Cytokine Gene Polymorphisms as Risk and Severity Factors for Juvenile Dermatomyositis

doi:10.1002/art.24039

Arthritis 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

PMCID: PMC2674642

NIHMSID: NIHMS87864

Cytokine Gene Polymorphisms as Risk and Severity Factors for Juvenile Dermatomyositis

Gulnara Mamyrova, MD, PhD,¹ Terrance P. O’Hanlon, PhD,¹ Laura Sillers, AB,¹ Karen Malley, BA,² Laura James-Newton, PhD, RN,¹ Christine G. Parks, PhD,³ Glinda S. Cooper, PhD,⁴ Janardan P. Pandey, PhD,⁵ Frederick W. Miller, MD, PhD,¹ Lisa G. Rider, MD,¹ and for the Childhood Myositis Heterogeneity Collaborative Study Group

¹Environmental Autoimmunity Group, National Institute of Environmental Health Sciences, National Institutes 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.

Corresponding Author and Reprint Requests: Lisa G. Rider, MD, Environmental Autoimmunity Group National Institute of Environmental 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: riderl/at/mail.nih.gov

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Abstract

Objective

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

Methods

TNFα and IL-1 cytokine polymorphism and HLA typing were performed in 221 Caucasian 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), and IL-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 the TNFα -308A (OR 3.8) and IL-1β+3953T (OR 1.7) alleles were risk factors and TNFα -238A (OR 0.29) and IL-1α+4845G (OR 0.46) were protective for juvenile DM. Random Forests classification analysis showed HLA DRB1*03 and TNFα -308A to have highest relative importance as risk factors for 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 (OR 0.41) alleles were protective for the development of calcinosis. TNFα-308AA (OR 7.0) was a possible risk factor, and carriage of the TNFα-308G allele (OR 0.14) was protective for the development of ulcerations. None of the studied TNFα, IL-1α and IL-β polymorphisms were associated with disease course, severity at diagnosis, or gender.

Conclusions

TNFα and IL-1 genetic polymorphisms contribute to the development of juvenile DM and may also be indicators of disease severity.

Juvenile dermatomyositis (DM) is a systemic autoimmune disease which is characterized by symmetric proximal weakness and distinctive skin rashes. The etiology of juvenile DM remains unknown, but recent evidence suggests combinations of genetic and environmental risk factors are involved (1). Cytokines appear to have an important role in the pathogenesis of inflammatory myopathies (2). The expression of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα), interleukin 1alpha (IL-1α) and interleukin 1beta (IL-1β) are increased in the mononuclear cell infiltrates and on muscle fibers of biopsies from myositis patients (2–4). The expression of IL-1α and IL-1β are also increased on the capillary endothelial cells of the affected muscle (2).

The TNFα-308A promoter polymorphism, which is associated with increased production of TNFα from peripheral blood mononuclear cells, is increased in frequency in Caucasian patients with juvenile and adult DM (5–8) and in Chinese patients with adult DM (9) compared to healthy controls. The TNFα-308A allele has also been associated with the development of calcinosis and a chronic disease course in juvenile DM patients (5). In terms of IL-1 polymorphisms, the pro-inflammatory intronic variable number tandem repeat of IL-1 receptor antagonist, IL1-RA A1, is a risk factor for juvenile idiopathic inflammatory myopathies in Caucasian 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 severity and susceptibility to systemic autoimmune diseases (11–13), with higher in-vitro stimulated production 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 cytokine polymorphisms for IL-1 and TNFα are risk or protective factors for juvenile DM, and to define their relative importance. We also aimed to examine these cytokine polymorphisms as severity factors for juvenile DM.

PATIENTS AND METHODS

Patients

DNA from 201 Caucasian patients with juvenile onset DM and 20 with juvenile DM in association with another autoimmune condition were used for IL-1α, IL-1β and TNFα polymorphism genotyping. All patients met probable or definite Bohan and Peter criteria for DM (19) and were diagnosed prior to 18 years of age. The diagnosis of juvenile overlap DM was defined by meeting both Bohan and Peter criteria for DM (1975), as well as American College 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 and immune thrombocytopenia purpura (2 each), ulcerative colitis, psoriasis and Hashimoto’s thyroiditis (1 each). Control cytokine polymorphism data were obtained from 203 ethnically - matched healthy volunteers who participated in the Carolina Lupus Study (20;21). All subjects were enrolled in investigational review board-approved NIH and Food and Drug Administration protocols. Patients were enrolled with a blood sample and a physician questionnaire of the illness features, which was reviewed by a pediatric rheumatologist (GM or LR).

Seventy-five percent of the juvenile DM patients were female. Twenty-five percent had a chronic 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 or gastrointestinal ulceration. Seventy six percent had a history of photosensitive rashes. The median age at the time of diagnosis was 7.5 years [25% 5 years, 75% 12 years] and the median physician global activity at diagnosis was 3 on a 0 – 4 point Likert scale.

Cytokine Polymorphism Genotyping

Single 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] were determined by PCR of peripheral blood leukocyte DNA, followed by restriction enzyme digestion and gel electrophoresis (20;21) in 221 juvenile DM patients and 203 controls. Assay failure 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 typing

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

Statistical Analysis

Analyses were performed using GraphPad InStat version 3.00 for Windows (GraphPad Software, 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 was determined by the number of cytokine polymorphism - positive subjects divided by the total number of subjects for which data from a particular allele were available. P values were adjusted for multiple testing using Holm’s procedure (23), which is a nonparametric step-down adjustment that strongly controls for family-wise error rate and makes no assumptions concerning the correlation structure of the data or the observed p-values. P values were determined 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 patients compared to controls prior to correction for multiple comparisons were termed possible risk or protective factors, respectively.

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

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

Haplotype analysis was performed using Haploview software (Haploview Software version 3.32, Broad Institute, Cambridge, MA, USA http://www.broad.mit.edu/mpg/haploview/) to determine the level of linkage disequilibrium between cytokine polymorphism markers by comparing patients and controls, and also analyzing control or patient population genotypes in isolation.

Results

Juvenile DM- associated risk and protective cytokine polymorphisms

The TNFα genotypes TNFα -308AG (odds ratio [OR] 3.6, P < 0.0001) and TNFα -238GG (OR 3.5, 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 (Table 1). Carriage of the TNFα -308A allele (OR 3.8, P < 0.0001) was a risk factor and carriage of the TNFα -238A allele (OR 0.29, P = 0.0009) was protective for juvenile DM (Table 1).

Table 1

Summary of cytokine polymorphisms in Caucasian patients with juvenile dermatomyositis (DM).

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 protective factors, respectively for the development of juvenile DM in Caucasian patients (Table 1). Two additional 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 not significant after adjustment for multiple comparisons. The HLA DRB1*03 allele was confirmed 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 in the 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 DM patients 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 rate of cytokine or DRB1 alleles between the juvenile DM patients and those who also had an associated autoimmune disease.

Relative importance of juvenile DM- associated risk and protective cytokine polymorphisms

The relative importance (RI) of cytokine polymorphisms as risk and protective factors for juvenile DM was examined using Random Forests Classification analysis. In examining specific cytokine polymorphism genotypes, TNFα -308AG (RI 100%) was found to have the highest RI for the development of juvenile DM in Caucasian patients, followed by the protective 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 DM were much lower (Table 2).

Table 2

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

In terms of the analysis of the relative importance of cytokine alleles in relationship to HLA DRB1*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 risk or protective factors for juvenile DM (Table 3).

Table 3

Relative importance of juvenile dermatomyositis associated risk and protective alleles in Caucasian patients as predicted by Random Forests Classification modeling.

The juvenile DM-associated risk and protective cytokine polymorphism alleles defined in univariate 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 protective factors, respectively, in the model. TNFα -308A (OR 1.9) had borderline significance as a risk factor in the model. None of the other risk and protective cytokine polymorphism alleles defined by univariate analysis were significant in the logistic regression model.

Table 4

Logistic regression analysis of risk and protective cytokine polymorphism alleles in Caucasian patients with juvenile dermatomyositis.

Allele interaction analyses

In order to assess possible linkage disequilibrium among the IL-1 or TNFα alleles, we examined whether 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 two TNFα 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’ range 0.116 – 0.676, r² range 0.005 – 0.253). The analysis was conducted separately in patients and controls, and in both groups combined; and similar results were obtained. The Hardy-Weinberg p values in controls ranged from 0.093 – 0.74.

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

Cytokine polymorphisms as disease severity factors for juvenile DM

To identify whether TNFα and IL-1 polymorphisms are disease severity factors, we compared juvenile DM cases with and without various disease features to see if these alleles contribute to the risk of certain illness complications. First, TNFα -308AA (OR 7.3) was found to be a risk 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 of the TNFα-308G (OR 0.14) and IL1α-889T (OR 0.41) alleles were protective factors for the development of calcinosis (Table 5). HLA DRB1*03 was not a severity factor for the development of calcinosis.

Table 5

Summary of cytokine polymorphisms as severity factors for the development of calcinosis in Caucasian patients with juvenile dermatomyositis (DM).

We also examined whether these IL-1 and TNFα polymorphisms were related to the development of gastrointestinal or cutaneous ulcerations. Only TNFα-308AA (OR 7.0) was found to be a possible risk factor, and carriage of the TNFα-308G allele (OR 0.14) was a protective 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 or protective factors for the development of ulcerations.

Table 6

Summary of TNFα -308 and HLA DRB 1^*03 alleles as severity factors for the development of gastrointestinal or cutaneous ulcerations in Caucasian patients with juvenile dermatomyositis (DM).

IL-1α+4845GG (OR 2.4, P = 0.003), and TNFα-238AG (OR 14.0, P = 0.016) were increased in Caucasian juvenile DM patients younger than 7.5 years of age at diagnosis, the median age of diagnosis of juvenile DM in this cohort, compared to patients older than 7.5 years at the time of diagnosis. IL-1α+4845TT (OR 0.32, P = 0.001) was less frequent in children with juvenile DM younger than 7.5 years of age compared to those older at diagnosis. Linear regression modeling confirmed IL-1α+4845GG and TNF-α-238A to be associated with a younger age at diagnosis, and carriage of the IL-1α+4845T allele to be associated with an older age 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) were possible 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 of photosensitive skin rashes. These findings were not significant after adjustment for multiple comparisons. Neither the TNF alleles nor HLA DRB1*03 were associated with photosensitive skin 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 at diagnosis, or with gender in patients with juvenile DM. There was no difference in the frequency of the TNFα-308AA genotype based on disease course: 1 of 53 (1.9%) patients with the TNFα-308AA genotype had a chronic course compared to 2 of 41 patients (4.9%) with a monocyclic 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 disease course: 31 of 53 (58.5%) patients with TNFα-308A allele had a chronic continuous disease course 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).

Discussion

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

Novel findings of this study also included the identification of IL-1 cytokine polymorphisms IL1α+4845TT and IL1β+3953T as risk factors and carriage of the IL1α+4845G allele as a protective factor for juvenile DM in Caucasians. We did not detect haplotypes among the four IL-1 cytokine polymorphisms, and the results of the Random Forests classification also suggest these 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 connective tissue diseases, including early-onset pauciarticular juvenile rheumatoid arthritis (25), psoriatic arthritis (26), Behcet’s disease (27), and systemic sclerosis (12). In patients with rheumatoid arthritis, IL-1β+3953T has been associated with more active and erosive disease, with higher in vitro stimulated production of IL-1β from monocytes stimulated with LPS (14) and with lower plasma levels of the anti-inflammatory IL-1RA (16;17).

The associations of multiple TNFα and IL-1 polymorphisms as risk and protective factors for juvenile DM suggest these cytokines are important contributors to the pathogenesis of juvenile DM. Both IL-1 and TNFα have been detected in the affected muscle of patients with DM and other idiopathic inflammatory myopathies (2–4). Stimulated peripheral blood mononuclear cells 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 -308A allele (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 also able to identify that the TNFα-308A allele has a much higher relative importance among all detected risk and protective TNFα and IL-1 cytokine polymorphisms. At the same time, the relative importance of the TNFα-308A allele is lower than HLA DRB1*03, the previously described major immunogenetic risk factor for juvenile DM (24;28), in Random Forests classification analysis. This finding of the stronger relative importance of DRB1*03 over TNFα was confirmed in logistic regression modeling, in which DRB1*03 was found to be a strong risk factor and TNFα-238A a protective factor, but the TNFα-308 A allele was not significant in the model. Because of the lack of availability of DRB1*0301 typing in control subjects, we were not able to perform haplotype analysis to examine linkage of TNFα alleles with DRB1*0301. However, our findings confirm the work of Chinoy et. al., who found that the TNFα-308A and DRB1*0301 alleles were independent risk factors for adult DM in logistic regression modeling and haplotype analysis, in contrast to subsets of adult myositis patients, including patients with polymyositis and with anti-synthetase and PM-Scl autoantibodies, in which linkage disequilibrium between TNFα and HLA alleles exists (8). Using association methodology, Werth et al, also reported independent risk associations for TNFα-308A and HLA DRB1*03 in a smaller adult DM population (6).

TNFα-308A has been reported to be associated with calcinosis and a chronic illness course in a small study of juvenile DM (5). Here, we confirmed TNFα-308AA as a risk factor for calcinosis and the TNFα-308G allele as protective, not only for calcinosis, but also for the development of gastrointestinal or cutaneous ulcerations in juvenile DM, a serious complication of illness that also marks severe disease (29;30). This dichotomous relationship of TNFα-308A as a risk factor for calcinosis in the absence of DRB1*03 further suggests independence 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 development of photosensitive skin rashes, but did find possible associations of photosensitive rashes with some of the IL-1 polymorphisms, which were not significant after adjustment for multiple comparisons. Interestingly, Werth et al. demonstrated an interaction of IL-1 and ultraviolet-B light 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 ancestral haplotype, was also seen as a risk factor for subacute cutaneous lupus, and the IL1β+3954 T allele 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 referral population of juvenile DM patients, enrolled from throughout the United States and Canada, primarily from tertiary care medical centers. The control population was also from the Carolinas, not from the entire United States (20;21). Confirmation of these findings in familial studies through transmission disequilibrium testing would be helpful. Also, the DRB1*03 typing available from control subjects was not resolved to the allelic level, and some patients were not genotyped for all of the cytokine polymorphism and DRB1 alleles, potentially introducing 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 accounted for 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 disease severity. TNFα appears to be more important than IL-1 polymorphisms in contributing to the risk 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.

Acknowledgments

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

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

Appendix

Members of the Childhood Myositis Heterogeneity Collaborative Study Group

Leslie 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 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, Gail D. 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, Melissa Hawkins-Holt, C Hendrics, Michael Henrickson, Gloria C. Higgins, J Roger Hollister, Russell Hopp, 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, Lawrence J. 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, Robert N. 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, Carol A. 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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