Rev. Bras. Reumatol. vol.57 número2

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

REVISTA

BRASILEIRA

DE

REUMATOLOGIA

Original

article

Vascular

endothelial

growth

factor

G1612A

(rs10434)

gene

polymorphism

and

neuropsychiatric

manifestations

in

systemic

lupus

erythematosus

patients

Sherif

Taha

_,

_Sherif

_Mohammed

_Gamal

,

Mohamed

Nabil

,

Nahla

Naeem

,

Dalia

Labib

_,

_Ibrahim

_Siam

_,

_Tamer

_Atef

_Gheita

b_{CairoUniversity,FacultyofMedicine,RheumatologyDepartment,Cairo,Egypt}

c_{Beni-SweifUniversity,FacultyofMedicine,InternalMedicineDepartment,Beni-Sweif,Egypt}

d_{CairoUniversity,FacultyofMedicine,NeurologyDepartment,Cairo,Egypt}

e_{NationalResearchCenter,InternalMedicineDepartment,Cairo,Egypt}

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Articlehistory: Received18May2016 Accepted29August2016

Availableonline14December2016

Keywords: SLE VEGF

G1612A(rs10434)gene Polymorphism

Neuropsychiatricmanifestations

a

b

s

t

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c

t

Aim: Toinvestigatetherelationbetweenvascularendothelialgrowthfactor(VEGF)gene polymorphisminsystemiclupuserythematosus(SLE)patientsandlupusrelated neuropsy-chiatricmanifestations.

Patientsandmethods:SixtyadultSLEpatientsrecruitedfromtheRheumatologyand Neurol-ogydepartmentsofCairoUniversityhospitalswereclassifiedintotwogroups;GroupA:30 patientswithneuropsychiatricmanifestations(NPSLE)andGroupB:30patientswithout. ForbothgroupstheSNPG1612A(rs10434)oftheVEGFgenewasgenotypedbyrealtime polymerasechainreaction(RT-PCR).

Results:Statistically significantdifferencewasfoundingenotypeandallele frequencies betweenbothgroups(AA[70%vs13.3%,p<0.001]andGG[10%vs66.7%,p<0.001]). Conclusion: PolymorphisminthegenecodingforVEGFmaybeassociatedwithincreased incidenceofneuropsychiatriclupusinSLEpatients.

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

∗ _{Correspondingauthor.}

E-mail:[email protected](S.M.Gamal).

http://dx.doi.org/10.1016/j.rbre.2016.11.007

Polimorfismo

genético

do

fator

de

crescimento

vascular

endotelial

G1612A

(rs10434)

e

manifestac¸ões

neuropsiquiátricas

em

pacientes

com

lúpus

eritematoso

sistêmico

Palavras-chave: LES

VEGF

GeneG1612A(rs10434) Polimorfismo

Manifestac¸ões neuropsiquiátricas

r

e

s

u

m

o

Objetivo: Investigararelac¸ãoentreopolimorfismogenéticodofatordecrescimento vascu-larendotelial(VEGF)empacientescomlúpuseritematososistêmico(LES)emanifestac¸ões neuropsiquiátricasrelacionadascomolúpus.

Pacientesemétodos: Foramrecrutados60pacientesadultoscomLESnosdepartamentos deReumatologiaeNeurologiadehospitaisuniversitáriosdoCairoeclassificadosemdois grupos;grupoA:30pacientescommanifestac¸õesneuropsiquiátricas(LESNP)egrupoB:30 pacientessemmanifestac¸õesneuropsiquiátricas.Genotipou-seoSNPG1612A(rs10434)do geneVEGFemambososgruposporreac¸ãoemcadeiadapolimeraseemtemporeal(RT-PCR). Resultados:Foiencontradadiferenc¸aestatisticamentesignificativanasfrequências genotípi-casealélicasentreosdoisgrupos(AA[70%vs.13,3%,p<0,001]eGG[10%vs.66,7%,p<0,001]). Conclusão: OpolimorfismonogenequecodificaoVEGFpodeestarassociadoaoaumento naincidênciadelúpusneuropsiquiátricoempacientescomLES.

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

Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease,1 _{withbothgeneticandenvironmentalfactors} play-ingsignificantroles inits pathogenesis.2_{Asaconsequence} ofitscompleximmunopathology,involvingtheproductionof autoantibodiesandimmunecomplexvasculitiswith endothe-lialcelldamage,3_{differentorgansandbloodvesselsmaybe} affectedbychronicinflammation.4

Thedamageand activation ofvascularendothelialcells are the initiating factors inthe pathogenesis ofSLE.5 Vas-cular endothelial growth factor (VEGF) is a key modulator ofangiogenesis,endothelialcellproliferationandmigration, chemotaxis,andcapillaryhyper-permeability,6_andwasfound tobeupregulatedinanumberofcollagendiseases includ-ingSLE.7 _{Furthermore,ithasbeenreportedthathigh VEGF} levelsmaybeassociatedwiththedisease activityinSLE.3,4 AlsopolymorphisminVEGFR2genehasalreadybeen corre-latedwithvasculardiseases andmayinfluenceendothelial integrity,repairandfunction.8

In addition to association of VEGF levels with disease activity in SLE, it was also found to be correlated with other disease manifestations as lupus nephritis, and with highermeancarotidintimamediathickness,9,10 _pulmonary hypertension,11 _{and inverselycorrelated to platelet count.}7 Althoughitwasreportedthatanti-ribosomalPantibodymay influencethepathologyofneuropsychiatriclupusthroughthe elevationofVEGFproductionfrommonocyticcells,12_however, the association between neuropsychiatric lupus and VEGF remainsunclear.

ThisstudyaimstoinvestigatetherelationbetweenVEGF singlenucleotidepolymorphism(SNP)G1612A(rs10434)gene polymorphisminSLEpatientsandlupusrelated neuropsychi-atricmanifestations.

Subjects

and

method

This isacross-sectional study thatincluded sixty patients who fulfilled theupdatedACR revisedcriteria forthe clas-sification of SLE.13 _{Thirty patients with neuropsychiatric} manifestationsofSLE(NPSLE)(GroupA)wererecruitedfrom Rheumatologyand NeurologydepartmentsofCairo Univer-sityHospitalsfromMay2013toMay2015.Neuropsychiatric manifestations of SLE (NPSLE) were defined by the pres-ence of current or past stroke, transient ischemic attack, psychosis, seizuredisorder,confusionalstate,and/or cogni-tivedysfunction.Another30consecutivepatientswithoutNP involvement(GroupB)wereselectedtomatchthesame num-berofpatientswithNPinvolvement.Informedconsentswere takenfromthepatientsandthestudywasapprovedbythe local ethics committee. Fullhistory taking, thorough clini-calexaminationwithspecialemphasisonneuropsychiatric involvementwasperformedforallpatients.Patientssuffering from activerenal disease, pulmonaryhypertension, hyper-tension,activearthritisandthrombocytopeniawereexcluded fromthestudy.

TheSNPG1612A(rs10434)inthe3′_{untranslatedregion(3}′

-UTR)ofVEGFgenewasgenotypedbyrealtimepolymerase chainreaction(RT-PCR)inallpatients.

DNAisolation

VEGFgenotypingbyRT-PCR

Real time PCR was performed by the Carousel-Based Sys-tem,Lightcycler2.0,usingthemastermixkit,Lightcyclerfast startMaster Hybprobekit (Roche DiagnosticsGmbH, 68298 Mannheim,Germany),together with the LightSNiPrs10434 VEGFAkit (TIB MOLBIOLGmbH –Eresburgstrasse22-23, D-12103Berlin,Germany)thatcontainedtheprimersnecessary forthereaction.

TheLightcycler fast start Master Hybprobe kit isa Hot StartReactionMixforPCRusingHybProbeprobesasdetection format.TheLightCyclerHybProbeformatisbasedonthe prin-cipleoffluorescenceresonanceenergytransfer(FRET),where twosequencespecificoligonucleotideprobes,labeledwith dif-ferentdyes(donorandacceptor),wereaddedtothereaction mixinadditiontoPCRprimers.Theamountoffluorescence generatedbythetagontheprobeisdirectlyproportionaltothe amountoftargetDNAgeneratedduringthePCRprocess.The PCRreactionwascarriedoutaccordingtothemanufacturers’ instructionsandwasfollowedbymeltingcurveanalysis.

Statisticalanalysis

Datawerestatisticallydescribedintermsofmean±standard deviation (±SD), median and range, or frequencies (num-berofcases)andpercentageswhenappropriate.Chi-square testwas usedtocompare qualitativevariables.Correlation betweenparameterswasperformedusingPearsoncorrelation coefficient.Differencesbetweengroupswereconsidered sta-tisticallysignificantwithpvalueslessthan0.05and highly significantifless than0.01.Allstatisticalcalculationswere doneusingcomputerprogramsSPSS(StatisticalPackagefor theSocialScience;SPSSInc.,Chicago,IL,USA)version16for MicrosoftWindows.

Results

Demographicdataandlaboratoryparametersofthepresent studyarepresentedinTable1.Bothgroupswereageandsex matchedwithnosignificantdifferencesindiseaseduration (p=0.9).

Neuropsychiatric manifestations in Group A (NPSLE) patientswereasfollows;9(30%)hadpsychosis,8(26.7%)had

Table1–Demographicfeaturesanddiseasedurationin SLEpatientsGroupA(NPSLE)andGroupBwithout neuropsychiatricmanifestations.

GroupA(n=30) GroupB(n=30) p

Age

Range 19–52 21–55.0 0.3

Mean±SD 31.9±7.6 34.3±8.6 NS

Sex

Male 1(3.3) 3(10) 0.6

Female 29(96.7) 27(90) NS

Diseaseduration

Range 1.0–12 1.0–11 0.9

Mean±SD 5.5±3.5 5.5±3.3 NS

seizures, 5 (16.7%)had transient ischemic attacks (TIAs),4 (13.3%)hadstroke,3(10%)hadtransversemyelitis(TM)and onepatient(3.3%)haddepression.Clinicalmanifestationsin GroupBincluded:mucocutaneousmanifestations,arthralgia, leucopenia,hemolyticanemiaandserositis.

Laboratoryfeaturesincludingthegenotypicdistributionof thestudypopulationareshowninTable2.

Discussion

SLE is a multi-system autoimmune disease.14 _{In lupus} chronicsystemicinflammationleadstoactivationofvascular endothelialcellswhichinturnleadstoasubstantialincrease inangiogenicfactorswhichplayasignificantrolein vascu-larpermeability,vasculargrowth,andinflammatoryresponse leadingtobloodvesseldestructionandseriousinternalorgan dysfunction.15

VEGFisalsoinvolvedinkidneyandlungfunctionaswell as serving as a survival factor for neuronal cells.16 Poly-morphismsinangiogenesis-regulatinggenesmayaffectthe responsetoanangiogenicstimulusandtherebyaffect suscep-tibilitytoand/ortheprogressionofangiogenesis-dependent disease. In previous gene studies,polymorphisms inVEGF and vascularendothelialgrowth factorreceptor2(VEGFR2) wereclearlyassociatedwiththedevelopmentof angiogenesis-dependentdisease.17

Circulating VEGF levels are highly heritable,18 _however, VEGF gene is highly polymorphic, with hundreds of poly-morphisms currently annotated in the Single Nucleotide Polymorphism database (dbSNP). Itincludes at least three polymorphisms thatare relativelycommon andmay affect VEGFexpression.Theinsertion/deletionpolymorphism(I/D) atthe−2549positionofthepromoterregionandthe−634G/C (rs2010963) polymorphism locatedinthe5′_{-UTRhave been}

consideredtobeassociatedwithincreasedVEGFexpression.19 Alsothe936C/T(rs3025039)polymorphismlocatedinthe3′

-UTRisassociatedwithsubstantiallyincreasedserumVEGF levels.20

OwingtotheimplicationofVEGFasamodulatorof angio-genesis,endothelialcellproliferationandmigration,theaim ofthecurrentstudywastoinvestigatethepossible relation-shipbetweenpolymorphismsinthegenecodingforVEGFand neuropsychiatricmanifestationsinSLEpatients.

Inourstudy,wefoundastatisticallysignificantdifference ingenotypeandallelefrequenciesbetweenpatientsinGroup A(thosewithneuropsychiatricmanifestations)andpatients inGroupB(thosewithoutneuropsychiatricinvolvement)(AA [70%vs13.3%,p<0.001]andGG[10%vs66.7%,p<0.001]).

Table2–ComparisonbetweenlaboratorydatainSLEpatientsGroupA(NPSLE)andGroupBwithoutneuropsychiatric manifestations.

GroupA(n=30) GroupB(n=30) p

ANA

+ve 28(93.3) 25(83.3) 0.4

−ve 2(6.7) 5(16.7) NS

DNA

+ve 11(36.7) 10(33.3) 1

−ve 19(63.3) 20(66.7) NS

VEGFgenotype

AA 21(70) 4(13.3) <0.001

AG 6(20) 6(20) 1

GG 3(10) 20(66.7) <0.001

ESR 48.8±32.3(5–12) 48.2±34.9(12–150) 0.9

Hb 12.3±1.4(9.5–15.3) 12.3±1.5(9.3–15.3) 0.9

WBC 8.4±2.5(3.8–16) 7.6±1.5(3.8–9.5) 0.1

PLT 246.2±77.4(80–466) 229.8±42.3(130–321) 0.3

ANA,antinuclearantibodies;VEGF,vascularendothelialgrowthfactor;ESR,erythrocytesedimentationrate;Hb,hemoglobin;WBC,whiteblood cells;PLT,platelet.

Resultsarepresentedasnumber(%)ormean±SD(range).

rs833070GGdecreasedthesusceptibilityofarthritisintheSLE patients.

Limitations

Thesmallnumberofpatientsincludedinthestudyandthe absenceofsimilarstudies focusingonthe relationofVEGF

genepolymorphismandlupusrelatedneuropsychiatric

man-ifestationsarethemostimportant.Howeverthisstudycanbe consideredasapilotstudy;whichmayopenthedoorinthe future,forstudiesconductedonlargepopulationstoestablish

therealimpactofVEGFgenepolymorphisminpathogenesis

orintheclinicalcharacteristicsofneuropsychiatric manifes-tationsofSLE.AlsofurtherstudieswithassessmentofVEGF serumlevelsinpatientswithneuropsychiatricmanifestations couldbebeneficial.

Key

message

TheSNP G1612A (rs10434)ofVEGF genemay represent an

increasedsusceptibility toneuropsychiatric involvementin patientswithSLE.

Conflict

of

interest

Theauthorsdeclarenoconflictsofinterest.

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