CD38 gene polymorphisms and genetic predisposition to multiple myeloma

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Praca oryginalna/Original research article

CD38 gene polymorphisms and genetic predisposition to multiple myeloma

Polimor ﬁzm genu CD38 a predyspozycja genetyczna do szpiczaka mnogiego

Zo ﬁa Szemraj-Rogucka

¹

, Janusz Szemraj

²

, Olga Grzybowska-Izydorczyk

³

, Tadeusz Robak

¹

, Krzysztof Jamroziak

^1,

*

1DepartmentofHematology,MedicalUniversityofLodz,Poland

2DepartmentofMedicalBiochemistry,MedicalUniversityofLodz,Poland

3DepartmentofExperimentalHematology,MedicalUniversityofLodz,Poland

Introduction

Multiple myeloma (MM) represents a B cell malignancy characterized by clonal plasma cells proliferation in the

bone marrow, monoclonal protein in the blood and associated organ dysfunction [1]. While the etiology of MM is largely unknown, there isstrong evidence for an inherited genetic susceptibilitytothis tumor.Presenceof thegenetic background is supported by the reports on MM cases in article info

Articlehistory:

Received:07.01.2013 Accepted:25.01.2013 Availableonline:23.02.2013

Keywords:

Multiplemyeloma

CD38

Polymorphism

Geneticsusceptibility

Słowakluczowe:

szpiczakplazmocytowy

CD38

polimorﬁzm

predyspozycjagenetyczna

abstract

Singlenucleotidepolymorphisms(SNPs)ofadhesionandsignalinggenesmayinfluence theetiopathogenesisofmultiplemyeloma(MM).CD38moleculeanditsligandCD31are expressed and interact in malignant plasma cells and MMmicroenvironment. In this studyweevaluatedallelefrequenciesanddistributionoftwopotentiallyfunctionalCD38 SNPs, intronic rs6449182(184C>G) and missense rs1800561 (418C>T, Arg¹⁴⁰Trp) in175 Caucasian patients with MM and 207 healthy blood donors. The carriers of variant Galleleofthers6449182SNPs werefound tohave significantlyelevatedrisk ofMMas comparedtonon-carriers;oddsratio=5.69(95%confidenceinterval=3.7–8.7),p<0.0001.

In contrast, rs1800561 SNP minor T allele was detected at very low and comparable frequenciesinpatientsandcontrolsgroups.Inconclusion,ourdatasuggestthatinher- itedgeneticvariationinCD38genemayimpactontheriskofMMdevelopment.

*Correspondingauthorat:DepartmentofHematology,MedicalUniversityofLodz,Ciolkowskiego2,93-510Lodz,Poland.

Tel.:+48426895191;fax:+48426895192.

E-mailaddress:krzysztof.jamroziak@wp.pl(K.Jamroziak).

ContentslistsavailableatSciVerseScienceDirect

Acta Haematologica Polonica

journal homepage:www.elsevier.com/locate/achaem

http://dx.doi.org/10.1016/j.achaem.2013.02.004

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monozygotictwinsaswellasthetwo-tofour-foldincreased MMriskin ﬁrst-degreerelativesof patientswithMM [2–4].

This heritable risk is likely a consequence of the co- inheritance of low-risk genetic variants, mainly single- nucleotide polymorphisms (SNPs). SNPs are a signiﬁcant source of genetic variation inhumans,and are thoughtto be responsible, at least partially, for the individual differ- encesingeneticsusceptibilitytocomplexdiseasesincluding MM[5].

Interestingly,ﬁndingson familialaggregationof casesof MM,chroniclymphocytic leukemia (CLL),non-Hodgkinlym- phomaandHodgkinlymphomaindicatethatthesedifferent B-cell malignancies share partially common genetic background[3,6–8].Ourrecentstudyshowedthattwopotentially functionalSNPsofCD38genecontributetotheincreasedrisk of CLL [9, 10]. Human CD38 is a type II transmembrane glycoprotein expressed in immature hematopoietic cells, down-regulated by mature cells, and re-expressed at high levels by activated B cells, T cells, natural killer cells, and dendriticcells[11,12].CD38displaystwo,probablyindepen- dent,functionsincludingatransmembranesignalingreceptor andanectoenzyme[13].ActingasareceptorCD38isinvolved incell-cellinteractions.EngagementbyitsligandCD31(also knownastheplateletendothelialcellularadhesionmolecule- 1, PECAM-1), followed by an increase in intracellular Ca²⁺, induces activation and differentiation signals in B, T and natural killer (NK) cells [14–16]. Furthermore, heterophilic (CD31/CD38) and homophilic (CD31/CD31) interactions of these adhesion molecules play a key role in lymphocyte adhesion and extravasation [17]. As an ectoenzyme CD38 exerts both cyclase and hydrolase activities, synthesizing moleculesinvolvedintheregulationof cytoplasmiccalcium levels [18]. The extracellular domain of CD38 contains an enzymaticsitethatcansynthesizecyclicADPribose(cADPR) fromnicotineadeninedinucleotide(NAD+)andiscapableto hydrolasecADPR and adenosine diphosphate ribose (ADPR) [18]. cADPR functions as a universal second messenger involvedinadistinctpathwayofintracellularCa²⁺mobiliza- tion, resulting in a variety of physiological effects such as activation, proliferation, differentiation and migration [18].

Importantly, the CD38 gene, located in the short arm of chromosome4(4p15),ishighlypolymorphic[19].

Interestingly,CD38isalsoexpressedonMMplasmacells andCD38/CD31interactionsareimportantforMMevolution [20]. However,little isknown onthe biologicaland clinical signiﬁcance of CD38 SNPs in the context of MM development.Inthis case-controlstudywecompared thefrequen- cies of two potentially functional CD38 SNPs, rs6449182 (184C>G) and rs1800561 (418C>T) in MM patients and unaffectedcontrolsofPolishCaucasianorigin.

Patients and methods

Studysubjects

Thestudyincluded175patientsdiagnosedwithMMatthe Department of Hematology, Medical University of Lodz or referred to this center for consultation between February 1993 and December 2006. Peripheral blood samples were

collected at the time of diagnosis in 5ml serum tubes and stored at 208C temperature until analyzed. Control individualswere 207 volunteerCaucasian blood donorsat the local blood bank, residents of the Lodz city region.

Fresh peripheral blood samples (2ml) were obtained from controls between 1999 and 2008. Ethnicity and cancer-free status wereself-reported atthe time ofblood collection.

The study has been reviewed and approved by the Bioethical Committee of the Medical University of Lodz (approval no. RNN/200/11/KE). All patients and controls providedinformedconsent.

DNAextractionandgenotyping

Blood DNA obtained from peripheral blood leukocyte frac- tionwaspurifiedonaQIAampspincolumn(Qiagen,Hilden, Germany).Theprotocol for DNA isolationfrom body fluids provided by the manufacturer was used withmodification asfollows:5mgofRNApoly(A)(PharmaciaBiotech,Uppsala, Sweden)wasaddedto1mlofserumtoserveasacarrierto improve therecovery of small amounts of DNA. Lysis was ensuredbyadding20mlofQiagenProteinaseKsolutionand 1ml of buffer AL (QIAamp^® DNA mini kit). After 10-min incubationat568C1mlofethanolwasadded.Themixture wasloadedonthe QIAampspincolumnandcentrifugedat 20000g for 1min. The column was washed twice by adding 500ml of buffer AW (QIAamp^® DNA mini kit) and centrifugedat20000gfor1min.Finally,DNAwasincubated for 5minat room temperaturewith50mlof AEbuffer and elutedbycentrifugation.

The investigated polymorphisms were analyzed using polymerase chain reaction – restriction fragment length polymorphism(PCR-RFLP)methodasdescribepreviously[9].

Brieﬂy,forrs6449182(184C>G) SNPdetectionassayforward primer 5⁰-CCGGGTGGTGCTGAGTAGGGAGTC-3⁰ and reverse primer5⁰-CTACGCAGCAGAGCCACCGAGCAG-3⁰ (IDT,Illinois) wereused.ThePCRreactionsweredoneina20mlPCRtube containing50ngofgenomicDNA,1.5pmol/lofeachprimer, 1.5mmol/lofMgCl2,250nmol/lofdNTPs,and2unitsofTaq polymerase (Promega) withampliﬁcationconditions asfollows: initialdenaturation at 958Cfor 5min,followed by35 cycles of 958C for 30s, 638C for 30s, and 728C for 30s followed by an extension step of 728C for 10min in a Biometra T Personal thermocycler (Biometra). The 128-bp amplicon was digested with 1 unit of enzyme PvuII (Pro- mega) analyzed on 6% paa gel overnight. Thepresence of the rs6449182C allele resulted in the digestion of the ampliconto63bpand65bpproducts.

The PCR-RFLP assay to detect rs1800561 (418C>T) SNP included forward primer 5⁰-GACATGCTACTAAATTGATCT- CAG-3⁰ andreverseprimer5⁰-CTACGCAGCAGAGCCACCGAG- CAG-3⁰ (IDT, Illinois). The PCR mixes consistedof 50ng of genomicDNA,5pmol/lofeachprimer,1.6mmol/lof MgCl2, 200nmol/l of dNTPs, and 1 unit of Taq polymerase (Pro- mega)ina20mlPCRtube.Theamplificationreactionswere performedwiththefollowingcyclingvariables:initialdena- turationat958Cfor5min,followedby30cyclesof948Cfor 30s,508Cfor30s,and728Cfor 30s,withafinalextension step at 728C for 10min. The amplified DNA product was

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digestedovernightwith1unitofenzymeAciI(NewEngland BioLabs). Digests were detected by electrophoresis on 6%

paagelasacombinationof uncut248-bpband(rs1800561T allele)and146-bpand102-bpboundsfordigestedfragments (rs1800561Callele).

Statisticalanalysis

The statistical analysis was performed using IBM SPSS Statistics 20 (IBM Corporation, Armonk, NY) statistical package. The permutation test was applied to assess accordance with the Hardy–Weinberg equilibrium within eachcaseandcontrolgroup.Unconditionallogisticregres- sion was usedto estimatethe odds ratios(ORs) with95%

conﬁdenceintervals(95%CI)forassociationsofcase-control status with the CD38 genotypes. Baseline laboratory and clinicalparametersatdiagnosisamongcarriersofdifferent CD38genotypeswerecomparedbythechi²ortheKruskal– Wallistestsasappropriate.Forallcalculationsp<0.05was consideredsigniﬁcant.

Results

Amongthe175MMpatientsincludedtothestudytherewere 93(53%)malesand81(46%)femalesofamedianage61years.

Detailsofthebaselineclinicalcharacteristicsatdiagnosisare listedinTableI.Foraproportionofpatients(n=57)whowere referred tothe Department of Hematology of Medical Uni- versityofLodzforanambulatoryconsultationsomeinforma- tiononlaboratoryresultsatdiagnosiswasmissing(TableI).

Genotypingwassuccessfulinallindividualsinvestigated within the study. Allele frequencies of the CD38 SNPs rs6449182 andrs1800561inpatientandcontrolpopulations are summarized in Table II. Both tested SNPs were in accordance with Hardy–Weinberg equilibrium regarding control group (Table II). Consistent with low rs1800561 T allele frequency observed previously among Caucasian populations, we detected only 3 heterozygous individuals (rs1800561 CT genotype) in the patient group, while only wild-type homozygous subjects (rs1800561 CC genotype) wereidentiﬁed intheMMpatientgroup. Therefore,further analyses were restricted to the rs6449182 polymorphism.

The allele frequency and distribution of rs6449182 SNP differed signiﬁcantly between patients and controls. We observed higherfrequencies of the variantallele rs6449182 G in patients with MM as compared to healthy controls, (0.411versus0.159,p<0.001)(TableII).Moreover,anassocia- tion betweenMMandheterozygous rs6449182GCgenotype was observed; OR=5.1 (95% CI=3.3–7.9). Interestingly, the MM risk was further elevated with rs6449182 GG homozygous genotype (OR=12.7; 95% CI=6.9–23.4) suggesting an allele-dose effect. The associations between CD38 SNPs genotypesandMMriskarepresentedinTableIII.

Subsequently, we analyzed potential relation between clinical and laboratory variables at diagnosis of MM and TableI–Baselinecharacteristicsatdiagnosisof175

Polishmultiplemyelomapatientsincludedtothestudy

Variable n(%)

Age(years) median range

61 39–87 Sex

male female

93(53) 81(46) Myelomaserotype

IgG IgA IgD

Lightchaindisease

91(52.0) 48(27.4) 1(0.6) 35(20.0) Hemoglobin(g/dL)

median range

9.2 5.6–15.4 Durie–Salmonstage

I II III

datanotavailable^a

9(5) 29(17) 81(46) 56(32) Renalinsufﬁciency

present absent

datanotavailable^a

31(18) 87(50) 57(32)

a Forpatients(n=57)referredtotheDepartmentofHematologyof MedicalUniversityofLodzforambulatoryconsultationsomedata fromthetimeofdiagnosiswerenotavailable.

TableII–AllelefrequenciesofCD38geneSNPrs6449182 andrs1800561inPolishCaucasianmultiplemyeloma patientsandcontrols.P-valuefordeviationfromHardy– Weinbergequilibriumwasderivedfromthepermutation test

CD38alleles MMcases(%) n=175

p Controls(%) n=207

p

rs6449128SNP

C 206(58.9) 0.01 348(84.1) 0.8

G 144(41.1) 66(15.9)

rs1800561SNP

C 347(99.1) 0.99 414(100.0) 0.99

T 3(0.9) 0

TableIII–AssociationsbetweenCD38geneSNPrs6449182genotypesandriskofmultiplemyeloma

rs6449182SNPgenotypes Cases Controls OR 95%CI p

CC 51 145 1.00

GC 104 58 5.10 (3.28–7.92) <0.0001

GG 20 4 14.22 (5.68–35.61) <0.0001

CC 51 145 1.00

GC+GG 124 62 5.69 (3.71–8.73) <0.0001

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CD38 alleles. Median age at diagnosis was comparable in carriersofrs6449182CC,CGandGGgenotypesandreached 64,60and62yearsrespectively,p=0.32.Similarly,patients' median age at MM diagnosis did not differ between the wild-typers1800561CChomozygotes(61years)andcarriers of heterozygous rs1800561 CT genotype (68 years), p=0.23.

Furthermore, we did not detect any signiﬁcant differences amongdifferentCD38 genotypesregardingsuchparameters assex (p=0.55 for rs6449182 SNP analysis and p=0.74 for rs1800561CTanalysis),Durie–Salmonclinicalstage(p=0.33 for rs6449182 SNP analysis and p=0.78 for rs1800561 CT analysis), baseline hemoglobin concentration (p=0.40 for rs6449182SNPanalysisandp=0.69for rs1800561CTanaly- sis) andpresence of renalinsufﬁciencyat diagnosis of MM (p=0.13 for rs6449182 SNP analysis and p=0.09 for rs1800561CTanalysis)(datanotshown).

Discussion

InthisstudywehypothesizedonthepotentialroleofCD38 SNPs as determinants of MM predisposition based on the dataonCD38andCD31expressioninMMandmicroenviron- ment cells and their interactions. We found a signiﬁcant association between genotypes containing common variant CD38rs6449182SNPGalleleandtheriskofMMdevelopment.

Toourknowledge,nostudiestestingtherelationofCD38 gene SNPs to MM have been reported to date. However, rs6449182CD38 genepolymorphism waslinked tosuscept- ibility to CLL as well as some clinical phenotypes in differentdiseases. Aydinet al., showed significantassocia- tion betweenrs6449182Galleleand advanced clinicalstage and elevated risk of Richter's transformation in patients withCLL[21].Subsequently,ourgroupfoundthatrs6449182 contributes to CLL predisposition and affects CD38 expres- sion [9]. In accordance with our finding Abramenko et al., described significant association between rs6449182 GG genotypeandCLLriskinUkrainianCaucasians[22].Finally, the metaanalysis of the three studies confirmed the influ- ence of this SNP on CLL predisposition[23]. Earlier, it had been reported that rs6449182 SNP influenced the mainte- nance of peak bone mineraldensity inwomen as well as clinicalcharacteristicsofsystemiclupuserythematosus[24, 25]. The biological mechanisms underlying these associa- tionsareunknown.However,theycanberelatedtoaltered CD38proteinexpressionasshowninourpreviousstudy[9].

Despitestrongepidemiologicaldatasupportingtheinher- itedbackgroundofMM,todatenolocihavebeenunambigu- ously established asrisk factors for MM development. The mostcommonapproach toidentifysuch loci wastoscreen potentiallyfunctionalorhaplotype-taggingSNPsincandidate genes involved in different pathways regulating malignant plasma cell proliferation or survival. Interleukin 6 (IL-6) is amaincytokinedrivingmyelomagrowthanddifferentiation [26]. In some trials IL-6 SNPs rs1800796 as well as IL-6 receptor rs6684439 and rs7529229 SNPs were found to sig- niﬁcantly alter risk to develop MM [27, 28]. Furthermore, tumor necrosis factor (TNF) haplotypesinvolving rs1800629 and rs909253 SNPs were associated with MM risk in some studies,butnotinothers[29,30].Anothersourceofpotential

susceptibilityallelestotumorigenesisarefunctionalvariants ingenesinvolvedinsuchprocessesasDNArepair,cellcycle control and apoptosis. The example of such ﬁndings is inﬂuence of rs963248 SNP of XRCC4 and rs1051685 SNP of XRCC5DNArepairgenesontherisktodevelopMM[31].

A limitation of our study, as well as majority of MM susceptibility studies reported to date, is relatively small samplesizedueinparttothelowincidenceofthedisease.In a consequence the statistical power is too low to detect variants associated withlow OR.This isparticularlyimpor- tantconsideringthefactthatthegeneticrisktodevelopMM is likely influenced bymany low-risk alleles. Even if meta- analyses can overcome the size limitations that occur in single studies, there are so far limited data on particular predispositionloci.Toovercometheseobstacleslargegroups of patients from different research groups can besimulta- neously studied in a consortium. Recently, at least two international consortia with objective to investigate MM genetic background have been founded, namely IMMEnSE (International Multiple Myeloma rESEarch) including 12 research groups across Europe and the MAGIC (MyelomA Genetics International Consortium), including 16 research groups inEurope,Asia, Australasia,theMiddleEastandthe Americas [32, 33]. Furthermore, hypotheses on candidate- genesinMMareproblematicasthereislittleunderstanding of thediseaseetiology.Anunbiasedapproachtodetectthe cancer associations is genome wide association studies (GWAS). Recently,ametaanalysis oftwo firstGWASinMM waspublished[34].Thestudiesinvestigatedthetotalof1675 MMcasesand5903controlsubjects,andidentifiedrisklociat 3p22.1 (rs1052501) and 7p15.3 (rs4487645) and a promising associationat 2p23.3(rs6746082).Thesenovelpredisposition variants,ifconfirmedbysubsequentassociationstudies,may leadtoimportantetiologicalinsightsinMM.

In conclusion, this study found that inherited polymorphism rs6449182 of CD38 gene is associated with apredispositiontoMM. Ourﬁndingmayhaveimplications for understanding the role of CD38 molecule inthe patho- genesisofMM.

Authors' contributions/Wkład autorów

ZS-R – study design, data collection and interpretation, manuscriptpreparation,literaturesearch,JS–datacollection, OG-I – data collection, manuscript preparation, TR – data interpretation, manuscript preparation, KJ – study design, statisticalanalysis,manuscriptpreparation,literaturesearch.

Financial support/Finansowanie

Supported in part by grants from the Polish Ministry of Science (NN402178334)and the Medical University of Lodz (502-11-680).

Conﬂict of interest/Konﬂikt interesu

Nonedeclared.

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Ethics/Etyka

Theworkdescribedinthis articlehavebeencarriedout in accordance with The Code of Ethics of the World Medical Association(Declaration of Helsinki)for experimentsinvol- ving humans; EU Directive 2010/63/EU for animal experi- ments;UniformRequirementsformanuscriptssubmittedto Biomedicaljournals.

TheownresearchwereconductedaccordingtotheGood Clinical Practiceguidelines and acceptedby local Bioethics Committee, all patients agreed in writing to participation andtheseresearches.

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