Research Article Osteoprotegerin Polymorphisms in a...

Research ArticleOsteoprotegerin Polymorphisms in a MexicanPopulation with Rheumatoid Arthritis and GeneralizedOsteoporosis A Preliminary Report

Maria Guadalupe Zavala-Cerna1 Maria Cristina Moran-Moguel2

Jesus Alejandro Cornejo-Toledo2 Norma Guadalupe Gonzalez-Montoya3

Jose Sanchez-Corona2 Mario Salazar-Paramo4

Arnulfo Hernan Nava-Zavala156 Erika Anita Aguilar-Chavez7

Miriam Fabiola Alcaraz-Lopez8 Alicia Guadalupe Gonzalez-Sanchez3

Laura Gonzalez-Lopez9 and Jorge Ivan Gamez-Nava10

1 Programa Internacional Facultad de Medicina Universidad Autonoma de Guadalajara 44100 Zapopan JAL Mexico2 Division de Medicina Molecular del Centro de Investigacion Biomedica de Occidente Instituto Mexicano del Seguro Social44340 Guadalajara JAL Mexico

3 Programa de Becarios en Investigacion del Instituto Mexicano del Seguro Social Programa de Doctorado en FarmacologiaCentro Universitario de Ciencias de la Salud Universidad de Guadalajara 44340 Guadalajara JAL Mexico

4 Division de Investigacion en Salud UMAE Hospital de Especialidades CMNO IMSS and Departamento de FisiologiaCUCS Universidad de Guadalajara 44340 Guadalajara JAL Mexico

5 Unidad de Investigacion en Epidemiologia Clinica Hospital de Especialidades Centro Medico Nacional de Occidente del InstitutoMexicano del Seguro Social 44340 Guadalajara JAL Mexico

6 Servicio de Medicina Interna Inmunologia y Reumatologia Hospital General de Occidente Secretaria de Salud Jalisco45170 Guadalajara JAL Mexico

7 Unidad de Medicina Familiar 2 Instituto Mexicano del Seguro Social 44340 Guadalajara JAL Mexico8 Departamento de Medicina Interna-Reumatologia Hospital General de Zona 14 Instituto Mexicano del Seguro Social44860 Guadalajara JAL Mexico

9 Departamento de Medicina Interna-Reumatologia del Hospital General Regional No 110 Instituto Mexicano del SeguroSocial y Programa de Doctorado en Salud Publica CUCS Universidad de Guadalajara 44340 Guadalajara JAL Mexico

10Unidad de Investigacion en Epidemiologia Clinica Hospital de Especialidades Centro Medico Nacional de Occidentedel Instituto Mexicano del Seguro Social y Programa de Doctorado en Salud Publica CUCS Universidad de Guadalajara44340 Guadalajara JAL Mexico

Correspondence should be addressed to Jorge Ivan Gamez-Nava drivangamezprodigynetmx

Received 17 July 2014 Revised 12 September 2014 Accepted 15 September 2014

Academic Editor Giacomina Brunetti

Copyright copy 2015 Maria Guadalupe Zavala-Cerna 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

Bone disease in rheumatoid arthritis (RA) is a complex phenomenon where genetic risk factors have been partially evaluated Thesystem formed by receptor activator for nuclear factor-120581B (RANK) receptor activator for nuclear factor-120581B ligand (RANKL) andosteoprotegerin (OPG) RANKRANKLOPG is a crucialmolecular pathway for coupling between osteoblasts and osteoclasts sinceOPG is able to inhibit osteoclast differentiation and activationWe aim to evaluate the association between SNPsC950T (rs2073617)C209T (rs3134069) T245G (rs3134070) in the TNFRSF11B (OPG) gene and osteoporosis in RA We included 81 women with RAand 52 healthy subjects in a cross-sectional study genotyped them and measured bone mineral density (BMD) at the lumbarspine and the femoral neck Mean age in RA was 50 plusmn 12 with disease duration of 12 plusmn 8 years According to BMD results 23(333) were normal and 46 (667) had osteopeniaosteoporosis We found a higher prevalence of C allele for C950T SNP in RA

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2015 Article ID 376197 7 pageshttpdxdoiorg1011552015376197

2 Journal of Immunology Research

Polymorphisms C209T and T245G did not reach statistical significance in allele distribution Further studies including patientsfrom other regions of Latin America with a multicenter design to increase the sample size are required to confirm our findings andelucidate if C950T SNP could be associated with osteoporosis in RA

1 Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory diseasethat affects synovial joints [1] Its prevalence in Mexico isconsiderably elevated ranging from 03 to 1 [2] Currentlythe diagnosis of osteoporosis is made on the basis of a mea-surement by dual-energy x-ray absorptiometry (DXA) at thespine and hip showing a decrement in bone mineral density(BMD) lower than 25 standard deviations in comparisonwith healthy young adults [3] However bone involvement inRA is complex an increased proportion of patientsmay sufferfrom osteoporosis nevertheless causal effects are consideredto bemultifactorial Previous studies performed in our centershowed a prevalence of axial osteoporosis inMexican womenwith RA of 31 [4]

Genetic risk factors constitute a variable that contributesto influencing the development of osteoporosis in RA in thiscontext some genetic polymorphisms may influence the rateof presentation of osteoporosis Nevertheless such geneticchanges do not explain entirely osteoporosis presence orfracture related events Strong evidence supports the fact thatosteoclasts have an important role in osteoporosis and boneerosion formation [5 6]

The system conformed by nuclear factor-120581B (RANK)receptor activator for nuclear factor-120581B ligand (RANKL) andosteoprotegerin (OPG) are crucial for the bone remodelingprocess Furthermore it appeared that not only osteoblastsbut also activated 119879 lymphocytes play a crucial role in thepathogenesis of rheumatoid arthritis (RA) and osteoporo-sis since they can produce RANKL which stimulates thedifferentiation and activation of osteoclasts [7] These threeproteins belong to the superfamily of the TNF-120572 and havedifferent patterns of expression RANKL is expressed inosteoblasts and its function is to participate in osteoclasts dif-ferentiation [7 8] Osteoprotegerin (OPG) lacks a transmem-brane domain and hence it is a receptor that can be secretedOPG recognizes and is able to bind RANKL blocking itsinteraction with RANK inhibiting osteoclastic differentiationand activation [9ndash11] Since OPG is homologous to RANK itacts as a false receptor for RANKL [12ndash14] OPG is secretedmainly by stromal cells but can also be secreted by other typesof cells [15] Different studies have been able to identify singlenucleotide polymorphisms (SNPs) inTNFSF11 (RANKL) andTNFRSF11B (OPG) genes in association with osteoporosis orlow bone density [12 16] It has been documented that OPGoverexpression induces an increase in bone density and pro-tects from the development to osteoporosis In contrast micethat are OPG-deficient develop osteoporosis very rapidly [1415 17] OPG overexpression blocks osteoclasts maturationcontrariwise an underexpression of this molecule resultsin an excessive increase of bone resorption and osteoporo-sis [18] Mutations in the promoter region of TNFRSF11B

could have influence on the transcription and translationrate of OPG A study in European population (Eslovenia)showed that two SNPs named rs3134069 and rs3134070 inthe TNFRSF11B could form a haplotype with susceptibility toosteoporosis [16 19] To date there is a lack of informationevaluating the relationship between polymorphisms of OPGgene and bone mineral density

Therefore the aim of the present study was to assess ifthere is an association between SNPs C950T (rs2073617)C2097 (rs3134069) andT245G (rs3134070) in theTNFRSF11B(OPG)gene andosteoporosis in rheumatoid arthritis patients

2 Patients and Methods

21 Study Subjects We carried out a cross-sectional studythat included 81 consecutive unrelated women who werediagnosed with RA from March 2007 to March 2009 Thesepatients were referred from an outpatient rheumatologyclinic in a secondary care center in Guadalajara Mexico(Hospital General Regional 110 IMSS) Inclusion criteriawere as follows age ge 18 years at entry self-identifiedrace Mexican Mestizo (defined as individuals who wereborn in Mexico and were descendants of the originalautochthonous inhabitants of the region and of individualsmainly Spaniards) fulfillment of the 1987 American Collegeof Rheumatology (ACR) classification criteria [20] and noprevious BMDmeasurement Patients were excluded if preg-nancy was present and if they had an overlap syndrome werereceiving bisphosphonates or parathyroid hormone therapyor had a comorbidity associated with low BMD such asdiabetes mellitus thyroid disease or chronic renal failurepatients were not related to each other

A healthy control group matched by age and sex wasobtained fromblood donorswho attended to ldquoCentroMedicoNacional de Occidenterdquo blood bank all blood donors were ofMexican Mestizo origin and denied at the time of the studyhaving any disease (acute or chronic)

22 Clinical Assessment Each patient was interviewed usinga structured questionnaire to record demographic informa-tion general risk factors for osteoporosis (ie age weightand height) clinical characteristics and RA treatment Atthe time of the evaluation two trained researchers assessedRA disease activity by systematical evaluation of the DAS-28 index [21] to determine patient disability the Spanishmodified version of the Health Assessment Questionnaire-Disability Index (HAQ-DI) [22] was used Global functionalstatus was evaluated according to the Steinbrocker clas-sification [23] Rheumatoid factor and C-reactive protein(CRP) were measured by nephelometry using commerciallyavailable kits Erythrocyte sedimentation rate (ESR mmh)was measured using the Wintrobe method

Journal of Immunology Research 3

Table 1 Genotyping strategies for TNFRSF11B polymorphism variants detection

SNP Primers Band size (bp) Restrictionenzyme

Recognizedsequence

Band size afterdigestion (bp)

C950Trs2073617

51015840-TGCGTCCGGATCTTGGCTGGATCGG-31015840 548 Hinc II GTY RAC C 248 and 287T 54851015840-ACTTACCACGAGCGCGCAGCACAGCAA-31015840

C209Trs3134070

51015840-CGAACCCTAGAGCAAAGTGC-31015840 271 Taq I T CGA C 212 31 and 28T 212 and 5951015840-TGTCTGATTGGCCCTAAAGC-31015840

T245Grs3134069

51015840-CGAACCCTAGAGCAAAGTGC-31015840 271 Hinf I G ANTC A 195 and 176C 127 76 and 6951015840-TGTCTGATTGGCCCTAAAGC-31015840

SNP single nucleotide polymorphism and bp base pair

23 BMD Measurement BMD was measured (gcm2) byDEXA using a Lunar Prodigy densitometer (GE MedicalSystems Lunar Madison WI software V 88 GE MedicalSystems) the anatomical regions assessed were lumbar spinein the posterior-anterior projection (L1-L4) and femoral neckThe coefficient of variation during the measurement of astandard phantom in our laboratory is 07The coefficient ofvariationwas 24at the lumbar spine and 16at the femoralneck All scans were performed by the same experiencedtechnician who was blinded to the characteristics of patientsEach patient was classified into one of the following cate-gories proposed by WHO normal BMD defined as havinga measurement of BMD within 1 SD of the BMD of normalyoung adult women (equivalent to 119879 score gt minus1) osteopeniadefined as having a 119879 score in BMD between minus1 and minus24 SDand osteoporosis defined as having a 119879 score in BMD leminus25 SD Patients with a 119879 score lt minus10 were considered tohave low BMD in consequence for analysis purposes in ourstudy we decided to include osteopenia and osteoporosis inone single group denominated as ldquolow BMDrdquo

24 Genotyping DNA from 81 patients with rheumatoidarthritis was extracted from blood samples using conven-tional methods [24] and stored frozen at minus80∘C

We chose three SNPs that are present in the promoterregion (51015840UTR) of the TNFRSF11B (OPG) gene such poly-morphisms were previously reported in association withosteoporosis in postmenopausal women or related conditions[19 25ndash28] We performed 3 polymerase chain reaction-restriction fragment length polymorphisms (PCR RFLPs)protocols as previously described [26 29 30] Table 1 showsprimer sequence products obtained after PCR restrictionenzymes used their recognition site and end products afterrestriction

Each PCR reaction was carried out in 10 120583L final volumecontaining (final concentrations) 1X buffer (200mM Tris-HCl pH 84 500mM KCl and 4mM MgCl

2) 5 pmolmL

each of the pair primers according to polymorphism(Table 1) 10mM each of the four deoxyribonucleosidetriphosphates 1 U of taqDNApolymerase (Invitrogen Carls-bad CA USA) and 200 to 300 ng DNA template ThePCR products were visualized by electrophoresis in 10polyacrylamide gels at 150V for 90min followed by silverstaining

25 Statistical Analysis Allelic and genotypic frequencies foreach SNP were determined by gene count Hardy-Weinbergequilibrium was tested using 1205942 test Genotypic differencesbetween RA patients with and without osteoporosis wereevaluated by Mantel-Haenszel test using the EPI INFO(version 604d) statistical program For allelic differences weused Fisherrsquos exact test Odds ratios (OR) and 95 confidenceinterval (95 CI) were computed to evaluate the risk forosteoporosis conferred by presence of the risk alleles Allanalyses used two-sided tails with a 119875 value of le005 used assignificance criterion

3 Results

31 Demographics and Clinical We included 81 patientsclassified as RA according to ACR classification criteria [20]and 52 healthy subjects For the RA group mean age was50 plusmn 12 years 100 of the studied subjects were womenmean duration disease was 12 plusmn 8 years and all of themwere receiving treatment some of them with monotherapyand others combined therapy the number and percentagesof patients taking a specific drug are listed in Table 2

RA patients were evaluated for BMD according to theclassification criteria of the WHO for osteoporosis Accord-ing to the results of bone densitometry 23 patients (333)had normal BMD 31 (449) had osteopenia (low bonedensity) and 15 (217) had osteoporosis For study purposeswe grouped osteopenia with osteoporosis in one groupconstituted by 46 patients (667) (see Table 2) From these23 patients (329) had osteopenia and 13 (186) hadosteoporosis in the lumbar spine whereas 35 patients (507)had osteopenia and 7 (101) had osteoporosis of the femoralneck Clinical risk factors and factors related to RA severityfor low BMD were registered and are listed in Table 2

32 Molecular Analysis We performed at least three PCR-RFLP tests for every subject included in the present study butwere able to obtain complete genotyping results in 77 DNAsamples from RA patients and 52 healthy subjects Healthysubjects group was in Hardy-Weinberg equilibrium (data notshown)

In Table 3 genotype and allele frequencies of three SNPsin the OPG gene in healthy subjects and rheumatoid arthritis


Table 2 Clinical characteristics of study patients

Characteristic 119899 = 81Age (years) median (range) 499 (21ndash79)Menopausal 119899 () 43 (531)Weight (kg) median (range) 658 (43ndash94)BMI median (range) 2687 (18ndash38)Oral contraceptives use 119899 () 41 (506)Current smoker 119899 () 17 (21)Family history of fractures 119899 () 6 (74)Alcohol consumption 3 or moreunitsday 119899 () 3 (337)

Duration of RA (years) median (range) 118 (15ndash35)DAS-28 score median (range) 38 (0ndash67)Global functional status III-IV 119899 () 7 (78)HAQ-DI score median (range) 07 (0ndash28)Medications 119899 ()Glucocorticoid 72 (889)Prednisone dosage (mgday) median(range) 44 (0ndash10)

Methotrexate 56 (709)Sulfasalazine 39 (494)Chloroquine 27 (342)Azathioprine 25 (316)D-Penicillamine 10 (127)Biologic agents 8 (101)BMD (WHO classification) 119899 ()Normal 23 (333)Osteopeniaosteoporosis 46 (667)BMI body mass index RA rheumatoid arthritis DAS-28 disease activityscore HAQ-DI health assessment questionnaire-disability index BMDbone mineral density and WHO World Health Organization

patients are shown we found significant difference in thedistribution of the C950T (rs2073617) alleles the C allelebeing more prevalent in the RA group PolymorphismsC209T and T245G did not reach statistical significance

We further analyzed genetic variants in RA patientsand divided this group with respect to bone mass indexstatus according to the WHO classification in normal orosteopeniaosteoporosis there was no significant differencein allele distribution between RA patients with and withoutosteoporosis Table 4 shows genotype and allele frequenciesin these subgroups

4 Discussion

Osteoporosis represents a major health problem throughoutthe world and the most serious consequence of osteoporosisis hip fracture which has a high associated morbidity andmortality [31] Systemic effects of RA in bone remodelinginclude loss of axial and appendicular bone mass associatedwith an increase in fracture risk (96 per 1000 person-years)

[32] Clinical patterns of bone mass loss include gener-alized osteoporosis (axial and peripheral) juxta-articularosteoporosis which is adjacent to synovial membrane andmarginal and subchondral erosions which are directly asso-ciated with inflamed synovial tissue [33] A high proportionof patients will suffer from hip and spine fractures causinglimitations in physical abilities and social capabilities Osteo-porosis presence in RA patients involves multiple factorssuch as disease activity the use of some prescribed drugsparticularly DMARDs and corticosteroids female sex olderage and menopause [34]

The development of erosive lesions as well as generalizedosteoporosis in RA results from an imbalance betweenbone mass formation and bone resorption process In RAthe unsteadiness between osteoblastic bone formation (mes-enchymal origin) and osteoclastic bone resorption (derivedfrommonocytesmacrophages) promotes bone resorption byosteoclasts and as a consequence loss in bone strength whichmay lead to the development of osteoporosis and fractures

Multiple interactions between bone and the immunesystem have been described bone is a major target of chronicinflammation since it increases bone resorption and resultsin suppressed local bone formation causing a wide spectrumof bone involvement in RA [35] Lymphocyte ormacrophage-derived cytokines are among the most potent mediatorsof inflammation involved in upregulation of osteoclastsformation activation and signaling pathways [36]

Osteoclasts progenitors are recruited under physiologicalconditions from the haematopoyetic system and can get stim-ulated by some cytokines and hormones to form precursorsthat will eventually express cell surface markers of a well-differentiated osteoclast furthermore some inflammatorysynovial cells present in RA particularly mononuclear cellsand giant multinucleated cells can be differentiated intoosteoclasts [5] under proinflammatory cytokines stimuliincluding tumor necrosis factor alpha (TNF-120572) interleukin1 (IL-1) and interleukin 6 (IL-6) [6]

Recently Pathak et al described that sera from RA activepatients contribute to bone loss by two mechanisms (1)directly inhibiting osteoblasts proliferation and differentia-tion and (2) enhancing osteoblast-mediated osteoclastogene-sis via RANKL and IL-6 [37]

Another key component that has influence over the osteo-clastic differentiation is RANK a receptor for TNF attachedto the membrane of osteoclast precursor cells that can bindto RNAKL through interactionwith osteoblastic stromal cells[9 38 39] RANK is essential for signal transduction that willlead to ostoclastic differentiation Osteoprotegerin (OPG)also known as inhibition factor for osteoclastogenesis is amember of the TNFRSF located in chromosome 8q24 [17]with a length of 29Kb [13] it is synthesized originally as apropeptide of 400 aa and is secreted as a soluble protein afterthe cleavage of 21 aa that corresponds to the transmembraneand cytoplasmic domains [17] OPG mRNA is expressed indifferent human tissues (lung heart kidney liver intestinesstomach cerebrum thyroid gland and bone marrow) apartfrom the bone where its primary function appears to be inhi-bition of osteoclasts maturation and activation In this studywe evaluated the association of three TNFRSF11B promoter


Table 3 Genotype and allele frequencies of polymorphisms in the TNFRSF11B (OPG) gene in healthy subjects and rheumatoid arthritis (RA)patients

SNP Genotypes HS RA Allele HS RA 119875

OR(95 CI)

C950Trs2073617

CC 9 (176) 28 (431) C 47 (461) 82 (631)001 050 (030ndash085)CT 29 (569) 26 (40) T 55 (539) 48 (369)

TT 13 (255) 11 (169)

C209Trs3134070

CC 39 (750) 52 (722) C 89 (856) 122 (847)10 11 (053ndash22)CT 11 (212) 18 (250) T 15 (144) 22 (153)

TT 2 (38) 2 (28)

T245Grs3134069

AA 41 (788) 66 (857) A 93 (894) 142 (922)051 071 (030ndash169)AC 11 (212) 10 (13) C 11 (106) 12 (78)

CC 0 (0) 1 (13)OPG osteoprotegerin SNP single nucleotide polymorphism RA rheumatoid arthritis HS healthy subjects OR odds ratios and 95 CI 95 confidenceintervals In order to compute OR (95 CI) the following alleles were used as reference in correspondence with the allele of risk rs2073617 T rs3134070 T andrs3134069 C

Table 4Genotype and allele frequencies of polymorphisms in theTNFRSF11B (OPG) gene in rheumatoid arthritis (RA) patients with normaland low bone mineral density (BMD)

SNP Genotypes RA withnormal BMD

RA with lowBMD Allele RA with

normal BMD RA with low BMD P OR(95 CI)

C950Trs2073617

CC 9 (500) 16 (444) C 25 (694) 46 (639)067 13 (055ndash303)CT 7 (389) 14 (389) T 11 (306) 26 (361)

TT 2 (111) 6 (167)

C209Trs3134070

CC 17 (810) 29 (690) C 37 (881) 70 (833)060 15 (049ndash443)CT 3 (143) 12 (286) T 5 (119) 14 (167)

TT 1 (48) 1 (24)

T245Grs3134069

AA 20 (909) 39 (886) A 42 (955) 82 (932)072 15 (029ndash795)AC 2 (91) 4 (91) C 2 (45) 6 (68)

CC 0 (0) 1 (23)OPG osteoprotegerin SNP single nucleotide polymorphism RA rheumatoid arthritis BMD bone mineral density OR odds ratios and 95 CI 95confidence intervals In order to compute OR (95 CI) the following alleles were used as reference in correspondence with the allele of risk rs2073617 Trs3134070 T and rs3134069 C

polymorphisms with presence of osteoporosis in RA Ourstudy demonstrated that the C allele of the C950T was morefrequent in RA compared with HS thereafter we analyzedthe RA group to seek for differences in allele distributionbetween patients with and without osteoporosis neverthelesswe did not observe a significant difference The other twopolymorphisms did not reach statistical significance

The observed association in the present study couldbe caused by direct functional effect of the SNP or tolinkage disequilibrium (LD) with another functional variantAccording to the HapMap data there is a certain amountof LD between these SNPs indicating that although it ispossible that different causative variants exist in TNFRSF11Bit is also possible that only one of them turns out tohave functional consequences The C950T SNP is locatedin the promoter region near the TGF-120573 response areaand 129 bp upstream the TATA box [40] A functionalstudy of TNFRSF11B polymorphisms through luciferasereporter assays revealed that gene expression was affected by

the C950T polymorphism since it was significantly higherin the presence of the C allele [27]

Previous association studies that include polymorphismsin the TNFRSF11B gene were performed in osteoporoticpatients In 2002 Ohmori et al showed a significant associ-ation between C950T SNP and lower BMD [25] in contrastwith such results in 2004 Brandstrom et al showed thatC950T was not associated with bone mineral density inelderly Swedish women [41] and later Arko et al in 2005tested the three polymorphisms included in the presentstudy and others searching for an association with bonemineral density in postmenopausal women the linkagedisequilibriumwas confirmed but no associationswere found[26] For SNP T245G similar frequencies as obtained inthis study have been described previously by Zajıckova et alin postmenopausal women [42] Only a few studies havelooked for an association between these SNPs andRA in 2010Assmann et al performed a case-control study and found anassociation between SNPs in the RANKandRANKL gene but


not in the OPG gene nevertheless none of the SNPs includedin our study were genotyped by them [43] In a recent studythe SNP A163G in the osteoprotegerin gene was associatedwith osteoporosis in RA [44] This polymorphism was notincluded in the present study but a lack of association withthis particular polymorphism and osteoporosis in RA waspreviously confirmed by our study group [45]

Because this work constitutes a preliminary report alimitation in our study is a small sample size that may limitour statistical power to detect small differences betweengroups although we consider that our sample would besufficient to identify polymorphisms that may confer a highrisk for lowBMD inRA and did not show any trend to suspectthat increasing the sample size a statistical significance wouldbe achieved We recognize that future multicenter studiesshould be performed increasing the sample size with ourresults being useful as a referent for Mexican patients withRA to identify that these polymorphisms probably do notconfer a high risk for low BMD in our population with RAFurther studies in other populations are required to confirmour findings

In conclusion we confirmed the association of C alleleof the C950T SNP in the TNFRSF11B gene with RA patientsbut we were not able to support our main hypothesis thatthis polymorphism in the OPG gene is associated withosteoporosis in RA Further studies including other regionsof Mexico and Latin America with an increased sample sizeare required to confirm our findings and further elucidateif C950T SNP in the OPG gene could be associated withosteoporosis and not only with RA presence

Ethical Approval

This research was conducted in accordance with the Decla-ration of Helsinki Informed consent was obtained from allparticipants the study protocol was approved by the LocalCommittees for Ethical and Health Research (CLIEIS 2007-1301-1) and all data was managed anonymously

Conflict of Interests

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

Acknowledgment

This work was supported by Grant from Instituto Mexicanodel Seguro Social through FISIMSSPROTC2007025

References

[1] M Akil and R S Amos ldquoRheumatoid arthritis I clinicalfeatures and diagnosisrdquo British Medical Journal vol 310 no6979 pp 587ndash590 1995

[2] MH Cardiel and J Rojas-Serrano ldquoCommunity based study toestimate prevalence burden of illness and help seeking behaviorin rheumatic diseases in Mexico City A COPCORD studyrdquoClinical and Experimental Rheumatology vol 20 no 5 pp 617ndash624 2002

[3] J A Kanis L J Melton III C Christiansen C C Johnston andN Khaltaev ldquoThe diagnosis of osteoporosisrdquo Journal of Boneand Mineral Research vol 9 no 8 pp 1137ndash1141 1994

[4] F Alcaraz-Lopez Miriam G-L L Aguilar-Chavez Erika ALopez-Olivo Maria C Loaiza-Cardenas and I Gamez-NavaJorge ldquoPerformance of albrand index for identifying low bonedensity in rheumatoid arthritisrdquo The Journal of Rheumatologyvol 33 no 2 p 408 2006

[5] E M Gravallese ldquoBone destruction in arthritisrdquo Annals of theRheumatic Diseases vol 61 supplement 2 pp ii84ndashii86 2002

[6] S Roux and P Orcel ldquoBone loss factors that regulate osteoclastdifferentiation an updaterdquo Arthritis Research vol 2 no 6 pp451ndash456 2000

[7] D L Lacey E Timms H-L Tan et al ldquoOsteoprotegerinligand is a cytokine that regulates osteoclast differentiation andactivationrdquo Cell vol 93 no 2 pp 165ndash176 1998

[8] N Udagawa N Takahashi H Yasuda et al ldquoOsteoprotegerinproduced by osteoblasts is an important regulator in osteoclastdevelopment and functionrdquo Endocrinology vol 141 no 9 pp3478ndash3484 2000

[9] H Hsu D L Lacey C R Dunstan et al ldquoTumor necrosisfactor receptor family member RANK mediates osteoclast dif-ferentiation and activation induced by osteoprotegerin ligandrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 96 no 7 pp 3540ndash3545 1999

[10] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005

[11] D H Jones Y-Y Kong and J M Penninger ldquoRole of RANKLand RANK in bone loss and arthritisrdquo Annals of the RheumaticDiseases vol 61 supplement 2 pp ii32ndashii39 2002

[12] S-S Dong X-G Liu Y Chen et al ldquoAssociation analyses ofRANKLRANKOPG gene polymorphisms with femoral neckcompression strength index variation in caucasiansrdquo CalcifiedTissue International vol 85 no 2 pp 104ndash112 2009

[13] Y-H Hsu T Niu H A Terwedow et al ldquoVariation in genesinvolved in theRANKLRANKOPGbone remodeling pathwayare associated with bone mineral density at different skeletalsites inmenrdquoHumanGenetics vol 118 no 5 pp 568ndash577 2006

[14] C Liu T S Walter P Huang et al ldquoStructural and functionalinsights of RANKL-RANK interaction and signalingrdquo Journalof Immunology vol 184 no 12 pp 6910ndash6919 2010

[15] A-P Trouvin and V Goeb ldquoReceptor activator of nuclearfactor-120581B ligand and osteoprotegerin maintaining the balanceto prevent bone lossrdquo Clinical Interventions in Aging vol 5 pp345ndash354 2010

[16] J B Richards F K Kavvoura F Rivadeneira et al ldquoCol-laborative meta-analysis associations of 150 candidate geneswith osteoporosis and osteoporotic fracturerdquo Annals of InternalMedicine vol 151 no 8 pp 528ndash537 2009

[17] P Narducci R Bareggi and V Nicolin ldquoReceptor Activator forNuclear Factor kappa B Ligand (RANKL) as an osteoimmunekey regulator in bone physiology and pathologyrdquo Acta Histo-chemica vol 113 no 2 pp 73ndash81 2011

[18] W J Boyle W S Simonet and D L Lacey ldquoOsteoclastdifferentiation and activationrdquo Nature vol 423 no 6937 pp337ndash342 2003

[19] B Arko J Preelj R Komel A Kocijani P Hudler and J MarcldquoSequence variations in the osteoprotegerin gene promoter inpatients with postmenopausal osteoporosisrdquo Journal of Clinical


Endocrinology and Metabolism vol 87 no 9 pp 4080ndash40842002

[20] F C Arnett S M Edworthy D A Bloch et al ldquoThe AmericanRheumatism Association 1987 revised criteria for the classifica-tion of rheumatoid arthritisrdquo Arthritis and Rheumatism vol 31no 3 pp 315ndash324 1988

[21] M L L Prevoo M A vanrsquoT Hof H H Kuper M A vanLeeuwen L B A van de Putte and P L C M van RielldquoModified disease activity scores that include twenty-eight-jointcounts development and validation in a prospective longitu-dinal study of patients with rheumatoid arthritisrdquo Arthritis ampRheumatism vol 38 no 1 pp 44ndash48 1995

[22] M H Cardiel M Abello-Banfi R Ruiz-Mercado and DAlarcon-Segovia ldquoHow to measure health status in rheuma-toid arthritis in non-English speaking patients validation ofa Spanish version of the Health Assessment QuestionnaireDisability Index (Spanish HAQ-DI)rdquo Clinical and ExperimentalRheumatology vol 11 no 2 pp 117ndash121 1993

[23] M C Hochberg R W Chang I Dwosh S Lindsey T Pincusand F Wolfe ldquoThe American College of Rheumatology 1991revised criteria for the classification of global functional statusin rheumatoid arthritisrdquo Arthritis and Rheumatism vol 35 no5 pp 498ndash502 1992

[24] S Gustincich G Manfioletti G del Sal C Schneider andP Carninci ldquoA fast method for high-quality genomic DNAextraction fromwhole human bloodrdquo BioTechniques vol 11 no3 pp 298ndash302 1991

[25] H Ohmori Y Makita M Funamizu et al ldquoLinkage andassociation analyses of the osteoprotegerin gene locus withhuman osteoporosisrdquo Journal of Human Genetics vol 47 no 8pp 400ndash406 2002

[26] B Arko J Prezelj A Kocijancic R Komel and J MarcldquoAssociation of the osteoprotegerin gene polymorphisms withbone mineral density in postmenopausal womenrdquo Maturitasvol 51 no 3 pp 270ndash279 2005

[27] C Vidal R Formosa and A Xuereb-Anastasi ldquoFunctionalpolymorphisms within the TNFRSF11B (osteoprotegerin) geneincrease the risk for low bone mineral densityrdquo Journal ofMolecular Endocrinology vol 47 no 3 pp 327ndash333 2011

[28] G Beyens A Daroszewska F de Freitas et al ldquoIdentificationof sex-specific associations between polymorphisms of theOsteoprotegerin gene TNFRSF11B and Pagets disease of bonerdquoJournal of Bone and Mineral Research vol 22 no 7 pp 1062ndash1071 2007

[29] M Soufi M Schoppet A M Sattler et al ldquoOsteoprotegeringene polymorphisms in men with coronary artery diseaserdquoJournal of Clinical Endocrinology and Metabolism vol 89 no8 pp 3764ndash3768 2004

[30] D Pitocco G Zelano G Gioffre et al ldquoAssociation betweenosteoprotegerin G1181C and T245G polymorphisms and dia-betic charcot neuroarthropathy a case-control studyrdquo DiabetesCare vol 32 no 9 pp 1694ndash1697 2009

[31] C Cooper G Campion and L J Melton III ldquoHip fracturesin the elderly a world-wide projectionrdquo Osteoporosis Interna-tional vol 2 no 6 pp 285ndash289 1992

[32] S Y Kim S Schneeweiss J Liu et al ldquoRisk of osteoporoticfracture in a large population-based cohort of patients withrheumatoid arthritisrdquo Arthritis Research and Therapy vol 12no 4 article R154 2010

[33] S R Goldring ldquoThe final pathogenetic steps in focal boneerosions in rheumatoid arthritisrdquo Annals of the RheumaticDiseases vol 59 no 1 pp i72ndashi74 2000

[34] R M R Pereira J F de Carvalho and E Canalis ldquoGlucocor-ticoid-induced osteoporosis in rheumatic diseasesrdquo Clinics vol65 no 11 pp 1197ndash1205 2010

[35] P Geusens andW F Lems ldquoOsteoimmunology and osteoporo-sisrdquo Arthritis Research and Therapy vol 13 no 5 article 2422011

[36] G Mori P DrsquoAmelio R Faccio and G Brunetti ldquoThe interplaybetween the bone and the immune systemrdquo Clinical andDevelopmental Immunology vol 2013 Article ID 720504 16pages 2013

[37] J L Pathak N Bravenboer P Verschueren et al ldquoInflammatoryfactors in the circulation of patients with active rheumatoidarthritis stimulate osteoclastogenesis via endogenous cytokineproduction by osteoblastsrdquo Osteoporosis International vol 25no 10 pp 2453ndash2463 2014

[38] J Li I Sarosi X-Q Yan et al ldquoRANK is the intrinsichematopoietic cell surface receptor that controls osteoclastoge-nesis and regulation of bone mass and calcium metabolismrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 97 no 4 pp 1566ndash1571 2000

[39] T L Burgess Y-X Qian S Kaufman et al ldquoThe ligand forosteoprotegerin (OPGL) directly activates mature osteoclastsrdquoJournal of Cell Biology vol 145 no 3 pp 527ndash538 1999

[40] K Thirunavukkarasu R R Miles D L Halladay et alldquoStimulation of osteoprotegerin (OPG) gene expression bytransforming growth factor-120573 (TGF-120573) Mapping of the OPGpromoter region that mediates TGF-120573 effectsrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36241ndash36250 2001

[41] H Brandstrom P Gerdhem F Stiger et al ldquoSingle nucleotidepolymorphisms in the human gene for osteoprotegerin are notrelated to bone mineral density or fracture in elderly womenrdquoCalcified Tissue International vol 74 no 1 pp 18ndash24 2004

[42] K Zajıckova A Zemanova M Hill and I Zofkova ldquoIs A163Gpolymorphism in the osteoprotegerin gene associated with heelvelocity of sound in postmenopausal womenrdquo PhysiologicalResearch vol 57 no 1 pp S153ndashS157 2008

[43] G Assmann J Koenig M Pfreundschuh et al ldquoGeneticvariations in genes encoding RANK RANKL and OPG inrheumatoid arthritis a case-control studyrdquo Journal of Rheuma-tology vol 37 no 5 pp 900ndash904 2010

[44] Y M Hussien A Shehata R A Karam S S Alzahrani HMagdy and A M El-Shafey ldquoPolymorphism in vitamin Dreceptor and osteoprotegerin genes in Egyptian rheumatoidarthritis patients with and without osteoporosisrdquo MolecularBiology Reports vol 40 no 5 pp 3675ndash3680 2013

[45] A J L Brambila-Tapia J Duran-Gonzalez L Sandoval-Ramırez et al ldquoMTHFR C677T MTHFR A1298C and OPGA163G polymorphisms in Mexican patients with rheumatoidarthritis and osteoporosisrdquo Disease Markers vol 32 no 2 pp109ndash114 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



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OphthalmologyJournal of


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Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Polymorphisms C209T and T245G did not reach statistical significance in allele distribution Further studies including patientsfrom other regions of Latin America with a multicenter design to increase the sample size are required to confirm our findings andelucidate if C950T SNP could be associated with osteoporosis in RA

1 Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory diseasethat affects synovial joints [1] Its prevalence in Mexico isconsiderably elevated ranging from 03 to 1 [2] Currentlythe diagnosis of osteoporosis is made on the basis of a mea-surement by dual-energy x-ray absorptiometry (DXA) at thespine and hip showing a decrement in bone mineral density(BMD) lower than 25 standard deviations in comparisonwith healthy young adults [3] However bone involvement inRA is complex an increased proportion of patientsmay sufferfrom osteoporosis nevertheless causal effects are consideredto bemultifactorial Previous studies performed in our centershowed a prevalence of axial osteoporosis inMexican womenwith RA of 31 [4]

Genetic risk factors constitute a variable that contributesto influencing the development of osteoporosis in RA in thiscontext some genetic polymorphisms may influence the rateof presentation of osteoporosis Nevertheless such geneticchanges do not explain entirely osteoporosis presence orfracture related events Strong evidence supports the fact thatosteoclasts have an important role in osteoporosis and boneerosion formation [5 6]

The system conformed by nuclear factor-120581B (RANK)receptor activator for nuclear factor-120581B ligand (RANKL) andosteoprotegerin (OPG) are crucial for the bone remodelingprocess Furthermore it appeared that not only osteoblastsbut also activated 119879 lymphocytes play a crucial role in thepathogenesis of rheumatoid arthritis (RA) and osteoporo-sis since they can produce RANKL which stimulates thedifferentiation and activation of osteoclasts [7] These threeproteins belong to the superfamily of the TNF-120572 and havedifferent patterns of expression RANKL is expressed inosteoblasts and its function is to participate in osteoclasts dif-ferentiation [7 8] Osteoprotegerin (OPG) lacks a transmem-brane domain and hence it is a receptor that can be secretedOPG recognizes and is able to bind RANKL blocking itsinteraction with RANK inhibiting osteoclastic differentiationand activation [9ndash11] Since OPG is homologous to RANK itacts as a false receptor for RANKL [12ndash14] OPG is secretedmainly by stromal cells but can also be secreted by other typesof cells [15] Different studies have been able to identify singlenucleotide polymorphisms (SNPs) inTNFSF11 (RANKL) andTNFRSF11B (OPG) genes in association with osteoporosis orlow bone density [12 16] It has been documented that OPGoverexpression induces an increase in bone density and pro-tects from the development to osteoporosis In contrast micethat are OPG-deficient develop osteoporosis very rapidly [1415 17] OPG overexpression blocks osteoclasts maturationcontrariwise an underexpression of this molecule resultsin an excessive increase of bone resorption and osteoporo-sis [18] Mutations in the promoter region of TNFRSF11B

could have influence on the transcription and translationrate of OPG A study in European population (Eslovenia)showed that two SNPs named rs3134069 and rs3134070 inthe TNFRSF11B could form a haplotype with susceptibility toosteoporosis [16 19] To date there is a lack of informationevaluating the relationship between polymorphisms of OPGgene and bone mineral density

Therefore the aim of the present study was to assess ifthere is an association between SNPs C950T (rs2073617)C2097 (rs3134069) andT245G (rs3134070) in theTNFRSF11B(OPG)gene andosteoporosis in rheumatoid arthritis patients

2 Patients and Methods

21 Study Subjects We carried out a cross-sectional studythat included 81 consecutive unrelated women who werediagnosed with RA from March 2007 to March 2009 Thesepatients were referred from an outpatient rheumatologyclinic in a secondary care center in Guadalajara Mexico(Hospital General Regional 110 IMSS) Inclusion criteriawere as follows age ge 18 years at entry self-identifiedrace Mexican Mestizo (defined as individuals who wereborn in Mexico and were descendants of the originalautochthonous inhabitants of the region and of individualsmainly Spaniards) fulfillment of the 1987 American Collegeof Rheumatology (ACR) classification criteria [20] and noprevious BMDmeasurement Patients were excluded if preg-nancy was present and if they had an overlap syndrome werereceiving bisphosphonates or parathyroid hormone therapyor had a comorbidity associated with low BMD such asdiabetes mellitus thyroid disease or chronic renal failurepatients were not related to each other

A healthy control group matched by age and sex wasobtained fromblood donorswho attended to ldquoCentroMedicoNacional de Occidenterdquo blood bank all blood donors were ofMexican Mestizo origin and denied at the time of the studyhaving any disease (acute or chronic)

22 Clinical Assessment Each patient was interviewed usinga structured questionnaire to record demographic informa-tion general risk factors for osteoporosis (ie age weightand height) clinical characteristics and RA treatment Atthe time of the evaluation two trained researchers assessedRA disease activity by systematical evaluation of the DAS-28 index [21] to determine patient disability the Spanishmodified version of the Health Assessment Questionnaire-Disability Index (HAQ-DI) [22] was used Global functionalstatus was evaluated according to the Steinbrocker clas-sification [23] Rheumatoid factor and C-reactive protein(CRP) were measured by nephelometry using commerciallyavailable kits Erythrocyte sedimentation rate (ESR mmh)was measured using the Wintrobe method




Recognizedsequence


C950Trs2073617


C209Trs3134070


T245Grs3134069







2) 5 pmolmL



3 Results












4 Discussion











OR(95 CI)

C950Trs2073617

CC 9 (176) 28 (431) C 47 (461) 82 (631)001 050 (030ndash085)CT 29 (569) 26 (40) T 55 (539) 48 (369)

TT 13 (255) 11 (169)

C209Trs3134070

CC 39 (750) 52 (722) C 89 (856) 122 (847)10 11 (053ndash22)CT 11 (212) 18 (250) T 15 (144) 22 (153)

TT 2 (38) 2 (28)

T245Grs3134069

AA 41 (788) 66 (857) A 93 (894) 142 (922)051 071 (030ndash169)AC 11 (212) 10 (13) C 11 (106) 12 (78)






C950Trs2073617

CC 9 (500) 16 (444) C 25 (694) 46 (639)067 13 (055ndash303)CT 7 (389) 14 (389) T 11 (306) 26 (361)

TT 2 (111) 6 (167)

C209Trs3134070

CC 17 (810) 29 (690) C 37 (881) 70 (833)060 15 (049ndash443)CT 3 (143) 12 (286) T 5 (119) 14 (167)

TT 1 (48) 1 (24)

T245Grs3134069

AA 20 (909) 39 (886) A 42 (955) 82 (932)072 15 (029ndash795)AC 2 (91) 4 (91) C 2 (45) 6 (68)










Ethical Approval




Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Recognizedsequence


C950Trs2073617


C209Trs3134070


T245Grs3134069







2) 5 pmolmL



3 Results












4 Discussion











OR(95 CI)

C950Trs2073617

CC 9 (176) 28 (431) C 47 (461) 82 (631)001 050 (030ndash085)CT 29 (569) 26 (40) T 55 (539) 48 (369)

TT 13 (255) 11 (169)

C209Trs3134070

CC 39 (750) 52 (722) C 89 (856) 122 (847)10 11 (053ndash22)CT 11 (212) 18 (250) T 15 (144) 22 (153)

TT 2 (38) 2 (28)

T245Grs3134069

AA 41 (788) 66 (857) A 93 (894) 142 (922)051 071 (030ndash169)AC 11 (212) 10 (13) C 11 (106) 12 (78)






C950Trs2073617

CC 9 (500) 16 (444) C 25 (694) 46 (639)067 13 (055ndash303)CT 7 (389) 14 (389) T 11 (306) 26 (361)

TT 2 (111) 6 (167)

C209Trs3134070

CC 17 (810) 29 (690) C 37 (881) 70 (833)060 15 (049ndash443)CT 3 (143) 12 (286) T 5 (119) 14 (167)

TT 1 (48) 1 (24)

T245Grs3134069

AA 20 (909) 39 (886) A 42 (955) 82 (932)072 15 (029ndash795)AC 2 (91) 4 (91) C 2 (45) 6 (68)










Ethical Approval




Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























4 Discussion











OR(95 CI)

C950Trs2073617

CC 9 (176) 28 (431) C 47 (461) 82 (631)001 050 (030ndash085)CT 29 (569) 26 (40) T 55 (539) 48 (369)

TT 13 (255) 11 (169)

C209Trs3134070

CC 39 (750) 52 (722) C 89 (856) 122 (847)10 11 (053ndash22)CT 11 (212) 18 (250) T 15 (144) 22 (153)

TT 2 (38) 2 (28)

T245Grs3134069

AA 41 (788) 66 (857) A 93 (894) 142 (922)051 071 (030ndash169)AC 11 (212) 10 (13) C 11 (106) 12 (78)






C950Trs2073617

CC 9 (500) 16 (444) C 25 (694) 46 (639)067 13 (055ndash303)CT 7 (389) 14 (389) T 11 (306) 26 (361)

TT 2 (111) 6 (167)

C209Trs3134070

CC 17 (810) 29 (690) C 37 (881) 70 (833)060 15 (049ndash443)CT 3 (143) 12 (286) T 5 (119) 14 (167)

TT 1 (48) 1 (24)

T245Grs3134069

AA 20 (909) 39 (886) A 42 (955) 82 (932)072 15 (029ndash795)AC 2 (91) 4 (91) C 2 (45) 6 (68)










Ethical Approval




Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















OR(95 CI)

C950Trs2073617

CC 9 (176) 28 (431) C 47 (461) 82 (631)001 050 (030ndash085)CT 29 (569) 26 (40) T 55 (539) 48 (369)

TT 13 (255) 11 (169)

C209Trs3134070

CC 39 (750) 52 (722) C 89 (856) 122 (847)10 11 (053ndash22)CT 11 (212) 18 (250) T 15 (144) 22 (153)

TT 2 (38) 2 (28)

T245Grs3134069

AA 41 (788) 66 (857) A 93 (894) 142 (922)051 071 (030ndash169)AC 11 (212) 10 (13) C 11 (106) 12 (78)






C950Trs2073617

CC 9 (500) 16 (444) C 25 (694) 46 (639)067 13 (055ndash303)CT 7 (389) 14 (389) T 11 (306) 26 (361)

TT 2 (111) 6 (167)

C209Trs3134070

CC 17 (810) 29 (690) C 37 (881) 70 (833)060 15 (049ndash443)CT 3 (143) 12 (286) T 5 (119) 14 (167)

TT 1 (48) 1 (24)

T245Grs3134069

AA 20 (909) 39 (886) A 42 (955) 82 (932)072 15 (029ndash795)AC 2 (91) 4 (91) C 2 (45) 6 (68)










Ethical Approval




Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Ethical Approval




Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Osteoprotegerin Polymorphisms in a...

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