Research Article The Effects of Gene ... - Hindawi

Research ArticleThe Effects of Gene Polymorphisms in Interleukin-4 andInterleukin-6 on the Susceptibility of Rheumatoid Arthritis ina Chinese Population

Xiang Li1 Wei Chai1 Ming Ni1 Meng Xu1 Zijian Lian1 Lewis Shi2

Yang Bai3 and Yan Wang1

1 Department of Orthopaedics General Hospital of Chinese Peoplersquos Liberation Army Fuxing Road No 28 Haidian DistrictBeijing 100853 China

2Department of Orthopaedics University of Chicago Hospital Maryland Avenue Chicago ll 60673 USA3Department of Stomatology General Hospital of Chinese Peoplersquos Liberation Army Fuxing Road No 28 Haidian DistrictBeijing 100853 China

Correspondence should be addressed to Yan Wang yanwang1961yahoocom

Received 1 November 2013 Revised 16 December 2013 Accepted 9 January 2014 Published 23 February 2014

Academic Editor Wei Chiao Chang

Copyright copy 2014 Xiang Li et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Interleukin-4 (IL-4) and interleukin-6 (IL-6) have been reported to associate with pathogenesis of rheumatoid arthritis(RA) however the role of IL-4 and IL-6 genetic polymorphisms in RA remains unknownMethod A total of 752 unrelated Chinesepatients with RA and 798 healthy Chinese volunteers with no family histories of any autoimmune diseases were recruited Thepromoter IL-4-590CT and IL-6-174GC polymorphismswere genotypedResultThe genotype distributions and allele frequenciesof IL-4-590 CT and IL-6-174 GC polymorphisms in RA patients were significantly different from healthy volunteers Statisticallysignificant differences were observed in genotypes for IL-4-590 and IL-6-174 The frequencies of both the T allele on the IL-4-590 and the C on the IL-6-174 were significantly increased in RA patients Conclusion The IL-4-590 and IL-6-174 promoterpolymorphisms may be associated with increased risk of RA and could be used as genetic marker for assessing the susceptibilityand severity of RA in Chinese

1 Introduction

Rheumatoid arthritis (RA) is a complex chronic inflam-matory disease that predominantly involves synovial jointsleading to cartilage and bone destruction [1 2] Althoughthe etiology of RA remains unknown numerous geneticfactors have been established to contribute as much as 60to RA susceptibility [1 3 4] Furthermore the HLA-DRloci were estimated to account for only about one-third ofthe genetic predisposition to RA [5] A single-nucleotidepolymorphismof ccr6 (rs3093024)was found to be associatedwith susceptibility to rheumatoid arthritis in Japanese andTaiwanese population [6 7] Many cytokine genes werealso playing an important role in its pathogenesis [8ndash12]Interleukin-4 (IL-4) and interleukin-6 (IL-6) are the twomost important cytokine genes associated with RA [4 13ndash18]

IL-4 is the first discovered B-cell pleiotropic cytokine thatpromotes proliferation of T cells and antibodies productionof B cells and plays an important role in the immune system[3 9 19ndash21] IL-6 is a multifunctional B-cell differentiationcytokine which is overexpressed in the affected tissues ofRA patients and induces the final maturation of activatedB cells into immunoglobulin-secreting plasma cells [8 1122ndash24] Therefore polymorphisms affecting genes of IL-4and IL-6 can be linked with RA risk and become of greatinterest to researchers [14 18 19] IL-4-590 promoter poly-morphism a C-to-T base substitution has been suggestedto be associated with RA especially with early pauciarticularjuvenile rheumatoid arthritis [25ndash28] Many previous studiesexamined the association of IL-4 gene polymorphisms withRA [9 12 14 15 20 21] but their data are conflicting so theassociation of IL-4 gene polymorphisms with RA in Chinese

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 265435 5 pageshttpdxdoiorg1011552014265435

2 BioMed Research International

Table 1The clinical and demographic characteristics of all subjects

Variables Cases (119899 = 752) Control (119899 = 798)Sex (femalemale) 354398 367431Age (years) 523 plusmn 163 521 plusmn 171Disease duration (range) 82 years (02ndash201) None

could not be deduced and needs further studies Several poly-morphisms have been revealed in the IL-6 gene includingone of the most important single-nucleotide polymorphisms(SNPs) in the promoter the -174G to C substitution whichaffect IL-6 levels and are associated with RA especially withsystemic juvenile chronic arthritis The association of IL-6-174GC with RA was studied in many populations suchas Europeans Turkish Koreans and Egyptians howeverbesides a very preliminary study in a few Han population inGuangdong there are not any systematic studies about theassociation of IL-6-174GC with RA in Chinese population

Although the association of IL-4-590 and IL-6-174gene polymorphisms with RA has been studied by manyresearchers its relation with RA in Chinese populationremains unknown and could not be deduced In this studywe enrolled 752 Chinese patients and 798 healthy Chinesevolunteers to explore the role of IL-4-590 and IL-6-174 genepolymorphisms in RA

2 Methods

21 Clinical Material A total of 752 unrelated patientswith RA diagnosed according to the American Criteria ofRheumatology (ACR-2011) classification criteria for rheuma-toid arthritis were recruited from the follow-up and inpatientunits The control group included 798 healthy Chinesesubjects with no family histories of any autoimmune diseasesBoth RA and control groups were interviewed to obtaindemographic data and all of the established risk factors Theclinical and demographic data are presented in Table 1 In thecases group 354 patients were females and 398 males themean agewas 523plusmn 163 with a range of 18ndash76 years themeandisease duration time was 82 with a range of 02ndash201 years

The control group consisted of 798 anonymous healthyChinese volunteers who did not show any clinical or labo-ratory signs of autoimmune diseases They were randomlyselected as to match the patients in age gender and ethnicity

22 Genetic Analysis The scientific investigation presentedin this paper has been carried out in accordance with theCode of Ethics of the World Medical Association (Declara-tion ofHelsinki) for experiments involving humans Reactionconditions for genotyping the two polymorphic loci (IL-4-590 and IL-6-174) were performed as follows genomic DNAwas extracted from peripheral venous blood by using theAxygen DNA isolation kit (Axygen CA) as recommended bythe supplier and then stored at minus80∘C until analyzed all poly-merase chain reaction (PCR) primers were synthesized byTaKaRa Biotechnology Co Ltd (Dalian China) as referenceslisted in Table 2 and Table 2 shows the primers sequencesannealing temperature fragment region and size All PCRs

were carried out in 20120583Lof reactionmixture containing 50 ngtemplate DNA 1 times buffer (Tris-HCl 100mmolL pH 83KCl 500mmolL) 025 120583molL primers 20mmolL MgCl2025mmolL dNTPs and 05UTaq polymerase (InvitrogenCorporation Carlsbad CAUSA)ThePCRswere performedon 94∘C for 5min followed by 40 cycles of 94∘C for 30 sannealing at 57∘C for 30 s and 72∘C for 35 s and a finalextension at 72∘C for 10minAll amplifiedPCRproductswerepreliminarily checked by electrophoresis on 20 agarosegel and then observed under UV light All SNPs of IL-4and IL-6 promoters were genotyped by PCR-RFLP Aliquotsof 5 120583L amplified PCR products were digested with 2Uselected restriction enzymes (MBI Fermentas St Leon-RotGermany Table 2) at 37∘C for 2 h following the supplierrsquosmanual Digested products were separated by 20 agarosegel electrophoresis and observed under UV light 10 of ran-dom samples were reanalyzed by DNA sequencing method(ABI3730xl DNA Analyzer Applied Biosystems Foster CityCA USA) to make sure concordance with the genotypingresults fromPCR-RFLPAllele and genotype frequencieswerecompared by 1205942 analysis

23 Statistical Analyses The chi-squared (1205942) test was uti-lized to evaluate the Hardy-Weinberg equilibrium in geno-typic distributions and clinical characteristics between casesand controls All statistical analyses to evaluate if each SNPwas independently associated with RA when adjusted for thepotential confounding effects of important clinical variableswere performed by using the Statistical Package for SocialSciences software (SPSS Windows version release 160 SPSSInc Chicago IL USA) A level of 119875 lt 005 was consideredstatistically significant

3 Results

As the demographic and clinical characteristics of all subjectsin the study were shown in Table 1 there were no significantdifferences in sex ratio and age between RA cases andcontrols

The single-nucleotide polymorphism (SNP) was foundto be in Hardy-Weinberg equilibrium and the genotypedistributions and allele frequencies of IL-4 and IL-6 promoterpolymorphisms in RA and control subjects are summarizedin Table 3 The genotype frequencies and allele frequenciesfor both IL-4 and IL-6 promoter polymorphisms are quitesignificantly different in RA subjects and controls underHardy-Weinberg equilibrium (119875 lt 0001)

As for the IL-4-590CT the frequency of the TT geno-type was significantly higher among RA patients (705)compared to controls (201) and the frequency of theCT genotype was also higher among RA patients (2900versus 2594) but the frequency of the CC genotypewas significantly lower among RA patients (6396) thancontrols (7206) Accordingly the T allele frequency wassignificantly higher in RA patients than controls (2154versus 1497 1205942 = 224713 119875 = 21330 times 10minus6 lt 0001)These results showed a significantly increased risk for RA for

BioMed Research International 3

Table 2 Primer pairs PCR-RFLP analysis for IL-4 and IL-6 promoter polymorphisms

SNP Primer sequences Annealingtemperature (∘C)

Amplificationfragment (bp)

Restrictionenzyme Genotype bp References

IL-4-590CT

51015840-ACTAGGCCTCACCTGATACG-3101584051015840-GTTGTAATGCAGTCCTCCTG-31015840 57 252 BsmFI

CC 192 60CT 252 192 60

TT 252[9]

IL-6-174GC

51015840-GGAGTCACACACTCCACCT-3101584051015840-CTGATTGGAAACCTTATTAAG-31015840 57 525 Hsp92II

GG 327 169GC 327 169 122CC 327 122

[23]

Table 3 The genotype and allele frequencies of IL-4 and IL-6 promoter polymorphisms in cases and controls

Genotype frequencies () Allele frequencies ()C gt T IL-4-590 CC CT TT C TCases (119899 = 752) 481 (6396) 218 (2900) 53 (705) 1180 (7846) 324 (2154)Controls (119899 = 798) 575 (7206) 207 (2594) 16 (201) 1357 (8503) 239 (1497)

1205942

= 271515 119875 = 12610 times 10minus6 1205942

= 224713 119875 = 21330 times 10minus6 OR = 06414G gt C IL-6-174 GG GC CC G CCases (119899 = 752) 613 (8152) 124 (1649) 15 (200) 1350 (8976) 154 (1024)Controls (119899 = 798) 786 (9850) 10 (125) 2 (025) 1582 (9912) 14 (088)

1205942

= 1270661 119875 = 25582 times 10minus28 1205942

= 1324104 119875 = 12168 times 10minus30 OR = 00776

the TT genotype and the T allele after adjustment with sexage BMI smoke status and history of heavy labor work

As for the IL-6-174GC the frequencies of the GGGC and CC genotypes were 8152 1649 and 200 inRA patients significantly different from those observed incontrols which were determined to be 9850 125 and025 respectively (1205942 = 1270661 119875 = 25582 times 10minus28 lt0001) Accordingly the allelic frequencies in the patientsand controls were also significantly different for G allele(8976 versus 9912) and C allele (1024 versus 088)respectively (1205942 = 1324104 119875 = 12168 times 10minus30 lt 0001)These results also showed a significantly increased risk for RAfor the CC genotype and the C allele after adjustment withsex age BMI smoke status and history of heavy labor work

4 Discussion

Rheumatoid arthritis (RA) is a common chronic autoim-mune disorder characterized by the destruction of articu-lar cartilage and bone which affects millions of patientsworldwide In this study we investigated whether IL-4 andIL-6 promoter polymorphisms influence the susceptibilityof RA in a Chinese population Our results showed thatthe TT genotype carriers had markedly higher risk for RAcompared with CC genotype carriers for IL-4 promoterpolymorphisms and the CC genotype carriers had markedlyhigher risk for RA compared with GG genotype carriers forIL-6 promoter polymorphisms besides the T allele of IL-4promoter polymorphisms and the C allele of IL-6 promoterpolymorphisms had shown an association with susceptibilityof RA in a Chinese population

IL-4 is a potent anti-inflammatory cytokine produced byactivated CD4+ lymphocytes mast cells and basophils and

exerts an important role in the immune system on differentcell types [27 29ndash33] In humans the IL-4 gene has beenmapped to chromosome 14q32 [34] The IL-4 gene promotercontains a number of polymorphic loci which were reportedto influence the susceptibility of many diseases includingthe IL-4-33CT [35] IL-4-589CT [36] and IL-4-590CT[20 37 38] especially the genotype and allele frequencies ofIL-4-590CT were well studied and reported to be associatedwith many diseases such as rheumatoid arthritis [3 20]liver disease [37] and gastric cancer [38] To our surprisealthough the role of the genotype and allele frequencies ofIL-4-590CT in association with rheumatoid arthritis hasbeen documented we did not find any reports with regard tothe genetic polymorphisms of IL-4-590CT with rheumatoidarthritis in Chinese population In this study we firstlyreported the role of genetic polymorphisms of IL-4 promoterin RA in Chinese population We found that IL-4-590CTpolymorphisms are associated with the RA risk and theT allele of IL-4 promoter polymorphisms has significantlyincreased the susceptibility of RA inChinese populationThisfinding suggests that the IL-4-590CT polymorphisms maybe used as a genetic marker for the onset and development ofRA in Chinese population

IL-6 is another multifunctional B-cell differentiationcytokine which also plays important role in inducing thefinal maturation of activated B cells into immunoglobulin-secreting plasma cells and influencing the susceptibility ofRA [7 8 11 22ndash24] dermatomyositis and systemic lupuserythematosus [39] liver cirrhosis and hepatocellular carci-noma [40] diabetic microvascular complications [41] coro-nary heart disease [42] acute appendicitis [43] and so onAlthough the association of IL-6-174 GC with RA was wellstudied in many populations such as Europeans Turkish


Koreans and Egyptians besides a very preliminary studyin a few Han population in Guangdong studies about theassociation of IL-6-174 GC with RA in Chinese populationdo not be reported In this study we firstly systematicallystudied the role of genetic polymorphisms of IL-6 promoterin RA in Chinese population We found that IL-6-174GCpolymorphisms are also associated with the RA risk and theC allele of IL-6 promoter polymorphisms has dramaticallyincreased the susceptibility of RA in Chinese populationThis finding suggests that besides the IL-4-590CT the IL-6-174CT polymorphisms may also be used as another geneticmarker for the onset and development of RA in Chinesepopulation

Although our study suggests that the genotype and allelefrequencies of IL-4-590CT and the IL-6-174CT polymor-phisms are associated with the susceptibility of RA in aChinese population to be honest it is also a preliminarystudy and the results need to be further confirmed in anideally larger-scale study

Conflict of Interests

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

Authorsrsquo Contribution

Xiang Li and Wei Chai contributed equally to this paper andshould be considered as cofirst authors

References

[1] C M Weyand and J J Goronzy ldquoPathogenesis of rheumatoidarthritisrdquoMedical Clinics of North America vol 81 no 1 pp 29ndash55 1997

[2] F H Epstein E H S Choy and G S Panayi ldquoCytokinepathways and joint inflamation in rheumatoid arthritisrdquo TheNew England Journal of Medicine vol 344 no 12 pp 907ndash9162001

[3] J Kurko T Besenyei J Laki T T Glant K Mikecz and ZSzekanecz ldquoGenetics of rheumatoid arthritismdasha comprehensivereviewrdquo Clinical Reviews in Allergy amp Immunology vol 45 no2 pp 170ndash179 2013

[4] JM Kim andH Y Kim ldquoPathogenesis of rheumatoid arthritisrdquoJournal of the Korean Medical Association vol 53 no 10 pp853ndash861 2010

[5] G T Nepom ldquoMajor histocompatibility complex-directed sus-ceptibility to rheumatoid arthritisrdquo Advances in Immunologyvol 68 pp 315ndash332 1998

[6] Y Kochi Y Okada A Suzuki et al ldquoA regulatory variant inCCR6 is associated with rheumatoid arthritis susceptibilityrdquoNature Genetics vol 42 no 6 pp 515ndash519 2010

[7] W C Chang P YWoon J C CWei et al ldquoA single-nucleotidepolymorphism of CCR6 (rs3093024) is associated with suscep-tibility to rheumatoid arthritis but not ankylosing spondylitisin a Taiwanese Populationrdquo The Journal of Rheumatology vol39 pp 1765ndash1765 2012

[8] J Jeon K Kim H Kim and C Suh ldquoThe interleukin 6receptor alpha gene polymorphisms are associated with clinical

manifestations of systemic lupus erythematosus in KoreansrdquoInternational Journal of Immunogenetics vol 40 pp 356ndash3602013

[9] Y M Hussein A S El-Shal N A Rezk S M Abdel Galil andS S Alzahrani ldquoInfluence of interleukin-4 gene polymorphismsand interleukin-4 serum level on susceptibility and severity ofrheumatoid arthritis in Egyptian populationrdquo Cytokine vol 61pp 849ndash855 2013

[10] L F da Rocha Junior M J B de Melo Rego M B Cavalcantiet al ldquoSynthesis of a novel thiazolidinedione and evaluationof its modulatory effect on IFN-120574 IL-6 IL-17A and IL-22 production in PBMCs from rheumatoid arthritis patientsrdquoBioMed Research International vol 2013 Article ID 926060 8pages 2013

[11] J Wang A Platt R Upmanyu et al ldquoIL-6 pathway-driveninvestigation of response to IL-6 receptor inhibition in rheuma-toid arthritisrdquo BMJ Open vol 3 no 8 Article ID e003199 2013

[12] A Krabben A Wilson D de Rooy et al ldquoA710 geneticvariants in the IL-4 and IL-4 receptor genes in association withthe severity of joint damage in rheumatoid arthritis a Studyin seven cohortsrdquo Annals of the Rheumatic Diseases vol 72supplement 1 p A51 2013

[13] G G Song S C Bae J H Kim et al ldquoAssociation betweenfunctional Fc receptor-like 3 (FCRL3)-169 CT polymorphismand susceptibility to seropositive rheumatoid arthritis inAsiansa meta-analysisrdquo Human Immunology vol 74 pp 1206ndash12132013

[14] H PengWWangM Zhou et al ldquoAssociations of interleukin-4receptor gene polymorphisms (Q551R I50V) with rheumatoidarthritis evidence from a meta-analysisrdquo Genetic Testing andMolecular Biomarkers vol 17 no 10 pp 768ndash774 2013

[15] A Inanir S Yigit A Tekcan S Tural and G Kismali ldquoIL-4 andMTHFR gene polymorphism in rheumatoid arthritis and theireffectsrdquo Immunology Letters vol 152 no 2 pp 104ndash108 2013

[16] A Crilly J M S Bartlett A White D Stirling H Capell andR Madhok ldquoInvestigation of novel polymorphisms within the31015840 region of the IL-6 gene in patients with rheumatoid arthritisusing Genescan analysisrdquo Cytokine vol 13 no 2 pp 109ndash1122001

[17] P Barrera S Faure J F Prudrsquohomme et al ldquoEuropeangenetic study on rheumatoid arthritis is there a linkage of theinterleukin-1 (IL-1) IL-10 or IL-4 genes to RArdquo Clinical andExperimental Rheumatology vol 19 no 6 pp 709ndash714 2001

[18] Y H Lee S C Bae S J Choi J D Ji and G G SongldquoThe association between interleukin-6 polymorphisms andrheumatoid arthritis a meta-analysisrdquo Inflammation Researchvol 61 no 7 pp 665ndash671 2012

[19] A Krabben AWilson D de Rooy et al ldquoAssociation of geneticvariants in the IL4 and IL4R genes with the severity of jointdamage in rheumatoid arthritis a study in seven cohortsrdquoArthritis amp Rheumatism vol 65 no 12 pp 3051ndash3057 2013

[20] A Pawlik JWrzesniewskaM Florczak B Gawronska-Szklarzand M Herczynska ldquoThe -590 IL-4 promoter polymorphismin patients with rheumatoid arthritisrdquo Rheumatology Interna-tional vol 26 no 1 pp 48ndash51 2005

[21] G G Song S C Bae J H Kim and Y H Lee ldquoInterleukin-4interleukin-4 receptor and interleukin-18 polymorphisms andrheumatoid arthritis ameta-analysisrdquo Immunological Investiga-tions vol 42 no 6 pp 455ndash469 2013

[22] S Lo C Huang H Lin W Chen C Tsai and F TsaildquoCytokine (IL-6) and chemokine (IL-8) gene polymorphisms


among rheumatoid arthritis patients in Taiwanrdquo Clinical andExperimental Rheumatology vol 26 no 4 pp 632ndash637 2008

[23] M Pascual A Nieto L Mataran A Balsa D Pascual-Salcedoand J Martın ldquoIL-6 promoter polymorphisms in rheumatoidarthritisrdquo Genes and Immunity vol 1 no 5 pp 338ndash340 2000

[24] L Fugger N Morling K Bendtzen et al ldquoIL-6 gene polymor-phism in rheumatoid arthritis pauciarticular juvenile rheuma-toid arthritis systemic lupus erythematosus and in healthyDanesrdquo Journal of Immunogenetics vol 16 no 6 pp 461ndash4651989

[25] V SuppiahGenetics of autoimmune diseases a study of immuno-regulatory genes CTLA4 IL-4 IL-4RA SOCS3 IFNG and IL-26in multiple sclerosis rheumatoid arthritis and juvenile idiopathicarthritis [PhD thesis] Queenrsquos University of Belfast BelfastNorthern Ireland 2006

[26] K J Murray A A Grom S D Thompson D Lieuwen M HPasso and D N Glass ldquoContrasting cytokine profiles in thesynovium of different forms of juvenile rheumatoid arthritisand juvenile spondyloarthropathy prominence of interleukin4 in restricted diseaserdquo Journal of Rheumatology vol 25 no 7pp 1388ndash1398 1998

[27] J Huang M Kuo I Hung C Wu L Ou and J ChengldquoLowered IL-4-producing T cells and decreased IL-4 secretionin peripheral blood from subjects with juvenile rheumatoidarthritisrdquo Chang Gung Medical Journal vol 24 no 2 pp 77ndash832001

[28] S DThompson L K Luyrink T B Graham et al ldquoChemokinereceptor CCR4 onCD4+ T cells in juvenile rheumatoid arthritissynovial fluid defines a subset of cells with increased IL-4IFN-120574mRNA ratiosrdquo Journal of Immunology vol 166 no 11 pp 6899ndash6906 2001

[29] R L Coffman ldquoConverging discoveries the first reports of IL-4rdquoThe Journal of Immunology vol 190 no 3 pp 847ndash848 2013

[30] V Gocheva H W Wang B B Gadea et al ldquoIL-4 inducescathepsin protease activity in tumor-associatedmacrophages topromote cancer growth and invasionrdquo Genes and Developmentvol 24 no 3 pp 241ndash255 2010

[31] I L King and M Mohrs ldquoIL-4-producing CD4+ T cells inreactive lymph nodes during helminth infection are T follicularhelper cellsrdquo Journal of Experimental Medicine vol 206 no 5pp 1001ndash1007 2009

[32] A M Arensdorf and D T Rutkowski ldquoEndoplasmic reticulumstress impairs IL-4IL-13 signaling through CEBP120573-mediatedtranscriptional suppressionrdquo Journal of Cell Science vol 126 no17 pp 4026ndash4036 2013

[33] Y Ji S Sun A Xu et al ldquoActivation of natural killer Tcells promotes M2 macrophage polarization in adipose tissueand improves systemic glucose tolerance via interleukin-4 (IL-4)STAT6 protein signaling axis in obesityrdquo Journal of BiologicalChemistry vol 287 no 17 pp 13561ndash13571 2012

[34] A Cantagrel and F Navaux ldquoInterleukin-1120573 interleukin-1receptor antagonist interleukin-4 and interleukin-10 genepolymorphisms relationship to occurrence and severity ofrheumatoid arthritisrdquo Arthritis amp Rheumatism vol 42 no 6pp 1093ndash1100 1999

[35] Y V Gervaziev L V Olenina J V Krasotkina A Y Lupatov SA Mazurina and V B Gervazieva ldquoOct-1 is responsible for theC-33T polymorphism effect in the IL-4 promoterrdquo InternationalJournal of Immunogenetics vol 37 no 1 pp 13ndash20 2010

[36] A Chatterjee A Rathore and T N Dhole ldquoAssociation of IL-4 589 CT promoter and IL-4R120572 I50V receptor polymorphism

with susceptibility to HIV-1 infection in North Indiansrdquo Journalof Medical Virology vol 81 no 6 pp 959ndash965 2009

[37] Z Zheng X Li Z Li and X C Ma ldquoIL-4minus 590CT polymor-phism and susceptibility to liver disease a meta-analysis andmeta-regressionrdquo DNA and Cell Biology vol 32 pp 443ndash4502013

[38] Z Sun Y Cui X Jin and J Pei ldquoAssociation between IL-4-590CgtT polymorphism and gastric cancer riskrdquoTumor Biologypp 1ndash5 2013

[39] M Hristova L Dourmishev Z Kamenarska et al ldquoRole of thepromoter polymorphism IL-6-174GC in dermatomyositis andsystemic lupus erythematosusrdquo BioMed Research Internationalvol 2013 Article ID 315365 5 pages 2013

[40] L Giannitrapani M Soresi A Giacalone et al ldquoIL-6-174GCpolymorphism and IL-6 serum levels in patients with livercirrhosis and hepatocellular carcinomardquo OMICS vol 15 no 3pp 183ndash186 2011

[41] G Rudofsky Jr A Schlotterer P Reismann et al ldquoThe -174GgtCIL-6 gene promoter polymorphism and diabetic microvascularcomplicationsrdquoHormone and Metabolic Research vol 41 no 4pp 308ndash313 2009

[42] G H Zheng H Y Chen and S Q Xiong ldquoPolymorphismsof -174GgtC and -572GgtC in the interleukin 6 (IL-6) geneand coronary heart disease risk a meta-analysis of 27 researchstudiesrdquo PLoS ONE vol 7 no 4 Article ID e34839 2012

[43] S Daneshmandi A Ghasemi and A A Pourfathollah ldquoIL-6-174GC promoter polymorphism in acute appendicitisrdquo Labo-ratory Medicine vol 40 no 10 pp 600ndash603 2009

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


Table 1The clinical and demographic characteristics of all subjects

Variables Cases (119899 = 752) Control (119899 = 798)Sex (femalemale) 354398 367431Age (years) 523 plusmn 163 521 plusmn 171Disease duration (range) 82 years (02ndash201) None

could not be deduced and needs further studies Several poly-morphisms have been revealed in the IL-6 gene includingone of the most important single-nucleotide polymorphisms(SNPs) in the promoter the -174G to C substitution whichaffect IL-6 levels and are associated with RA especially withsystemic juvenile chronic arthritis The association of IL-6-174GC with RA was studied in many populations suchas Europeans Turkish Koreans and Egyptians howeverbesides a very preliminary study in a few Han population inGuangdong there are not any systematic studies about theassociation of IL-6-174GC with RA in Chinese population

Although the association of IL-4-590 and IL-6-174gene polymorphisms with RA has been studied by manyresearchers its relation with RA in Chinese populationremains unknown and could not be deduced In this studywe enrolled 752 Chinese patients and 798 healthy Chinesevolunteers to explore the role of IL-4-590 and IL-6-174 genepolymorphisms in RA

2 Methods

21 Clinical Material A total of 752 unrelated patientswith RA diagnosed according to the American Criteria ofRheumatology (ACR-2011) classification criteria for rheuma-toid arthritis were recruited from the follow-up and inpatientunits The control group included 798 healthy Chinesesubjects with no family histories of any autoimmune diseasesBoth RA and control groups were interviewed to obtaindemographic data and all of the established risk factors Theclinical and demographic data are presented in Table 1 In thecases group 354 patients were females and 398 males themean agewas 523plusmn 163 with a range of 18ndash76 years themeandisease duration time was 82 with a range of 02ndash201 years

The control group consisted of 798 anonymous healthyChinese volunteers who did not show any clinical or labo-ratory signs of autoimmune diseases They were randomlyselected as to match the patients in age gender and ethnicity

22 Genetic Analysis The scientific investigation presentedin this paper has been carried out in accordance with theCode of Ethics of the World Medical Association (Declara-tion ofHelsinki) for experiments involving humans Reactionconditions for genotyping the two polymorphic loci (IL-4-590 and IL-6-174) were performed as follows genomic DNAwas extracted from peripheral venous blood by using theAxygen DNA isolation kit (Axygen CA) as recommended bythe supplier and then stored at minus80∘C until analyzed all poly-merase chain reaction (PCR) primers were synthesized byTaKaRa Biotechnology Co Ltd (Dalian China) as referenceslisted in Table 2 and Table 2 shows the primers sequencesannealing temperature fragment region and size All PCRs

were carried out in 20120583Lof reactionmixture containing 50 ngtemplate DNA 1 times buffer (Tris-HCl 100mmolL pH 83KCl 500mmolL) 025 120583molL primers 20mmolL MgCl2025mmolL dNTPs and 05UTaq polymerase (InvitrogenCorporation Carlsbad CAUSA)ThePCRswere performedon 94∘C for 5min followed by 40 cycles of 94∘C for 30 sannealing at 57∘C for 30 s and 72∘C for 35 s and a finalextension at 72∘C for 10minAll amplifiedPCRproductswerepreliminarily checked by electrophoresis on 20 agarosegel and then observed under UV light All SNPs of IL-4and IL-6 promoters were genotyped by PCR-RFLP Aliquotsof 5 120583L amplified PCR products were digested with 2Uselected restriction enzymes (MBI Fermentas St Leon-RotGermany Table 2) at 37∘C for 2 h following the supplierrsquosmanual Digested products were separated by 20 agarosegel electrophoresis and observed under UV light 10 of ran-dom samples were reanalyzed by DNA sequencing method(ABI3730xl DNA Analyzer Applied Biosystems Foster CityCA USA) to make sure concordance with the genotypingresults fromPCR-RFLPAllele and genotype frequencieswerecompared by 1205942 analysis

23 Statistical Analyses The chi-squared (1205942) test was uti-lized to evaluate the Hardy-Weinberg equilibrium in geno-typic distributions and clinical characteristics between casesand controls All statistical analyses to evaluate if each SNPwas independently associated with RA when adjusted for thepotential confounding effects of important clinical variableswere performed by using the Statistical Package for SocialSciences software (SPSS Windows version release 160 SPSSInc Chicago IL USA) A level of 119875 lt 005 was consideredstatistically significant

3 Results

As the demographic and clinical characteristics of all subjectsin the study were shown in Table 1 there were no significantdifferences in sex ratio and age between RA cases andcontrols

The single-nucleotide polymorphism (SNP) was foundto be in Hardy-Weinberg equilibrium and the genotypedistributions and allele frequencies of IL-4 and IL-6 promoterpolymorphisms in RA and control subjects are summarizedin Table 3 The genotype frequencies and allele frequenciesfor both IL-4 and IL-6 promoter polymorphisms are quitesignificantly different in RA subjects and controls underHardy-Weinberg equilibrium (119875 lt 0001)

As for the IL-4-590CT the frequency of the TT geno-type was significantly higher among RA patients (705)compared to controls (201) and the frequency of theCT genotype was also higher among RA patients (2900versus 2594) but the frequency of the CC genotypewas significantly lower among RA patients (6396) thancontrols (7206) Accordingly the T allele frequency wassignificantly higher in RA patients than controls (2154versus 1497 1205942 = 224713 119875 = 21330 times 10minus6 lt 0001)These results showed a significantly increased risk for RA for






IL-4-590CT


CC 192 60CT 252 192 60

TT 252[9]

IL-6-174GC


GG 327 169GC 327 169 122CC 327 122

[23]



1205942

= 271515 119875 = 12610 times 10minus6 1205942


1205942

= 1270661 119875 = 25582 times 10minus28 1205942

= 1324104 119875 = 12168 times 10minus30 OR = 00776



4 Discussion












References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






IL-4-590CT


CC 192 60CT 252 192 60

TT 252[9]

IL-6-174GC


GG 327 169GC 327 169 122CC 327 122

[23]



1205942

= 271515 119875 = 12610 times 10minus6 1205942


1205942

= 1270661 119875 = 25582 times 10minus28 1205942

= 1324104 119875 = 12168 times 10minus30 OR = 00776



4 Discussion












References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of








References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article The Effects of Gene ... - Hindawi

Documents

Transcript of Research Article The Effects of Gene ... - Hindawi