(anenzyme involvedinhydrolizationofoxidizedphospholipids)ina popula-tionof1919olderindividuals.Theyconcludedthatsigniﬁcantly increasedlevelsofisoprostanesandLpPLA2masswererelatedto greateroddsoffrailty,sincetheyfoundindividualassociationof frailtywithelevatedlevelsofisoprostanesandLpPLA2mass,and ofpre-frailtywithelevatedlevelsofLpPLA2activity.

Inglésetal.(2009)employedMDAandproteincarbonylation(a circulatingindicatorofoxidativedamagetoproteins)toassessthe relationshipbetweenfrailtyandoxidativestress,andthe capac-ityofthesetwoparametersaspossiblebiomarkersoffrailty.Their resultsshowedsigniﬁcantlyhigherlevelsofMDAandcarbonylated proteinsinolderindividualsclassiﬁedasfrail,accordingtoFried’s criteria,thaninnon-frail;norelationshipwithageorsexwasfound inanycase.

Also,Pereiraetal.(2016)foundinasmallpopulation(n=15) thatolderindividualswithahigherdegreeoffrailty,measuredby FIorHARPcriteria,showedhigherlevelsoflipidperoxidation.In thisstudy,proteinoxidation levels(MDAproteinadducts)were alsoevaluated,showinghigherlevelsinindividuals withhigher riskoffrailtydeterminedbyHARPcriteria,butnotbyFI.

Collerton et al. (2012) carried out a study with 552 older adults classiﬁed by means of both Fried’s criteria and FI (40 items),evaluatingthepossibleassociationbetweenfrailtystatus and biomarkersof lipidperoxidation, namelyisoprostanes iPF2

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M.Sánchez-Floresetal./AgeingResearchReviews37(2017)1–15 11

alpha-IIIandiPF2alpha-VI.Noassociationbetweenfrailtyandthe oxidativestressbiomarkerswasfoundinthiscase.

As previously indicated, ROS may react with different biomoleculesin cells,and oneof themaintargetsis DNA.This kindofdamageisoftenreferredtoasoxidativeDNAdamage.The consequencesincludedifferenttypesofDNAalterations,ranging fromsimpleoxidationofbasestolargedeletions,throughsingle anddoublestrandbreaks(Rao,2009).PathogenicrolesforDNA oxidationincludetheinductionofmitochondrialdysfunction, pro-motionofcytotoxicityandmodulationofinflammatoryresponses (EvansandCooke,2006).Accordingtoourrevision,onlytwo stud-ieshaveevaluatedconsequencesofoxidativestressonDNAinfrail andnon-frailolderpeople.Wuetal.(2009)evaluatedthe relation-shipbetweenoxidativestressandfrailtybymeasuringthelevelof serum8-hidroxy-2-deoxyguanosine(8-OHdG)inasampleof Chi-neseolderindividuals(n=90).Thefrailtystatuswasdetermined byusingtheFried’scriteria.Resultsshowedsignificantlyincreased serum8-OHdGlevelsinfrailindividualswithrespecttothe pre-frailandnon-frailsubjects.Morerecently,Namiokaetal.(2016) observed that urinary excretions of 8-OHdG and 8-isoprostane weresignificantlyhigherinfrailandpre-frailindividualsthanthose determinedinthenon-frailgroup.

Togetherwiththese9selectedstudies,twoadditional epidemi-ologicalstudies,notincludedinthefinalreviewedworksbecause theydidnotstrictlyfulfilltheinclusioncriteria,addressedthe rela-tionshipbetweenfrailtyandoxidativestressinanindirectway. Firstly,Caballeroetal.(2014)assessedtheroleofoxidativestresson deficienciesoffunctionalphysicalperformanceofthelowerand/or upper body limbs,that could affecta future pre-frailty pheno-type(Fried’scriteria).Tothisaim,theystudiedtheconcentrations ofplasmaproteins,carbonylatedproteins,lipidperoxidationand plasmatotalantioxidantactivity,asmeasuresofoxidative dam-age,inapopulationof200individualsaged≥70years.Theirresults showedsignificantlylowerlevelsoftotalantioxidantcapacityin women_≤76yearsoldwithdeficienciesinthephysicalperformance ofbothlowerandupperbodylimbs,butnotinwomen>76years oldorinmenofanyage,suggestingthatdeficientoxidativedefense intheelderlycouldsignificantlyaffectthefunctionalphysical per-formanceandfutureoutcomesofpre-frailindividuals.

Secondly,Baptistaetal.(2012)usedthegaitspeedasameasure ofphysicalperformanceinolderadults,inapopulationof280 indi-viduals.Theobjectiveofthisstudywastoevaluatethesuperoxide anionproduction,anditsinteractionwithphysicalfrailtymeasured bygaitspeed in olderadults. Theyfoundnodifferences inthe baselinelevelsofsuperoxideanionproductionbetweensubjects withslowerandfastergaitspeed.However,afterstimulationwith PMA(lucigeninandphorbol12-myristate13-acetate),the super-oxideanionproductionwassigniﬁcantlyhigherinslowwalkers (gaitspeed<0.8m/s).Hence,theysuggestedthattheproduction ofthesuperoxideanioncanbeinvolvedinthedeclineofphysical performanceintheolderageand,therefore,inthefrailtyprocess. 3.2. Genomicbiomarkers

Thecontributionoftheindividualgeneticproﬁletothe devel-opmentoffrailisstilluncertain.Whetherandtowhatextentthe individualgeneticfeatures affecttheindividualsusceptibilityto frailtyisnotwellestablishedyet.Inthisregard,studiesinvolving analysisofthecontributionofgeneticbackgroundtofrailtyandin particularevaluatingtheassociationofgenomicinstabilitytothis conditionmayhelptoprovideinsightsintobiologicallyrelevant pathwaysthatcontributetofrailty.Amongthestudiesreviewed hereaddressinggeneticalterations,sixofthem(35.3%)evaluated geneticbackgroundoffrailindividuals,includingvariationsinboth nuclearandmitochondrialDNA,eight(47%)investigatedthe rela-tionshipbetweengenomicinstabilityandfrailty,andthree(17,7%)

addressedthepossibleepigeneticcharacteristicsoffrailtystatus (Table2).

3.2.1. Individualgeneticbackground

Threeoutofthe17studiesreviewedhereevaluatedthe indi-vidualgeneticdifferencesassociatedtofrailty. Kimetal.(2015) studiedtheheritabilityof healthy ageingasanattempt tofind themorebeneficialgeneticvariantsorlessdisadvantageous vari-antspresentinhealthy,long-livedpeople.Theauthorsperformeda genome-widelinkageanalysisandfine-scaledassociationmapping oflinkageregions,usingtheFI(34deficits)asaquantitative mea-sureoffrailtyinapopulationof320olderadults.Threehealthy ageingsites(HAS)werefoundat12q13-14inintergenicregions (HAS-1and−2withenhanceractivity,HAS-3withsilencer activ-ity).

Hoetal.(2011)studieddifferentbiologicalpathwaysinvolved infrailtytotrytofindvariationsingenesrelatedtothefrailty syn-drome.Tothisaim,theygenotyped1354SNPfrom134candidate genesinvolvedininflammationandmusclemaintenance,twoof themainphenotypiccharacteristicsoffrailty,inapopulationof348 subjectsclassifiedaccordingtoFried’scriteria.TwentySNP, indi-rectlyrelatedtoinflammatoryprocess,werefoundtobeassociated withfrailty,althoughstatisticalsignificancewasnotreached.

Matteini et al. (2010) also investigated genetic variants of sixcandidategenes(MTHFR,MTR,MTRR,CBS,TCN1,andTCN2) involvedinVitaminB12metabolicpathwayandtheirassociation withfrailty(Fried’scriteria),in416olderwomen.Forthispurpose 56SNPfromthosesixcandidategenesweregenotyped.Theresults foundSNPintheTCN2geneshowingsigniﬁcantassociationwith frailty.Moreover,twoSNPinMTRRgeneshowed2–4timesgreater oddsofbeingfrailcomparedtorobust.

Mitochondriaareinvolvedinseveralmajorcellfunctions,such ascellproductionofenergy,metabolicandapoptoticprocesses,as wellasbeingamajorsiteofROSgeneration.Mitochondrialfunction experiencesalterationswithageandthesechangesareassociated withseveralage-relateddiseases.Thus,age-relatedchangesand variationsinthemtDNAhavebeenproposedasplausible candi-datestoplayaroleindegenerativeandsenescentprocesses,and tocontributetoincreasesinvulnerabilityinlatelife(Mooreetal., 2010);hencetheyarelikelytobeassociatedwithfrailtysyndrome. Threestudieswerefoundintheliteraturesearchthatevaluatedthe possibleassociationbetweenmtDNAalterations(incopynumber orinsequence)andfrailty.Firstly,Collertonetal.(2013) evalu-atedtheassociationbetweencommongeneticvariantsofmtDNA andfrailtyasameantostudyheritabilityofhumanlongevityand healthyageing,sincefrailtyisconsideredan“unhealthyageing” phenotype.Theyevaluatedapopulationof1173olderadults.No associationbetweenmtDNAhaplogroupsandfrailty,assessedby bothFried’scriteriaandFI(40deﬁcits),wasobserved.

Onthecontrary,Asharetal.(2015)evaluatedtheassociation betweenmtDNAcopynumberandprevalentfrailty(Fried’s crite-ria;n=4892),reportinglowermtDNAcopynumberinfrailwhite, butnotinfrailblack,individuals.Theyalsoobservedasignificant inverseassociationofmtDNAcopynumberwithage,andhigher mtDNAcopynumberinwomenrelativetomen.Finally,Mooreetal. (2010)studiedhowmtDNAvariations(SNP)withagemayincrease thesusceptibilitytofrailty.Inapilotstudyof315individuals classi-fiedaccordingtoFried’scriteria,threemtDNASNPwereassociated withfrailty(mt146,mt204,and mt228).Fromthesethree SNP, mt204Cwasconfirmedtobethealleleassociatedwithgreater like-lihoodoffrailtyinthecross-sectionalfurtherstudyincluding5275 subjects.

Aftercelldamage ordeath,DNAis releasedintothe circula-tion;thisiswhyplasmacellfreeDNA(cf-DNA)reflectssystematic inflammationandcelldeath,whatmakesitapotentialbiomarker ofageingandfrailty.Forthatreason,Jylhavaetal.(2013)quantified

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theplasma levelsof total cf-DNA, unmethylatedcf-DNA, gene-codingcf-DNA,andAlurepeatcf-DNA,andmtDNAcopynumber to study their potential as biomarkers of frailty (Fried’s crite-ria;n=174). Theirresults showedincreasedlevels oftotal and unmethylatedcf-DNA associatedwithfrailty. AlsomtDNA copy numberwascorrelatedwithfrailty.However,neithergene-coding cf-DNAnorAlurepeatcf-DNAwereassociatedwithfrailty. Simi-larly,thesamegroup(Jylhavaetal.,2014)studiedtheassociationof thesebiomarkerswithmortality,ﬁndingthatfrailtyisdetrimental forsurvivalwhenusedtoadjusttheresultsobtained.

3.2.2. Genomicinstability

Asmentionedbefore,eightoutof17studiesevaluatedgenomic instabilityinfrailty.Genomicinstabilityreferstoasetofgenetic eventscapableofcausingtemporaryorpermanentunscheduled alterationswithinthegenome, includingdiversetypes of chro-mosomalalterations (e.g.inversions,deletions,duplicationsand translocationsof largechromosomal segments) (Migliore et al., 2011;Valdiglesiasetal.,2015).Thus,genomeinstabilitycouldlead toalteredgenedosageandgeneexpressionaswellascontribute totherisk ofacceleratedcelldeathinneuronal tissue(Thomas andFenech,2007).Inaccordance,genomicinstabilityisconsidered ahallmarkofanumberofageing-relateddiseasesincluding can-cer,andisrelatedtotheageingphenotypeandneurodegenerative disorders(CoppedèandMigliore,2010;Miglioreetal.,2011),

Amongthestudiesevaluatinggenomicinstability,allbutone (Valdiglesiasetal.,2015)analyzedtelomerelength(TL)intheolder subjects.TLanalysisisacommonapproachtoevaluategenomic instabilityin bloodsamples.Telomeresareregionsofrepetitive nucleotidesequenceateachendofthechromosomes,which con-tributetomaintaintheirintegrity.Theyprogressivelyshortenas the cell divides, limiting the number of divisions that normal somaticcellscanundergo(Marzettietal.,2014).Theobservation thattelomeresshortenoverthelifecoursehasledtothehypothesis thattelomereattritionmaybeamechanismdrivingtheageing pro-cess(MikhelsonandGamaley,2012).Nevertheless,allthestudies reviewedherefailedinfindinganassociationbetweenfrailtyand TLshortening:Collertonetal.(2012),usingbothFried’scriteriaand FI(40deficits;n=552),Marzettietal.(2014)(n=142)andYuetal. (2015)(n=2006)employingtheFried’scriteria,Saumetal.(2014) (n=3537)andWooetal.(2008)(n=2006)usingtheFI(34and17 deficits,respectively).LackofassociationbetweenTLandfrailty (Fried’scriteria)wasalsoobtainedbyBraultetal.(2014)inolder subjectswithcardiovasculardisease(n=53);however,inthiscase, theyfoundanunexpectedassociationbetweenlongerleukocyte andaorticT/Sratio(meantelomererepeatcopytosinglegenecopy number)andgreaternumberofclinicalfrailtycriteria.Together withthese studies, Breitling et al. (2016) evaluated the possi-bleassociationbetweenTLandDNAmethylationageacceleration (methylationageminuschronologicalage)inapopulationof851 olderadults.Theyfoundnocorrelationbetweenthetwo param-eters,supportingthepreviouslymentionedobservationsandthe ideathatTLisnotagoodbiomarkerfortheidentificationoffrailty. Thefrequencyofmicronucleus(MN)inperipheralblood lym-phocytesisalsoabiomarkerofgenomicstabilitywidelyemployed inmolecularepidemiology.SimilarlytoTL,MNfrequencyhasbeen previouslyassociatedwithage-relateddiseases andtheprocess ofageing(Bonassietal.,2001;Miglioreetal.,2011).Valdiglesias etal.(2015)evaluatedthefrequencyofMNtostudythe associa-tionofgenomicinstabilityandfrailtyinapopulationof180older adultsclassifiedaccordingtoFried’scriteria.Again,noassociation betweenMNfrequencyandfrailtywasobservedinthiscase.

Thus,despitethewell-reasonedworkinghypothesis(genomic instabilityis involved in thepathogenesis of frailtysyndrome), allthesestudiesresultedsurprisingly fruitless,withnopositive associationbetweengenomicinstabilityandfrailtyseverity.Two

possibleexplanationsweresuggestedforthislackofassociation (Valdiglesiasetal.,2015).Firstly,thenegativeﬁndingsreported in all these studies may be caused by the high rate of basal genomicdamagepresentinhealthyolderindividualsandreported in previous studies (Mladinic et al., 2010). And secondly, this lackofassociationmaybeduetothephysiologicalaccumulation ofgenomedamageintheelderly,whichcouldlimittherateof genomicdamage production.Thisconditioncould,forinstance, limittherateofMNformation.

3.2.3. Epigenetics

It hasbeen previouslyreported that thegeneticmaterial of cellsexperiencesepigeneticvariationsduringtheageingprocess (reviewedinSenetal.,2016).Atotalof3studiesoutof17 eval-uatinggenomic biomarkers,addressed thestudyof epigenetics infrailolderadults.Thethreeofthemshowedpositive correla-tionbetweenfrailty statusand DNA methylation. Bellizziet al. (2012)reportedthefirststudyinvestigating thepossible corre-lation between age-related functional decline, including frailty status,andepigeneticmodifications.TheymeasuredglobalDNA methylation levelsin a sample of 318older people,divided in middle/advanced-agedsubjects(medianage75years)and ultra-nonagenarians(medianage99years)inacross-sectionalstudy, and in a 7yearfollow-up of a subsample of pre-frail and non-frail middle agedsubjects. Frailtystatus was determinedusing theMontesantoetal. (2010)scale,a hierarchicalcluster analy-sis.Higher valuesof global DNAmethylation wereobserved in frailmiddleaged subjectsrespecttopre-frailand non-frail,but nodifference betweenvery frail and frail was observedin the ultranonagenarians.Inthefollow-upstudy,thoseindividualsthat becamefrailafter7years showeda significantincrease ofDNA methylationlevels.

More recently, Breitling et al. (2016) evaluated the associa-tionbetweenDNAmethylationageaccelerationand frailtyin a populationof969older adults,classifiedaccording theirfrailty statusbyusingtheFI(34deficits).Theirfindingsshowed signif-icantlyincreasingaccumulationoffrailtydeficitswithincreasing methylationageacceleration,supportinganassociationbetween epigeneticageaccelerationandfrailtystatus.Alsoonthisregard, Gaoetal.(2017)examinedtheassociationsofsmoking-relatedDNA methylationbiomarkersandfrailtyinapopulationofolderadults classifiedaccordingtoFI,andobservedthatmethylationintensity ofeachlocusinthevalidationpanelwassignificantlylowerinthe frail,whencomparedtonon-frail,population,whereas intermedi-atelevelsofmethylationintensitywereobservedinthepre-frail subjects.Onthebasisoftheirresults,authorssuggestedthatCpG sitesidentifiedcouldhavethepotentialtobeprognostic biomark-ersoffrailtyorfrailty-relatedhealthoutcomes.

FollowingthesameassumptionthatDNAmethylationchanges withage,especiallyingenepromoterregions,Collertonetal.(2014) studiedtheimportanceofaltered DNAmethylationinfrailtyin 552subjects(Fried’scriteria).Tothisend,methylationatspecific cytosineresidueswithinCpGislandsassociatedwithgene tran-scriptionalstartsiteswasquantified.Furthermore,toestimatethe genome-wideDNAmethylationlevels,theyquantifiedmethylation atLINE-1repetitiveelementsasasurrogate,showingno associa-tionwithfrailty.However,aclearassociationbetweenCpGisland methylationandfrailtywasobserved,suggestingapotentialrole forage-relatedrangesinCpGislandmethylationinthe develop-mentoffrailty.

3.3. DNArepairability

The DNA repair system has been recognized as one of the mostimportantcellulardefensemechanismsresponsibleforthe integrityofDNA.DecreasedDNArepairabilityisexhibitedin

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vari-M.Sánchez-Floresetal./AgeingResearchReviews37(2017)1–15 13

ousclinicalconditionsandassociatedwithincreasedfrequencyof carcinogenesis,sinceinefﬁcientrepairleadstoanaccumulationof aberrationsinthegenomethatculminateinthegeneticinstability typicalofmanymalignanciesandotherpathologies(Valdiglesias etal.,2011).

Daily exposure to environmental agents (such as oxidizing chemicals,methylatingagents,UVlight,and ionizingradiation), andevennormalphysiologicalprocesses(suchasreplicationand recombination),allmaydamagecellularcomponents,including DNA. While modified proteinsand lipids can bedegraded and resynthesized,DNAmustberepairedbeforereplicationandcell division take place (Klungland and Bjelland, 2007). Toxic and mutagenicconsequencesareminimizedbydistinctpathwaysof cellularrepairthat includedifferentenzymesandprotein com-plexesencodedbyanumberofhumanDNArepairgenes.Genomic instability,previouslydescribed,ishighlyrelatedtofailuresin cel-lularrepairsinceaberrantDNApolymerasesandothercomponents ofthetranscriptionalandtranslationalmachineryareaccumulated withage(Rattan,2012).Thus,duringageing,accumulationof mis-takestakesplaceinthegeneticmaterialduetothelossofefficiency oftheseDNArepairsystems,andcontributestothedevelopmentof genomicinstability.Indeed,mostage-relateddiseasesandageing signsareassociatedwithgenomicinstabilityandwithunrepaired orerroneouslyrepairedgenomedamage(Bürkle,2001).Besides,it isknownthattheaccumulationofDNAdamageisinvolvedin pre-matureageingandneurodegenerativeprocesses,suggestingthat alterationsintheDNArepairmechanismsmayberelevanttothese disorders(CoppedèandMigliore,2010).Indeed,theimpactof cellu-larsenescenceonageingoforganismswaspreviouslyevaluatedin differentstudiesrevealinganaccumulationofDNAdamageinboth senescentcellsandageingorganisms(Sedelnikovaetal.,2008).In particular,severalworksdescribedaccumulated␥H2AXfoci,which revealpersistentDNAdouble-strandedbreaks,insenescinghuman cellculturesandinageingmice(Sedelnikovaetal.,2004),inearly thymocytesubsetsofagedascomparedtoyoungmice(Hesseetal., 2009),indifferentorgansfromageingC57Bl6mice(Wangetal., 2009)andinfibroblaststakenfrompatientswithWernersyndrome (Sedelnikovaetal.,2008).

Toourknowledge,theonlystudythatevaluatesDNAdamage andrepaircapacityinassociationwithfrailtyinhuman population-basedstudiesistheonereportedbyCollertonetal.(2012),where noassociationwasobservedusingbothFried’scriteriaandFI(40 deﬁcits)toidentifyfrailtystatus.

4. Conclusions

Frailtyisgainingattentioninthelastdecadesingeriatricsand researchareas,withmoreandmoreprofessionalsclaimingfrailty measurementtobeincorporatedintoclinicalpracticeaspartof routinecareforolderpatients.However,thecurrentlyused crite-riaidentifyfrailtyonlyafterclinicalmanifestationsareobvious. Increasing evidence suggest that the clinical concept of frailty −basedmainlyonphenotypicalsignsandsymptomsandbarely consideringitsbiologicalbasis−isobsoletesincenosinglealtered systemalonedeﬁnesfrailty,butmultiplesystemsareinvolvedin thissyndrome.

Inordertoachieveamorethoroughandobjectiveassessment forearlyidentiﬁcationoffrailty, itis necessarytodevelopnew toolsthatallowtorecognizethoseindividualsmorevulnerableand morepronetodevelop thefrailtysyndrome.Withinthis frame-work,cellularandmolecularbiomarkerscouldbeusedtoreacha moreaccurateidentiﬁcationoffrailty,aswellasthoseindividuals inearlyandpotentiallyreversiblefrailtystages(pre-frail individ-uals).Thedevelopmentofthesenewtoolsandtheirinclusionin thecriteriatoidentifyfrailtywouldfacilitatetheimplementation

ofpersonalizedcareandtreatments,aswellasimproveoutcomes bymeansofpreventionandinterventionprogrammes.Additional researchisneededtofurtherexplorethepathophysiologicalbases offrailty.

Inthisreview,populationstudiesevaluatingalterations associ-atedwithfrailtystatusatcellularandmolecularlevel−bymeans ofoxidativestress,genomicandDNA repairbiomarkers− were revisedandcomparedinordertogatherallthisinformationandto identifypotentialbiomarkersthatcouldbeusefulinfrailty iden-tification, as wellas to point out gaps of knowledge and new researchareasneededinthisfield.Resultsofthisrevisionshowed thatseveraloxidativestressbiomarkers−includingalterationsin antioxidantsystems,increasedlevelsoflipidperoxidationandDNA oxidativedamage,aswellasDNAmethylationandsomespecific geneticpolymorphisms−areassociatedwithfrailtystatusinolder people.Onthecontrarygenomicinstability,oratleastthetwo biomarkerstestedsofar(telomerelengthandMNrate)seemsnot tobelinkedtofrailty.Theonlystudywhichaddressedthe possi-blerelationshipbetweenDNArepairmodulationsandfrailtystatus alsofailedinfindingassociations.

Despitethenumberofcellularalterationsinitiallyassociated withfrailty,studiesonthisregard arestill veryscarceand lim-itedtosomeofthepossiblecellulartargets.Additionalresearchis neededtofurtherexplorethesealterationspriortoincludeanyof theminthefrailtyassessmentcriteria.However,giventhesolid linkbetweenDNArepairability,genomicinstability,andageand age-relateddisorders,deeperinvestigationsinthislinemustbe carriedoutbeforereachingsolidconclusions.

Acknowledgments

ResearchfundedbyXuntadeGalicia(ED431B2016/013)andby EUCOSTActionIS1402.V.ValdiglesiaswassupportedbyaXuntade Galiciapostdoctoralfellowship(referenceED481B2016/190-0).D. Marcos-PérezandM.Sánchez-Floresweresupportedby INDITEX-UDCfellowships.TheworkofS.Bonassiwassupportedbygrants fundedbytheAssociazioneItalianaperlaRicercasulCancro(AIRC).

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