Cytokine gene polymorphisms as risk and severity factors for juvenile dermatomyositis

Cytokine Gene Polymorphisms as Risk and Severity Factors forJuvenile Dermatomyositis

Gulnara Mamyrova, MD, PhD1, Terrance P. O’Hanlon, PhD1, Laura Sillers, AB1, Karen Malley,BA2, Laura James-Newton, PhD, RN1, Christine G. Parks, PhD3, Glinda S. Cooper, PhD4,Janardan P. Pandey, PhD5, Frederick W. Miller, MD, PhD1, Lisa G. Rider, MD1, and for theChildhood Myositis Heterogeneity Collaborative Study Group1Environmental Autoimmunity Group, National Institute of Environmental Health Sciences, NationalInstitutes of Health, DHHS, Bethesda, MD

2Malley Research Programming Inc, Rockville, MD

3National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, Durham, NC

4National Center for Environmental Assessment, U.S. Environmental Protection Agency, NW Washington,DC

5Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC.

AbstractObjective—To study TNFα and IL-1 cytokine polymorphisms as possible risk and protectivefactors, define their relative importance and examine these as severity factors in patients with juveniledermatomyositis (DM).

Methods—TNFα and IL-1 cytokine polymorphism and HLA typing were performed in 221Caucasian patients with juvenile DM and compared to 203 ethnically matched healthy volunteers.

Results—The genotypes TNFα -308AG (odds ratio [OR] 3.6), TNFα -238GG (OR 3.5), andIL-1α+4845TT (OR 2.2) were risk factors, and TNFα -308GG (OR 0.26) as well as TNFα -238AG(OR 0.22) were protective for the development of juvenile DM. Carriage of a single copy of theTNFα -308A (OR 3.8) and IL-1β+3953T (OR 1.7) alleles were risk factors and TNFα -238A (OR0.29) and IL-1α+4845G (OR 0.46) were protective for juvenile DM. Random Forests classificationanalysis showed HLA DRB1*03 and TNFα -308A to have highest relative importance as risk factorsfor juvenile DM compared to the other alleles (Gini scores 100% and 90.7%, respectively). TNFα-308AA (OR 7.3) was a risk factor, and carriage of the TNFα-308G (OR 0.14) and IL1α-889T (OR0.41) alleles were protective for the development of calcinosis. TNFα-308AA (OR 7.0) was a possiblerisk factor, and carriage of the TNFα-308G allele (OR 0.14) was protective for the development ofulcerations. None of the studied TNFα, IL-1α and IL-β polymorphisms were associated with diseasecourse, severity at diagnosis, or gender.

Conclusions—TNFα and IL-1 genetic polymorphisms contribute to the development of juvenileDM and may also be indicators of disease severity.

Juvenile dermatomyositis (DM) is a systemic autoimmune disease which is characterized bysymmetric proximal weakness and distinctive skin rashes. The etiology of juvenile DM remainsunknown, but recent evidence suggests combinations of genetic and environmental risk factors

Corresponding Author and Reprint Requests: Lisa G. Rider, MD, Environmental Autoimmunity Group National Institute ofEnvironmental Sciences, National Institutes of Health, DHHS, Clinical Research Center Room 4-2352, 10 Center Drive, MSC 1301,Bethesda, MD 20892-1301, Phone: (301) 451- 6272, Fax: (301) 451-5588, Email: E-mail: [email protected].

NIH Public AccessAuthor ManuscriptArthritis Rheum. Author manuscript; available in PMC 2009 December 1.

Published in final edited form as:Arthritis Rheum. 2008 December ; 58(12): 3941–3950. doi:10.1002/art.24039.

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are involved (1). Cytokines appear to have an important role in the pathogenesis ofinflammatory myopathies (2). The expression of pro-inflammatory cytokines such as tumornecrosis factor alpha (TNFα), interleukin 1alpha (IL-1α) and interleukin 1beta (IL-1β) areincreased in the mononuclear cell infiltrates and on muscle fibers of biopsies from myositispatients (2–4). The expression of IL-1α and IL-1β are also increased on the capillary endothelialcells of the affected muscle (2).

The TNFα-308A promoter polymorphism, which is associated with increased production ofTNFα from peripheral blood mononuclear cells, is increased in frequency in Caucasian patientswith juvenile and adult DM (5–8) and in Chinese patients with adult DM (9) compared tohealthy controls. The TNFα-308A allele has also been associated with the development ofcalcinosis and a chronic disease course in juvenile DM patients (5). In terms of IL-1polymorphisms, the pro-inflammatory intronic variable number tandem repeat of IL-1 receptorantagonist, IL1-RA A1, is a risk factor for juvenile idiopathic inflammatory myopathies inCaucasian patients (10). Polymorphic determinants of IL-1α and β, including IL-1α-889,IL-1α+4845, IL-1 β -511 and IL-1β+3953, have been associated with increased disease severityand susceptibility to systemic autoimmune diseases (11–13), with higher in-vitro stimulatedproduction of these cytokines (14;15) and with altered plasma cytokine levels (16–18).

The objective of our study was to determine whether a broader number of cytokinepolymorphisms for IL-1 and TNFα are risk or protective factors for juvenile DM, and to definetheir relative importance. We also aimed to examine these cytokine polymorphisms as severityfactors for juvenile DM.

PATIENTS AND METHODSPatients

DNA from 201 Caucasian patients with juvenile onset DM and 20 with juvenile DM inassociation with another autoimmune condition were used for IL-1α, IL-1β and TNFαpolymorphism genotyping. All patients met probable or definite Bohan and Peter criteria forDM (19) and were diagnosed prior to 18 years of age. The diagnosis of juvenile overlap DMwas defined by meeting both Bohan and Peter criteria for DM (1975), as well as AmericanCollege of Rheumatology (ACR) or other criteria for another connective tissue disease.Associated autoimmune diseases included systemic lupus erythematosus (4 patients),scleroderma (7 patients), insulin dependent diabetes mellitus, juvenile rheumatoid arthritis andimmune thrombocytopenia purpura (2 each), ulcerative colitis, psoriasis and Hashimoto’sthyroiditis (1 each). Control cytokine polymorphism data were obtained from 203 ethnically -matched healthy volunteers who participated in the Carolina Lupus Study (20;21). All subjectswere enrolled in investigational review board-approved NIH and Food and DrugAdministration protocols. Patients were enrolled with a blood sample and a physicianquestionnaire of the illness features, which was reviewed by a pediatric rheumatologist (GMor LR).

Seventy-five percent of the juvenile DM patients were female. Twenty-five percent had achronic continuous disease course, with persistent disease activity for > 2 years, and 19.5%each had a monocyclic and polycyclic illness course; illness course was undefined in 36%.Thirty-one percent developed calcinosis, and 20.8% had a history of cutaneous orgastrointestinal ulceration. Seventy six percent had a history of photosensitive rashes. Themedian age at the time of diagnosis was 7.5 years [25% 5 years, 75% 12 years] and the medianphysician global activity at diagnosis was 3 on a 0 – 4 point Likert scale.

Mamyrova et al. Page 2

Arthritis Rheum. Author manuscript; available in PMC 2009 December 1.

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Cytokine Polymorphism GenotypingSingle nucleotide polymorphisms for TNFα at positions -238 (A→G) [rs361525] and -308(A→G) [rs1800629], as well as IL-1α-889 (C→T) [rs1800587], IL-1α+4845 (G→T)[rs17561], IL-1β-511 (C→T) [rs16944], and IL-1β+3953 (C→T) [rs1143634] weredetermined by PCR of peripheral blood leukocyte DNA, followed by restriction enzymedigestion and gel electrophoresis (20;21) in 221 juvenile DM patients and 203 controls. Assayfailure rate ranged from 0.6 to 5.2%. All six cytokine polymorphism SNPs met Hardy-Weinberg equilibrium in the control subjects (p = 0.10 – 0.74).

HLA typingHLA DRB1*03 allele typing from DNA extracted from peripheral blood mononuclear cellswas available in 143 juvenile DM and 202 control individuals. HLA typing was performed byPCR-mediated sequence-specific oligonucleotide probe hybridization and sequence-specificpriming techniques as described by O’Hanlon et al. (22). The assay failure rate ranged from 5– 10%.

Statistical AnalysisAnalyses were performed using GraphPad InStat version 3.00 for Windows (GraphPadSoftware, San Diego California USA, www.graphpad.com), the SAS (R) System for Windows,version 9.1.3 (SAS Institute, Cary, NC) and SAS Enterprise Guide, version 4.1 (SAS Institute,Cary, NC). Fisher’s exact test was used to calculate P values for 2 × 2 tables. Carriage rate wasdetermined by the number of cytokine polymorphism - positive subjects divided by the totalnumber of subjects for which data from a particular allele were available. P values wereadjusted for multiple testing using Holm’s procedure (23), which is a nonparametric step-downadjustment that strongly controls for family-wise error rate and makes no assumptionsconcerning the correlation structure of the data or the observed p-values. P values weredetermined to be significant when the adjusted P values were at or below the 0.05 level.Cytokine polymorphisms which were of higher or lower frequency in juvenile DM patientscompared to controls prior to correction for multiple comparisons were termed possible riskor protective factors, respectively.

The relative importance of each individual cytokine polymorphism as a genetic risk orprotective factor for juvenile DM was estimated using a statistical learning machine with theRandom Forests algorithm (http://stat-www.berkeley.edu/users/breiman/RandomForests/), asdescribed previously (24). Each Random Forests Classification analysis consisted of 100random forests of 1000 trees each. The number of variables tried at each split was first tunedusing a single 500-tree forest, and the tuned number used for all 100 forests. The tuning forestwas not included in the final 100 forests. Classification error rates for each analysis werecomputed as the within-forest error rate averaged over the 100 forests. Class weights wereadjusted to approximately equalize the analysis error rate between the two groups. An intervalestimate of the variability of variable importance was calculated as ± 2 times the estimatedstandard error of the classification error rates.

Logistic regression analysis was performed as an independent method of confirming theassociations identified by univariate analysis. Hosmer-Lemeshow goodness-of-fit statisticswere used to assess how well the logistic regression model fit the data. Linear regressionanalysis was performed to study correlation between cytokine polymorphisms and age atdiagnosis. The R-square and adjusted R-square were used in assessing the fit of models inlinear regression analysis.

Haplotype analysis was performed using Haploview software (Haploview Software version3.32, Broad Institute, Cambridge, MA, USA http://www.broad.mit.edu/mpg/haploview/) to



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http://www.graphpad.com

http://stat-www.berkeley.edu/users/breiman/RandomForests/

http://www.broad.mit.edu/mpg/haploview/

determine the level of linkage disequilibrium between cytokine polymorphism markers bycomparing patients and controls, and also analyzing control or patient population genotypesin isolation.

ResultsJuvenile DM- associated risk and protective cytokine polymorphisms

The TNFα genotypes TNFα -308AG (odds ratio [OR] 3.6, P < 0.0001) and TNFα -238GG (OR3.5, P = 0.0009) were risk factors and TNFα -308GG (OR 0.26, P < 0.0001) and TNFα -238AG(OR 0.22, P = 0.0002) were protective factors for juvenile DM in Caucasian patients (Table1). Carriage of the TNFα -308A allele (OR 3.8, P < 0.0001) was a risk factor and carriage ofthe TNFα -238A allele (OR 0.29, P = 0.0009) was protective for juvenile DM (Table 1).

IL-1α+4845TT (OR 2.2, P = 0.006) was a risk factor for juvenile DM. Carriage of IL-1β+3953T(OR 1.7, P = 0.019) and the IL-1α+4845G allele (OR 0.46, P = 0.006) were risk and protectivefactors, respectively for the development of juvenile DM in Caucasian patients (Table 1). Twoadditional IL-1 genotypes, IL-1β+3953CT (OR 1.7, P = 0.022) and IL-1β+3953CC (OR 0.59,P = 0.019), were possible risk and protective factors, respectively (Table 1), but these were notsignificant after adjustment for multiple comparisons. The HLA DRB1*03 allele wasconfirmed as a risk factor for juvenile DM, using the control subjects in the present study(20;21;24), and its strength of association was comparable to that of the TNF-α -308A allele.

No significant differences were detected between juvenile DM patients and control subjects inthe frequency of the following genotypes: TNFα-308AA, TNFα-238AA, IL-1α-889CC,IL-1α-889CT, IL-1α-889TT, IL-1α+4845GG, IL-1α+4845GT, IL-1-α-511CC, IL-1-α-511CT,and IL-1-α-511TT. Carriage of the following alleles also did not differ between juvenile DMpatients and controls: TNFα-308G, TNFα-238G, IL-1α-889C, IL-1α-889T, IL-1α+4845T,IL-1β+3953C, IL-1α-511C, and IL-1α-511T. No differences were detected in the carriage rateof cytokine or DRB1 alleles between the juvenile DM patients and those who also had anassociated autoimmune disease.

Relative importance of juvenile DM- associated risk and protective cytokine polymorphismsThe relative importance (RI) of cytokine polymorphisms as risk and protective factors forjuvenile DM was examined using Random Forests Classification analysis. In examiningspecific cytokine polymorphism genotypes, TNFα -308AG (RI 100%) was found to have thehighest RI for the development of juvenile DM in Caucasian patients, followed by theprotective genotype TNFα -308GG (RI 87.7%). The relative importance of the IL-1β+3953CT,IL-1β+3953CC and IL-1α+4845TT genotypes as risk or protective factors for juvenile DMwere much lower (Table 2).

In terms of the analysis of the relative importance of cytokine alleles in relationship to HLADRB1*03, previously found to be the major MHC class II risk factor for juvenile DM (24),HLA DRB1*03 had the highest relative importance as a risk factor for juvenile DM (RI 100%),followed by carriage of the TNFα -308A allele (RI 90.7%) using Random Forests classification.Again, carriage of the TNFα-238A allele (RI 53.1%) as well as carriage of the IL-1β+3953T(RI 59.4%) and IL-1α+4845G (RI 38.9%) alleles had much lower relative importance as riskor protective factors for juvenile DM (Table 3).

The juvenile DM-associated risk and protective cytokine polymorphism alleles defined inunivariate analysis were further examined by multiple logistic regression analysis (Table 4).HLA DRB1*03 (OR 3.5) and TNFα -238A (OR 0.19) were the primary risk and protectivefactors, respectively, in the model. TNFα -308A (OR 1.9) had borderline significance as a risk



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factor in the model. None of the other risk and protective cytokine polymorphism allelesdefined by univariate analysis were significant in the logistic regression model.

Allele interaction analysesIn order to assess possible linkage disequilibrium among the IL-1 or TNFα alleles, we examinedwhether presumed haplotypes composed of combinations of two TNFα or four IL-1 alleles,respectively, existed in juvenile DM patients and healthy control subjects. Although the twoTNFα alleles were in linkage disequilibrium (D’ =1.0), the correlation was low (r2 = 0.017).No evidence for linkage disequilibrium was observed among the four IL-1 alleles (D’ range0.116 – 0.676, r2 range 0.005 – 0.253). The analysis was conducted separately in patients andcontrols, and in both groups combined; and similar results were obtained. The Hardy-Weinbergp values in controls ranged from 0.093 – 0.74.

Potential interaction between the two risk alleles HLA DRB1*03 and TNFα-308A wasexamined in a multiplicative logistic regression model. No significant multiplicativeinteraction of these two risk factors was found (P = 0.28).

Cytokine polymorphisms as disease severity factors for juvenile DMTo identify whether TNFα and IL-1 polymorphisms are disease severity factors, we comparedjuvenile DM cases with and without various disease features to see if these alleles contributeto the risk of certain illness complications. First, TNFα -308AA (OR 7.3) was found to be arisk factor for the development of calcinosis. Another cytokine polymorphism, IL-1α-889CC(OR 2.4), was found to be a possible risk factor for the development of calcinosis. Carriage ofthe TNFα-308G (OR 0.14) and IL1α-889T (OR 0.41) alleles were protective factors for thedevelopment of calcinosis (Table 5). HLA DRB1*03 was not a severity factor for thedevelopment of calcinosis.

We also examined whether these IL-1 and TNFα polymorphisms were related to thedevelopment of gastrointestinal or cutaneous ulcerations. Only TNFα-308AA (OR 7.0) wasfound to be a possible risk factor, and carriage of the TNFα-308G allele (OR 0.14) was aprotective factor for the development of ulcerations in Caucasian patients with juvenile DM(Table 6). Neither TNFα -238 nor the IL-1 cytokine polymorphisms were found to be risk orprotective factors for the development of ulcerations.

IL-1α+4845GG (OR 2.4, P = 0.003), and TNFα-238AG (OR 14.0, P = 0.016) were increasedin Caucasian juvenile DM patients younger than 7.5 years of age at diagnosis, the median ageof diagnosis of juvenile DM in this cohort, compared to patients older than 7.5 years at thetime of diagnosis. IL-1α+4845TT (OR 0.32, P = 0.001) was less frequent in children withjuvenile DM younger than 7.5 years of age compared to those older at diagnosis. Linearregression modeling confirmed IL-1α+4845GG and TNF-α-238A to be associated with ayounger age at diagnosis, and carriage of the IL-1α+4845T allele to be associated with an olderage at diagnosis. Other cytokine polymorphisms were unrelated to age at onset.

Genotypes IL-1α-889CC (OR 2.6, P = 0.032) and IL-1β+3953TT (OR 9.7, P = 0.04) werepossible risk factors and carriage of the IL-1α-889T (OR 0.38, P = 0.032) and IL-1β+3953C(OR 0.10, P = 0.04) alleles were possible protective factors for the development ofphotosensitive skin rashes. These findings were not significant after adjustment for multiplecomparisons. Neither the TNF alleles nor HLA DRB1*03 were associated with photosensitiveskin rashes.

None of the studied IL-1α, IL-β and TNFα polymorphisms were associated with disease course(monocyclic, polycyclic or chronic continuous), physician rating of overall disease severity atdiagnosis, or with gender in patients with juvenile DM. There was no difference in the



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frequency of the TNFα-308AA genotype based on disease course: 1 of 53 (1.9%) patients withthe TNFα-308AA genotype had a chronic course compared to 2 of 41 patients (4.9%) with amonocyclic course and 1 of 41 patients (2.4%) with a polycyclic course of illness (P = 0.61).Also, no difference was detected in the carriage rate of the TNFα-308A allele based on diseasecourse: 31 of 53 (58.5%) patients with TNFα-308A allele had a chronic continuous diseasecourse compared to 29 of 41 (70.7%) with a monocyclic course and 23 of 41 patients (56.1%)with a polycyclic course of diseases (P= 0.73).

DiscussionWe studied TNFα and IL-1 cytokine polymorphisms in a large group of Caucasian patientswith juvenile DM, and confirmed carriage of the TNFα-308A allele, and specifically theTNFα-308 AG genotype, is a risk factor, as reported previously in studies of adult DM and asmaller study of juvenile DM (5–8). We also found TNFα-238GG to be a risk factor, andTNFα-238AG as well as carriage of the TNFα-238A allele as protective factors for juvenileDM.

Novel findings of this study also included the identification of IL-1 cytokine polymorphismsIL1α+4845TT and IL1β+3953T as risk factors and carriage of the IL1α+4845G allele as aprotective factor for juvenile DM in Caucasians. We did not detect haplotypes among the fourIL-1 cytokine polymorphisms, and the results of the Random Forests classification also suggestthese cytokine polymorphism associations are independent and not in linkage disequilibrium.The IL-1 gene cluster has been important in susceptibility to a number of systemic connectivetissue diseases, including early-onset pauciarticular juvenile rheumatoid arthritis (25), psoriaticarthritis (26), Behcet’s disease (27), and systemic sclerosis (12). In patients with rheumatoidarthritis, IL-1β+3953T has been associated with more active and erosive disease, with higherin vitro stimulated production of IL-1β from monocytes stimulated with LPS (14) and withlower plasma levels of the anti-inflammatory IL-1RA (16;17).

The associations of multiple TNFα and IL-1 polymorphisms as risk and protective factors forjuvenile DM suggest these cytokines are important contributors to the pathogenesis of juvenileDM. Both IL-1 and TNFα have been detected in the affected muscle of patients with DM andother idiopathic inflammatory myopathies (2–4). Stimulated peripheral blood mononuclearcells from patients with juvenile DM carrying the TNFα -308A allele produce more TNFα(5), and serum levels of the TNFα to IL-10 ratio are higher in patients with the TNFα -308Aallele (7), suggesting the TNFα-308A allele is associated with higher production of TNFα.

With a large patient population and use of multivariable statistical approaches, we were alsoable to identify that the TNFα-308A allele has a much higher relative importance among alldetected risk and protective TNFα and IL-1 cytokine polymorphisms. At the same time, therelative importance of the TNFα-308A allele is lower than HLA DRB1*03, the previouslydescribed major immunogenetic risk factor for juvenile DM (24;28), in Random Forestsclassification analysis. This finding of the stronger relative importance of DRB1*03 overTNFα was confirmed in logistic regression modeling, in which DRB1*03 was found to be astrong risk factor and TNFα-238A a protective factor, but the TNFα-308 A allele was notsignificant in the model. Because of the lack of availability of DRB1*0301 typing in controlsubjects, we were not able to perform haplotype analysis to examine linkage of TNFα alleleswith DRB1*0301. However, our findings confirm the work of Chinoy et. al., who found thatthe TNFα-308A and DRB1*0301 alleles were independent risk factors for adult DM in logisticregression modeling and haplotype analysis, in contrast to subsets of adult myositis patients,including patients with polymyositis and with anti-synthetase and PM-Scl autoantibodies, inwhich linkage disequilibrium between TNFα and HLA alleles exists (8). Using association



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methodology, Werth et al, also reported independent risk associations for TNFα-308A andHLA DRB1*03 in a smaller adult DM population (6).

TNFα-308A has been reported to be associated with calcinosis and a chronic illness course ina small study of juvenile DM (5). Here, we confirmed TNFα-308AA as a risk factor forcalcinosis and the TNFα-308G allele as protective, not only for calcinosis, but also for thedevelopment of gastrointestinal or cutaneous ulcerations in juvenile DM, a seriouscomplication of illness that also marks severe disease (29;30). This dichotomous relationshipof TNFα-308A as a risk factor for calcinosis in the absence of DRB1*03 further suggestsindependence in the TNFα-308A and HLA DRB1*03 associations.

We did not find the TNFα alleles to be associated with disease course or with the developmentof photosensitive skin rashes, but did find possible associations of photosensitive rashes withsome of the IL-1 polymorphisms, which were not significant after adjustment for multiplecomparisons. Interestingly, Werth et al. demonstrated an interaction of IL-1 and ultraviolet-Blight resulting in an increase in TNFα-308A transcription in an in vitro fibroblast model (31).TNFα-308A, in linkage disequilibrium with the HLA A*01, B*08, DRB1*0301 ancestralhaplotype, was also seen as a risk factor for subacute cutaneous lupus, and the IL1β+3954 Tallele was possibly protective, in contrast to our patients with juvenile DM (32).

A limitation of our study is that it is not a population-based study, but rather based on a referralpopulation of juvenile DM patients, enrolled from throughout the United States and Canada,primarily from tertiary care medical centers. The control population was also from theCarolinas, not from the entire United States (20;21). Confirmation of these findings in familialstudies through transmission disequilibrium testing would be helpful. Also, the DRB1*03typing available from control subjects was not resolved to the allelic level, and some patientswere not genotyped for all of the cytokine polymorphism and DRB1 alleles, potentiallyintroducing bias as a result of the missing data. Regarding alleles that modify disease severity,such as calcinosis or ulcerations, the impact of therapy on these outcomes was not accountedfor in these analyses.

Taken together, our results suggest TNFα and IL-1 genetic polymorphisms that are pro-inflammatory contribute to the development of juvenile DM and also are indicators of diseaseseverity. TNFα appears to be more important than IL-1 polymorphisms in contributing to therisk and severity of disease. TNFα and DRB1*03 are independent risk factors for juvenile DM,but the relative importance of DRB1*03 is greater than that of TNFα.

AcknowledgmentsWe thank Drs. Elaine Remmers and Sharon Adams for critical review of the manuscript. The authors gratefullyacknowledge Drs. Joan Bailey-Wilson and Priya Duggal (Statistical Genetic Section Inherited Disease ResearchBranch, National Human Genome Research Institute, NIH) for their helpful advice with the haplotype analysis. Wealso thank members of the Childhood Myositis Heterogeneity Collaborative Study Group who contributed to this studyas listed in the appendix.

Grant Support: Gulnara Mamyrova is a research fellow of the Cure JM Foundation. This work was supported in partby the Intramural Research Programs of NIEHS, NIH and CBER, Food and Drug Administration and in part by theU.S. Department of Energy cooperative agreement DE-FC09-02CH11109.

Appendix

Members of the Childhood Myositis Heterogeneity Collaborative Study GroupLeslie S. Abramson, Barbara S. Adams, Elizabeth M. Adams, F Paul Alepa (post-humus),Kathy Amoroso, Elif Arioglu, Frank C. Arnett, E Arthur, Balu H. Athreya, Alan N. Baer, Susan



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Hyatt Ballinger, Karyl S. Barron, April C. Bingham, William P. Blocker, John Bohn, John F.Bohnsack, Gilles Boire, Michael S. Borzy, Gary R. Botstein, Susanne L. Bowyer, Richard W.Brackett, Elizabeth B. Brooks, Christine Brunet, Thomas Bunch, Victoria W. Cartwright, GailD. Cawkwell, Stephen J. Chanock, Chun Peng T. Chao, Darryl Crisp, Randy Q. Cron, R Culp,John Daigh, Luminita David, Frederick C. Delafield, Andrew H. Eichenfield, John F. Eggert,Melissa Elder, J Ellman, Janet E. Ellsworth, C Etheridge, S Evans, Kathleen Fearn, Terri H.Finkel, Charles B. Foster, Robert C. Fuhlbrigge, Vernon F. Garwood, Abraham Gedalia,Natalie Gehringer, Stephen W. George, Harry L. Gewanter, Ellen A. Goldmuntz, Donald P.Goldsmith, Gary V. Gordon, Larry M. Greenbaum, Katherine R. Gross, Hillary Haftel, MelissaHawkins-Holt, C Hendrics, Michael Henrickson, Gloria C. Higgins, J Roger Hollister, RussellHopp, Bruce Hudson, E Huh, Norman T. Ilowite, Lisa F. Imundo, Jerry C. Jacobs (post humus),Rita S. Jerath, Courtney R. Johnson, Mary Jones, Olcay Jones, Lawrence K. Jung, LawrenceJ. Kagen, Stuart J. Kahn, Thomas G. Kantor, Ildy M. Katona, Gregory F. Keenan, Edward C.Keystone, Yukiko Kimura, Daniel J. Kingsbury, Steven J. Klein, C. Michael Knee, J Koenig,Bianca A. Lang, Andrew Lasky, Alexander Lawton, Johanan Levine, Carol B. Lindsley, RobertN. Lipnick, Seth H. Lourie, Elizabeth Love, Max S. Lundberg, Katherine L. Madson, Peter N.Malleson, Donna Maneice, A Mariano, Harold Marks, Alan L. Martin, F Matthew, John Miller,S. Ray Mitchell, Hamid J. Moallem, Penelope A. Morel, Chihiro Morishima, Frederick T.Murphy, Henry Nathan, Ann Neumeyer, Chester V. Oddis, Judyann C. Olson, Karen Onel,Barbara E. Ostrov, Lauren M. Pachman, Ramesh Pappu, Murray H. Passo, Maria D. Perez,Donald A. Person, Karin S. Peterson, Paul H. Plotz, Marilynn G. Punaro, Charles D. Radis,Linda I. Ray, Peter D. Reuman, Robert M. Rennebohm, John D. Reveille, Rafael F. Rivas-Chacon, Alan L. Rosenberg, Deborah Rothman, Peter A. Schlesinger, Kenneth C. Schuberth,Donald W. Scott, D Seamon, Bracha Shaham, Robert M. Sheets, David D. Sherry, Sara H.Sinal, Frances J Stafford, Howard Stang, Robert P. Sundel, Ilona S. Szer, Ira N. Targoff,Simeon Taylor, Elizabeth S. Taylor-Albert, Donald E. Thomas, Richard K. Vehe, Maria-Lourdes Villalba, Scott A. Vogelgesang, Larry B. Vogler, Emily Von Scheven, S Wahl, CarolA. Wallace, Harry J. Wander, Arthur Weinstein, Jana Wells, Patience H. White, Grace C.Wright, John Yee, Christianne M. Yung, Lawrence S. Zemel.

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12. Kawaguchi Y, Tochimoto A, Ichikawa N, Harigai M, Hara M, Kotake S, et al. Association of IL1Agene polymorphisms with susceptibility to and severity of systemic sclerosis in the Japanesepopulation. Arthritis Rheum 2003;48:186–192. [PubMed: 12528118]

13. Kaijzel EL, van Dongen H, Bakker AM, Breedveld FC, Huizinga TWF, Verweij CL. Relationshipof polymorphisms of the interleukin-1 gene cluster to occurence and severity of rheumatoid arthritis.Tissue Antigen 2002;59:122–126.

14. Pociot F, Molvig J, Wogensen L, Worsaae H, Nerup J. A TaqI polymorphism in the humaninterleukin-1 beta (IL-1 beta) gene correlates with IL-1 beta secretion in vitro. Eur J Clin Invest1992;22(6):396–402. [PubMed: 1353022]

15. Wen A, Wang J, Feng K, Zhu P, Wang Z, Jiang J. Effects of haplotypes in the interleukin 1b promoteron lipopolysaccharide-induced interleukin 1b expression. Shock 2006;26:25–30. [PubMed:16783194]

16. Buchs N, di Giovine FS, Silvestri T, Vannier E, Duff GW, Miossec P. IL-1B and IL-1Ra genepolymorphisms and disease severity in rheumatoid arthritis: interaction with their plasma levels.Genes Immun 2001;2(4):222–228. [PubMed: 11477478]

17. Tolusso B, Pietrapertosa D, Morelli A, De SM, Gremese E, Farina G, et al. IL-1B and IL-1RN genepolymorphisms in rheumatoid arthritis: relationship with protein plasma levels and response totherapy. Pharmacogenomics 2006;7(5):683–695. [PubMed: 16886894]

18. Hall SK, Perregaux DG, Gabel CA, Woodworth T, Durham LK, Huizinga TW, et al. Correlation ofpolymorphic variation in the promoter region of the interleukin-1 beta gene with secretion ofinterleukin-1 beta protein. Arthritis Rheum 2004;50:1976–1983. [PubMed: 15188375]

19. Bohan A, Peter JB. Polymyositis and dermatomyositis. Parts 1 and 2. N Engl J Med 1975;292:344–347. 3403–3407. [PubMed: 1090839]

20. Parks CG, Pandey JP, Dooley MA, Treadwell EL, St CE, Gilkeson GS, et al. Genetic polymorphismsin tumor necrosis factor (TNF)-alpha and TNF-beta in a population-based study of systemic lupuserythematosus: associations and interaction with the interleukin-1alpha-889 C/T polymorphism.Hum Immunol 2004;65(6):622–631. [PubMed: 15219382]

21. Parks CG, Cooper GS, Dooley MA, Treadwell EL, St Clair EW, Gilkeson GS, et al. Systemic lupuserythematosus and genetic variation in the interleukin 1 gene cluster: a population based study in thesoutheastern United States. Ann Rheum Dis 2004;63(1):91–94. [PubMed: 14672899]

22. O'anlon TP, Carrick DM, Targoff IN, Arnett FC, Reveille JD, Carrington M, et al. HLA-A, -B, -DRB1 and -DQA1 allelic profiles for the idiopathic inflammatory myopathies: Distinctimmunogenetic risk and protective factors distinguish European American patients with differentmyositis autoantibodies. Medicine 2006;85:111–127. [PubMed: 16609350]

23. Holm S. A simple sequentially rejective multiple test procedure. Scand J Statist 1979;6:65–70.24. Mamyrova G, O'Hanlon TP, Monroe JB, Carrick DM, Malley JD, Adams S, et al. Immunogenetic

risk and protective factors for juvenile dermatomyositis in Caucasians. Arthritis and Rheumatism2006;54:3979–3987. [PubMed: 17133612]

25. McDowell TL, Symons JA, Ploski R, Forre O, Duff GW. A genetic association between juvenilerheumatoid arthritis and a novel interleukin-1 alpha polymorphism. Arthritis Rheum 1995;38:221–228. [PubMed: 7848312]



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26. Rahman P, Sun S, Peddle L, Snelgrove T, Melay W, Greenwood C, et al. Association between theinterleukin-1 family gene cluster and psoriatic arthritis. Arthritis Rheum 2006;54:2321–2325.[PubMed: 16918024]

27. Karasneh J, Hajeer AH, Barrett J, Ollier WE, Thornhill M, Gul A. Association of specific interleukin1 gene cluster polymorphisms with increased susceptibility for Behcet's disease. Rheumatology(Oxford) 2003;42(7):860–864. [PubMed: 12730545]

28. Friedman JM, Pachman LM, Maryjowski ML, Radvany RM, Crowe WE, Hanson H, et al.Immunogenetic studies of juvenile dermatomyositis, HLA-DR antigen frequencies. Arthritis Rheum1983;26:210–216. [PubMed: 6600616]

29. Crowe WE, Bove KE, Levinson JE, Hilton PK. Clinical and Pathogenetic Implications ofHistopathology in Childhood Polydermatomyositis. Arthritis Rheum 1982;25:126–139. [PubMed:7066042]

30. Mamyrova G, Kleiner DE, James-Newton L, Shaham B, Miller FM, Rider LG. Late-onsetgastrointestinal pain in juvenile dermatomyositis as a manifestation of ischemic ulceration fromchronic endarteropathy. Arthritis Rheum 2007;57:881–884. [PubMed: 17530691]

31. Werth VP, Zhang W, Dortzbach K, Sullivan K. Association of a promoter polymorphism of tumornecrosis factor-alpha with subacute cutaneous lupus erythematosus and distinct photoregulation oftranscription. J Invest Dermatol 2000;115(4):726–730. [PubMed: 10998151]

32. Millard TP, Kondeatis E, Cox A, Wilson AG, Grabczynska SA, Carey BS, et al. A candidate geneanalysis of three related photosensitivity disorders: cutaneous lupus erythematosus, polymorphiclight eruption and actinic prurigo. Br J Dermatol 2001;145(2):229–236. [PubMed: 11531784]



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Mamyrova et al. Page 11Ta

ble

1Su

mm

ary

of c

ytok

ine

poly

mor

phis

ms i

n C

auca

sian

pat

ient

s with

juve

nile

der

mat

omyo

sitis

(DM

).

Cyt

okin

ePo

lym

orph

ism

Juve

nile

DM

N (%

)C

ontr

ols

N (%

)P

Pcor

OR

95%

CI

TNFα

-308

N=

221

N=

203

Gen

otyp

e

AA

8 (3

.6)

4 (2

.0)

0.39

AG

132

(59.

7)59

(29.

1)<

0.00

010.

0003

3.6

2.4

– 5.

4

GG

81 (3

6.7)

140

(69.

0)<

0.0

001

0.00

030.

260.

17 –

0.3

9

Alle

le

A14

0 (6

3.3)

63 (3

1.0)

< 0.

0001

0.00

023.

82.

6 –

5.8

G21

3 (9

6.4)

199

(98.

0)0.

39

TNFα

-238

N =

206

N=

203

Gen

otyp

e

AA

2 (1

.0)

0 (0

.0)

0.50

AG7

(3.4

)28

(13.

8)0.

0002

0.00

060.

220.

09 –

0.5

2

GG

197

(95.

6)17

5 (8

6.2)

0.00

090.

002

3.5

1.6

– 7.

6

Alle

le

A9

(4.4

)28

(13.

8)0.

0009

0.00

20.

290.

13 –

0.6

2

G20

4 (9

9.0)

203

(100

.0)

0.50

IL-1α-

889

N =

152

N =

202

Gen

otyp

e

CC

53 (3

4.9)

68 (3

3.7)

0.82

CT

84 (5

5.3)

109

(54.

0)0.

83

TT15

(9.9

)25

(12.

4)0.

50

Alle

le

C13

7 (9

0.1)

177

(87.

6)0.

50

T99

(65.

1)13

4 (6

6.3)

0.82

IL-1α+

4845

N =

203

N =

202

Gen

otyp

e


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Mamyrova et al. Page 12C

ytok

ine

Poly

mor

phis

mJu

veni

le D

MN

(%)

Con

trol

sN

(%)

PPc

orO

R95

% C

I

GG

81 (3

9.9)

95 (4

7.0)

0.16

GT

76 (3

7.4)

83 (4

1.1)

0.48

TT

46 (2

2.7)

24 (1

1.9)

0.00

60.

017

2.2

1.3

– 3.

7

Alle

le

G15

7 (7

7.3)

178

(88.

1)0.

006

0.01

10.

460.

27 –

0.7

9

T12

2 (6

0.1)

107

(53.

0)0.

16

IL-1β-

511

218

202

Gen

otyp

e

CC

76 (3

4.9)

89 (4

4.1)

0.06

CT

111

(50.

9)87

(43.

1)0.

12

TT31

(14.

2)26

(12.

8)0.

78

Alle

le

C18

7 (8

5.8)

176

(87.

1)0.

78

T14

2 (6

5.1)

113

(55.

9)0.

06

IL-1β+

3953

N =

158

N=

201

Gen

otyp

e

CC

74 (4

6.8)

120

(59.

7)0.

019

0.05

60.

590.

39 –

0.9

1

CT

73 (4

6.3)

68 (3

3.8)

0.02

20.

056

1.7

1.1

– 2.

6

TT11

(7.0

)13

(6.5

)1.

00

Alle

le

C14

7 (9

3.0)

188

(93.

5)1.

00

T84

(53.

2)81

(40.

3)0.

019

0.03

81.

71.

1 –

2.6

HLA

DR

B1

Alle

leN

= 1

43N

= 2

02

DR

B1* 03

70 (4

8.95

)43

( 21

.3)

<0.0

001

0.00

013.

52.

2 –

5.7

* Abb

revi

atio

ns: D

M, d

erm

atom

yosi

tis; O

R, o

dds r

atio

; CI,

conf

iden

ce in

terv

al; T

NFα

, tum

or n

ecro

sis f

acto

r alp

ha; I

L-1,

inte

rleuk

in 1

; P, p

val

ue; P

cor,

p va

lue

afte

r Hol

m’s

adj

ustm

ent f

or m

ultip

leco

mpa

rison

s with

in e

ach

geno

type

or a

llele

, usi

ng fa

mily

– w

ise

erro

r rat

es o

f 5%

.

**A

llele

car

riage

rate

s are

show

n. S

igni

fican

t ris

k fa

ctor

s are

show

n in

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dfac

e; si

gnifi

cant

pro

tect

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fact

ors a

re sh

own

in it

alic

s.


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Table 2Relative importance of juvenile dermatomyositis associated risk and protective cytokine polymorphism genotypes inCaucasian patients as predicted by Random Forests Classification modeling.

Genotype Gini Score (%) −2SE +2SE

TNFα-308AG* 100.0 100.0 - 100.0

TNFα-308GG** 87.7 86.8 – 88.6

IL-1β+3953CT 38.1 37.7 – 38.4

IL-1β+3953CC 36.2 35.8 – 36.5

IL-1α+4845TT 35.1 34.8 – 35.4

TNFα-238GG 33.4 33.1 – 33.7

TNFα-238AG 31.0 30.7 – 31.2*Abbreviations: SE, standard error; TNFα, tumor necrosis factor alpha; IL-1, interleukin 1.

**Significant risk factors are shown in boldface; protective factors are shown in italics. Only genotypes significant from the univariate analysis were

included in this modeling, performed 202 control subjects. Error Rate = 39.7%.


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Table 3Relative importance of juvenile dermatomyositis associated risk and protective alleles in Caucasian patients aspredicted by Random Forests Classification modeling.

Allele GINI Score (%) −2SE +2SE

HLA DRB1*03 100.0 100.0 - 100.0

TNFα-308A 90.7 90.2– 91.3

IL-1β+3953T 59.4 59.1 –59.7

TNFα-238A 53.1 52.9– 53.4

IL-1a+4845G 38.9 38.7– 39.1*Abbreviations and conventions: same as Table 2A. Only alleles significant from the univariate analysis, including HLA DRB1*03, were included in this

modeling, performed using 65 juvenile DM and 202 control subjects. Error Rate = 36.7%.


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Table 4Logistic regression analysis of risk and protective cytokine polymorphism alleles in Caucasian patients with juveniledermatomyositis.

Allele Coefficient P value OR 95% Wald confidence limits

HLA DRB1*03 1.2 0.0006 3.5 1.7 – 7.0

TNFα-238A −1.6 0.035 0.19 0.04 – 0.89

TNFα-308A 0.66 0.06 1.9 0.97 – 3.9

IL-1β+3953T 0.52 0.10 1.7 0.91 – 3.1

IL-1a+4845G −0.42 0.34 0.66 0.28 – 1.6

Likelihood Ratio Chi Square 40.9, P < 0.0001; Per cent concordant 69.6

Hosmer-Lemeshow Goodness-of-it test Chi-square = 3.1, P = 0.80*Abbreviations and conventions: the same as Table 2A. The significant cytokine polymorphisms from univariate analysis after Holm’s adjustment for

multiple comparisons were used in this modeling performed using 65 for juvenile DM patients and 201 control subjects.


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ble

5Su

mm

ary

of c

ytok

ine

poly

mor

phis

ms

as s

ever

ity f

acto

rs f

or t

he d

evel

opm

ent

of c

alci

nosi

s in

Cau

casi

an p

atie

nts

with

juv

enile

derm

atom

yosi

tis (D

M).

Cyt

okin

e Po

lym

orph

ism

Juve

nile

DM

with

Cal

cino

sis N

(%)

Juve

nile

DM

with

out

Cal

cino

sis N

(%)

PPc

orO

R95

% C

I

TNFα

-308

N =

68

N =

153

Gen

otyp

e

AA

6 (8

.8)

2 (1

.3)

0.01

50.

045

7.3

1.4

– 37

.2

AG

37 (5

4.4)

95 (6

2.1)

0.30

GG

25 (3

6.8)

56 (3

6.6)

1.00

Alle

le

A43

(63.

2)97

(63.

4)1.

00

G62

(91.

2)15

1 (9

8.7)

0.01

50.

030

0.14

0.03

– 0

.70

TNFα

-238

N =

64

N =

142

Gen

otyp

e

AA

0 (0

.0)

3 (1

.4)

0.55

AG

0 (0

.0)

6 (6

.3)

0.18

GG

64 (1

00.0

)13

3 (9

3.3)

0.06

Alle

le

A0

(0.0

)9

(6.3

)0.

06

G64

(100

.0)

139

(97.

9)0.

55

IL-1α-

889

N =

46

N =

106

Gen

otyp

e

CC

25 (5

4.4)

35 (3

3.0)

0.01

90.

057

2.4

1.2

– 4.

9

CT

16 (3

4.8)

56 (5

2.8

)0.

052

TT5

(10.

9)15

(14.

2 )

0.79

Alle

le

C41

(89.

1)91

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8)0.

79

T21

(45.

7)71

(67.

0)0.

019

0.03

80.

410.

20 –

0.8

4

IL-1α+

4845

N =

64

N =

139


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Mamyrova et al. Page 17C

ytok

ine

Poly

mor

phis

mJu

veni

le D

M w

ithC

alci

nosi

s N (%

)Ju

veni

le D

M w

ithou

tC

alci

nosi

s N (%

)P

Pcor

OR

95%

CI

Gen

otyp

e

GG

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0.6)

57 (4

1.0)

1.00

GT

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0.6)

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6.0)

0.54

TT12

(18.

8)31

(23.

0)0.

58

Alle

le

G52

(81.

3)10

7 (7

7.0)

0.29

T38

(59.

4)82

(59.

0)1.

00

IL-1β-

511

N =

68

N =

150

Gen

otyp

e

CC

13 (1

9.1)

25 (1

6.7)

0.70

CT

41 (6

0.3)

96 (6

4.0)

0.77

TT14

(20.

6)29

(19.

3)0.

86

Alle

le

C54

(79.

4)12

1 (8

0.7)

0.86

T55

(80.

9)12

5 (8

3.3)

0.70

IL-1β+

3953

N =

50

N=

108

Gen

otyp

e

CC

25 (5

0.0)

49 (4

5.4)

0.61

CT

23 (4

6.0)

50 (4

6.3)

1.00

TT2

(4.0

)9

(8.3

)0.

50

Alle

le

C48

(25.

0)99

(91.

7)0.

51

T25

(50.

0)59

(54.

6)0.

61

HLA

DR

B1

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leN

= 5

1N

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2

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30 (5

8.8)

40 (4

3.4)

0.08

* Abb

revi

atio

ns: D

M, d

erm

atom

yosi

tis; O

R, o

dds r

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; CI,

conf

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.

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n. S

igni

fican

t ris

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ctor

s are

show

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bol

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e; si

gnifi

cant

pro

tect

ive

fact

ors a

re sh

own

in it

alic

s.


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NIH



ble

6Su

mm

ary

of T

NFα

-308

and

HLA

DR

B 1

* 03

alle

les a

s sev

erity

fact

ors f

or th

e de

velo

pmen

t of g

astro

inte

stin

al o

r cut

aneo

us u

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atio

nsin

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casi

an p

atie

nts w

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le d

erm

atom

yosi

tis (D

M).

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mor

phis

mJu

veni

le D

M w

ithU

lcer

atio

nsN

(%)

Juve

nile

DM

with

out

Ulc

erat

ions

N (%

)

PP

cor

OR

95%

CI

TNFα

-308

N =

46

N =

175

Gen

otyp

e

AA

5 (1

0.9)

3 (1

.7)

0.01

90.

057

7.0

1.6

– 30

.5

AG

25 (5

4.4)

107

(61.

1)0.

4

GG

16 (3

4.8)

65 (3

7.1)

0.86

Alle

le

A30

(65.

2)11

0 (6

2.9)

0.86

G41

(89.

1)17

2 (9

8.3)

0.01

10.

022

0.14

0.00

3 –

0.62

HLA

DR

B1

Alle

leN

= 3

6N

= 1

07

DR

B1* 03

22 (6

1.1)

48 (4

4.9)

0.12

* Abb

revi

atio

ns: D

M, d

erm

atom

yosi

tis; O

R, o

dds r

atio

; CI,

conf

iden

ce in

terv

al; T

NF-α,

tum

or n

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sis f

acto

r alp

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L-1,

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; P, p

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in e

ach

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type

or a

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, usi

ng fa

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ise

erro

r rat

es o

f 5%

.

**A

llele

car

riage

rate

s are

show

n. S

igni

fican

t ris

k fa

ctor

s are

show

n in

bol

dfac

e; p

rote

ctiv

e fa

ctor

s are

show

n in

ital

ics.

For I

L1α-

889,

IL1β

-511

, IL1α+

4845

, IL1β+

3953

and

TN

Fα-2

38 c

ytok

ine

poly

mor

phis

ms,

P >

0.05

(dat

a no

t sho

wn)


Documents

Cytokine gene polymorphisms as risk and severity factors for juvenile dermatomyositis