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Tissue Antigens ISSN 0001-2815 BRIEF COMMUNICATION The CD3Z 844 T > Apolymorphismwithin the3 # -UTRof CD3Z confersincreasedriskofincidenceofsystemiclupuserythematosus T. Warchoł 1 , P. Piotrowski 1 , M. Lianeri 1 , D. Cies´lak 2 , M. Wudarski 3 , P. Hrycaj 2 ,J. K. Ła˛cki 3 & P. P. Jagodzin´ski 1 1 Department of Biochemistry and Molecular Biology, Poznan University of Medical Sciences, Poznan´, Poland2 Department of Rheumatology and Clinical Immunology, Poznan University of Medical Sciences, Poznan´, Poland3 Institute of Rheumatology, Warsaw, Poland Key words SLE; CD3Z ; polymorphism Correspondence Dr Paweł P. JagodzinskiDepartment of Biochemistry and MolecularBiologyPoznan´ University of Medical Sciences,6 S´wie˛cickiego Street60-781 Poznan´PolandTel: (48 61) 8546519Fax: (48 61) 8546510e-mail:
[email protected] 2 December 2008; revised 17February 2009; accepted 19 March 2009doi: 10.1111/j.1399-0039.2009.01264.x Abstract Recently, a family-based association analysis showed that the haplotype carryinga low expression of the variant CD3Z 844 T > A (rs1052231) polymorphism locatedinthe3 # -untranslatedregionof CD3Z predisposestosystemiclupuserythematosus(SLE)incidence. Weanalyzedtheprevalenceofthe CD3Z 844T > Apolymorphismin SLE patients ( n ¼ 152) and controls ( n ¼ 304) in Poland. We observed thatwomen with the CD3Z AA and CD3Z AT genotypes exhibited a 1.845-foldincreased risk of SLE [95% confidence intervals (95% CI) ¼ 1.222–2.787, P ¼ 0.0038]. However, we did not find an increased risk for the homozygousCD3Z AA genotype (odds ratio ¼ 1.204, 95% CI ¼ 0.2838–5.108, P ¼ 1.0000).ThisobservationconfersthatgeneticfactorscausingadecreasedlevelofCD3- z inTcells may predispose to SLE incidence.Systemic lupus erythematosus (SLE) is an autoimmunerheumatic disease characterized by a large number of immunologic abnormalities that appear to result fromdefects in T and B cells and monocytes (1, 2).It hasbeen reported thatthe signal transductionpathwayusing the T-cell receptor (TCR) is altered in the T cells of patients with SLE (2–4).Stimulation of T cells is triggered by binding of the TCR/CD3 complex to an antigen bound to the major histocompat-ibility complex located on the surface of antigen-presentingcells (5). The interaction of T-helper CD4 1 cells with Blymphocytes during antigen presentation is an essential step inB-cell differentiation into plasma cells and antibody bio-synthesis (6). T-cell stimulation results in the activation of nonreceptor tyrosine kinases, which phosphorylate the cyto-plasmicdomainoftheimmunoreceptortyrosine-basedactiva-tion motif of CD3-zeta ( z ) and other signal molecules (5, 7, 8).Abnormalities in signaling through the TCR/CD3complex may result in T-cell dysfunction and developmentof autoimmunity in NOD mice, MRL lpr/lpr mice, andhumans (8–16). Moreover, the CD3- z -chain is downregu-latedinmanychronicinfectiousandinflammatorydiseases,including SLE (12–19).It has been reported that SLE incidence exhibits a stronggenetic component (20–25). Genome-wide screening of multicase families has indicated that chromosome 1q22–24is linked to SLE (25). Because CD3Z is within this linkageregion, it has been suggested that polymorphisms at the CD3Z locus may contribute to the pathogenesis of SLE.The CD3- z from SLE patients contains polymorphismsand aberrantly spliced forms more frequently than non-SLEindividuals (26, 27). It hasbeen reported thatthe CD3Z generegion corresponding to amino acids in patients with SLE isnonpolymorphic (28). However, the 3 # -untranslated region(3 # -UTR) of the CD3Z gene in patients with SLE containsmany single nucleotide polymorphisms (SNPs), and manytranscriptsofthisregionexistasalternativelysplicedvariants(3, 26–29).Recently, it has been reported that 3 # -UTR SNPs instrong disequilibrium CD3Z 844 T > A (rs1052231) and 68 ª 2009 John Wiley & Sons A/S Tissue Antigens 74 , 68–72 CD3Z 837 G > C (rs1052230) SNPs are associated with lowCD3- z expressioninSLEpatientsandhealthycontrols(29).Moreover, a family-based association analysis showed thatthehaplotypecarryingthelow-expressionvariantsof CD3Z 844 T > A polymorphism predisposes to SLE (29).Therefore,weanalyzedtheprevalenceofCD3Z844T > Apolymorphic variants in SLE patients ( n ¼ 152) andcontrols ( n ¼ 304).Thedatafor152patients(onlywomen)inpursuanceoftheAmericanCollegeofRheumatologyclassificationcriteriaforSLE (30, 31) were collected from Institute of Rheumatologyin Warsaw and from Department of Rheumatology andClinical Immunology at the Poznan ´ University of MedicalSciences in Poznan ´, Poland. The protocol of the study wasapproved by the Local Ethical Committee of Poznan ´University of Medical Sciences. Informed written consentwas obtained from all participating subjects.Bothpatientsandcontrols ( n ¼ 304, only women) were of Polish Caucasian srcin. The mean age of SLE patients atdiagnosiswas 35 12 years and for controls34 10 years.Clinical findings of SLE in the patient group includedcentralnervoussystem(17%),vascular(11%),renal(49%),musculoskeletal (64%), serositis (19%), cutaneous mani-festations (51%), immunologic (39%), constitutional(fever) (15%), and hematologic (33%) components.DNAwasobtainedfromperipheralbloodlymphocytesbysalt extraction method. The presence of the CD3Z 844 T > Apolymorphic variant was determined by polymerase chainreaction (PCR)–restriction fragment length polymorphism.PCR was conducted with the primer pair 5 # AGTGTGAA-TAAAGTGCTGCGG 3 # and 5 # CCTGTGCCCTGTAAT-GACG 3 # , with subsequent digestion using Hin 1II (NewEngland Biolabs, Ipswich, MA).The844Aallelewascleavedinto105-and91-bpfragments,whereas the 844T allele was uncut into 196-bp fragments.DNAfragmentswereseparated on 10%polyacrylamidegelsand stained by silver method. Polymorphism confirmationwas performed by sequencing analysis.The distribution of genotypes in all groups was tested fordeviation from Hardy–Weinberg equilibrium. Fisher exacttest was applied to examine differences in the genotypic andallelic distribution between patients and controls. The oddsratio (OR) and 95% confidence intervals (95% CI) werecalculated with statistical significance at P value < 0.05.PoweranalysiswasperformedusingFisherexacttest,whichis available at an online Internet service, http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/PowerSampleSize.Genotypic distribution of the CD3Z 844 T > A poly-morphism showed no significant deviation form Hardy– Weinberg equilibrium between patients and controls.Table 1presentsthealleleandgenotypedistributionofthe CD3Z 844 T > A polymorphism in SLE patients ( n ¼ 152)andcontrols( n ¼ 304).Weobservedacontributionof CD3Z 844AA and CD3Z 844AT genotypes to the development of SLE. The frequency of the CD3Z 844AA genotype wassimilar in patients and in controls and amounted toapproximately 2% (Table 1). However, the CD3Z 844ATheterozygous frequency was 1.5-fold times higher in patientsthan in controls and reached 38% and 25% in these groups,respectively (Table 1). Moreover, the distribution of the CD3Z 844A allele was also 1.5-fold times higher in SLEpatients than in controls and reached 21% and 14%,respectively (Table 1).We observed that women with the CD3Z 844AA andCD3Z 844AT genotypes exhibited a 1.845-fold increasedrisk of SLE (95% CI ¼ 1.222–2.787, P ¼ 0.0038). How-ever, we did not find an increased risk for the homozygousCD3Z 844AA genotype (OR ¼ 1.204, 95% CI ¼ 0.2838– 5.108, P ¼ 1.0000). The statistical power of this study was80% for the CD3Z 844AA and CD3Z 844AT genotypes.We did not find a significant linkage between clinicalmanifestations of SLE and disease activity and distributionof CD3Z 844 T > A polymorphic variants.It has been believed that T cells are central in thepathogenesis of SLE, and dysfunctions in their regulatoryaction may result in changes in immune response (32).Numerous investigations have demonstrated that T cellsfrom patients with SLE display both quantitative andqualitative differences in their levels and function of CD3- z compared with T cells from healthy controls (12, 33–41).However,themolecularmechanismsofareducedexpressionof CD3- z mRNA and protein in SLE T cells are still unclear. Table 1 Association of the CDZ (rs1052231) polymorphisms in SLE patients and controlsnGenotypedistributionabsolutenumber (frequency %)Allele absolute number(frequency %)Odds ratio (95% CI, P c )T/T A/T A/A T A P C CDZ rls 1052231 ControlsTotal304 223 (73) 76 (25) 5 (2) 522 (86) 86 (14) 0.0103 1.204 (0.2838-5.108, 1.0000) a PatientsTotal152 91 (60) 58 (38) 3 (2) 240 (79) 64 (21) 1.845 (1.222-2.787, 0.0038) b The Odds ratio was calculated for a (A/A vs T/T or A/T), and b (A/A and A/T vs T/T). c Fisher exact test.T. Warchoł et al. CD3Z 844 T > A polymorphism ª 2009 John Wiley & Sons A/S Tissue Antigens 74 , 68–72 69 The human CD3Z gene spans 88 kb of DNA (42). Thespliced mRNA product is 1.492-kb long, composed of a 492-bp coding region, and a 3 # -UTR of 906 bp (43). Ex-pression of the CD3Z gene is regulated at transcriptional,posttranscriptional, and posttranslational levels (37–40, 44).However, the expression of CD3- z is mainly regulated at theposttranscriptional level through the 3 # -UTR. This CD3- z transcript region plays an essential role in that it changesmRNA stability, transport, and localization (37–40). Down-regulation of CD3- z expression has also been linked toincreaseddegradationofthisproteinthroughubiquitination,granzyme B, and caspase actions (33–36, 41, 45).We presume that the CD3Z 844 T > A polymorphicvariant may indirectly change the secondary structure of RNA, which affects RNA-binding proteins. This maydecrease the stability of mRNAs, leading to the reductionof CD3- z protein. Further investigations are required todetermine the effect of CD3Z 844 T > A polymorphism inthe reduction of CD3- z mRNA level.To date, sequence analysis of CD3Z has shown anincreased frequency of an alternatively spliced variant thatis missing various exons and a splice site insertion in CD3- z mRNA isolated from SLE patients’ T cells (26). Tsuzakaet al . reported that variants of CD3- z mRNA lacking the36-bp exon 7 were associated with the downregulation of CD3- z protein expression in T cells and reducedInterleukin2 (IL-2) production after stimulation with anti-CD3antibody (46). Reduced mRNA stability was also observedfor the 3 # UTR 344-bp product (compared with the wild-type 906-bp fragment) form with a deletion of nucleotidesfrom672to1233ofexon8ofthemRNA(37–40).However,none of these splicing variant abnormalities have beenclearly associated with SLE susceptibility.We found that the T > A polymorphic variant located in3 # -UTR of CD3Z contributes to the incidence of SLE.Employing flow cytometry analysis, we also observed thatSLE patients with AT and AA genotypes had significantlylowerCD3- z proteinlevelsinTcellscomparedwithpatientswith TT genotype (results not shown). Ourobservations forSLE patients support the findings of Gorman et al . , whodemonstrated an association of the haplotype bearing the844A variants of CD3Z with familial SLE (29). Moreover,Gorman et al . showed that this polymorphism was signifi-cantlyassociatedwithareducedexpressionofCD3- z mRNAand protein in SLE patients and healthy individuals (29).It has been reported that chronic exposure of T cells totumour necrosis factor-alpha reversibly downregulates theexpression of the CD3- z chain in part through a reduction ingene transcription (47). The reduced densityofT-cell surfaceCD3- z wasalsoobservedindistinctchronicinfectionssuchasleprosy, tuberculosis (18), HIV (19), gastric and colonicmalignancies (17), and autoimmune diseases such as SLE(12–14) and rheumatoid arthritis (15, 16).However, it is yet unknown whether a decreased CD3- z density in T cells is a primary mechanism or the result of chronic inflammatory response.AttenuatedTCRsignalingassociatedwiththepresenceof autoreactive T cells was observed in SLE patients bearingnonfunctional polymorphic variants of protein tyrosinephosphatase nonreceptor 22 (48, 49). Moreover, it has beenreported that mice with a mutation in the TCR proximalprotein tyrosine kinase ZAP-70 exhibit a systemic autoim-mune inflammatory polyarthritis resembling rheumatoidarthritis.Inthisanimalmodel,refractoryTCRsignalsresultin an abnormal selection in thymic T-cell development andoccurrence of autoreactive peripheral T cells (50). Theseobservations suggest that genetic factors reducing CD3- z levels in T cells may also predispose to decreased TCRsignaling, favoring positive selection of autoreactive Teffector cells in the thymus.Our findings, together with the observations of Gormanetal . ,suggestthatCD3- z downregulationmaybeoneoftheprimary factors, along with inflammatory mediators orother factors, contributing to SLE pathogenesis.Further investigations of the distribution of this poly-morphism in other populations are necessary to moreprecisely establish the contribution of the CD3Z 844 T > Apolymorphism to SLE incidence. 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