Rev. Bras. Reumatol. vol.57 número6

r e v b r a s r e u m a t o l . 2017;57(6):633–636

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

REVISTA

BRASILEIRA

DE

REUMATOLOGIA

Brief

communication

Case-control

study

of

vitamin

D

receptor

gene

polymorphism

in

Pakistani

rheumatoid

arthritis

patients

Estudo

caso-controle

do

polimorfismo

do

gene

receptor

da

vitamina

D

em

pacientes

paquistaneses

com

artrite

reumatoide

Peter

John

,

Attya

Bhatti

,

Noor

ul

Ain

,

Tahir

Iqbal

,

Tayyaba

Sadaf

,

Javed

Mehmood

Malik

a_{NationalUniversityofSciences&Technology(NUST),Atta-Ur-RahmanSchoolofAppliedBiosciences(ASAB),Departmentof}

HealthcareBiotechnology,Islamabad,Pakistan

b_{ShifaTameer-e-MillatUniversity(STMU),FacultyofMedicine,Islamabad,Pakistan}

c_{ArthritisResearchCenter,RahmatNoorClinic,Rawalpindi,Pakistan}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received13May2014 Accepted28January2015 Availableonline20August2015

Introduction

Rheumatoidarthritis(RA)isthemostcommonchronic inflam-matorydisease,characterizedbyautoantibodyformationand progressivejointdestruction.InRA,synovialmembrane pri-marilygetsaffectedbut itcanaffectother organsaswell.1 RAisadevastatingandcommondiseasehavingprevalence ofapproximately1%inworldpopulation,and0.14–0.3% Paki-stanipopulation,withwomenaffectedthreetimesmoreoften thanmen.2 _{TheexactetiologyofRAisyetunknownbut it} dependsontheinteractionofnumberofenvironmentaland geneticfactors.3_{InrecentyearsstarringroleofvitaminD,a} secosteroidhormonecarriesout theactivitiesthroughVDR

∗ _{Correspondingauthor.}

E-mail:[email protected](P.John).

andVDRitselfhasbeenfoundinimmunomodulation,thuswe selectedthegeneVDRtoinvestigateitsimpactinRA suscep-tibility.TheVDRproteinistranslatedfromVDRgenewhichis extremelypolymorphic.ThefourimportantVDR gene vari-ants,rs2228570in exon2,rs1544410,rs7975232inintron 8 andrs731236inexon9havebeenwidelystudiedandarealso known tobeassociatedwithautoimmunediseases includ-ingsystemiclupuserythematosus,Addison’sdisease,typeI diabetesandRA.4

Study of differences in genotype distribution would be helpful to identify the consequences ofethnic allele vari-ation because ofdistinct genetic backgrounds, additionally haveunlikeenvironmentalelements.Italsohelpsin interpre-tinganddetectingphenotypicvariabilityintermsofdisease

http://dx.doi.org/10.1016/j.rbre.2015.07.003

634

Table1–Generalcharacteristicsofpopulationstudied.

Characteristics Cases(n=100) Control(n=100)

Male 37 31

Female 63 69

Age(mean) 44.2 43

severity. Based on genotype distinctions, biological inter-pretation could beclaimed by disease association studies. Polymorphism rs1544410 located in intron 8 near 3′ _UTR

region, affects the VDR gene transcription level, transcript stabilityand posttranscriptionalmodifications.5 _Some pre-viouslyreporteddatashowedfunctionalsignificanceinthe distributionofrs1544410genotype,ourstudywas accompa-niedtoevaluatetheassociationofVDRpolymorphismwith RAinourgroupofstudy.Thereisagreatneedtodiscoverthe potentialgeneticriskmarkersofRAsusceptibility,inour Paki-stanipopulation,butnosuchworkhavepreviouslybeendone forPakistaniRApatients.

Materials

and

methods

A total of 100 patients and 100 healthy controls (non-symptomaticindividuals)matchedforageandsex(Table1) wererecruitedforthis case–controlstudy.Patientsfulfilling the 2011 classification criteria of the American College of Rheumatology(ACR-2011)werediagnosedbyrheumatologists fromRehmatNoorClinic,Rawalpindi,workingin collabora-tion with our research group. Thestudy was approved by InstitutionalReviewBoard(IRB)ofASAB-NUST,followingthe rulesoftheDeclarationofHelsinkiandtheinformedconsent wastaken.BloodsamplesofalltheincludedRApatientsas wellasfromthehealthycontrolswerecollectedin0.5MEDTA (ethylenediaminetetraaceticacid)tubes.SNPthatcauses sin-glenucleotidechange frommajorallele frequencyguanine (G)tominorallelefrequencyadenine(A)wasinvestigatedto checkthepresenceofpolymorphisminourstudygroup.

Genomic DNA wasextracted from collected blood sam-ples using phenol–chloroform method, quantified by nan-odrop (eppendrof Biophotometer Plus). Manually designed two forward and a common reverse primer (VDR-F1-5′_{GCCACAGACAGGCCTGCG3}′_{, VDR-F2-5}′ GCCACAGACAGGCC-TGCA3′_{, VDR-R-5}′_{GTCACTGCACATTGCCTCCAA3}′_{)were used} for VDR gene non-coding variant (rs1544410) genotyp-ing through Amplification Refractory Mutation System-PolymeraseChainReaction(ARMS-PCR). PCRproductswere

resolved on 2% agarose gel after amplification in96 wells thermocycler 2720 (Applied Biosystems). Statistical analy-sis wasperformed using GraphpadPrism 6Vsoftware, Chi square(X2_{)andtwo-tailedFisher’sexacttestswereappliedfor} associationanalysisofrs1544410polymorphisminourstudy population.Oddsratio(OR)with95%confidenceintervalwas calculatedandpvalue<0.05wasconsideredstatistically sig-nificant.

Results

Hardy–Weinberg equilibrium (HWE) calculations made for control group shows no significant difference between observed valuesandthe expectedvalues, withthe p-value of 0.0034 and showed to be in HWE. This indicates that therewasnosignificantdriftinobservedallelefrequenciesof patients,whencomparedwithcontrols,thusboththegroups weresuitableforfurtheranalysisandassociationstudies.The frequencyofallpossiblegenotypesdidnotalsoshowany sig-nificantdifferenceinpatientsandhealthyindividuals(Table2 and Fig.1A).ThevalueofX2 _{was foundtobe5.662witha} probabilityoferror(p-value)of0.0590.Allelefrequenciesfor RA patientswere also notsignificantlydifferent from con-trols(Table2andFig.1B).Theobservedresultsindicatedno significant association betweenthe SNP and RA.Thus, the datanullifiedanysignificantassociationofVDRgene polymor-phismwithRAinourgroupofstudy.Sexspecificassociation withrs1544410wasalsocarriedoutbutnosignificant associa-tionhasbeenshownbetweenRAaffectedfemalesandmales (datanotshown).

Discussion

Intherecentyearsgenepolymorphismhasbeenoneofthe utmostdiscussedtopicofgenomicvariations.RAisasystemic inflammatorydiseasethataffectstheboneandcartilageof the patient. Vitamin D exerts several immunomodulatory effectsandthusmayplayaroleinthecourseofautoimmune diseases.6 _{A considerable association between VD} insuffi-ciencyandanincreasedoccurrenceofautoimmunedisorders hasbeendetermined.7_{IthasbeenobservedthatinRApatients} lymphocytesexpressVDR.8_{Incytoplasmhydroxylatedform} of vitamin D 1,25[OH2]D binds to VDR which then move towardnucleus,whereitincreasestheVDdependentgenes transcriptionessentialincalciumandbonemetabolism,9_also inhibitingT-cellproliferationandthereleaseofTh1cytokine

Table2–Genotypeandallelefrequenciesdistributionincasesandcontrols.

Genotype Genotypefrequencies X2_{statistics Allele Allelefrequencies Statistics}

Cases (n=100)

Control (n=100)

X2_{value df p-value} (alpha<0.05)

Case (n=100)(%)

Control (n=70)(%)

Oddsratio (OR)(95%CI)

p-value

GG 31.0 40.0

5.662 2 0.0590

G 56.0 66.0 0.6556(0.4375–0.9825) 0.0513

GA 50.0 52.0

AA 19.0 8.0 A 44.0 34.0

rev bras reumatol.2017;57(6):633–636

635

60

40

20

0

AA GA

GG G A

150

100

50

0

Cases Controls

Genotype frequency Allele frequency

Genotypes Allele

rs1 544 410 rs1 544 410

A

B

Fig.1–A,GraphshowinggenotypedistributionofVDRpolymorphism(rs1544410)inRAcasesandhealthycontrols.B, GraphshowingallelicdistributionofVDRpolymorphism(rs1544410)inRAcasesandhealthycontrols.

suchasIL-2,IFN-␥andTNF-␣.10Variousstudieshaveshown

thattheriskofautoimmunediseaseshavebeenincreaseddue tovitaminDdeficiency, additionallya considerableclinical improvementwasobservedintheVD-treatedRApatients.11 InourstudyVDRgeners1544410polymorphisminvestigation showsnostatisticallysignificantdifferencesingenotypeand allele frequenciesofRApatients andhealthy controls.Our observationsareinconsistencewiththefindings ofMaalej et al.1 _{who demonstrated no association of the rs1544410} polymorphism with the development ofRA inFrench and Tunisianpopulations.However,thispolymorphismwas asso-ciatedwithotherautoimmunediseasesliketype1diabetes (T1D)inmanycountries,includingdifferentpopulationslike Hungary, Japan, Greece, Bangladesh, Taiwan and Chile.12 Significant association was also found between rs1544410 polymorphismandosteoporosis.13_{Furthermore,thestudies} havebeenconductedonothervariousautoimmunediseases includinglupus,cirrhosis,hepatitis,Crohn’s,Gravesdisease andmultiplesclerosisinrelationshipwithrs1544410 polymor-phismandtheincidenceofsystemiclupuserythematosusin JapaneseandChinesewasfound.Sothepolymorphismcan beinvestigatedinassociationwithotherimmunediseases.14 PolymorphismsinVDRgenehavebeenobservedtobe associ-atedinasexdependentmanner,i.e.moreprevalentinfemales affectedwithRA.15_{Sexspecificassociationwithrs1544410in} ourgrouphasindicatedthatpolymorphismdoesnoteffect insexdependentmannerincaseofRAandthereisnolinkof thispolymorphismtotheincreasedincidenceofRAamong females.

Although finding of this study shows no evidence of rs1544410associationwithRAinourgroupofstudy,butthere mightbeotherpolymorphismsfoundthatwouldhavebeen recognizedasriskfactorsforRA.Thereisaneedtoinvestigate othermoleculesimplicatedintheinflammatorypathwayof RAforgeneticassociationwithRApathogenesis.Other poly-morphismsintheVDRgenemayhavesignificantassociation towardRA,solargerpopulationstudyisrequiredtodelineate theassociation.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheworkpresentedinthisstudywasfundedbyHigher Edu-cationCommission.

r

e

f

e

r

e

n

c

e

s

1.MaalejA,Petit-TeixeiraE,MichouL,RebaiA,CornelisF,Ayadi H.AssociationstudyofVDRgenewithrheumatoidarthritis intheFrenchpopulation.GenesImmun.2005;6:707–11. 2.DaiSE,HanXH,ZhaoDB,ShiYQ,LiuY,MengJM.Prevalence

ofrheumaticsymptoms,rheumatoidarthritis,ankylosing spondylitis,andgoutinShanghai,China:aCopcordstudy.J Rheumatol.2003;30:2245–51.

3.KlareskogL,PadyukovL,RonnelidJ,AlfredssonL.Genes, environmentandimmunityinthedevelopmentof rheumatoidarthritis.CurrOpinImmunol.2006;18:650–5. 4.KiranG,DebashishD.VitaminDandrheumatoidarthritis:is

therealink?IntJRheumDis.2008;11:206–11.

5.UitterlindenAG,FangY,VanMeursJB,PolsHA,VanLeeuwen JP.VitaminDreceptorgenepolymorphismsinrelationto VitaminDrelateddiseasestates.JSteroidBiochemMolBiol. 2004;89–90:187–93.

6.KaletaB,BogaczewiczJ,RobakE,Sysa-J ˛edrzejowskaA, WrzosekM,SzubierajskaW,etal.VitaminDreceptorgene BsmIpolymorphisminPolishpatientswithsystemiclupus erythematosus.ISRNEndocrinol.2013.

7.AdoriniL,PennaG.Controlofautoimmunediseasesbythe vitaminDendocrinesystem.NatClinPractRheumatol. 2008;4:404–12.

636

9. KhazaiN,JuddSE,TangprichaV.CalciumandvitaminD: skeletalandextraskeletalhealth.CurrRheumatolRep. 2008;10:110–7.

10.LemireJM.Immunomodulatoryroleof1,25-dihydroxyvitamin D3.JCellBiochem.1992;49:26–31.

11.AndjelkovicZ,VojinovicJ,PejnovicN,PopovicM,DujicA, MitrovicD,etal.Diseasemodifyingandimmunomodulatory effectsofhighdose1alpha(OH)D3inrheumatoidarthritis patients.ClinExpRheumatol.1999;17:453–6.

12.ChakhtouraM,AzarST.TheroleofvitaminDdeficiencyin theincidence,progression,andcomplicationsoftype1 diabetesmellitus.IntJEndocrinol.2013.ArticleID148673.

13.PouresmaeiliF,JamshidiJ,AzargashbE,SamangoueeS. AssociationbetweenvitaminDreceptorgeneBsmI polymorphismandbonemineraldensityinapopulationof 146Iranianwomen.CellJ.2013;15:75–82.

14.VogelA,StrassburgCP,MannsMP.Geneticassociationof vitaminDreceptorpolymorphismwithprimarybiliary cirrhosisandautoimmunehepatitis.Hepatology. 2002;35:126–31.