Research ArticleAbsence of Association between CCR5 rs333 Polymorphism andChildhood Acute Lymphoblastic Leukemia

Carlos Eduardo Coral de Oliveira1 Marla Karine Amarante2 Aparecida de Lourdes Perim2

Patricia Midori Murobushi Ozawa1 Carlos Hiroki1 Glauco Akelinghton Freire Vitiello1

Roberta Losi Guembarovski1 and Maria Angelica Ehara Watanabe1

1 Laboratory of Study and Application of DNA Polymorphisms Department of Pathological Sciences Biological Sciences CenterState University of Londrina Rodovia Celso Garcia Cid (PR 445) Km 380 86051-970 Londrina PR Brazil

2 Laboratory of Hematology Department of Pathology Clinical and Toxicological Analysis Health Sciences CenterState University of Londrina Londrina PR Brazil

Correspondence should be addressed to Maria Angelica Ehara Watanabe maewatuelgmailcom

Received 27 January 2014 Revised 19 March 2014 Accepted 21 March 2014 Published 13 April 2014

Academic Editor Helen A Papadaki

Copyright copy 2014 Carlos Eduardo Coral de Oliveira et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Acute lymphoblastic leukemia (ALL) is a malignant disorder that originates from one single hematopoietic precursor committedto B- or T-cell lineage Ordinarily these cells express CCR5 chemokine receptor which directs the immune response to a cellularpattern and is involved in cancer pathobiology The genetic rs333 polymorphism of CCR5 (Δ32) results in a diminished receptorexpression thus leading to impaired cell trafficking The objective of the present study was to investigate the effect of CCR5chemokine receptor rs333 polymorphism in the pathogenesis of ALL The genotype distribution was studied in 79 patients andcompared with 80 control subjects in a childhood population of Southern Brazil Genotyping was performed using DNA samplesamplified by polymerase chain reaction with sequence-specific primers (PCR-SSP) The homozygous (Δ32Δ32) deletion was notobserved in any subject involved in the study Heterozygous genotype was not associated with ALL risk (OR 07 95 CI 021ndash232 119875 gt 005) nor recurrence status of ALL (OR 086 95 CI 013ndash548 119875 gt 005) This work demonstrated for the first timeno significant differences in the frequency of the CCR5Δ32 genotype between ALL and control groups indicating no effect of thisgenetic variant on the ALL susceptibility and recurrence risk

1 Introduction

Leukemia is the most common childhood cancer althoughoverall incidence is rare Within this population acutelymphoblastic leukemia (ALL) occurs approximately fivetimes more frequently than acute myelogenous leukemia(AML) and accounts for approximately 78 of all childhoodleukemia diagnoses [1] In Brazil the National Cancer Insti-tute (INCA) estimated 9370 new cases of leukemia in 2014with the highest age incidence of 1ndash4 years [2]

The specific biological and molecular mechanisms thataccount for the aggressiveness and poor therapy response ofsome ALL cases remain to be elucidated Once chemokines

and their receptors have been discovered as essential andselective mediators in leukocyte migration to inflammatorysites and to secondary lymphoid organs it is reasonable thatthey also play a critical role in tumor initiation promotionand progression [3 4] Moreover updated research indicatesthat cancer cells subvert the normal chemokine system andthese molecules and their receptors become important con-stituents of the tumor microenvironment with very differentways to exert tumor-promoting roles [5]

The CC chemokine receptor 5 (CCR5) belongs to thetrimeric guanine nucleotide-binding-protein-coupled seven-transmembrane receptor superfamily which comprises thelargest superfamily of human proteins [6] It exerts its activity

Hindawi Publishing CorporationAdvances in HematologyVolume 2014 Article ID 924030 5 pageshttpdxdoiorg1011552014924030

2 Advances in Hematology

via G protein and binds to the chemokines RANTES (CCL5)MIP-1120572 (CCL3) and MIP-1120573 (CCL4) [7] This receptor isinvolved in the chemotaxis of leucocytes to inflammationsites [8] and plays important function in the recruitment ofmacrophages T cells and monocytes [9]

The importance of CCR5 for immune response is depen-dent on the type of stimuli moreover in some casescompensating mechanisms override the absence of CCR5expression and function Noteworthy CCR5 may exert a farmore important role in the immune response than in immunecells traffic regulation [10]

It has been shown that CCR5 expression in stromal cellsas well as hematopoietic cells contributes to tumor metas-tasis For instance CCR5 is involved in chondrosarcomasmetastasization [11] and oral cancer cells migration [12] Itsexpression correlates with multiple myeloma cell growthbone marrow homing and osteolysis [13]

Aster and colleagues [14] showed that T-cell ALL is a dis-ease primarily caused by aberrant activation of the NOTCH1signaling pathway In this context expression and functionof important chemokine receptors such as CCR5 and CCR9are partially controlled by the oncogenic NOTCH1 isoformin T-cell ALL regulating blast malignant properties andlocalization of extramedullary infiltrations [15]

Additionally CCR5 has been related to play a key rolein metastasis of aggressive NK-cells leukemia to the liverof patients contributing to hepatosplenomegaly and hepaticfailure [16 17] Also Davies et al [18] showed that the Gallele of rs1799987 polymorphism in CCR5 was associatedwith more favorable minimal residual disease status thanthe A allele when comparing ldquobestrdquo and ldquoworstrdquo risk groupsof B-cell ALL adjusted for prognostic features Consideringthe lower activity of CCR5 promoter in the presence ofthis polymorphism [19] this evidence indicated that bothacquired and host genetics influence response to cancertherapy Thus it is plausible that CCR5 might play a role inALL pathogenesis and prognosis

It is known that the polymorphism rs333 in CCR5 acommon 32-base pair deletion (Δ32) causes truncation andloss of this receptor on lymphoid cell surface with completeretention in the endoplasmic reticulum within homozygousor diminished expression in heterozygous genotype [20]The CCR5 studies have demonstrated the importance of Δ32mutation particularly in the susceptibility to HIV infection[21] since CCR5 is a coreceptor in the primary stage ofinfection that is essential for the AIDS onset [22]

Our research grouphas reported polymorphic allelic vari-ants related to the immune system and tumor development indifferent cancer types Nevertheless there are no data relatingCCR5Δ32 polymorphism to ALL population In this contextthe present work analyzed rs333 polymorphism of CCR5 inALL patients from the southern region of Brazil

2 Materials and Methods

21 Human Subjects Following approval from the HumanEthics Committee of the State University of Londrina

Parana Brazil (CAAE number 17123113400005231) inclu-sion of the individuals to the study was conditioned byan obtained written informed consent form from parentsregarding the use of their children and adolescents bloodsamples Seventy-nine ALL patients were enrolled and diag-nostic criteria were based on the guidelines proposed byHematology Department of the University Hospital of Lon-drina Recurrence risk status of ALL patients was evaluatedthrough the GBTLI Protocol (Brazilian Group of Child-hood Leukemia Treatment Protocol-99) which is based onthe Cancer Therapy Evaluation Program proposed by theNational Cancer Institute and takes into account age at diag-nosis leukocyte count immunophenotyping involvementof tissues other than bone marrow and responsiveness totreatment The control group is comprised of 80 healthyindividuals free of neoplasia matched by age and gender

22 Genomic DNA Extraction Genomic DNA was extractedfrom whole blood by Biopur Mini Spin Plus Kit (BiometrixDiagnostica Curitiba Brazil) according to the manufac-turerrsquos instructions DNA was eluted in 50 120583L of milliQwater and quantified byNanoDrop 2000c spectrophotometer(Thermo Fisher Scientific Inc Wilmington USA) at a wave-length of 260280 nm Final preparation was stored at minus20∘Cand used as templates in polymerase chain reactions (PCR)

23 Optimization of PCR for CCR5 The method of geno-typing (rs333) was optimized in the Laboratory of Study andApplication ofDNAPolymorphisms of the StateUniversity ofLondrina using genomicDNAand specific primers forCCR5Primer sense 51015840 ACC AGA TCT CAA AAA GAA 31015840 andPrimer antisense 51015840 CAT GAT GGT GAA GAT AAG CCTCA 31015840 (GenBank accession AF009962) Genotyping of CCR5was determined by PCR-SSP The samples were amplifiedusing 125 units of Taq polymerase (Invitrogen CarlsbadUSA) in aMastercyclerGradientThermal Cycler (EppendorfHamburg Germany) PCR conditions were denaturationstep at 94∘C for 5min 35 cycles of 1min at 94∘C 1min at58∘C and 1min at 72∘C and 10min of elongation at 72∘C PCRproducts (225 bp or 193 bp) were analyzed on polyacrylamidegel (10) stained with silver nitrate (AgNO

3)

24 Statistical Analysis Contingency tables andFisherrsquos exacttest were used to calculate differences in genotype distribu-tions and allele frequencies 119875 lt 005 was considered toindicate a statistically significant difference Goodness of fitof Hardy-Weinberg equilibrium was tested by calculating theexpected frequencies of each genotype and comparing themwith the observed value using a chi-square test

3 Results

The distribution of CCR5 alleles in both ALL and controlgroups was in accordance with the assumption of Hardy-Weinberg equilibrium (119875 gt 0 05) The mean age of controlsand ALL patients was 108 years plusmn 565 and 87 years plusmn620 respectively all of whom were predominantly fromCaucasoid population due to European colonization

Advances in Hematology 3

225 bp193bp

L 1 2 3 bl

Figure 1 CCR5 genotype profile The PCR products were detectedusing silver staining method after polyacrylamide gel electrophore-sis Lane LDNALadder 100 bp lane 1 CCR5wild-type homozygousgenotype (CCR5CCR5 225 bp) lane 2 heterozygous genotype(CCR5Δ32 225 bp and 193 bp) and lane 3 variant allele homozy-gous genotype (Δ32Δ32 193 bp) bl represents blank reaction(without DNA)

Table 1 Genotype distribution of CCR5 rs333 polymorphism andrecurrence risk status analysis in ALL and control groups

Genotypes OR 95 CI 119875lowastCCR5CCR5 CCR5Δ32

Control (80) 73 (9125) 7 (875) 07 021ndash232 076ALL (79) 74 (925) 5 (75)

High risk (50) 47 (94) 3 (6) 086 013ndash548 100Low risk (29) 27 (931) 2 (69)

lowastFisherrsquos exact test 119875 gt 005 OR odds ratio CI confidence interval

The patients and controls were matched for sex andgender although there was a modest higher frequency ofmales in ALL group (5316) than in controls (4875) Fifty(6329) ALL patients were classified in high recurrence riskgroup and 29 (3671) in low recurrence risk group Thepossible observed genotypes for CCR5 rs333 polymorphismare shown in Figure 1

Genotyping results did not show any homozygous indi-viduals for Δ32 deletion in both groups The heterozygousCCR5Δ32 genotypes were observed in 875 (119899 = 7) ofcontrols and 75 (119899 = 5) of ALL patients To determineif there was a statistically significant increased risk of ALLdevelopment related to the 1198621198621198775 genotypes we conductedlogistic regression analysis (Table 1) which showed thatindividuals with one copy ofΔ32 variant allele did not exhibitALL-associated risk No statistical difference was observedwhen allelic frequency of Δ32 at rs333 in ALL patients wasassociated with control subjects (OR = 071 95CI =022ndash230 119875 = 077)

In addition we compared the CCR5 genotype distri-bution in ALL patients classified in high risk or low riskaccording to recurrence status From five (75) ALL Δ32carriers three were classified as high-risk patients However

association study between both recurrence statuses did notreach statistical significance

The CCR5 genotypes distribution in ALL patients andcontrols was stratified by gender Subgroup analyses revealedthat the effect of gender was not significantly different amongCCR5 genotypes (female ALL versus female control OR =052 95CI = 009ndash307 and male ALL versus male controlOR = 097 95CI = 018ndash514)

4 Discussion

Chemokines and their receptors are key regulators ofimmune activities and in parallel could play conflicting rolesin malignancy While most combinations of these receptorsand chemokines are active in cancer many findings in thefield have emphasized the chemokine CCL5 and its cognatereceptor CCR5 [23]

The gene variants of the chemokine and chemokinereceptor genes associated with inflammationmay be involvedin cancer initiation and progression [24] Considering theremarkable difference in CCR5Δ32 allele frequency amongworldwide populations we aimed to survey the geneticvariations in CCR5 in ALL patients and control individuals

The patientsrsquo age in this study was the expected forALL which is frequent in children and younger patientsMoreover as previouslymentioned both sample groups werecomposed predominantly of Caucasian individuals fromSouthern Brazil However due to high degree of miscegena-tion of Brazilian population and the demand to use geneticmarkers for correct characterization of individuals [25 26] inour country these data have not been explored in relation tothe variants analyzed

Chemokines and chemokine receptors are among factorsthat may influence ALL progression and localization [15] A32-base pair nucleic acid deletion in CCR5 exists and causesa frameshift mutation in the amino acids comprising thesecond extracellular loop This deletion leads to prematuretruncation of the protein disabling its ability to translocateto the membrane impairing expression and ligand bindingat the cell surface and causingmembrane receptor deficiencythat may influence leukocyte trafficking [27]

Based on this we hypothesized that the ability of CCR5to bind its ligands and signal recruitment of pathogenic Tcells into target tissues may be impaired thus impartingALL protection Although there were no differences in thefrequency of CCR5CCR5 andCCR5Δ32 genotypes betweenpatient and control groups the variant genotype had no effecton the ALL susceptibility

These results corroborated with studies in different dis-orders such as leishmaniasis breast laryngeal thyroid andbrain carcinoma which also identified no differences in thefrequency of these alleles among healthy subjects and patientsof Southern Brazilian population [28ndash30] andworldwide [31ndash33]

Intensive multiagent chemotherapy regimens and intro-duction of risk-stratified therapy have substantially improvedcure rates for children with ALL Current risk allocationschemas are imperfect as some children are classified as

4 Advances in Hematology

lower-risk and treated with less intensive therapy relapsewhile others deemed higher-risk are probably overtreated[34] In this context genetic polymorphisms in chemokinereceptors could predict outcome and be considered an inde-pendent risk factor to stratify and allocate therapy in ALL

More than half of the patients with ALL were classified inhigher-risk group according to the clinical and laboratorialfindings at diagnosis as defined by GBTLI LLA-99 protocol[2] When the genotype data were analyzed for stratifiedgroup of ALL the results indicated that the presence ofΔ32 did not influence this clinical parameter Similarly arecent study conducted by our research group has not foundassociation among tumor suppressor TP53 and chemokineCXCL12 polymorphisms and ALL recurrence risk status [35]

5 Conclusion

The comprehension about cellular and molecular mecha-nisms of ALL is critically important for the developmentof new approaches to hematological neoplasia treatmentAlthough any association of rs333 polymorphism of CCR5was verified we believe that the current researchmust lead toa better definition of the host-tumor relationship particularlywith respect to immunologic response and interrelation ofCCR5 and ALL development Given the sample size of thepresent case-control association study strong conclusions arenot possible however future investigation involving muchlarger cases may determine the absence of clinical implica-tions for CCR5Δ32 alleles in relation to ALL pathogenesis

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to acknowledge the volunteerswho made this study possible the University Hospital ofLondrina and Londrina Cancer Institute PR Brazil fortheir collaboration This study was supported by Con-selho Nacional de Desenvolvimento Cientıfico e Tecnologico(CNPq) Coordenacao de Aperfeicoamento de Pessoal deNıvel Superior (CAPES) Fundacao Araucaria do ParanaSecretaria da Ciencia Tecnologia e Ensino Superior (SETI)Fundo Estadual para a Infancia e Adolescencia (FIAPR) eSecretaria da Famılia e Desenvolvimento Social (SEDS) andCoordination of Undergraduate Studies of Londrina StateUniversity (PROPPG-UEL)

References

[1] M Belson B Kingsley and A Holmes ldquoRisk factors foracute leukemia in children a reviewrdquo Environmental HealthPerspectives vol 115 no 1 pp 138ndash145 2007

[2] INCA Estimate 2014 Cancer Incidence in Brazil INCA Rio deJaneiro Brazil 2013

[3] F Balkwill ldquoCancer and the chemokine networkrdquo NatureReviews Cancer vol 4 no 7 pp 540ndash550 2004

[4] J Vandercappellen J Van Damme and S Struyf ldquoThe role ofCXC chemokines and their receptors in cancerrdquo Cancer Lettersvol 267 no 2 pp 226ndash244 2008

[5] D Aldinucci andA Colombatti ldquoThe inflammatory chemokineCCL5 and cancer progressionrdquoMediators of Inflammation vol2014 Article ID 292376 12 pages 2014

[6] C J Raport J Gosling V L Schweickart P W Gray and I FCharo ldquoMolecular cloning and functional characterization ofa novel human CC chemokine receptor (CCR5) for RANTESMIP-1120573 and MIP-1120572rdquo Journal of Biological Chemistry vol 271no 29 pp 17161ndash17166 1996

[7] M Samson O Labbe C Mollereau G Vassart and MParmentier ldquoMolecular cloning and functional expression of anew human CC-chemokine receptor generdquo Biochemistry vol35 no 11 pp 3362ndash3367 1996

[8] P Proost AWuyts and J vanDamme ldquoThe role of chemokinesin inflammationrdquo Clinical and Experimental Medicine vol 26no 4 pp 211ndash223 1996

[9] P Spagnolo E A Renzoni A U Wells et al ldquoC-C chemokinereceptor 5 gene variants in relation to lung disease in sarcoido-sisrdquoAmerican Journal of Respiratory and Critical CareMedicinevol 172 no 6 pp 721ndash728 2005

[10] E-M Weiss A Schmidt D Vobis et al ldquoFoxp3-mediatedsuppression of cd95l expression confers resistance to activation-induced cell death in regulatory T cellsrdquo Journal of Immunologyvol 187 no 4 pp 1684ndash1691 2011

[11] C-H Tang A Yamamoto Y-T Lin Y-C Fong and T-W TanldquoInvolvement of matrix metalloproteinase-3 in CCL5CCR5pathway of chondrosarcomas metastasisrdquo Biochemical Pharma-cology vol 79 no 2 pp 209ndash217 2010

[12] J-Y Chuang W-H Yang H-T Chen et al ldquoCCL5CCR5 axispromotes the motility of human oral cancer cellsrdquo Journal ofCellular Physiology vol 220 no 2 pp 418ndash426 2009

[13] E Menu E De Leenheer H De Raeve et al ldquoRole of CCR1 andCCR5 in homing and growth of multiple myeloma and in thedevelopment of osteolytic lesions a study in the 5TMMmodelrdquoClinical and Experimental Metastasis vol 23 no 5-6 pp 291ndash300 2006

[14] J C Aster W S Pear and S C Blacklow ldquoNotch signaling inleukemiardquo Annual Review of Pathology Mechanisms of Diseasevol 3 pp 587ndash613 2008

[15] L Mirandola M Chiriva-Internati DMontagna et al ldquoNotch1regulates chemotaxis and proliferation by controlling the CC-chemokine receptors 5 and 9 in T cell acute lymphoblasticleukaemiardquo Journal of Pathology vol 226 no 5 pp 713ndash7222012

[16] H Makishima T Ito N Asano et al ldquoSignificance ofchemokine receptor expression in aggressive NK cell leukemiardquoLeukemia vol 19 no 7 pp 1169ndash1174 2005

[17] HMakishima T Ito KMomose et al ldquoChemokine system andtissue infiltration in aggressive NK-cell leukemiardquo Leukemiaresearch vol 31 no 9 pp 1237ndash1245 2007

[18] S M Davies M J Borowitz G L Rosner et al ldquoPharmaco-genetics of minimal residual disease response in children withB-precursor acute lymphoblastic leukemia a report from thechildrenrsquos oncology grouprdquo Blood vol 111 no 6 pp 2984ndash29902008

Advances in Hematology 5

[19] D H McDermott P A Zimmerman F Guignard C AKleeberger S F Leitman and P M Murphy ldquoCCR5 promoterpolymorphism and HIV-1 disease progressionrdquoThe Lancet vol352 no 9131 pp 866ndash870 1998

[20] M Chelli and M Alizon ldquoDeterminants of the trans-dominantnegative effect of truncated forms of the CCR5 chemokinereceptorrdquo Journal of Biological Chemistry vol 276 no 50 pp46975ndash46982 2001

[21] E M V Reiche M A E Watanabe A M Bonametti etal ldquoFrequency of CCR5-Δ32 deletion in human immunode-ficiency virus type 1 (HIV-1) in healthy blood donors HIV-1-exposed seronegative and HIV-1-seropositive individuals ofsouthern Brazilian populationrdquo International Journal of Molec-ular Medicine vol 22 no 5 pp 669ndash675 2008

[22] A P Galvani and J Novembre ldquoThe evolutionary historyof the CCR5-Delta32 HIV-resistance mutationrdquo Microbes andInfection vol 7 no 2 pp 302ndash309 2005

[23] P Weitzenfeld and A Ben-Baruch ldquoThe chemokine systemand its CCR5 and CXCR4 receptors as potential targets forpersonalized therapy in cancerrdquo Cancer Letters 2013

[24] C Kucukgergin F K Isman S Dasdemir et al ldquoThe roleof chemokine and chemokine receptor gene variants on thesusceptibility and clinicopathological characteristics of bladdercancerrdquo Gene vol 511 no 1 pp 7ndash11 2012

[25] V R Arruda C E Grignolli M S Goncalves et al ldquoPreva-lence of homozygosity for the deleted alleles of glutathione S-transferase mu (GSTM1) and theta (GSTT1) among distinctethnic groups from Brazil relevance to enviromental carcino-genesisrdquo Clinical Genetics vol 54 no 3 pp 210ndash214 1998

[26] F C Parra R C Amado J R Lambertucci et al ldquoColor andgenomic ancestry in Braziliansrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no1 pp 177ndash182 2003

[27] J SMaier-Moore C A Canas G Tobon et al ldquoTheCCR5 delta32 polymorphism (rs333) is not associated with Sjogrenrsquos syn-drome or type 1 diabetes in colombiansrdquo Clinical Immunologyvol 148 no 2 pp 206ndash208 2013

[28] S M Muxel S D Borelli M K Amarante et al ldquoAssociationstudy of CCR5 delta 32 polymorphism among the HLA-DRB1Caucasian population in northern Parana Brazilrdquo Journal ofClinical Laboratory Analysis vol 22 no 4 pp 229ndash233 2008

[29] K Brajao de Oliveira E M Vissoci Reiche H KaminamiMorimoto et al ldquoAnalysis of the CC chemokine receptor 5delta32 polymorphism in a Brazilian populationwith cutaneousleishmaniasisrdquo Journal of Cutaneous Pathology vol 34 no 1 pp27ndash32 2007

[30] M N Aoki A C D S do Amaral Herrera M K Amarante JL do Val Carneiro M H P Fungaro and M A E WatanabeldquoCCR5 and p53 codon 72 gene polymorphisms implications inbreast cancer developmentrdquo International Journal of MolecularMedicine vol 23 no 3 pp 429ndash435 2009

[31] N Degerli E Yilmaz and F Bardakci ldquoThe Δ32 allele distribu-tion of the CCR5 gene and its relationship with certain cancersin a Turkish populationrdquoClinical Biochemistry vol 38 no 3 pp248ndash252 2005

[32] K Guleria S Sharma M Manjari et al ldquopR72P PIN3 Ins16bppolymorphisms of TP53 and CCR532 in north Indian breastcancer patientsrdquo Asian Pacific Journal of Cancer Prevention vol13 no 7 pp 3305ndash3311 2012

[33] A Zafiropoulos N Crikas A M Passam and D A SpandidosldquoSignificant involvement of CCR2-64I and CXCL12-3a in the

development of sporadic breast cancerrdquo Journal of MedicalGenetics vol 41 no 5 p e59 2004

[34] D T Teachey and S P Hunger ldquoPredicting relapse risk inchildhood acute lymphoblastic leukaemiardquo British Journal ofHaematology vol 162 no 5 pp 606ndash620 2013

[35] A de Lourdes Perim R L Guembarovski J M Oda et alldquoCXCL12 and TP53 genetic polymorphisms as markers ofsusceptibility in a Brazilian children population with acutelymphoblastic leukemia (ALL)rdquoMolecular Biology Reports vol40 no 7 pp 4591ndash4596 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Advances in Hematology

via G protein and binds to the chemokines RANTES (CCL5)MIP-1120572 (CCL3) and MIP-1120573 (CCL4) [7] This receptor isinvolved in the chemotaxis of leucocytes to inflammationsites [8] and plays important function in the recruitment ofmacrophages T cells and monocytes [9]

The importance of CCR5 for immune response is depen-dent on the type of stimuli moreover in some casescompensating mechanisms override the absence of CCR5expression and function Noteworthy CCR5 may exert a farmore important role in the immune response than in immunecells traffic regulation [10]

It has been shown that CCR5 expression in stromal cellsas well as hematopoietic cells contributes to tumor metas-tasis For instance CCR5 is involved in chondrosarcomasmetastasization [11] and oral cancer cells migration [12] Itsexpression correlates with multiple myeloma cell growthbone marrow homing and osteolysis [13]

Aster and colleagues [14] showed that T-cell ALL is a dis-ease primarily caused by aberrant activation of the NOTCH1signaling pathway In this context expression and functionof important chemokine receptors such as CCR5 and CCR9are partially controlled by the oncogenic NOTCH1 isoformin T-cell ALL regulating blast malignant properties andlocalization of extramedullary infiltrations [15]

Additionally CCR5 has been related to play a key rolein metastasis of aggressive NK-cells leukemia to the liverof patients contributing to hepatosplenomegaly and hepaticfailure [16 17] Also Davies et al [18] showed that the Gallele of rs1799987 polymorphism in CCR5 was associatedwith more favorable minimal residual disease status thanthe A allele when comparing ldquobestrdquo and ldquoworstrdquo risk groupsof B-cell ALL adjusted for prognostic features Consideringthe lower activity of CCR5 promoter in the presence ofthis polymorphism [19] this evidence indicated that bothacquired and host genetics influence response to cancertherapy Thus it is plausible that CCR5 might play a role inALL pathogenesis and prognosis

It is known that the polymorphism rs333 in CCR5 acommon 32-base pair deletion (Δ32) causes truncation andloss of this receptor on lymphoid cell surface with completeretention in the endoplasmic reticulum within homozygousor diminished expression in heterozygous genotype [20]The CCR5 studies have demonstrated the importance of Δ32mutation particularly in the susceptibility to HIV infection[21] since CCR5 is a coreceptor in the primary stage ofinfection that is essential for the AIDS onset [22]

Our research grouphas reported polymorphic allelic vari-ants related to the immune system and tumor development indifferent cancer types Nevertheless there are no data relatingCCR5Δ32 polymorphism to ALL population In this contextthe present work analyzed rs333 polymorphism of CCR5 inALL patients from the southern region of Brazil

2 Materials and Methods

21 Human Subjects Following approval from the HumanEthics Committee of the State University of Londrina

Parana Brazil (CAAE number 17123113400005231) inclu-sion of the individuals to the study was conditioned byan obtained written informed consent form from parentsregarding the use of their children and adolescents bloodsamples Seventy-nine ALL patients were enrolled and diag-nostic criteria were based on the guidelines proposed byHematology Department of the University Hospital of Lon-drina Recurrence risk status of ALL patients was evaluatedthrough the GBTLI Protocol (Brazilian Group of Child-hood Leukemia Treatment Protocol-99) which is based onthe Cancer Therapy Evaluation Program proposed by theNational Cancer Institute and takes into account age at diag-nosis leukocyte count immunophenotyping involvementof tissues other than bone marrow and responsiveness totreatment The control group is comprised of 80 healthyindividuals free of neoplasia matched by age and gender

22 Genomic DNA Extraction Genomic DNA was extractedfrom whole blood by Biopur Mini Spin Plus Kit (BiometrixDiagnostica Curitiba Brazil) according to the manufac-turerrsquos instructions DNA was eluted in 50 120583L of milliQwater and quantified byNanoDrop 2000c spectrophotometer(Thermo Fisher Scientific Inc Wilmington USA) at a wave-length of 260280 nm Final preparation was stored at minus20∘Cand used as templates in polymerase chain reactions (PCR)

23 Optimization of PCR for CCR5 The method of geno-typing (rs333) was optimized in the Laboratory of Study andApplication ofDNAPolymorphisms of the StateUniversity ofLondrina using genomicDNAand specific primers forCCR5Primer sense 51015840 ACC AGA TCT CAA AAA GAA 31015840 andPrimer antisense 51015840 CAT GAT GGT GAA GAT AAG CCTCA 31015840 (GenBank accession AF009962) Genotyping of CCR5was determined by PCR-SSP The samples were amplifiedusing 125 units of Taq polymerase (Invitrogen CarlsbadUSA) in aMastercyclerGradientThermal Cycler (EppendorfHamburg Germany) PCR conditions were denaturationstep at 94∘C for 5min 35 cycles of 1min at 94∘C 1min at58∘C and 1min at 72∘C and 10min of elongation at 72∘C PCRproducts (225 bp or 193 bp) were analyzed on polyacrylamidegel (10) stained with silver nitrate (AgNO

3)

24 Statistical Analysis Contingency tables andFisherrsquos exacttest were used to calculate differences in genotype distribu-tions and allele frequencies 119875 lt 005 was considered toindicate a statistically significant difference Goodness of fitof Hardy-Weinberg equilibrium was tested by calculating theexpected frequencies of each genotype and comparing themwith the observed value using a chi-square test

3 Results

The distribution of CCR5 alleles in both ALL and controlgroups was in accordance with the assumption of Hardy-Weinberg equilibrium (119875 gt 0 05) The mean age of controlsand ALL patients was 108 years plusmn 565 and 87 years plusmn620 respectively all of whom were predominantly fromCaucasoid population due to European colonization

Advances in Hematology 3

225 bp193bp

L 1 2 3 bl

Figure 1 CCR5 genotype profile The PCR products were detectedusing silver staining method after polyacrylamide gel electrophore-sis Lane LDNALadder 100 bp lane 1 CCR5wild-type homozygousgenotype (CCR5CCR5 225 bp) lane 2 heterozygous genotype(CCR5Δ32 225 bp and 193 bp) and lane 3 variant allele homozy-gous genotype (Δ32Δ32 193 bp) bl represents blank reaction(without DNA)

Table 1 Genotype distribution of CCR5 rs333 polymorphism andrecurrence risk status analysis in ALL and control groups

Genotypes OR 95 CI 119875lowastCCR5CCR5 CCR5Δ32

Control (80) 73 (9125) 7 (875) 07 021ndash232 076ALL (79) 74 (925) 5 (75)

High risk (50) 47 (94) 3 (6) 086 013ndash548 100Low risk (29) 27 (931) 2 (69)

lowastFisherrsquos exact test 119875 gt 005 OR odds ratio CI confidence interval

The patients and controls were matched for sex andgender although there was a modest higher frequency ofmales in ALL group (5316) than in controls (4875) Fifty(6329) ALL patients were classified in high recurrence riskgroup and 29 (3671) in low recurrence risk group Thepossible observed genotypes for CCR5 rs333 polymorphismare shown in Figure 1

Genotyping results did not show any homozygous indi-viduals for Δ32 deletion in both groups The heterozygousCCR5Δ32 genotypes were observed in 875 (119899 = 7) ofcontrols and 75 (119899 = 5) of ALL patients To determineif there was a statistically significant increased risk of ALLdevelopment related to the 1198621198621198775 genotypes we conductedlogistic regression analysis (Table 1) which showed thatindividuals with one copy ofΔ32 variant allele did not exhibitALL-associated risk No statistical difference was observedwhen allelic frequency of Δ32 at rs333 in ALL patients wasassociated with control subjects (OR = 071 95CI =022ndash230 119875 = 077)

In addition we compared the CCR5 genotype distri-bution in ALL patients classified in high risk or low riskaccording to recurrence status From five (75) ALL Δ32carriers three were classified as high-risk patients However

association study between both recurrence statuses did notreach statistical significance

The CCR5 genotypes distribution in ALL patients andcontrols was stratified by gender Subgroup analyses revealedthat the effect of gender was not significantly different amongCCR5 genotypes (female ALL versus female control OR =052 95CI = 009ndash307 and male ALL versus male controlOR = 097 95CI = 018ndash514)

4 Discussion

Chemokines and their receptors are key regulators ofimmune activities and in parallel could play conflicting rolesin malignancy While most combinations of these receptorsand chemokines are active in cancer many findings in thefield have emphasized the chemokine CCL5 and its cognatereceptor CCR5 [23]

The gene variants of the chemokine and chemokinereceptor genes associated with inflammationmay be involvedin cancer initiation and progression [24] Considering theremarkable difference in CCR5Δ32 allele frequency amongworldwide populations we aimed to survey the geneticvariations in CCR5 in ALL patients and control individuals

The patientsrsquo age in this study was the expected forALL which is frequent in children and younger patientsMoreover as previouslymentioned both sample groups werecomposed predominantly of Caucasian individuals fromSouthern Brazil However due to high degree of miscegena-tion of Brazilian population and the demand to use geneticmarkers for correct characterization of individuals [25 26] inour country these data have not been explored in relation tothe variants analyzed

Chemokines and chemokine receptors are among factorsthat may influence ALL progression and localization [15] A32-base pair nucleic acid deletion in CCR5 exists and causesa frameshift mutation in the amino acids comprising thesecond extracellular loop This deletion leads to prematuretruncation of the protein disabling its ability to translocateto the membrane impairing expression and ligand bindingat the cell surface and causingmembrane receptor deficiencythat may influence leukocyte trafficking [27]

Based on this we hypothesized that the ability of CCR5to bind its ligands and signal recruitment of pathogenic Tcells into target tissues may be impaired thus impartingALL protection Although there were no differences in thefrequency of CCR5CCR5 andCCR5Δ32 genotypes betweenpatient and control groups the variant genotype had no effecton the ALL susceptibility

These results corroborated with studies in different dis-orders such as leishmaniasis breast laryngeal thyroid andbrain carcinoma which also identified no differences in thefrequency of these alleles among healthy subjects and patientsof Southern Brazilian population [28ndash30] andworldwide [31ndash33]

Intensive multiagent chemotherapy regimens and intro-duction of risk-stratified therapy have substantially improvedcure rates for children with ALL Current risk allocationschemas are imperfect as some children are classified as

4 Advances in Hematology

lower-risk and treated with less intensive therapy relapsewhile others deemed higher-risk are probably overtreated[34] In this context genetic polymorphisms in chemokinereceptors could predict outcome and be considered an inde-pendent risk factor to stratify and allocate therapy in ALL

More than half of the patients with ALL were classified inhigher-risk group according to the clinical and laboratorialfindings at diagnosis as defined by GBTLI LLA-99 protocol[2] When the genotype data were analyzed for stratifiedgroup of ALL the results indicated that the presence ofΔ32 did not influence this clinical parameter Similarly arecent study conducted by our research group has not foundassociation among tumor suppressor TP53 and chemokineCXCL12 polymorphisms and ALL recurrence risk status [35]

5 Conclusion

The comprehension about cellular and molecular mecha-nisms of ALL is critically important for the developmentof new approaches to hematological neoplasia treatmentAlthough any association of rs333 polymorphism of CCR5was verified we believe that the current researchmust lead toa better definition of the host-tumor relationship particularlywith respect to immunologic response and interrelation ofCCR5 and ALL development Given the sample size of thepresent case-control association study strong conclusions arenot possible however future investigation involving muchlarger cases may determine the absence of clinical implica-tions for CCR5Δ32 alleles in relation to ALL pathogenesis

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to acknowledge the volunteerswho made this study possible the University Hospital ofLondrina and Londrina Cancer Institute PR Brazil fortheir collaboration This study was supported by Con-selho Nacional de Desenvolvimento Cientıfico e Tecnologico(CNPq) Coordenacao de Aperfeicoamento de Pessoal deNıvel Superior (CAPES) Fundacao Araucaria do ParanaSecretaria da Ciencia Tecnologia e Ensino Superior (SETI)Fundo Estadual para a Infancia e Adolescencia (FIAPR) eSecretaria da Famılia e Desenvolvimento Social (SEDS) andCoordination of Undergraduate Studies of Londrina StateUniversity (PROPPG-UEL)

References

[1] M Belson B Kingsley and A Holmes ldquoRisk factors foracute leukemia in children a reviewrdquo Environmental HealthPerspectives vol 115 no 1 pp 138ndash145 2007

[2] INCA Estimate 2014 Cancer Incidence in Brazil INCA Rio deJaneiro Brazil 2013

[3] F Balkwill ldquoCancer and the chemokine networkrdquo NatureReviews Cancer vol 4 no 7 pp 540ndash550 2004

[4] J Vandercappellen J Van Damme and S Struyf ldquoThe role ofCXC chemokines and their receptors in cancerrdquo Cancer Lettersvol 267 no 2 pp 226ndash244 2008

[5] D Aldinucci andA Colombatti ldquoThe inflammatory chemokineCCL5 and cancer progressionrdquoMediators of Inflammation vol2014 Article ID 292376 12 pages 2014

[6] C J Raport J Gosling V L Schweickart P W Gray and I FCharo ldquoMolecular cloning and functional characterization ofa novel human CC chemokine receptor (CCR5) for RANTESMIP-1120573 and MIP-1120572rdquo Journal of Biological Chemistry vol 271no 29 pp 17161ndash17166 1996

[7] M Samson O Labbe C Mollereau G Vassart and MParmentier ldquoMolecular cloning and functional expression of anew human CC-chemokine receptor generdquo Biochemistry vol35 no 11 pp 3362ndash3367 1996

[8] P Proost AWuyts and J vanDamme ldquoThe role of chemokinesin inflammationrdquo Clinical and Experimental Medicine vol 26no 4 pp 211ndash223 1996

[9] P Spagnolo E A Renzoni A U Wells et al ldquoC-C chemokinereceptor 5 gene variants in relation to lung disease in sarcoido-sisrdquoAmerican Journal of Respiratory and Critical CareMedicinevol 172 no 6 pp 721ndash728 2005

[10] E-M Weiss A Schmidt D Vobis et al ldquoFoxp3-mediatedsuppression of cd95l expression confers resistance to activation-induced cell death in regulatory T cellsrdquo Journal of Immunologyvol 187 no 4 pp 1684ndash1691 2011

[11] C-H Tang A Yamamoto Y-T Lin Y-C Fong and T-W TanldquoInvolvement of matrix metalloproteinase-3 in CCL5CCR5pathway of chondrosarcomas metastasisrdquo Biochemical Pharma-cology vol 79 no 2 pp 209ndash217 2010

[12] J-Y Chuang W-H Yang H-T Chen et al ldquoCCL5CCR5 axispromotes the motility of human oral cancer cellsrdquo Journal ofCellular Physiology vol 220 no 2 pp 418ndash426 2009

[13] E Menu E De Leenheer H De Raeve et al ldquoRole of CCR1 andCCR5 in homing and growth of multiple myeloma and in thedevelopment of osteolytic lesions a study in the 5TMMmodelrdquoClinical and Experimental Metastasis vol 23 no 5-6 pp 291ndash300 2006

[14] J C Aster W S Pear and S C Blacklow ldquoNotch signaling inleukemiardquo Annual Review of Pathology Mechanisms of Diseasevol 3 pp 587ndash613 2008

[15] L Mirandola M Chiriva-Internati DMontagna et al ldquoNotch1regulates chemotaxis and proliferation by controlling the CC-chemokine receptors 5 and 9 in T cell acute lymphoblasticleukaemiardquo Journal of Pathology vol 226 no 5 pp 713ndash7222012

[16] H Makishima T Ito N Asano et al ldquoSignificance ofchemokine receptor expression in aggressive NK cell leukemiardquoLeukemia vol 19 no 7 pp 1169ndash1174 2005

[17] HMakishima T Ito KMomose et al ldquoChemokine system andtissue infiltration in aggressive NK-cell leukemiardquo Leukemiaresearch vol 31 no 9 pp 1237ndash1245 2007

[18] S M Davies M J Borowitz G L Rosner et al ldquoPharmaco-genetics of minimal residual disease response in children withB-precursor acute lymphoblastic leukemia a report from thechildrenrsquos oncology grouprdquo Blood vol 111 no 6 pp 2984ndash29902008

Advances in Hematology 5

[19] D H McDermott P A Zimmerman F Guignard C AKleeberger S F Leitman and P M Murphy ldquoCCR5 promoterpolymorphism and HIV-1 disease progressionrdquoThe Lancet vol352 no 9131 pp 866ndash870 1998

[20] M Chelli and M Alizon ldquoDeterminants of the trans-dominantnegative effect of truncated forms of the CCR5 chemokinereceptorrdquo Journal of Biological Chemistry vol 276 no 50 pp46975ndash46982 2001

[21] E M V Reiche M A E Watanabe A M Bonametti etal ldquoFrequency of CCR5-Δ32 deletion in human immunode-ficiency virus type 1 (HIV-1) in healthy blood donors HIV-1-exposed seronegative and HIV-1-seropositive individuals ofsouthern Brazilian populationrdquo International Journal of Molec-ular Medicine vol 22 no 5 pp 669ndash675 2008

[22] A P Galvani and J Novembre ldquoThe evolutionary historyof the CCR5-Delta32 HIV-resistance mutationrdquo Microbes andInfection vol 7 no 2 pp 302ndash309 2005

[23] P Weitzenfeld and A Ben-Baruch ldquoThe chemokine systemand its CCR5 and CXCR4 receptors as potential targets forpersonalized therapy in cancerrdquo Cancer Letters 2013

[24] C Kucukgergin F K Isman S Dasdemir et al ldquoThe roleof chemokine and chemokine receptor gene variants on thesusceptibility and clinicopathological characteristics of bladdercancerrdquo Gene vol 511 no 1 pp 7ndash11 2012

[25] V R Arruda C E Grignolli M S Goncalves et al ldquoPreva-lence of homozygosity for the deleted alleles of glutathione S-transferase mu (GSTM1) and theta (GSTT1) among distinctethnic groups from Brazil relevance to enviromental carcino-genesisrdquo Clinical Genetics vol 54 no 3 pp 210ndash214 1998

[26] F C Parra R C Amado J R Lambertucci et al ldquoColor andgenomic ancestry in Braziliansrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no1 pp 177ndash182 2003

[27] J SMaier-Moore C A Canas G Tobon et al ldquoTheCCR5 delta32 polymorphism (rs333) is not associated with Sjogrenrsquos syn-drome or type 1 diabetes in colombiansrdquo Clinical Immunologyvol 148 no 2 pp 206ndash208 2013

[28] S M Muxel S D Borelli M K Amarante et al ldquoAssociationstudy of CCR5 delta 32 polymorphism among the HLA-DRB1Caucasian population in northern Parana Brazilrdquo Journal ofClinical Laboratory Analysis vol 22 no 4 pp 229ndash233 2008

[29] K Brajao de Oliveira E M Vissoci Reiche H KaminamiMorimoto et al ldquoAnalysis of the CC chemokine receptor 5delta32 polymorphism in a Brazilian populationwith cutaneousleishmaniasisrdquo Journal of Cutaneous Pathology vol 34 no 1 pp27ndash32 2007

[30] M N Aoki A C D S do Amaral Herrera M K Amarante JL do Val Carneiro M H P Fungaro and M A E WatanabeldquoCCR5 and p53 codon 72 gene polymorphisms implications inbreast cancer developmentrdquo International Journal of MolecularMedicine vol 23 no 3 pp 429ndash435 2009

[31] N Degerli E Yilmaz and F Bardakci ldquoThe Δ32 allele distribu-tion of the CCR5 gene and its relationship with certain cancersin a Turkish populationrdquoClinical Biochemistry vol 38 no 3 pp248ndash252 2005

[32] K Guleria S Sharma M Manjari et al ldquopR72P PIN3 Ins16bppolymorphisms of TP53 and CCR532 in north Indian breastcancer patientsrdquo Asian Pacific Journal of Cancer Prevention vol13 no 7 pp 3305ndash3311 2012

[33] A Zafiropoulos N Crikas A M Passam and D A SpandidosldquoSignificant involvement of CCR2-64I and CXCL12-3a in the

development of sporadic breast cancerrdquo Journal of MedicalGenetics vol 41 no 5 p e59 2004

[34] D T Teachey and S P Hunger ldquoPredicting relapse risk inchildhood acute lymphoblastic leukaemiardquo British Journal ofHaematology vol 162 no 5 pp 606ndash620 2013

[35] A de Lourdes Perim R L Guembarovski J M Oda et alldquoCXCL12 and TP53 genetic polymorphisms as markers ofsusceptibility in a Brazilian children population with acutelymphoblastic leukemia (ALL)rdquoMolecular Biology Reports vol40 no 7 pp 4591ndash4596 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Advances in Hematology 3

225 bp193bp

L 1 2 3 bl

Figure 1 CCR5 genotype profile The PCR products were detectedusing silver staining method after polyacrylamide gel electrophore-sis Lane LDNALadder 100 bp lane 1 CCR5wild-type homozygousgenotype (CCR5CCR5 225 bp) lane 2 heterozygous genotype(CCR5Δ32 225 bp and 193 bp) and lane 3 variant allele homozy-gous genotype (Δ32Δ32 193 bp) bl represents blank reaction(without DNA)

Table 1 Genotype distribution of CCR5 rs333 polymorphism andrecurrence risk status analysis in ALL and control groups

Genotypes OR 95 CI 119875lowastCCR5CCR5 CCR5Δ32

Control (80) 73 (9125) 7 (875) 07 021ndash232 076ALL (79) 74 (925) 5 (75)

High risk (50) 47 (94) 3 (6) 086 013ndash548 100Low risk (29) 27 (931) 2 (69)

lowastFisherrsquos exact test 119875 gt 005 OR odds ratio CI confidence interval

The patients and controls were matched for sex andgender although there was a modest higher frequency ofmales in ALL group (5316) than in controls (4875) Fifty(6329) ALL patients were classified in high recurrence riskgroup and 29 (3671) in low recurrence risk group Thepossible observed genotypes for CCR5 rs333 polymorphismare shown in Figure 1

Genotyping results did not show any homozygous indi-viduals for Δ32 deletion in both groups The heterozygousCCR5Δ32 genotypes were observed in 875 (119899 = 7) ofcontrols and 75 (119899 = 5) of ALL patients To determineif there was a statistically significant increased risk of ALLdevelopment related to the 1198621198621198775 genotypes we conductedlogistic regression analysis (Table 1) which showed thatindividuals with one copy ofΔ32 variant allele did not exhibitALL-associated risk No statistical difference was observedwhen allelic frequency of Δ32 at rs333 in ALL patients wasassociated with control subjects (OR = 071 95CI =022ndash230 119875 = 077)

In addition we compared the CCR5 genotype distri-bution in ALL patients classified in high risk or low riskaccording to recurrence status From five (75) ALL Δ32carriers three were classified as high-risk patients However

association study between both recurrence statuses did notreach statistical significance

The CCR5 genotypes distribution in ALL patients andcontrols was stratified by gender Subgroup analyses revealedthat the effect of gender was not significantly different amongCCR5 genotypes (female ALL versus female control OR =052 95CI = 009ndash307 and male ALL versus male controlOR = 097 95CI = 018ndash514)

4 Discussion

Chemokines and their receptors are key regulators ofimmune activities and in parallel could play conflicting rolesin malignancy While most combinations of these receptorsand chemokines are active in cancer many findings in thefield have emphasized the chemokine CCL5 and its cognatereceptor CCR5 [23]

The gene variants of the chemokine and chemokinereceptor genes associated with inflammationmay be involvedin cancer initiation and progression [24] Considering theremarkable difference in CCR5Δ32 allele frequency amongworldwide populations we aimed to survey the geneticvariations in CCR5 in ALL patients and control individuals

The patientsrsquo age in this study was the expected forALL which is frequent in children and younger patientsMoreover as previouslymentioned both sample groups werecomposed predominantly of Caucasian individuals fromSouthern Brazil However due to high degree of miscegena-tion of Brazilian population and the demand to use geneticmarkers for correct characterization of individuals [25 26] inour country these data have not been explored in relation tothe variants analyzed

Chemokines and chemokine receptors are among factorsthat may influence ALL progression and localization [15] A32-base pair nucleic acid deletion in CCR5 exists and causesa frameshift mutation in the amino acids comprising thesecond extracellular loop This deletion leads to prematuretruncation of the protein disabling its ability to translocateto the membrane impairing expression and ligand bindingat the cell surface and causingmembrane receptor deficiencythat may influence leukocyte trafficking [27]

Based on this we hypothesized that the ability of CCR5to bind its ligands and signal recruitment of pathogenic Tcells into target tissues may be impaired thus impartingALL protection Although there were no differences in thefrequency of CCR5CCR5 andCCR5Δ32 genotypes betweenpatient and control groups the variant genotype had no effecton the ALL susceptibility

These results corroborated with studies in different dis-orders such as leishmaniasis breast laryngeal thyroid andbrain carcinoma which also identified no differences in thefrequency of these alleles among healthy subjects and patientsof Southern Brazilian population [28ndash30] andworldwide [31ndash33]

Intensive multiagent chemotherapy regimens and intro-duction of risk-stratified therapy have substantially improvedcure rates for children with ALL Current risk allocationschemas are imperfect as some children are classified as

4 Advances in Hematology

lower-risk and treated with less intensive therapy relapsewhile others deemed higher-risk are probably overtreated[34] In this context genetic polymorphisms in chemokinereceptors could predict outcome and be considered an inde-pendent risk factor to stratify and allocate therapy in ALL

More than half of the patients with ALL were classified inhigher-risk group according to the clinical and laboratorialfindings at diagnosis as defined by GBTLI LLA-99 protocol[2] When the genotype data were analyzed for stratifiedgroup of ALL the results indicated that the presence ofΔ32 did not influence this clinical parameter Similarly arecent study conducted by our research group has not foundassociation among tumor suppressor TP53 and chemokineCXCL12 polymorphisms and ALL recurrence risk status [35]

5 Conclusion

The comprehension about cellular and molecular mecha-nisms of ALL is critically important for the developmentof new approaches to hematological neoplasia treatmentAlthough any association of rs333 polymorphism of CCR5was verified we believe that the current researchmust lead toa better definition of the host-tumor relationship particularlywith respect to immunologic response and interrelation ofCCR5 and ALL development Given the sample size of thepresent case-control association study strong conclusions arenot possible however future investigation involving muchlarger cases may determine the absence of clinical implica-tions for CCR5Δ32 alleles in relation to ALL pathogenesis

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to acknowledge the volunteerswho made this study possible the University Hospital ofLondrina and Londrina Cancer Institute PR Brazil fortheir collaboration This study was supported by Con-selho Nacional de Desenvolvimento Cientıfico e Tecnologico(CNPq) Coordenacao de Aperfeicoamento de Pessoal deNıvel Superior (CAPES) Fundacao Araucaria do ParanaSecretaria da Ciencia Tecnologia e Ensino Superior (SETI)Fundo Estadual para a Infancia e Adolescencia (FIAPR) eSecretaria da Famılia e Desenvolvimento Social (SEDS) andCoordination of Undergraduate Studies of Londrina StateUniversity (PROPPG-UEL)

References

[1] M Belson B Kingsley and A Holmes ldquoRisk factors foracute leukemia in children a reviewrdquo Environmental HealthPerspectives vol 115 no 1 pp 138ndash145 2007

[2] INCA Estimate 2014 Cancer Incidence in Brazil INCA Rio deJaneiro Brazil 2013

[3] F Balkwill ldquoCancer and the chemokine networkrdquo NatureReviews Cancer vol 4 no 7 pp 540ndash550 2004

[4] J Vandercappellen J Van Damme and S Struyf ldquoThe role ofCXC chemokines and their receptors in cancerrdquo Cancer Lettersvol 267 no 2 pp 226ndash244 2008

[5] D Aldinucci andA Colombatti ldquoThe inflammatory chemokineCCL5 and cancer progressionrdquoMediators of Inflammation vol2014 Article ID 292376 12 pages 2014

[6] C J Raport J Gosling V L Schweickart P W Gray and I FCharo ldquoMolecular cloning and functional characterization ofa novel human CC chemokine receptor (CCR5) for RANTESMIP-1120573 and MIP-1120572rdquo Journal of Biological Chemistry vol 271no 29 pp 17161ndash17166 1996

[7] M Samson O Labbe C Mollereau G Vassart and MParmentier ldquoMolecular cloning and functional expression of anew human CC-chemokine receptor generdquo Biochemistry vol35 no 11 pp 3362ndash3367 1996

[8] P Proost AWuyts and J vanDamme ldquoThe role of chemokinesin inflammationrdquo Clinical and Experimental Medicine vol 26no 4 pp 211ndash223 1996

[9] P Spagnolo E A Renzoni A U Wells et al ldquoC-C chemokinereceptor 5 gene variants in relation to lung disease in sarcoido-sisrdquoAmerican Journal of Respiratory and Critical CareMedicinevol 172 no 6 pp 721ndash728 2005

[10] E-M Weiss A Schmidt D Vobis et al ldquoFoxp3-mediatedsuppression of cd95l expression confers resistance to activation-induced cell death in regulatory T cellsrdquo Journal of Immunologyvol 187 no 4 pp 1684ndash1691 2011

[11] C-H Tang A Yamamoto Y-T Lin Y-C Fong and T-W TanldquoInvolvement of matrix metalloproteinase-3 in CCL5CCR5pathway of chondrosarcomas metastasisrdquo Biochemical Pharma-cology vol 79 no 2 pp 209ndash217 2010

[12] J-Y Chuang W-H Yang H-T Chen et al ldquoCCL5CCR5 axispromotes the motility of human oral cancer cellsrdquo Journal ofCellular Physiology vol 220 no 2 pp 418ndash426 2009

[13] E Menu E De Leenheer H De Raeve et al ldquoRole of CCR1 andCCR5 in homing and growth of multiple myeloma and in thedevelopment of osteolytic lesions a study in the 5TMMmodelrdquoClinical and Experimental Metastasis vol 23 no 5-6 pp 291ndash300 2006

[14] J C Aster W S Pear and S C Blacklow ldquoNotch signaling inleukemiardquo Annual Review of Pathology Mechanisms of Diseasevol 3 pp 587ndash613 2008

[15] L Mirandola M Chiriva-Internati DMontagna et al ldquoNotch1regulates chemotaxis and proliferation by controlling the CC-chemokine receptors 5 and 9 in T cell acute lymphoblasticleukaemiardquo Journal of Pathology vol 226 no 5 pp 713ndash7222012

[16] H Makishima T Ito N Asano et al ldquoSignificance ofchemokine receptor expression in aggressive NK cell leukemiardquoLeukemia vol 19 no 7 pp 1169ndash1174 2005

[17] HMakishima T Ito KMomose et al ldquoChemokine system andtissue infiltration in aggressive NK-cell leukemiardquo Leukemiaresearch vol 31 no 9 pp 1237ndash1245 2007

[18] S M Davies M J Borowitz G L Rosner et al ldquoPharmaco-genetics of minimal residual disease response in children withB-precursor acute lymphoblastic leukemia a report from thechildrenrsquos oncology grouprdquo Blood vol 111 no 6 pp 2984ndash29902008

Advances in Hematology 5

[19] D H McDermott P A Zimmerman F Guignard C AKleeberger S F Leitman and P M Murphy ldquoCCR5 promoterpolymorphism and HIV-1 disease progressionrdquoThe Lancet vol352 no 9131 pp 866ndash870 1998

[20] M Chelli and M Alizon ldquoDeterminants of the trans-dominantnegative effect of truncated forms of the CCR5 chemokinereceptorrdquo Journal of Biological Chemistry vol 276 no 50 pp46975ndash46982 2001

[21] E M V Reiche M A E Watanabe A M Bonametti etal ldquoFrequency of CCR5-Δ32 deletion in human immunode-ficiency virus type 1 (HIV-1) in healthy blood donors HIV-1-exposed seronegative and HIV-1-seropositive individuals ofsouthern Brazilian populationrdquo International Journal of Molec-ular Medicine vol 22 no 5 pp 669ndash675 2008

[22] A P Galvani and J Novembre ldquoThe evolutionary historyof the CCR5-Delta32 HIV-resistance mutationrdquo Microbes andInfection vol 7 no 2 pp 302ndash309 2005

[23] P Weitzenfeld and A Ben-Baruch ldquoThe chemokine systemand its CCR5 and CXCR4 receptors as potential targets forpersonalized therapy in cancerrdquo Cancer Letters 2013

[24] C Kucukgergin F K Isman S Dasdemir et al ldquoThe roleof chemokine and chemokine receptor gene variants on thesusceptibility and clinicopathological characteristics of bladdercancerrdquo Gene vol 511 no 1 pp 7ndash11 2012

[25] V R Arruda C E Grignolli M S Goncalves et al ldquoPreva-lence of homozygosity for the deleted alleles of glutathione S-transferase mu (GSTM1) and theta (GSTT1) among distinctethnic groups from Brazil relevance to enviromental carcino-genesisrdquo Clinical Genetics vol 54 no 3 pp 210ndash214 1998

[26] F C Parra R C Amado J R Lambertucci et al ldquoColor andgenomic ancestry in Braziliansrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no1 pp 177ndash182 2003

[27] J SMaier-Moore C A Canas G Tobon et al ldquoTheCCR5 delta32 polymorphism (rs333) is not associated with Sjogrenrsquos syn-drome or type 1 diabetes in colombiansrdquo Clinical Immunologyvol 148 no 2 pp 206ndash208 2013

[28] S M Muxel S D Borelli M K Amarante et al ldquoAssociationstudy of CCR5 delta 32 polymorphism among the HLA-DRB1Caucasian population in northern Parana Brazilrdquo Journal ofClinical Laboratory Analysis vol 22 no 4 pp 229ndash233 2008

[29] K Brajao de Oliveira E M Vissoci Reiche H KaminamiMorimoto et al ldquoAnalysis of the CC chemokine receptor 5delta32 polymorphism in a Brazilian populationwith cutaneousleishmaniasisrdquo Journal of Cutaneous Pathology vol 34 no 1 pp27ndash32 2007

[30] M N Aoki A C D S do Amaral Herrera M K Amarante JL do Val Carneiro M H P Fungaro and M A E WatanabeldquoCCR5 and p53 codon 72 gene polymorphisms implications inbreast cancer developmentrdquo International Journal of MolecularMedicine vol 23 no 3 pp 429ndash435 2009

[31] N Degerli E Yilmaz and F Bardakci ldquoThe Δ32 allele distribu-tion of the CCR5 gene and its relationship with certain cancersin a Turkish populationrdquoClinical Biochemistry vol 38 no 3 pp248ndash252 2005

[32] K Guleria S Sharma M Manjari et al ldquopR72P PIN3 Ins16bppolymorphisms of TP53 and CCR532 in north Indian breastcancer patientsrdquo Asian Pacific Journal of Cancer Prevention vol13 no 7 pp 3305ndash3311 2012

[33] A Zafiropoulos N Crikas A M Passam and D A SpandidosldquoSignificant involvement of CCR2-64I and CXCL12-3a in the

development of sporadic breast cancerrdquo Journal of MedicalGenetics vol 41 no 5 p e59 2004

[34] D T Teachey and S P Hunger ldquoPredicting relapse risk inchildhood acute lymphoblastic leukaemiardquo British Journal ofHaematology vol 162 no 5 pp 606ndash620 2013

[35] A de Lourdes Perim R L Guembarovski J M Oda et alldquoCXCL12 and TP53 genetic polymorphisms as markers ofsusceptibility in a Brazilian children population with acutelymphoblastic leukemia (ALL)rdquoMolecular Biology Reports vol40 no 7 pp 4591ndash4596 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Advances in Hematology

lower-risk and treated with less intensive therapy relapsewhile others deemed higher-risk are probably overtreated[34] In this context genetic polymorphisms in chemokinereceptors could predict outcome and be considered an inde-pendent risk factor to stratify and allocate therapy in ALL

More than half of the patients with ALL were classified inhigher-risk group according to the clinical and laboratorialfindings at diagnosis as defined by GBTLI LLA-99 protocol[2] When the genotype data were analyzed for stratifiedgroup of ALL the results indicated that the presence ofΔ32 did not influence this clinical parameter Similarly arecent study conducted by our research group has not foundassociation among tumor suppressor TP53 and chemokineCXCL12 polymorphisms and ALL recurrence risk status [35]

5 Conclusion

The comprehension about cellular and molecular mecha-nisms of ALL is critically important for the developmentof new approaches to hematological neoplasia treatmentAlthough any association of rs333 polymorphism of CCR5was verified we believe that the current researchmust lead toa better definition of the host-tumor relationship particularlywith respect to immunologic response and interrelation ofCCR5 and ALL development Given the sample size of thepresent case-control association study strong conclusions arenot possible however future investigation involving muchlarger cases may determine the absence of clinical implica-tions for CCR5Δ32 alleles in relation to ALL pathogenesis

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to acknowledge the volunteerswho made this study possible the University Hospital ofLondrina and Londrina Cancer Institute PR Brazil fortheir collaboration This study was supported by Con-selho Nacional de Desenvolvimento Cientıfico e Tecnologico(CNPq) Coordenacao de Aperfeicoamento de Pessoal deNıvel Superior (CAPES) Fundacao Araucaria do ParanaSecretaria da Ciencia Tecnologia e Ensino Superior (SETI)Fundo Estadual para a Infancia e Adolescencia (FIAPR) eSecretaria da Famılia e Desenvolvimento Social (SEDS) andCoordination of Undergraduate Studies of Londrina StateUniversity (PROPPG-UEL)

References

[1] M Belson B Kingsley and A Holmes ldquoRisk factors foracute leukemia in children a reviewrdquo Environmental HealthPerspectives vol 115 no 1 pp 138ndash145 2007

[2] INCA Estimate 2014 Cancer Incidence in Brazil INCA Rio deJaneiro Brazil 2013

[3] F Balkwill ldquoCancer and the chemokine networkrdquo NatureReviews Cancer vol 4 no 7 pp 540ndash550 2004

[4] J Vandercappellen J Van Damme and S Struyf ldquoThe role ofCXC chemokines and their receptors in cancerrdquo Cancer Lettersvol 267 no 2 pp 226ndash244 2008

[5] D Aldinucci andA Colombatti ldquoThe inflammatory chemokineCCL5 and cancer progressionrdquoMediators of Inflammation vol2014 Article ID 292376 12 pages 2014

[6] C J Raport J Gosling V L Schweickart P W Gray and I FCharo ldquoMolecular cloning and functional characterization ofa novel human CC chemokine receptor (CCR5) for RANTESMIP-1120573 and MIP-1120572rdquo Journal of Biological Chemistry vol 271no 29 pp 17161ndash17166 1996

[7] M Samson O Labbe C Mollereau G Vassart and MParmentier ldquoMolecular cloning and functional expression of anew human CC-chemokine receptor generdquo Biochemistry vol35 no 11 pp 3362ndash3367 1996

[8] P Proost AWuyts and J vanDamme ldquoThe role of chemokinesin inflammationrdquo Clinical and Experimental Medicine vol 26no 4 pp 211ndash223 1996

[9] P Spagnolo E A Renzoni A U Wells et al ldquoC-C chemokinereceptor 5 gene variants in relation to lung disease in sarcoido-sisrdquoAmerican Journal of Respiratory and Critical CareMedicinevol 172 no 6 pp 721ndash728 2005

[10] E-M Weiss A Schmidt D Vobis et al ldquoFoxp3-mediatedsuppression of cd95l expression confers resistance to activation-induced cell death in regulatory T cellsrdquo Journal of Immunologyvol 187 no 4 pp 1684ndash1691 2011

[11] C-H Tang A Yamamoto Y-T Lin Y-C Fong and T-W TanldquoInvolvement of matrix metalloproteinase-3 in CCL5CCR5pathway of chondrosarcomas metastasisrdquo Biochemical Pharma-cology vol 79 no 2 pp 209ndash217 2010

[12] J-Y Chuang W-H Yang H-T Chen et al ldquoCCL5CCR5 axispromotes the motility of human oral cancer cellsrdquo Journal ofCellular Physiology vol 220 no 2 pp 418ndash426 2009

[13] E Menu E De Leenheer H De Raeve et al ldquoRole of CCR1 andCCR5 in homing and growth of multiple myeloma and in thedevelopment of osteolytic lesions a study in the 5TMMmodelrdquoClinical and Experimental Metastasis vol 23 no 5-6 pp 291ndash300 2006

[14] J C Aster W S Pear and S C Blacklow ldquoNotch signaling inleukemiardquo Annual Review of Pathology Mechanisms of Diseasevol 3 pp 587ndash613 2008

[15] L Mirandola M Chiriva-Internati DMontagna et al ldquoNotch1regulates chemotaxis and proliferation by controlling the CC-chemokine receptors 5 and 9 in T cell acute lymphoblasticleukaemiardquo Journal of Pathology vol 226 no 5 pp 713ndash7222012

[16] H Makishima T Ito N Asano et al ldquoSignificance ofchemokine receptor expression in aggressive NK cell leukemiardquoLeukemia vol 19 no 7 pp 1169ndash1174 2005

[17] HMakishima T Ito KMomose et al ldquoChemokine system andtissue infiltration in aggressive NK-cell leukemiardquo Leukemiaresearch vol 31 no 9 pp 1237ndash1245 2007

[18] S M Davies M J Borowitz G L Rosner et al ldquoPharmaco-genetics of minimal residual disease response in children withB-precursor acute lymphoblastic leukemia a report from thechildrenrsquos oncology grouprdquo Blood vol 111 no 6 pp 2984ndash29902008

Advances in Hematology 5

[19] D H McDermott P A Zimmerman F Guignard C AKleeberger S F Leitman and P M Murphy ldquoCCR5 promoterpolymorphism and HIV-1 disease progressionrdquoThe Lancet vol352 no 9131 pp 866ndash870 1998

[20] M Chelli and M Alizon ldquoDeterminants of the trans-dominantnegative effect of truncated forms of the CCR5 chemokinereceptorrdquo Journal of Biological Chemistry vol 276 no 50 pp46975ndash46982 2001

[21] E M V Reiche M A E Watanabe A M Bonametti etal ldquoFrequency of CCR5-Δ32 deletion in human immunode-ficiency virus type 1 (HIV-1) in healthy blood donors HIV-1-exposed seronegative and HIV-1-seropositive individuals ofsouthern Brazilian populationrdquo International Journal of Molec-ular Medicine vol 22 no 5 pp 669ndash675 2008

[22] A P Galvani and J Novembre ldquoThe evolutionary historyof the CCR5-Delta32 HIV-resistance mutationrdquo Microbes andInfection vol 7 no 2 pp 302ndash309 2005

[23] P Weitzenfeld and A Ben-Baruch ldquoThe chemokine systemand its CCR5 and CXCR4 receptors as potential targets forpersonalized therapy in cancerrdquo Cancer Letters 2013

[24] C Kucukgergin F K Isman S Dasdemir et al ldquoThe roleof chemokine and chemokine receptor gene variants on thesusceptibility and clinicopathological characteristics of bladdercancerrdquo Gene vol 511 no 1 pp 7ndash11 2012

[25] V R Arruda C E Grignolli M S Goncalves et al ldquoPreva-lence of homozygosity for the deleted alleles of glutathione S-transferase mu (GSTM1) and theta (GSTT1) among distinctethnic groups from Brazil relevance to enviromental carcino-genesisrdquo Clinical Genetics vol 54 no 3 pp 210ndash214 1998

[26] F C Parra R C Amado J R Lambertucci et al ldquoColor andgenomic ancestry in Braziliansrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no1 pp 177ndash182 2003

[27] J SMaier-Moore C A Canas G Tobon et al ldquoTheCCR5 delta32 polymorphism (rs333) is not associated with Sjogrenrsquos syn-drome or type 1 diabetes in colombiansrdquo Clinical Immunologyvol 148 no 2 pp 206ndash208 2013

[28] S M Muxel S D Borelli M K Amarante et al ldquoAssociationstudy of CCR5 delta 32 polymorphism among the HLA-DRB1Caucasian population in northern Parana Brazilrdquo Journal ofClinical Laboratory Analysis vol 22 no 4 pp 229ndash233 2008

[29] K Brajao de Oliveira E M Vissoci Reiche H KaminamiMorimoto et al ldquoAnalysis of the CC chemokine receptor 5delta32 polymorphism in a Brazilian populationwith cutaneousleishmaniasisrdquo Journal of Cutaneous Pathology vol 34 no 1 pp27ndash32 2007

[30] M N Aoki A C D S do Amaral Herrera M K Amarante JL do Val Carneiro M H P Fungaro and M A E WatanabeldquoCCR5 and p53 codon 72 gene polymorphisms implications inbreast cancer developmentrdquo International Journal of MolecularMedicine vol 23 no 3 pp 429ndash435 2009

[31] N Degerli E Yilmaz and F Bardakci ldquoThe Δ32 allele distribu-tion of the CCR5 gene and its relationship with certain cancersin a Turkish populationrdquoClinical Biochemistry vol 38 no 3 pp248ndash252 2005

[32] K Guleria S Sharma M Manjari et al ldquopR72P PIN3 Ins16bppolymorphisms of TP53 and CCR532 in north Indian breastcancer patientsrdquo Asian Pacific Journal of Cancer Prevention vol13 no 7 pp 3305ndash3311 2012

[33] A Zafiropoulos N Crikas A M Passam and D A SpandidosldquoSignificant involvement of CCR2-64I and CXCL12-3a in the

development of sporadic breast cancerrdquo Journal of MedicalGenetics vol 41 no 5 p e59 2004

[34] D T Teachey and S P Hunger ldquoPredicting relapse risk inchildhood acute lymphoblastic leukaemiardquo British Journal ofHaematology vol 162 no 5 pp 606ndash620 2013

[35] A de Lourdes Perim R L Guembarovski J M Oda et alldquoCXCL12 and TP53 genetic polymorphisms as markers ofsusceptibility in a Brazilian children population with acutelymphoblastic leukemia (ALL)rdquoMolecular Biology Reports vol40 no 7 pp 4591ndash4596 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Advances in Hematology 5

[19] D H McDermott P A Zimmerman F Guignard C AKleeberger S F Leitman and P M Murphy ldquoCCR5 promoterpolymorphism and HIV-1 disease progressionrdquoThe Lancet vol352 no 9131 pp 866ndash870 1998

[20] M Chelli and M Alizon ldquoDeterminants of the trans-dominantnegative effect of truncated forms of the CCR5 chemokinereceptorrdquo Journal of Biological Chemistry vol 276 no 50 pp46975ndash46982 2001

[21] E M V Reiche M A E Watanabe A M Bonametti etal ldquoFrequency of CCR5-Δ32 deletion in human immunode-ficiency virus type 1 (HIV-1) in healthy blood donors HIV-1-exposed seronegative and HIV-1-seropositive individuals ofsouthern Brazilian populationrdquo International Journal of Molec-ular Medicine vol 22 no 5 pp 669ndash675 2008

[22] A P Galvani and J Novembre ldquoThe evolutionary historyof the CCR5-Delta32 HIV-resistance mutationrdquo Microbes andInfection vol 7 no 2 pp 302ndash309 2005

[23] P Weitzenfeld and A Ben-Baruch ldquoThe chemokine systemand its CCR5 and CXCR4 receptors as potential targets forpersonalized therapy in cancerrdquo Cancer Letters 2013

[24] C Kucukgergin F K Isman S Dasdemir et al ldquoThe roleof chemokine and chemokine receptor gene variants on thesusceptibility and clinicopathological characteristics of bladdercancerrdquo Gene vol 511 no 1 pp 7ndash11 2012

[25] V R Arruda C E Grignolli M S Goncalves et al ldquoPreva-lence of homozygosity for the deleted alleles of glutathione S-transferase mu (GSTM1) and theta (GSTT1) among distinctethnic groups from Brazil relevance to enviromental carcino-genesisrdquo Clinical Genetics vol 54 no 3 pp 210ndash214 1998

[26] F C Parra R C Amado J R Lambertucci et al ldquoColor andgenomic ancestry in Braziliansrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no1 pp 177ndash182 2003

[27] J SMaier-Moore C A Canas G Tobon et al ldquoTheCCR5 delta32 polymorphism (rs333) is not associated with Sjogrenrsquos syn-drome or type 1 diabetes in colombiansrdquo Clinical Immunologyvol 148 no 2 pp 206ndash208 2013

[28] S M Muxel S D Borelli M K Amarante et al ldquoAssociationstudy of CCR5 delta 32 polymorphism among the HLA-DRB1Caucasian population in northern Parana Brazilrdquo Journal ofClinical Laboratory Analysis vol 22 no 4 pp 229ndash233 2008

[29] K Brajao de Oliveira E M Vissoci Reiche H KaminamiMorimoto et al ldquoAnalysis of the CC chemokine receptor 5delta32 polymorphism in a Brazilian populationwith cutaneousleishmaniasisrdquo Journal of Cutaneous Pathology vol 34 no 1 pp27ndash32 2007

[30] M N Aoki A C D S do Amaral Herrera M K Amarante JL do Val Carneiro M H P Fungaro and M A E WatanabeldquoCCR5 and p53 codon 72 gene polymorphisms implications inbreast cancer developmentrdquo International Journal of MolecularMedicine vol 23 no 3 pp 429ndash435 2009

[31] N Degerli E Yilmaz and F Bardakci ldquoThe Δ32 allele distribu-tion of the CCR5 gene and its relationship with certain cancersin a Turkish populationrdquoClinical Biochemistry vol 38 no 3 pp248ndash252 2005

[32] K Guleria S Sharma M Manjari et al ldquopR72P PIN3 Ins16bppolymorphisms of TP53 and CCR532 in north Indian breastcancer patientsrdquo Asian Pacific Journal of Cancer Prevention vol13 no 7 pp 3305ndash3311 2012

[33] A Zafiropoulos N Crikas A M Passam and D A SpandidosldquoSignificant involvement of CCR2-64I and CXCL12-3a in the

development of sporadic breast cancerrdquo Journal of MedicalGenetics vol 41 no 5 p e59 2004

[34] D T Teachey and S P Hunger ldquoPredicting relapse risk inchildhood acute lymphoblastic leukaemiardquo British Journal ofHaematology vol 162 no 5 pp 606ndash620 2013

[35] A de Lourdes Perim R L Guembarovski J M Oda et alldquoCXCL12 and TP53 genetic polymorphisms as markers ofsusceptibility in a Brazilian children population with acutelymphoblastic leukemia (ALL)rdquoMolecular Biology Reports vol40 no 7 pp 4591ndash4596 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom