Research Article MUTYH Gene Polymorphisms as …downloads.hindawi.com/journals/bmri/2015/893796.pdf · Research Article MUTYH Gene Polymorphisms as Risk Factors for Rheumatoid Arthritis

Research ArticleMUTYH Gene Polymorphisms as Risk Factors forRheumatoid Arthritis

Yung-Jen Kung1 Kun Shi Tsai2 Chung-Ming Huang34 Hui-Ju Lin5 Ter-Hsin Chen6

Yu-An Hsu7 Ching-Yao Chang89 Yong-San Huang1 and Lei Wan91011

1 Department of Veterinary Medicine National Chung Hsing University Taichung 40227 Taiwan2Department of Biological Science and Technology National Chiao Tung University Hsinchu 30010 Taiwan3Graduate Institute of Integrated Medicine China Medical University Taichung 40402 Taiwan4Division of Immunology and Rheumatology Department of Internal Medicine China Medical University HospitalTaichung 40402 Taiwan

5Department of Ophthalmology China Medical University Hospital Taichung 40402 Taiwan6Graduate Institute of Veterinary Pathobiology National Chung Hsing University Taichung 40227 Taiwan7Department of Life Science National Tsing Hua University Hsinchu 30013 Taiwan8Department of Medical Research China Medical University Hospital Taichung 40402 Taiwan9Department of Biotechnology Asia University Taichung 41354 Taiwan10School of Chinese Medicine China Medical University No 91 Hsueh-Shih Road Taichung 40402 Taiwan11Department of Gynecology China Medical University Hospital Taichung 40402 Taiwan

Correspondence should be addressed to Lei Wan gome897yahoocomtw

Received 14 June 2014 Revised 2 September 2014 Accepted 3 September 2014

Academic Editor James C C Wei

Copyright copy 2015 Yung-Jen Kung et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objectives MUTYH glycosylase involved in DNA repair pathways may be associated with the risk of autoimmune diseases suchas rheumatoid arthritis (RA) Therefore the association between polymorphisms in the MUTYH gene and RA was evaluatedMethods We recruited 192 RA patients and 192 healthy subjects in Taiwan The 4MUTYH polymorphisms (rs3219463 rs3219476rs3219489 and rs3219493) were detected and haplotype analysis was performed using the BayesianmethodThe genotype and allelicfrequency distributions of the polymorphisms in both RA patients and healthy patients were compared by the chi-square testResults Comparison of the genotypeallele frequencies between individuals with RA and the control groups revealed significantdifferences in 2 MUTYH gene polymorphisms rs3219463 and rs3219476 After we performed a haplotype-specific analysis thehaplotypes Ht6-GTGC and Ht8-GGCG had lower presenting rates in RA patients than in the control groups Furthermore thegenotype frequency of rs3219463 Gminus was significantly increased among patients with immunoglobulin M rheumatoid factorswhereas that of rs3219476 was not Conclusion We demonstrated that the rs3219463 and rs3219476 polymorphisms in RA patientsfrom a Taiwan Chinese population were associated with disease susceptibility These data indicate that theMUTYH gene may playa role in the progression of RA

1 Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune diseasethat can lead to deformities and severe disability becauseof irreversible damage to the tendons joints and bonesAlthough RA is a common type of arthritis with a prevalenceof 1 the pathogenesis is still unknown [1 2] RA can

be considered to be a complex genetic disease and manyldquodisease-riskrdquo genes or ldquodisease-protectiverdquo genes may beinvolved [3 4]

Numerous studies have reported an association betweenHLA-DR gene polymorphisms [4] and RA and CD4 Tcells play important roles in the development of RA [5] Inaddition the chronic inflammatory process of RA ismediated

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 893796 8 pageshttpdxdoiorg1011552015893796

2 BioMed Research International

through the cytokines network which induces the expressionof some destructive enzymes such as matrix metallopro-teinases in the cellular synovial tissue of joints [1] Althoughthere is no conclusive evidence there are hints that genes andenzymes of DNA repair may be involved in some types ofautoimmune diseases It had been suggested that the T cells inRA patients fail to produce sufficient transcripts and proteinsof the DNA repair kinase ataxia telangiectasia mutated(ATM) gene [6] Therefore we are interested in whetherthere are other mechanisms of DNA repair related to theetiology of RA It has been reported that p53 mutations havebeen observed in microdissected RA synovial tissue [7] Thisstrongly suggests that somatic DNA mutations induced byoxidative stress enhance the transformation of cells involvedin the invasive excess growth of the RA joints and that a loss ofgenomic stability is one of the key mechanisms related to theseverity of RA Additionally because inflammatory responsesare the main characteristics of RA [4] we have focused onthe deficient DNA repair enzymes related to severe or chronicinflammation

Inflammation can elicit a variety of cellular signals andinduce the formation of reactive oxygen species (ROS)which threaten the integrity of cellular DNA The oxidizedbase 78-dihydroxy-8-oxoguanine (8-oxo-G) is one of themutagenic products of oxidative DNA damage howevermisincorporated 8-oxo-G can be excised by the base excisionrepair (BER) pathway [8] MUTYH glycosylase which isencoded by MUTYH (mutY Homolog (Escherichia coli)) isinvolved in this pathwayWhen incorrect pairing 8-oxo-GsdotAoccurs MUTYH glycosylase intercepts the 8-oxo-GsdotA basepair and removes the inappropriate A base and provides anopportunity for creation of an 8-oxo-GsdotC substrate for repairby 8-oxo-G glycosylase (OGG1) [9ndash11] MUTYH glycosylasedirectly interacts with various proteins involved in otherDNA repair pathways [12] and defective MUTYH functionis associated with various forms of cancer including lungcancer [13] gastric cancer [14] head and neck squamous cellcarcinomas [15] and colorectal cancer (CRC) [16] Becausethere is evidence which suggests that ROS-induced DNAdamage contributes to tumor development in a mouse modelof chronic inflammation [9] we sought to determine whethermutations inMUTYH are risk factors of RA

We hypothesized that genetic variations in MUTYH (inits promoter region introns or exons)may be associatedwiththe risk of autoimmune diseases such as RA Therefore weanalyzed the genetic and allelic frequencies of 4 polymor-phisms in MUTYH and the haplotypes among RA patientsand healthy controls in the Taiwan Chinese population Wealso compared the genotypes and haplotypes among RApatients with various clinical variables to investigate whethera relationship exists between MUTYH polymorphisms andthe clinical manifestation of RA

2 Methods

21 Patients The study subjects including a total of 192patients with RA and 192 healthy subjects were recruitedfrom China Medical University Hospital in Taiwan Patientswith RA according to the revised America College of

Rheumatology criteria were enrolled [17] Nephelometry wasused to detect rheumatoid factor (RF) Values ≧ 30 IUmLwere defined as positive The presence or history of extra-articular manifestations in patients with RA was recorded[18] The healthy control from the general population wasselected from health examination All individualsrsquo sampleswere collected by venipuncture for genomic DNA isolationInformed consent was obtained from all participants and wasapproved by the local Ethics Committee

22 Genomic DNA Extraction and Genotyping GenomicDNA was prepared from peripheral blood according tostandard protocols of the DNA extraction kit (QiagenValencia CA USA) The four MUTYH single nucleotidepolymorphisms (SNPs) (rs3219463 rs3219476 rs3219489 andrs3219493) were detected by a polymerase chain reaction-restriction fragment length polymorphism technique (PCR-RFLP) PCRs forMUTYH gene polymorphisms were carriedout in a 50 120583L reaction mixture containing 50 ng of genomicDNA 2 to 6 pmole of each primer 1 times Taq polymerase buffer(15mM MgCl

2) and 025U of AmpliTaq DNA polymerase

(Applied Biosystems) The primers PCR conditions andrestriction enzyme cutting sites used to determine MUTYHpolymorphisms were listed in Table 1

23 Haplotype Analysis Haplotypes were inferred from un-phased genotype data using the Bayesian statistical methodavailable in the software program Phase 21 [19 20] All fourSNPs were analyzed with the Phase 21 software and thepopulation datawere divided into 12 groups fromHt1 toHt12A linkage disequilibrium (LD) map was constructed usingHaploview software (version 42 httpwwwbroadinstituteorgscientific-communityscienceprogramsmedical-and-population-geneticshaploviewhaploview) to estimate thecorrelation coefficient between those SNPs

24 Statistical Analysis The chi-square test was utilized toevaluate the Hardy-Weinberg equilibrium in genotypic dis-tributions Also the genotype frequency and allelic frequencydistributions of the polymorphisms in both RA patients andhealthy subjects were compared by the chi-square method(2 times 3 table for genotype frequency 2 times 2 table for allelefrequency) by using the SPSS Version 100 software (SPSSInc Chicago IL USA) Results were considered significantwhen the 119875 values were lt005 The Bonferroni method wasused to correct for multiple testingThe odd ratios (OR) werealso calculated from the genomic and allelic frequencies witha 95 confidence interval (95 CI)

3 Results

31 Demographic Characteristics of Control Subjects and RAPatients A case and control association study was per-formed As shown in the Supplementary Table 1 availableonline at httpdxdoiorg1011552015893796 there are nosignificant differences in gender and age between controlsubjects and RA patients Within the 192 RA patients 151people were female and the mean age was 534 years In the

BioMed Research International 3

Table1Prim

ersa

ndPC

Rcond

ition

susedin

thisstu

dyto

determ

ineM

UTY

Hgene

polymorph

isms

Gene

SNPrs

number

Position

Alleles

Prim

ers

PCR

prod

uct

size(bp

)

PCRcond

ition

s(ann

ealin

gtemperature)

Restric

tion

enzymes

ite

DNA

fragmentsize

(bp)

MUTY

H

rs3219463

145579019

(promoter

region

)AG

Forw

ard51015840-TCC

CACA

AGCC

TTTG

TAAC

C-31015840

Reverse51015840-C

GTA

TTGGGAG

CTCT

GGTG

T-31015840

347

60∘C

SbfI

A347

G161

+186

rs3219476

145575257(in

tron)

GT

Forw

ard51015840-G

TTGCA

GTG

AGCC

AAG

ATCA

-31015840

Reverse51015840-AAC

CTGCC

TTTT

GTG

AGCT

G-31015840

311

58∘C

BtsC

IG58+172+81

T58

+253

rs3219489

145570092(exon)

GC

Forw

ard51015840-TGTG

GTG

AGCA

CCAAAC

CTA-

31015840Re

verse51015840-G

GCT

GTT

CCAG

AAC

ACAG

GT-31015840

319

62∘C

HpyCH

4III

G193

+126

C319

rs3219493

145568856(in

tron)

GC

Forw

ard51015840-C

TCAAG

TGAT

CCAC

CCGAC

T-31015840

Reverse51015840-AGTG

AAG

CCTG

GAG

TGGAG

A-31015840

332

58∘C

HpyCH

4VG332

C139+193


healthy controls 151 peoplewere female and themean agewas505

32 Allele and Genotype Frequency of MUTYH Polymor-phisms Of the four tested SNPs three SNPs were foundto be in Hardy-Weinberg equilibrium (119875 value gt 005)however the genotype distribution of the rs3219476 was notcompatible withHardy-Weinberg equilibriumThe genotypeallele dominant and recessive distributions of the 4 polymor-phisms are shown in Table 2

Comparison of the genotypes between individuals withRA and the control groups revealed significant differences inthe frequency of 2MUTYH gene polymorphisms rs3219463and rs3219476 The 119875 values were corrected for multiplecomparison by Bonferroni correction represented as 119888119875value For the rs3219463GApolymorphism the 119888119875 valuewas000024 and the odds ratio (OR) was 116 (95 confidenceinterval (CI) 062ndash217) for the heterozygous mutant GAand 041 (95CI 021ndash082) for the homozygousmutant GGThere was also a significant difference for the polymorphismrs3219476 TG (119888119875 = 00036) the OR was 212 (95 CI126ndash359) for the heterozygous mutant TG and 381 (95CI 181ndash804) for the homozygous mutant TT Howeverthere was no significant difference between the RA patientsand control individuals for rs3219489 or rs3219493 Thedifferences in allele frequencies of these polymorphismsbetween individuals with and without RA were similar tothe results of the genotypes frequencies (Table 2) There weresignificant differences in allele frequencies for the rs3219463(119888119875 = 00064 OR 063 and 95 CI 047ndash084) and rs3219476polymorphism (119888119875 = 00096 OR 156 and 95 CI 117ndash207)while the allele frequencies of the rs3219489 and rs3219493polymorphisms were not significantly different (119888119875 = 0318and 2684 resp) The dominant and recessive distributionsof these two RA-related SNPs also presented significant dif-ferences of the frequencies except for dominant distributionof rs319463These results demonstrate a significant differencein the allele genotype dominant and recessive distributionfor the rs3219463 and rs3219476 polymorphisms between theRA and control groups

33 Distribution of MUTYH Haplotypes To further inves-tigate whether haplotypes of MUTYH were correlated withRA the LD map was constructed (Figure 1) and haplotypefrequencies differences were estimated for the 4 identifiedpolymorphisms The pattern of LD in the MUTYH locuswas measured by 1198631015840 score among the 4 SNPs Linkagedisequilibrium indicated that rs3219493 and rs3219489 had ahigh LD score (1198631015840 gt 80) and so did rs3219493 and rs3219476However rs3219463 and rs3219476 had a low LD score (1198631015840 =61)

Of the 12 observed haplotypes haplotypes with frequencygreater than 5 were presented (Table 3) and were furtheranalyzed After performing a haplotype-specific analysisthe haplotypes Ht6-GTGC and Ht8-GGCG demonstratedsignificantly lower frequencies in RA patients than in thecontrol groups (Ht6-GTGC 119875 = 00076 OR 041 and 95CI 021ndash081 Ht8-GGCG 119875 = 00070 OR 032 and 95 CI

1 2 3 4

rs3219493

rs3219489

rs3219476

83

93

63

69

68

61

rs3219463

Figure 1 Four SNPs on the LD map pf MUTYH gene LD coeffi-cients (1198631015840) between four SNPs of MUTYH gene were graphicallyindicated

013ndash076) while after Bonferroni correction the differenceswere not significant There were no differences in haplotypefrequency between the RA and control groups for the otherhaplotypes

34MUTYHPolymorphisms andClinical Features of RA Theassociation between the clinical features of patients with RAwith the genotypes rs3219463 and rs3219476 was analyzed(Table 4) Among the patients with RA the genotype fre-quency of rs3219463 Gminus was significantly greater in patientswith immunoglobulinMrheumatoid factors (RF) (119875 = 0019OR 270 and 95 CI 1155ndash6312) However a comparison ofthe genotype frequency of rs3219476 and the clinical featuresrevealed no significant difference among RA patients Therewas no significant association between the haplotypes Ht6-GTGC and Ht8-GGCG and the clinical features among RApatients (Table 5)

4 Discussions

In the present study we had found two SNPs of the119872119880119879119884119867gene associated with RA The geneticallelic frequenciesof rs3219463 (in the promoter region) and rs3219476 (inan intron) showed significant differences between RA andcontrol patients Our results provide the evidence for geneticassociation coordinated by these polymorphisms with theclinical features of RA There have been some reports thatshowed the relationship between the DNA repair pathwayand autoimmune diseases For example dysfunctional T cells


Table 2 Association between genotypic and allelic distributions ofMUTYH gene polymorphisms and individuals with RA

SNP database ID RAnumber ()

Controlsnumber () 119875 value119888119875 value Odds ratio (95 CI)

rs3219463GG 37 (193) 76 (396) 000006lowast 041 (021ndash082)GA 129 (672) 94 (490) 000024lowast 116 (062ndash217)AA 26 (135) 22 (114) 100G allele 203 (529) 246 (641) 00016lowast 063 (047ndash084)A allele 181 (471) 138 (359) 00064lowast 100GG + GA 166 (865) 170 (885) 05371 083 (045ndash152)AA 26 (135) 22 (115) 21496 100GG 37 (193) 76 (396) 000001lowast 036 (023ndash058)GA + AA 155 (807) 116 (604) 000006lowast

rs3219476TT 33 (172) 17 (89) 00009lowast 381 (181ndash804)TG 132 (688) 122 (635) 00036lowast 212 (126ndash359)GG 27 (140) 53 (276) 100T allele 198 (516) 156 (406) 00024lowast 156 (117ndash207)G allele 186 (484) 228 (594) 00096lowast 100TT + TG 165 (86) 139 (724) 00011lowast 233 (139ndash390)GG 27 (140) 53 (276) 00044lowast 100TT 33 (172) 17 (89) 0013lowast 217 (117ndash405)TG + GG 159 (828) 178 (911) 00650 100

rs3219489GG 52 (271) 70 (365) 01383 061 (033ndash113)GC 106 (552) 94 (490) 05532 093 (052ndash165)CC 34 (177) 28 (145) 100G allele 210 (547) 234 (609) 00795 077 (058ndash103)C allele 174 (453) 150 (391) 03180 100GG + GC 158 (823) 164 (855) 04053 079 (046ndash137)CC 34 (177) 28 (145) 16212 100GG 52 (271) 70 (365) 00485lowast 065 (042ndash100)GC + CC 140 (729) 122 (635) 01940 100

rs3219493GG 164 (854) 161 (839) 0671 113 (065ndash197)GC 28 (146) 31 (161) 26840 100CC 0 (0) 0 (0)G allele 356 (927) 353 (919) 06844 112 (066ndash190)C allele 28 (73) 31 (81) 27376 100GG 164 (854) 161 (839) 06712 113 (065ndash197)GC + CC 28 (146) 31 (161) 13424 100

95 CI 95 confidence intervalslowast119875 value of lt005 was determined as statistically significant The chi-square test was performed to obtain the 119875 valuelowast119888119875 value 119875 value corrected by Bonferroni correction

show chronic inflammatory immune responses in the syn-ovium of RA patients and the DNA repair kinase ATMmay be involved [6] Furthermore it has been postulatedthat SNPs in the X-ray repair cross-complementing gene 1(XRCC1) one of the BER proteins may correlate with RA[21] To our knowledge this study is the first investigation

to demonstrate that gene polymorphisms inMUTYH whichencodes a glycosylase member of BER may contribute to thepathogenesis of RA

ROS-associated base damage can be repaired by the BERpathway which is initiated by many damage-specific glyco-sylases that cleave the glycosylic bond between the damaged


Table 3 Odds ratios and 95 confidence intervals for the association betweenMUTYH gene haplotypes and RA

Haplotype rs3219463 rs3219476 rs3219489 rs3219493 RA Control 119875 value119888119875 value Odds ratio (95 CI)Ht1 G G G G 150 (391) 173 (451) 0092707416 078 (059ndash104)Ht2 A T C G 124 (323) 102 (266) 008150652 132 (097ndash180)Ht3 G T C G 19 (49) 11 (29) 0136210896 177 (083ndash376)Ht4 A G C G 17 (44) 15 (39) 07185744 114 (056ndash232)Ht5 G T G G 13 (34) 12 (31) 0838967112 109 (049ndash241)Ht6 G T G C 13 (34) 30 (78) 00076lowast00608 041 (021ndash081)Ht7 A G G G 11 (29) 19 (49) 0136210896 057 (027ndash121)Ht8 G G C G 7 (18) 21 (55) 0007lowast0056 032 (013ndash076)95 CI 95 confidence intervalslowast119875 value of lt005 was determined as statistically significant

The chi-square test was performed to obtain the 119875 valuelowast119888119875 value 119875 value corrected by Bonferroni correction

Table 4 Frequencies of rs3219463 genotypes of RA patients with various clinical features

rs3219463 genotype RF Extra-articular Erosion rs3219476 genotype RF Extra-articular Erosion119899 () 119899 () 119899 () 119899 () 119899 () 119899 ()

Gminus (119899 = 166) 126 (759) 81 (488) 83 (500) Tminus (119899 = 165) 119 (721) 75 (455) 87 (527)AA (119899 = 26) 14 (538) 10 (385) 14 (538) GG (119899 = 27) 22 (815) 16 (593) 10 (370)P 0019lowast 0326 0715 119875 0307 0183 0131OR 2700 1525 0857 OR 0588 0573 189695 CI 1155ndash6312 0654ndash3556 0374ndash1964 95 CI 021ndash165 025ndash131 082ndash439Aminus (119899 = 155) 109 (703) 76 (490) 79 (510) Gminus (119899 = 159) 120 (755) 77 (484) 77 (484)GG (119899 = 37) 31 (838) 15 (405) 18 (486) TT (119899 = 33) 21 (636) 14 (424) 20 (606)P 0098 0353 0800 119875 0161 0530 0203OR 0459 1411 1097 OR 1758 1274 061095 CI 0179ndash1174 0681ndash22922 0535ndash2249 95 CI 079ndash390 060ndash272 028ndash13195 CI 95 confidence intervals OR odds ratiolowast119875 value of lt005 was determined as statistically significant The chi-square test was performed to obtain the 119875 value

RF rheumatoid factors

base and the sugar phosphate backbone [8]MUTYH whichremoves adenine paired with 8-oxo-G is important in sup-pressing GsdotC to TsdotA transversions and in preventing CRCin people and mice In murine studies MUTYH-deficientmice were more susceptible to spontaneous tumors [22]and oxidative stress-induced intestinal tumors MUTYH-associated polyposis (MAP) an autosomal recessive disorderis associated with biallelic germline mutations in MUTYH[11 23] and was first reported in 2002 Since then manyaspects of MAP have been investigated The possibility thatsome mutations are nonpathogenic polymorphisms that arefound coincidentally in patients with colorectal adenomasand carcinomas cannot be ruled out [8]

RA a chronic inflammatory disease with tissue-destructive potential is now recognized as a complex geneticdisorder Although numerous studies have reported anassociation between RA and HLA-DRB1lowast04 cytokinesand CD4 cells other genes may contribute to diseasesusceptibility [4] Disease risk genes may increase the riskof pathological reactions which suggests a gene-dose effectOne effect involves a snowballing mechanism that leads toincreased levels of cellular damage and death resulting inmore inflammation that induces the production of more

ROS [9] Several SNPs of various genes were found related tothe risk of RA among Taiwanese or Chinese population Forexample an SNP (rs7135617) located in the intron region ofORAI1 gene was associated with a risk of RA in the Taiwanesepopulation [24] The polymorphism of FCGR2B was relatedto early-onset of RA and the polymorphism of FCGR3Amay influence RF production [25 26] Furthermore therewere many studies discussing the IL-family associated withpathogenesis of RA in Chinese population such as IL-4IL-6 and IL-18 [27 28] The question remains whetherdefects in DNA damage repair render individuals susceptibleto RA or whether this is a consequence of the diseaseThe accumulation of DNA damage may also have broaderimplications on impairing diverse cellular functions

In haplotype studies 2 haplotypes of the MUTYH geneshowed differences between the RA and control groups(Table 3) The haplotypes Ht6-GTGC and Ht8-GGCG hadlower presenting rates in RA patients than in the controlgroups these appeared to be ldquoprotectiverdquo haplotypes Thers3219463 polymorphism positioned in the MUTYH genepromoter region the rs3219476 and rs3219493 polymor-phisms positioned in the MUTYH gene intron region andrs3219489 polymorphism positioned in the MUTYH gene


Table 5 Frequencies ofMUTYH haplotype (Ht7) of RA patients with various clinical features

Ht6 genotype RF Extra-articular Erosion Ht8 genotype RF Erosion119899 () 119899 () 119899 () 119899 () 119899 ()

Ht6non-ht6 (119899 = 13) 10 (769) 7 (538) 9 (692) Ht8non-ht8 (119899 = 7) 6 (857) 3 (429)Non-ht6non-ht6 (119899 = 179) 130 (726) 84 (469) 88 (492) Non-ht8non-ht8 (119899 = 185) 134 (724) 94 (508)P 0736 0630 0162 119875 0438 0679OR 1256 1319 2327 OR 2284 072695 CI 033ndash476 043ndash408 069ndash783 95 CI 027ndash1944 016ndash33395 CI 95 confidence intervals OR odds ratioRF rheumatoid factors

exon region (Gln338His) We found that investigation of thehaplotype of the MUTYH gene gave an additional sign fordetermining whether this gene is associated with RA

Previous studies have shown that patients with erosiveRF-positive joint disease are characterized by the genotypeHLA-DRB1lowast4RA nonassociated allele [4] In this study wefound that the Gminus frequency of rs3219463 was significantlyincreased in patients with RF (Table 3) Therefore we pos-tulated that rs3219463 may be a risk factor for RF in RApatients How the RF factor contributes to the disease processis unknown but it has been known to facilitate antigenpresentation Immune complexes of RF and immunoglobulinG may contribute to disease activity by activating comple-ments and stimulating cytokine synthesis [1] From a clinicalperspective the dissection of the phenotypic and genotypicvariants of RA is critical for further exploration of targetedtherapeutic treatment

There were two major limitations in this study Firstlybecause of the noncompatibility in Hardy-Weinberg equi-librium of rs3219476 genotype distributions we minimizederrors in genotyping and validated our findings by repeatingthe genotyping analysis several times and obtaining con-sistent results Therefore occurrence of genotyping errorsin this study was kept to a minimum Moreover the TGgenotype compared with TT genotype of rs3219476 has beenrevealed as a protective genotype of cholangiocarcinomaamong individuals of Han Chinese living in Jiangsu provincein China [29] Based on the genotype frequencies in thecontrols the 119875 value is 00859 which is in Hardy-Weinbergequilibrium but very close to 005 Compared to our data weregard the occurrence frequency of rs3219476 as consistentFurthermore we tried to trace the ancestry backgroundof controls and patients and found that study subjectswere a mixed population of Minnan Hakka and Cantondescendants According to the paper published by Pan etal [30] they mentioned that the SNP profiles in the majorhistocompatibility complex (MHC) region (6p213) showedno significant difference amongMinnan descendants Hakkadescendants and mixed population of Minnan and Hakkadescendants which indicate the homogeneity of the popu-lation Thus population stratification should not produce inthis study [30]

Secondly due to the limited subject number from onemedical center in this study many comparisons revealed nosignificant differences after Bonferroni correction Besidesneither the genotype of rs3219463 and rs3219476 nor the

haplotypes were significantly associated in patients withextra-articular RA erosion However we had evaluated thepower in this sample size For rs3219463 and rs3219476the statistical powers were 721 and 699 respectivelyAnd these two SNPs represent low linkage disequilibrium(Figure 1) Thus the MUTYH polymorphisms did associatewith susceptibility of RA Even so our data may be biasedby the relatively small number of subjects and larger-scalestudies are needed to confirm our findings

In conclusion our findings demonstrate that thers3219463 and rs3219476 polymorphisms in the MUTYHgene in patients with RA in a Taiwanese populationwere associated with disease susceptibility Although theexact function of the MUTYH polymorphisms cannot beconfirmed in this study we suggest that genetic variations inthe MUTYH gene may affect BER efficiency and play a rolein the progression of RA

Conflict of Interests

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

Authorsrsquo Contribution

Yung-Jen Kung and Kun Shi Tsai contributed equally to thework and therefore should be considered equivalent authors

Acknowledgments

This study was supported by a grant from the National Sci-ence Council (NSC101-2320-B-039-038 and NSC102-2320-B-039-043-MY3) Taipei Taiwan grants from the ChinaMedical University Hospital (DMR-103-064 and CMU102-ASIA-09) Taichung Taiwan and a grant in part by TaiwanMinistry of Health and Welfare Clinical Trial and ResearchCenter of Excellence (DOH102-TD-B-111-004)

References

[1] M Feldmann F M Brennan and R N Maini ldquoRheumatoidarthritisrdquo Cell vol 85 no 3 pp 307ndash310 1996

[2] M Ziff and A Smith ldquoRheumatoid arthritismdashits present andfuturerdquo Journal of Rheumatology vol 17 no 2 pp 127ndash133 1990

[3] A H Lynn C K Kwoh C M Venglish C E Aston and AChakravarti ldquoGenetic epidemiology of rheumatoid arthritisrdquo


American Journal of Human Genetics vol 57 no 1 pp 150ndash1591995

[4] C M Weyand P A Klimiuk and J J Goronzy ldquoHeterogene-ity of rheumatoid arthritis from phenotypes to genotypesrdquoSpringer Seminars in Immunopathology vol 20 no 1-2 pp 5ndash22 1998

[5] M R Ehrenstein J G Evans A Singh et al ldquoCompromisedfunction of regulatory T cells in rheumatoid arthritis and rever-sal by anti-TNF120572 therapyrdquo Journal of Experimental Medicinevol 200 no 3 pp 277ndash285 2004

[6] L Shao H Fujii I Colmegna H Oishi J J Goronzy and CM Weyand ldquoDeficiency of the DNA repair enzyme ATM inrheumatoid arthritisrdquo Journal of Experimental Medicine vol206 no 6 pp 1435ndash1449 2009

[7] Y Yamanishi D L Boyle S Rosengren D R Green N JZvaifler and G S Firestein ldquoRegional analysis of p53mutationsin rheumatoid arthritis synoviumrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no15 pp 10025ndash10030 2002

[8] S S David V L OrsquoShea and S Kundu ldquoBase-excision repair ofoxidative DNA damagerdquo Nature vol 447 no 7147 pp 941ndash9502007

[9] L B Meira J M Bugni S L Green et al ldquoDNA damageinduced by chronic inflammation contributes to colon carcino-genesis in micerdquo The Journal of Clinical Investigation vol 118no 7 pp 2516ndash2525 2008

[10] R C Niessen R H Sijmons J Ou et al ldquoMUTYH and themismatch repair system partners in crimerdquo Human Geneticsvol 119 no 1-2 pp 206ndash211 2006

[11] M L M Poulsen and M L Bisgaard ldquoMUTYH AssociatedPolyposis (MAP)rdquo Current Genomics vol 9 no 6 pp 420ndash4352008

[12] A R Parker and J R Eshleman ldquoHumanMutY gene structureprotein functions and interactions and role in carcinogenesisrdquoCellular and Molecular Life Sciences vol 60 no 10 pp 2064ndash2083 2003

[13] A Miyaishi K Osawa Y Osawa et al ldquoMUTYH Gln324Hisgene polymorphism and genetic susceptibility for lung cancerin a Japanese populationrdquo Journal of Experimental and ClinicalCancer Research vol 28 no 1 article 10 2009

[14] Y Zhang X Liu Y Fan et al ldquoGermline mutations and poly-morphic variants in MMR E-cadherin and MYH genes associ-ated with familial gastric cancer in Jiangsu of Chinardquo Interna-tional Journal of Cancer vol 119 no 11 pp 2592ndash2596 2006

[15] T Sliwinski L Markiewicz P Rusin et al ldquoPolymorphisms ofthe DNA base excision repair gene MUTYH in head and neckcancerrdquo Experimental Oncology vol 31 no 1 pp 57ndash59 2009

[16] HChen L XuQQi Y YaoM Zhu andYWang ldquoAhaplotypevariation affecting the mitochondrial transportation of hMYHprotein could be a risk factor for colorectal cancer in ChineserdquoBMC Cancer vol 8 article 269 2008

[17] 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

[18] J-H Yen J-R Chen W-J Tsai J-J Tsai and H-W LiuldquoHLA-DRB1 genotyping in patients with rheumatoid arthritisin Taiwanrdquo Journal of Rheumatology vol 22 no 8 pp 1450ndash1454 1995

[19] M Stephens and P Donnelly ldquoA comparison of bayesian meth-ods for haplotype reconstruction from population genotype

datardquo The American Journal of Human Genetics vol 73 no 5pp 1162ndash1169 2003

[20] M Stephens N J Smith and P Donnelly ldquoA new statisticalmethod for haplotype reconstruction from population datardquoAmerican Journal of Human Genetics vol 68 no 4 pp 978ndash989 2001

[21] AKoyama YKubota T Shimamura and SHoriuchi ldquoPossibleassociation of the X-ray cross complementing gene 1 (XRCC1)Arg280His polymorphism as a risk for rheumatoid arthritisrdquoRheumatology International vol 26 no 8 pp 749ndash751 2006

[22] K Sakamoto Y Tominaga K Yamauchi et al ldquoMUTYH-null mice are susceptible to spontaneous and oxidative stress-induced intestinal tumorigenesisrdquo Cancer Research vol 67 no14 pp 6599ndash6604 2007

[23] J P Cheadle and J R Sampson ldquoMUTYH-associated poly-posismdashfrom defect in base excision repair to clinical genetictestingrdquo DNA Repair vol 6 no 3 pp 274ndash279 2007

[24] J-H Yen C-M Chang Y-W Hsu et al ldquoA polymorphism ofORAI1 rs7135617 is associated with susceptibility to rheumatoidarthritisrdquo Mediators of Inflammation vol 2014 Article ID834831 5 pages 2014

[25] J-Y Chen C-M Wang C-C Ma et al ldquoA transmembranepolymorphism in Fc120574RIIb (FCGR2B) is associated with theproduction of anti-cyclic citrullinated peptide autoantibodies inTaiwanese RArdquo Genes amp Immunity vol 9 no 8 pp 680ndash6882008

[26] J-Y Chen C-M Wang J-M Wu H-H Ho and S-F LuoldquoAssociation of rheumatoid factor production with Fc120574RIIIapolymorphism in Taiwanese rheumatoid arthritisrdquo Clinical andExperimental Immunology vol 144 no 1 pp 10ndash16 2006

[27] S Chen F Jiang J Ren J Liu and W Meng ldquoAssociation ofIL-18 polymorphisms with rheumatoid arthritis and systemiclupus erythematosus in Asian populations a meta-analysisrdquoBMCMedical Genetics vol 13 article 107 2012

[28] X Li W Chai M Ni et al ldquoThe effects of gene polymorphismsin interleukin-4 and interleukin-6 on the susceptibility ofrheumatoid arthritis in a Chinese populationrdquo BioMed ResearchInternational vol 2014 Article ID 265435 5 pages 2014

[29] S-H You X Wang S Huang et al ldquoMYH rs3219476 andrs3219472 polymorphisms and risk of cholangiocarcinomardquoMolecular Medicine Reports vol 7 no 1 pp 347ndash351 2013

[30] W H Pan C S J Fann J Y Wu et al ldquoHan Chinese cell andgenome bank in Taiwan purpose design and ethical consider-ationsrdquo Human Heredity vol 61 no 1 pp 27ndash30 2006

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



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


through the cytokines network which induces the expressionof some destructive enzymes such as matrix metallopro-teinases in the cellular synovial tissue of joints [1] Althoughthere is no conclusive evidence there are hints that genes andenzymes of DNA repair may be involved in some types ofautoimmune diseases It had been suggested that the T cells inRA patients fail to produce sufficient transcripts and proteinsof the DNA repair kinase ataxia telangiectasia mutated(ATM) gene [6] Therefore we are interested in whetherthere are other mechanisms of DNA repair related to theetiology of RA It has been reported that p53 mutations havebeen observed in microdissected RA synovial tissue [7] Thisstrongly suggests that somatic DNA mutations induced byoxidative stress enhance the transformation of cells involvedin the invasive excess growth of the RA joints and that a loss ofgenomic stability is one of the key mechanisms related to theseverity of RA Additionally because inflammatory responsesare the main characteristics of RA [4] we have focused onthe deficient DNA repair enzymes related to severe or chronicinflammation

Inflammation can elicit a variety of cellular signals andinduce the formation of reactive oxygen species (ROS)which threaten the integrity of cellular DNA The oxidizedbase 78-dihydroxy-8-oxoguanine (8-oxo-G) is one of themutagenic products of oxidative DNA damage howevermisincorporated 8-oxo-G can be excised by the base excisionrepair (BER) pathway [8] MUTYH glycosylase which isencoded by MUTYH (mutY Homolog (Escherichia coli)) isinvolved in this pathwayWhen incorrect pairing 8-oxo-GsdotAoccurs MUTYH glycosylase intercepts the 8-oxo-GsdotA basepair and removes the inappropriate A base and provides anopportunity for creation of an 8-oxo-GsdotC substrate for repairby 8-oxo-G glycosylase (OGG1) [9ndash11] MUTYH glycosylasedirectly interacts with various proteins involved in otherDNA repair pathways [12] and defective MUTYH functionis associated with various forms of cancer including lungcancer [13] gastric cancer [14] head and neck squamous cellcarcinomas [15] and colorectal cancer (CRC) [16] Becausethere is evidence which suggests that ROS-induced DNAdamage contributes to tumor development in a mouse modelof chronic inflammation [9] we sought to determine whethermutations inMUTYH are risk factors of RA

We hypothesized that genetic variations in MUTYH (inits promoter region introns or exons)may be associatedwiththe risk of autoimmune diseases such as RA Therefore weanalyzed the genetic and allelic frequencies of 4 polymor-phisms in MUTYH and the haplotypes among RA patientsand healthy controls in the Taiwan Chinese population Wealso compared the genotypes and haplotypes among RApatients with various clinical variables to investigate whethera relationship exists between MUTYH polymorphisms andthe clinical manifestation of RA

2 Methods

21 Patients The study subjects including a total of 192patients with RA and 192 healthy subjects were recruitedfrom China Medical University Hospital in Taiwan Patientswith RA according to the revised America College of

Rheumatology criteria were enrolled [17] Nephelometry wasused to detect rheumatoid factor (RF) Values ≧ 30 IUmLwere defined as positive The presence or history of extra-articular manifestations in patients with RA was recorded[18] The healthy control from the general population wasselected from health examination All individualsrsquo sampleswere collected by venipuncture for genomic DNA isolationInformed consent was obtained from all participants and wasapproved by the local Ethics Committee

22 Genomic DNA Extraction and Genotyping GenomicDNA was prepared from peripheral blood according tostandard protocols of the DNA extraction kit (QiagenValencia CA USA) The four MUTYH single nucleotidepolymorphisms (SNPs) (rs3219463 rs3219476 rs3219489 andrs3219493) were detected by a polymerase chain reaction-restriction fragment length polymorphism technique (PCR-RFLP) PCRs forMUTYH gene polymorphisms were carriedout in a 50 120583L reaction mixture containing 50 ng of genomicDNA 2 to 6 pmole of each primer 1 times Taq polymerase buffer(15mM MgCl

2) and 025U of AmpliTaq DNA polymerase

(Applied Biosystems) The primers PCR conditions andrestriction enzyme cutting sites used to determine MUTYHpolymorphisms were listed in Table 1

23 Haplotype Analysis Haplotypes were inferred from un-phased genotype data using the Bayesian statistical methodavailable in the software program Phase 21 [19 20] All fourSNPs were analyzed with the Phase 21 software and thepopulation datawere divided into 12 groups fromHt1 toHt12A linkage disequilibrium (LD) map was constructed usingHaploview software (version 42 httpwwwbroadinstituteorgscientific-communityscienceprogramsmedical-and-population-geneticshaploviewhaploview) to estimate thecorrelation coefficient between those SNPs

24 Statistical Analysis The chi-square test was utilized toevaluate the Hardy-Weinberg equilibrium in genotypic dis-tributions Also the genotype frequency and allelic frequencydistributions of the polymorphisms in both RA patients andhealthy subjects were compared by the chi-square method(2 times 3 table for genotype frequency 2 times 2 table for allelefrequency) by using the SPSS Version 100 software (SPSSInc Chicago IL USA) Results were considered significantwhen the 119875 values were lt005 The Bonferroni method wasused to correct for multiple testingThe odd ratios (OR) werealso calculated from the genomic and allelic frequencies witha 95 confidence interval (95 CI)

3 Results

31 Demographic Characteristics of Control Subjects and RAPatients A case and control association study was per-formed As shown in the Supplementary Table 1 availableonline at httpdxdoiorg1011552015893796 there are nosignificant differences in gender and age between controlsubjects and RA patients Within the 192 RA patients 151people were female and the mean age was 534 years In the


Table1Prim

ersa

ndPC

Rcond

ition

susedin

thisstu

dyto

determ

ineM

UTY

Hgene

polymorph

isms

Gene

SNPrs

number

Position

Alleles

Prim

ers

PCR

prod

uct

size(bp

)

PCRcond

ition

s(ann

ealin

gtemperature)

Restric

tion

enzymes

ite

DNA

fragmentsize

(bp)

MUTY

H

rs3219463

145579019

(promoter

region

)AG

Forw

ard51015840-TCC

CACA

AGCC

TTTG

TAAC

C-31015840

Reverse51015840-C

GTA

TTGGGAG

CTCT

GGTG

T-31015840

347

60∘C

SbfI

A347

G161

+186

rs3219476

145575257(in

tron)

GT

Forw

ard51015840-G

TTGCA

GTG

AGCC

AAG

ATCA

-31015840

Reverse51015840-AAC

CTGCC

TTTT

GTG

AGCT

G-31015840

311

58∘C

BtsC

IG58+172+81

T58

+253

rs3219489

145570092(exon)

GC

Forw

ard51015840-TGTG

GTG

AGCA

CCAAAC

CTA-

31015840Re

verse51015840-G

GCT

GTT

CCAG

AAC

ACAG

GT-31015840

319

62∘C

HpyCH

4III

G193

+126

C319

rs3219493

145568856(in

tron)

GC

Forw

ard51015840-C

TCAAG

TGAT

CCAC

CCGAC

T-31015840


AAG

CCTG

GAG

TGGAG

A-31015840

332

58∘C

HpyCH

4VG332

C139+193







1 2 3 4

rs3219493

rs3219489

rs3219476

83

93

63

69

68

61

rs3219463




4 Discussions








































Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table1Prim

ersa

ndPC

Rcond

ition

susedin

thisstu

dyto

determ

ineM

UTY

Hgene

polymorph

isms

Gene

SNPrs

number

Position

Alleles

Prim

ers

PCR

prod

uct

size(bp

)

PCRcond

ition

s(ann

ealin

gtemperature)

Restric

tion

enzymes

ite

DNA

fragmentsize

(bp)

MUTY

H

rs3219463

145579019

(promoter

region

)AG

Forw

ard51015840-TCC

CACA

AGCC

TTTG

TAAC

C-31015840

Reverse51015840-C

GTA

TTGGGAG

CTCT

GGTG

T-31015840

347

60∘C

SbfI

A347

G161

+186

rs3219476

145575257(in

tron)

GT

Forw

ard51015840-G

TTGCA

GTG

AGCC

AAG

ATCA

-31015840

Reverse51015840-AAC

CTGCC

TTTT

GTG

AGCT

G-31015840

311

58∘C

BtsC

IG58+172+81

T58

+253

rs3219489

145570092(exon)

GC

Forw

ard51015840-TGTG

GTG

AGCA

CCAAAC

CTA-

31015840Re

verse51015840-G

GCT

GTT

CCAG

AAC

ACAG

GT-31015840

319

62∘C

HpyCH

4III

G193

+126

C319

rs3219493

145568856(in

tron)

GC

Forw

ard51015840-C

TCAAG

TGAT

CCAC

CCGAC

T-31015840


AAG

CCTG

GAG

TGGAG

A-31015840

332

58∘C

HpyCH

4VG332

C139+193







1 2 3 4

rs3219493

rs3219489

rs3219476

83

93

63

69

68

61

rs3219463




4 Discussions








































Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















1 2 3 4

rs3219493

rs3219489

rs3219476

83

93

63

69

68

61

rs3219463




4 Discussions








































Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















































Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article MUTYH Gene Polymorphisms as …downloads.hindawi.com/journals/bmri/2015/893796.pdf · Research Article MUTYH Gene Polymorphisms as Risk Factors for Rheumatoid Arthritis