Rev. Bras. Reumatol. vol.57 número4

r e v b r a s r e u m a t o l . 2017;57(4):286–293

ww w . r e u m a t o l o g i a . c o m . b r

REVISTA

BRASILEIRA

DE

REUMATOLOGIA

Original

article

Analysis

of

four

serum

biomarkers

in

rheumatoid

arthritis:

association

with

extra

articular

manifestations

in

patients

and

arthralgia

in

relatives

Flávia

R.

Nass

_,

_Thelma

_L.

_Skare

_,

_Isabela

_Goeldner

_,

_Renato

_Nisihara

,

Iara

T.

Messias-Reason

_,

_Shirley

_R.R.

_Utiyama

a_{UniversidadeFederaldoParaná,LaboratóriodeImunopatologia,Curitiba,PR,Brazil} b_{HospitalUniversitárioEvangélicodeCuritiba,UnidadedeReumatologia,Curitiba,PR,Brazil} c_{UniversidadePositivo,DepartamentodeMedicina,Curitiba,PR,Brazil}

d_{UniversidadeFederaldoParaná,DepartamentodeAnálisesClínicas,Curitiba,PR,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received17June2015 Accepted18November2015 Availableonline22March2016

Keywords:

Rheumatoidarthritis Biomarkers

Rheumatoidfactor AntiCCp

Anticitrulinatedvimentin

a

b

s

t

r

a

c

t

Objectives:ToevaluatethefrequencyoffourserumbiomarkersinRApatientsandtheir relativesandidentifypossibleassociationswithclinicalfindingsofthedisease.

Methods:Thiswasatransversalanalyticalstudy.Anti-cycliccitrullinatedpeptide(anti-CCP), anti-mutatedcitrullinatedvimentin(anti-MCV)andIgA-rheumatoidfactor(RF)were deter-minedbyELISAandIgM-RFbylatexagglutinationin210RApatients,198relativesand92 healthycontrolsfromSouthernBrazil.Clinicalanddemographicdatawereobtainedthrough chartsreviewandquestionnaires.

Results:AhigherpositivityforallantibodieswasobservedinRApatientswhencompared torelativesandcontrols(p<0.0001).IgA-RFwasmorefrequentinrelativescomparedto controls(14.6%vs.5.4%,p=0.03,OR=2.98;95%CI=1.11–7.98)whereasanti-CCPwasthe mostcommonbiomarkeramongRApatients(75.6%).Concomitantpositivityforthefour biomarkerswasmorecommoninpatients(46.2%,p<0.0001).Relativesandcontrolswere mostlypositiveforjustonebiomarker(20.2%,p<0.0001and15.2%,p=0.016,respectively). Noassociationwasobservedbetweenthenumberofpositivebiomarkersandageofdisease onset,functionalclassortobaccoexposure.Inseronegativepatientspredominateabsence ofextraarticularmanifestations(EAMs)(p=0.01;OR=3.25;95%CI=1.16–10.66).Arthralgia waspresentinpositiverelatives,regardlessthetypeofbiomarker.

Conclusions:AhighernumberofbiomarkerswaspresentinRApatientswithEAMs.Positivity ofbiomarkerswasrelatedtoarthralgiainrelatives.Thesefindingsreinforcethelinkbetween distinctbiomarkersandthepathophysiologicmechanismsofAR.

©2016ElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:renatomitsu@yahoo.com.br(R.Nisihara). http://dx.doi.org/10.1016/j.rbre.2016.03.001

Análise

de

quatro

marcadores

sorológicos

na

artrite

reumatoide:

associac¸ão

com

manifestac¸ões

extra-articulares

no

paciente

e

artralgia

em

familiares

Palavras-chave: Artritereumatoide Biomarcadores Fatorreumatoide AntiCCP

Antivimentinacitrulinada

r

e

s

u

m

o

Objetivos: AvaliarafrequênciadequatromarcadoressorológicosempacientescomARe familiareseidentificarpossíveisassociac¸õescomachadosclínicosdadoenc¸a.

Métodos: Estudo analítico transversal. Determinaram-se os níveis de anticorpos antipeptídeocitrulinadocíclico(anti-CCP),anticorposantivimentina citrulinada-mutada (anti-MCV)efatorreumatoide(FR)IgAporElisaedeFR-IgMporaglutinac¸ãoemlátexem 210pacientescomAR,198parentese92controlessaudáveisdosuldoBrasil.Coletaram-se dadosclínicosedemográficospormeiodarevisãodeprontuáriosequestionários. Resultados: Observou-semaiorpositividadeparatodososanticorposempacientescom ARemcomparac¸ãocomosfamiliaresecontroles(p<0,0001).OFR-IgAeramaisfrequente emfamiliaresquandocomparadoscomoscontroles(14,6%versus5,4%,p=0,03,OR=2,98; IC95%=1,11a7,98).Oanti-CCPfoiobiomarcadormaiscomumentrepacientescomAR (75,6%).Apositividadeconcomitanteparaosquatrobiomarcadoresfoimaiscomumnos pacientes(46,2%,p<0,0001).Familiaresecontroleserampositivosemsuamaioriapara apenasumbiomarcador(20,2%,p<0,0001e15,2%,p=0,016,respectivamente).Nãofoi obser-vadaassociac¸ãoentreonúmerodebiomarcadorespositivoseaidadedeiníciodadoenc¸a, classefuncionalouexposic¸ãoaofumo.Empacientessoronegativos,predominouaausência demanifestac¸õesextra-articulares(MEA)(p=0,01;OR=3,25;IC95%=1,16a10,66).Aartralgia estavapresenteemfamiliarespositivos,independentementedotipodebiomarcador. Conclusões:UmamaiorquantidadedebiomarcadoresestavapresenteempacientescomAR comMEA.Apositividadedosbiomarcadoresestavarelacionadacomaartralgiaem famil-iares.Essesachadosreforc¸amaligac¸ãoentreosdiferentesbiomarcadoreseosmecanismos fisiopatológicosdaAR.

©2016ElsevierEditoraLtda.Este ´eumartigoOpenAccesssobumalicenc¸aCC BY-NC-ND(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Rheumatoidarthritis(RA)isachronicinflammatorydisease thatoccursin0.2–1%oftheworldpopulation.1_{Itsprevalence} infirstdegreerelativesofRApatientsisof2–4%,characterizing them as a risk group for RA.2–5 _{In this context,} investiga-tion of serological markers in healthy relatives may be of value to identify earlier cases of RA6,7 _{and to understand} the pathophysiologic mechanisms underlying the disease process.

Rheumatoidfactor (RF)is a classical serological marker used for RA diagnosis, with IgM-RF being the most com-monisoform.In contrast,IgA-RFhasbeen associatedwith erosivearthritisand seems tobe abetter indicatorof dis-ease severity than IgM-RF or IgG-RF.8,9 _{Antibodies against} citrullinated peptides (ACPAs), such as anti-cyclic citrul-linated peptide (anti-CCP) and anti-mutated citrullinated vimentin (anti-MCV) are considered highly specific mark-ersfor RA.10 _{Anti-CCP has both diagnostic and prognostic} value and can be detected before clinical manifestations of the disease.11 _{Recently, anti-MCV has also been} pro-posed as a diagnostic marker for early arthritis, with the samespecificitybuthighersensitivityofanti-CCP.12_In addi-tion, anti-MCV has been detected in healthy relatives of RA patients13 _{suggesting it as a novel prognostic marker} forRA.

PositivityformorethanoneRAserologicalmarkerinthe samepatientmayindicateworseprognosisor,inthecaseof relatives,earlydiseaseonset.14,15_{Infact,positivityformore} thanoneautoantibodyhasbeenreportedamongunaffected relativesofRApatients,especiallyinfamilieswithmultiple cases.16,17

Todate,therearenoreportsconsideringthesimultaneity ofthesebiomarkersinRApatientsandtheirrelativesinthe Brazilianpopulation. Thus,inthepresentwork we investi-gatedthefrequencyofanti-CCP,anti-MCV,IgA-RFandIgM-RF inRApatientsandtheirrelatives,andtriedtoidentify possi-bleassociationsbetweenthesimultaneityofthesebiomarkers andclinicalfindingsordiagnosisofRA.

Methods

Thiswasatransversalandanalyticalstudyapprovedbythe localEthics CommitteeinResearch. Informedconsentwas obtainedfromallsubjects.

Subjects

288

Table1–DemographicandclinicalfeaturesofRA patients.

%(N)

Meanage(years) 51.1±11.8

Female 84.8(178/210)

Ageatdiseaseonset–(years;median) 45.0±12.8

Diseaseduration(years;median) 7.0±8.5

Eurodescendents(autodeclared) 72.8(131/180)

Afrodescendents(autodeclared) 26.7(48/180)

Tobaccoexposure 46.0(87/189)

Functionalclass

ClassI 47.4(99/209)

ClassII 37.7(83/209)

ClassIII 10.5(22/209)

ClassIV 2.4(5/209)

Extraarticularmanifestations

Rheumatoidnodules 8.9(17/190)

SecondarySjögrensyndrome 30.2(49/162)

Pulmonaryfibrosis 8.6(15/175)

consecutivelyenrolledinthestudyfromAugust2007toApril 2009.DemographicandclinicalfeaturesfromRApatientsare showninTable1.

Atotalof198 relativeswere investigated(61.1%female, 38.9% male; mean age 36.8 years; range 7–91 years, 94% first-degree), representing altogether 78 families (2.54 rela-tives/family).Additionally, sera from 92 healthy volunteers from the same geographicalarea,matched forgenderand agewithpatients,wereusedascontrols(82.6%female,17.4% male;meanage45.8years;range23–81years).Noneofcontrols hadfamilialRAcases.

Autoantibodiesevaluation

Anti-CCP and IgA-RF were assessed by enzyme-linked immunosorbentassay(ELISA)fromINOVADiagnostics(San Diego, USA). Anti-MCV was determined using the ORGEN-TEC Diagnostika ELISA kit (Mainz, Germany). IgM-RF was measured using latex agglutination test (BioSystems S.A., Barcelona,Spain).Cut-offvalueswereestablishedaccording tothemanufacturer’sspecifications.

Datacollection

Demographicand clinical datafrom patientsand relatives wereobtainedthroughmedicalrecordreviewandindividual standardquestionnairesappliedbyarheumatologistatthe timeofenrollment (Tables1and 2).InRApatientsthe fol-lowingextraarticularmanifestations(EAM)weretakeninto account: rheumatoid nodules, serositis, pericarditis, valvu-lar disease, vasculitis, lung fibrosis, pneumonitis, Felty’s syndrome and secondary Sjögren syndrome. Steinbrocker functional classificationwasappliedinorder toaccessthe extentofphysicaldisabilityinRApatients.Diagnosisof sec-ondarySjogren’ssyndromefollowedtheAmericanEuropean criteria.19_{Clinicalfollow-upofthepositiverelativeshasbeen} carriedoutforthemediantimeof7years(range2–8;IQR=6–7 years).

Positivity (

%

)

Relatives Patients

P=.004 P=.03

80

70

60

50

40

30

20

10

0

Anti-CCP FR-IgM FR-IgA Anti-MCV Controls

Fig.1–Positivityofbiomarkersinthestudiedgroups. Patients:anti-CCP+vs.IgA-RF+:p=0.03;OR=1.62;95%

CI=1.06–2.48.Allothercomparisons:p=ns.Relatives:

anti-CCP+vs.IgA-RF+:p=0.004;OR=0.34;95%

CI=0.16–0.70.Allothercomparison:p=ns.Controls:all

comparisonsp=ns.RelativesIgA-RF+vs.ControlsIgA-RF+: p=0.03;OR=2.98;95%CI=1.11–7.98.Allother

comparisons:p=ns.ns,notsignificant.

Statisticalanalyses

StatisticalanalyseswereperformedusingGraphPadPrism4.0 (GraphPadSoftwareInc.,LaJolla,USA)andStatistic5.5 (Stat-Soft Inc.,Tulsa,USA).Comparisonsbetweenautoantibodies levels weredoneapplyingnonparametric Kruskal–Wallisor Mann–Whitneytests.Chi-squareandFisher’sexacttestswere applied for the analysesof the positivity between groups. Spearman’stestwasusedforcorrelationanalysis.The signif-icancelevelwassetat0.05.

Results

PrevalenceofbiomarkersinRApatients,relativesand

controls

TheprevalenceofeachbiomarkerinRApatients,their rela-tivesandcontrolscanbeseenonFig.1,whereitcanbenoted thatthemostprevalentbiomarkerinRApatientsisantiCCP andthemostcommoninrelativeswasIgARF.

Themediantiterofeachbiomarkerwasstudiedinthe3 groupsandresultsareshowedinFig.2whereitispossibleto seethatrelativeshadanintermediatevaluebetweenpatients andcontrols.

In RA patients a significant correlation was observed among the titers of the four investigated biomarkers, but thecorrelationbetweenanti-MCVandanti-CCP(r=0.73),and IgM-RF and IgA-RF (r=0.71) were both stronger compared to other associations (anti-MCV/IgM-RF, r=0.38; anti-MCV/ IgA-RF, r=0.41; IgM-RF/anti-CCP, r=0.46; IgA-RF/anti-CCP, r=0.47).

r

e

v

b

r

a

s

r

e

u

m

a

t

o

l

.

2

0

1

7;

5

7(4)

:286–293

289

0(n=28) 1(n=15) 2(n=28) 3(n=42) 4(n=97)

Femalegender 26/28(92.8) 9/15(60) 25/28(89.3) 42/42(100) 76/97(78.4) 0.0006a

Ageatdiseaseonset(median) 18–75(41.5;IQR27.2–48.7) 24–69(46.0;IQR35.0–53.0) 19–69(43.5;IQR31.5–50.0) 18–83(43.5;IQR30–49.7) 16–71(46;IQR32.7–52.0) 0.50c

Age(mean) 48.2±10.4 50.3±12.9 50.1±11.9 52.9±13.79 51.7±11.35 0.55d

Diseaseduration(median/years) 1–28(7.0;IQR3.0–12.8) 1–27(4.0;IQR2.0–7.0) 1–35(7.5;IQR3.2–14.2) 1–60(7.0;IQR3.0–11.7) 1–36(6.5;IQR3.2–12.0) 0.36c

Functionalclass

ClassI 15/28(53.6) 8/15(53.3) 13/28(46.4) 22/42(52.4) 41/96(42.7) 0.75a

ClassII 10/28(35.7) 6/15(40) 11/28(39.3) 17/42(40.5) 39/96(40.6) 0.99a

ClassIII+IV 3/28(10.7) 1/15(6.7) 4/28(14.3) 3/42(7.1) 16/96(16.7) 0.53a

Eurodescendent 18/24(75) 10/13(76.9) 17/26(65.4) 28/38(73.7) 58/79(73.4) 0.91a

Afrodescendent 6/24(25) 3/13(23.1) 9/26(34.6) 10/38(26.3) 20/79(25.3) 0.90a

Tobaccoexposure 13/25(52) 7/12(58.3) 12/26(46.1) 16/39(41.02) 39/87(44.8) 0.82a

Nodules 2/24(8.33) 0/13(0) 4/28(14.28) 1/37(2.7) 10/88(11.36) 0.32a

Sjögrensyndrome 1/19(5.2) 3/11(27.2) 4/24(16.6) 16/33(48.4) 25/75(33.3) 0.009a

Lungfibrosis 1/22(4.50) 1/11(9.0) 2/24(8.3) 5/32(15.6) 6/72(8.3) 0.68a

IQR,interquartilerange.

a _Chi-square.

b _Fishertest.

c _{Kruskal–Wallistest.}

290

300

P<.0001

P=.0204

P=.0098

P<.0001 P=.0003

P=.0126

A

B

D

C

250

200

150

100

50

0

200

150

100

50

0 3000

2500

2000

1500

1000

500

0

4000

3000

2000

1000

0

Patients

Anti-CCP titer (U/ml)

Anti-MCV titer (U/ml)

IgM RF titer (U/ml) IgA RF titer (U/ml)

Relatives Controls∗ Patients Relatives Controls

Relatives

Patients Controls

Relatives

Patients Controls

Fig.2–Anti-CCP,anti-MCV,IgM-RFandIgA-RFinpatients,relativesandcontrols.*Analysisnotapplicable.

Associationbetweenbiomarkersandclinicalfindingsin

RApatientsandrelatives

Table2showsdemographicandclinicaldataofRApatients

andtheirassociationwiththenumberofpositivebiomarkers. EAM were detected in 34.8% (69/198) of the patients. Patientsthat were negativeforall biomarkers had alower frequency of EAM (5.8% vs. 17.8%, p=0.01; OR=3.25; 95% CI=1.16–10.66).Anincreaseintheconcomitantpositivityfor 3biomarkerswasobservedinpatientswithEAMcompared topatientswithout it (Table3).In theonebyone analysis of the different EAM, only the p value for secondary Sjö-grensyndromeremainedsignificant (p=0.01;OR=2.73;95% CI=1.24–6.08).

Table4showsassociationbetweendemographic,clinical

and serologicaldata and biomarkerspositivityinrelatives. Among the relatives 11.7% (22/197) had arthralgia, and a significantfrequencyofarthralgiawas observedinpositive relatives,regardlessofthetypeofbiomarker.Eachbiomarker presentedsignificant association with the others autoanti-bodiestested(Table4).

Follow-upofpositiverelatives

Therelativeswerefollowedprospectivelyforamediantimeof 7years(range2–8;IQR=6–7years)withbiannualevaluations. Atmoment,threepositiverelatives(twowomenandoneman, meanage43.3years)hadthediagnosisforRAconfirmed;all ofthem withhigh biomarkertiters.Amongthoserelatives, onewaspositiveforall4biomarkersandhissibling(withRA) hadhightitersofanti-CCP.Inaddition,sevenunaffected rela-tiveswithhighautoantibodytitersand somesymptomsof

thediseasearebeingfollowed.Twoofthemarepositivefor4 biomarkersconcomitantly,twofor2biomarkersandthreefor onebiomarker.Noneoftheotherpositiverelativesfortested biomarkershaddiagnosiscriterionsforRA.

NoRAcasesweredetectedamongrelativesnegativesfor autoantibodies.

Discussion

Thisisapioneerstudyinwhichfourserologicalbiomarkers wereevaluatedconcomitantlyinpatientswithRAandtheir relativesinaSouthern Brazilianpopulation.Previous stud-ieshaveshowedanincreasedpositivityforautoantibodiesin unaffectedfamilymemberswhencomparedtohealthy con-trolsubjects5,17,20,21_{andthepresentresultscorroboratethese} findings.

AlthoughIgA-RFspecificityforRAisnotashighasanti-CCP, IgA-RFcanbedetectedseveralyearsbeforethesymptomsof thedisease,suggestingaprimaryroleofIgA-RFinthe patho-genesisofRA.14_{Thisisaveryinterestingobservationsince} inthepresentstudy IgA-RFwasthe biomarkerwithhigher positivityamongrelatives.Inaddition,severalrelativeswere positiveforRFandanti-MCV,butthethreerelativesthathad confirmedRAwereall positiveforanti-CCP,reinforcing the highspecificity(99%)andpositivepredictivevalue(99%)for thisautoantibody.Supportingthisobservation,anti-CCPwas theonlybiomarkerwithnosignificantlydifferenttiterswhen patientsandrelativeswerecompared(Fig.2A).

Table3–Extraarticularmanifestationsaccordingtothenumberofpositivebiomarkers.

Numberofpositivebiomarkers WithEAM(n=69)

N(%)

WithoutEAM(n=129)

N(%)

p

4(n=90) 34(49.3) 56(43.4) ns

3(n=39) 19(27.5) 20(15.5) 0.05

2(n=28) 9(13.1) 19(14.7) ns

1(n=14) 3(4.3) 11(8.5) ns

Nobiomarker(n=27) 4(5.8) 23(17.8) 0.01

EAM,extraarticularmanifestation;ns,notsignificant.

Note:3biomarkerswithEAMvs.3biomarkerswithoutEAM;p=0.059;OR=1.26;95%CI=0.70–2.27.NobiomarkerwithEAMvs.Nobiomarker

withoutEAM;p=0.0179;OR=3.25;95%IC=1.16–10.66.Fishertest.

Table4–Associationbetweendemographic,clinicalandserologicaldataandbiomarkerspositivityintheRApatients relatives.

IgM-RF+(n=16) IgM-RF−(n=182) p

Euro-descendent 14/16 140/182 0.53a

Afrodescendent 2/16 42/182 0.53a

Tobaccoexposure 3/16 54/182 0.56a

Female 13/16 108/182 0.11a

Meanage(years) 40.3±12.3 36.5±15.6 0.34c

Arthralgia 6/16 16/178 0.004a

Anti-CCP+ 5/16 6/182 0.0006a

IgA-RF+ 6/16 23/182 0.01a

Anti-MCV+ 5/16 11/182 0.004a

IgA-RF+(n=29) IgA-RF−(n=169) p

Euro-descendent 24/29 130/169 0.63a

Afrodescendent 5/29 39/169 0.63a

Tobaccoexposure 9/29 48/169 0.77b

Female 17/29 104/169 0.76b

Meanage(years) 40.6±15.4 36.1±15.3 0.15c

Arthralgia 6/29 16/165 0.08b

Anti-CCP+ 4/29 7/169 0.059a

IgM-RF+ 6/29 10/169 0.007b

Anti-MCV+ 7/29 9/169 0.0006b

Anti-CCP+(n=11) Anti-CCP−(n=187) p

Euro-descendent 9/11 145/187 1.00a

Afrodescendent 2/11 42/187 1.00a

Tobaccoexposure 3/11 54/187 1.00a

Female 9/11 112/187 0.37a

Meanage(years) 39.3±15.9 36.6±15.4 0.57c

Arthralgia 4/11 18/183 0.02a

IgM-RF+ 5/11 11/187 0.0006a

IgA-RF+ 4/11 25/187 0.05a

Anti-MCV+ 5/11 11/187 0.0006a

Anti-MCV+(n=16) Anti-MCV−(n=182) p

Euro-descendent 12/16 142/182 0.75a

Afrodescendent 4/16 40/182 0.75a

Tobaccoexposure 4/16 53/182 1.00a

Female 14/16 107/182 0.03a

Meanage(years) 33.5±16.9 37.1±15.2 0.37c

Arthralgia 6/16 22/178 0.0006a

Anti-CCP+ 5/16 6/182 <0.0001b

IgM-RF+ 5/16 11/182 0.004a

IgA-RF+ 7/16 22/182 0.002b

a _Fishertest.

b _Chi-square.

292

thisoutcomemaybetheepitopespreadingphenomenonthat causesincreasingantibodylevelsjustpriortotheflareof clin-icaldisease.23

Somestudieshavesuggestedthatanti-MCVhasa com-parablevaluetoanti-CCPforRAdiagnosis,withevenhigher sensitivity.24,25 _{Thiswas notthe case inthe present study} whentheprevalence,sensitivityandspecificityofanti-MCV werelowerthanthoseofanti-CCP.

A strong correlation between RA biomarkers has been previouslydisclosed.PoulsomandCharles26_showedagood correlationbetweenanti-MCVandanti-CCPandbetween anti-MCVandIgM-RF,butnotbetweenanti-CCPandIgM-RF.We foundsignificantcorrelationbetweenallmarkers,especially between anti CCP and anti MCV and between RF IgA and IgM.ThispatternwaspreviouslydescribedinfirstRAdegree relatives27_{andmaybeassociatedwiththepresenceof} com-monepitopes.

OurcasuisticpresentedahighprevalenceofEAMs(34.8%) that may be explained by geneticand environmental fac-tors that influence the immune reactions leading to their development.28_{Weobservedpositivityforahighernumberof} biomarkersamongpatientswithEAMs,inspecialsecondary SS.

Thepresent resultsfor relativesofRA patients demon-stratedthatserologicalscreeningisfeasibleandusefultool forthescreeningofthoseindividualsatrisk,asemphasized byotherauthors.17,21,22_{Thehighprevalenceofarthralgiain} relativespositivetoanyofthefourbiomarkershighlightsthis hypothesis,sincesomeoftheserelatives(three)havealready evolvedtofullblownRA.AlltherelativeswhodevelopedRA presentedhighbiomarkerstiters;onewaspositiveforallfour biomarkersandhad oneextra siblingpositive foranti-CCP. Amongtheothertwo,onewaspositiveforbothanti-CCPand IgM-RF,whilethe otherwasonlypositiveforanti-CCP.The majorityofrelativeswithautoantibodieswhodidnotdevelop thediseasesofarwerepositiveforjustonebiomarker. How-everthisdoesnotruleoutthegreatriskforthemtodevelop thedisease.Itispossiblethattheymayposteriorly serocon-verttotwo,threeorevenmorebiomarkersasshowninthe patientswithestablishedRA.

Finally, the number of biomarkers was higher in RA patients with EAMs but it was not linked to duration of thediseaseand/orthefunctionalclass.Ontheother hand, seronegative patientspresentedless EAMs. Highfrequency ofarthralgiawasobservedinpositiverelatives,regardlessof thebiomarkertype.Futurestudiesmayhighlightthe fascinat-inglinkbetweenthepresenceofdistinctbiomarkersandthe pathophysiologicmechanismsofAR.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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