Genome-wide association study identifies HLA 8.1 ancestral haplotype alleles as major genetic risk factors for myositis phenotypes.

Autoimmune muscle diseases (myositis) comprise a group of complex phenotypes influenced by genetic and environmental factors. To identify genetic risk factors in patients of European ancestry, we conducted a genome-wide association study (GWAS) of the major myositis phenotypes in a total of 1710 cases, which included 705 adult dermatomyositis, 473 juvenile dermatomyositis, 532 polymyositis and 202 adult dermatomyositis, juvenile dermatomyositis or polymyositis patients with anti-histidyl-tRNA synthetase (anti-Jo-1) autoantibodies, and compared them with 4724 controls. Single-nucleotide polymorphisms showing strong associations (P<5×10(-8)) in GWAS were identified in the major histocompatibility complex (MHC) region for all myositis phenotypes together, as well as for the four clinical and autoantibody phenotypes studied separately. Imputation and regression analyses found that alleles comprising the human leukocyte antigen (HLA) 8.1 ancestral haplotype (AH8.1) defined essentially all the genetic risk in the phenotypes studied. Although the HLA DRB1*03:01 allele showed slightly stronger associations with adult and juvenile dermatomyositis, and HLA B*08:01 with polymyositis and anti-Jo-1 autoantibody-positive myositis, multiple alleles of AH8.1 were required for the full risk effects. Our findings establish that alleles of the AH8.1 comprise the primary genetic risk factors associated with the major myositis phenotypes in geographically diverse Caucasian populations.

Pathological calcification in juvenile dermatomyositis (JDM): microCT and synchrotron x-ray diffraction reveal hydroxyapatite with varied microstructures.

The objective of this study was to begin to relate the microstructure of calcinosis samples to clinical and laboratory characteristics of the juvenile dermatomyositis (JDM) patients. Laboratory x-ray microCT (micro-Computed Tomography) noninvasively mapped microstructure for the first time in JDM calcifications. Synchrotron x-ray diffraction (transmission geometry) identified the mineral phase and crystallite size in the deposits. Samples were obtained from four children who had active JDM longer than 80 months and who were typed for TNFalpha-308 allele polymorphisms. Uniform mineral (giving the appearance of an extruded solid) was observed in one patient, and irregular blocks of differing sizes filled the samples from two other patients. The sample from the fourth patient appeared to combine features of the other two types. These spatial distributions of mineral were quite different from those in a bone reference sample. The only mineral observed in the JDM samples was hydroxyapatite (HAP), and the diffraction peaks of the JDM samples were slightly narrower than those of a trabecular bone reference sample. Diffraction peak widths of the JDM specimens revealed crystallite sizes (approximately 220-240 A) that are comparable to values reported in the literature for bone. Three children were positive for TNFalpha-308 GA polymorphism. The data suggest several possible origins for blocky vs. uniform structure of the JDM calcifications, including differences in duration of untreated inflammation, in TNFalpha-308 polymorphism, and in mechanical constraint at the calcification site. Information from additional samples is required to determine the relative role of each of these factors. Taken together, non-invasive microCT and x-ray diffraction characterization on the same samples offer an informative window into the dystrophic mineralization process in JDM.

Juvenile dermatomyositis: the association of the TNF alpha-308A allele and disease chronicity.

Little is known concerning factors associated with the outcome of juvenile dermatomyositis (JDM), which can be variable and lethal. Previous work has documented that the association of DQA1*0501 with JDM is higher than in control groups and that the first symptoms (rash and weakness) of JDM appear to follow evidence of an infectious process--most frequently upper respiratory in nature. Preliminary data show that a long period of symptoms being left untreated before starting therapy and the TNF alpha-308A allele are associated with prolonged JDM symptoms requiring > or = 36 months of immunosuppressive therapy. A short duration of untreated disease is associated with a relative increase in CD8(+) T cells and CD56(+) natural killer (NK) cells in the untreated JDM muscle biopsy compared with a longer duration of untreated disease. The TNF alpha-308A allele is overrepresented in white children with JDM. In addition, it is associated with pathologic calcifications, increased production of TNF alpha by peripheral blood mononuclear cells in vitro and JDM muscle fibers in vivo, and occlusion of capillaries, which may be mediated in part by elevated circulating levels of thrombospondin-1, a potent anti-angiogenic factor. We speculate that DQA1*0501 is associated with JDM susceptibility to an infectious process, eliciting and activating NK cells early in the disease course. We conclude that the TNF alpha-308A allele indicates directly (or is a surrogate marker of) children with JDM who produce higher concentrations of TNF alpha in response to this undefined inflammatory stimulus, as well as increased concentrations of TSP-1 with resultant small vessel occlusion, contributing to subsequent disease chronicity.

Expression of TNFalpha by muscle fibers in biopsies from children with untreated juvenile dermatomyositis: association with the TNFalpha-308A allele.

Juvenile dermatomyositis (JDM) is the most common pediatric inflammatory myopathy. In patients with JDM, the A --> G polymorphism in the tumor necrosis factor alpha (TNFalpha)-308 promoter region (TNFalpha-308A) is associated with prolonged disease course and increased production of TNFalpha by peripheral blood mononuclear cells (Arthritis Rheum. 43, 2368-2377, 2000). Magnetic resonance imaging directed biopsies from 21 white children with untreated JDM were evaluated for TNFalpha expression. Using monoclonal antibody to TNFalpha, fresh frozen sections were processed by the standard immunohistochemical technique. We investigated the association among the expression of TNFalpha by muscle fibers, disease activity, duration of untreated disease, and the TNFalpha-308 polymorphism. Untreated children with JDM who had the TNFalpha-308A allele had an increased number of TNFalpha stained muscle fibers than children with the TNFalpha-308G allele (P = 0.001). There was no association with disease activity or duration of untreated disease. We speculate that muscle fiber production of TNFalpha provides a microenvironment in which TNFalpha acts synergistically with other mediators to prolong muscle fiber damage.

TNFalpha-308A allele in juvenile dermatomyositis: association with increased production of tumor necrosis factor alpha, disease duration, and pathologic calcifications.

OBJECTIVE: To characterize the association between the TNFalpha-308A allele and 1) duration of active disease, 2) peripheral blood mononuclear cell (PBMC) synthesis of tumor necrosis factor alpha (TNFalpha) in vitro, and 3) pathologic calcifications in patients with juvenile dermatomyositis (DM). METHODS: The TNFalpha-308 alleles were determined by polymerase chain reaction in 37 white patients with juvenile DM and in 29 control subjects. Patients were grouped according to duration of immunosuppressive therapy: long (> or =36 months) or short (<36 months). Unstimulated PBMC were examined by enzyme-linked immunosorbent assay for TNFalpha production in vitro. Sixty-five white patients with juvenile DM were examined for pathologic calcifications. RESULTS: TNFalpha-308A was identified in 18 of 37 patients with juvenile DM, in contrast with 5 of 29 controls (P = 0.009). Sixteen of the 18 patients with juvenile DM who had the TNFalpha-308A allele had a disease course > or =36 months, compared with 6 of 19 patients with TNFalpha-308G (P = 0.001). PBMC from 16 of the 18 juvenile DM patients with TNFalpha-308A synthesized more TNFalpha (median 53 pg/ml) compared with PBMC from 9 of 19 patients with TNFalpha-308G (median 19 pg/ml) (P = 0.007). Nineteen of 22 juvenile DM patients requiring therapy for > or =36 months produced more TNFalpha (median 20.5 pg/ml) in comparison with 6 of 15 juvenile DM patients with a <36-month treatment course (median TNFalpha 0.0 pg/ml) (P = 0.005). Detectable calcifications were present in 3 of 8 children with juvenile DM who had TNFalpha-308AA, compared with 2 of 21 children with TNFalpha-308AG and 1 of 36 children who had TNFalpha-308GG (P = 0.017). CONCLUSION: A long course of juvenile DM and the presence of pathologic calcifications were associated with the TNFalpha-308A allele and with the increased production of TNFalpha, which may perpetuate the inflammatory response.

Decreased levels of CD54 (ICAM-1)-positive lymphocytes in the peripheral blood in untreated patients with active juvenile dermatomyositis.

Significant abnormalities are observed in the peripheral blood of juvenile dermatomyositis (JDM) patients with active disease. In this study, we confirm that there is a significant increase in the relative percentage of B lymphocytes in the peripheral blood of a group of untreated children with newly diagnosed active JDM compared to healthy children (P < 0.0001). In order to investigate if properties intrinsic to B cells contributed to their relative increase in JDM, the percentage of B cells expressing activation markers (CD23, CD25, CD54, and CD69) was measured and compared to pediatric controls. Compared to healthy children less than 10 years of age (not significantly different from the JDM group), the JDM patients had an increase in the proportion of lymphocytes expressing CD19 (B cells; P = 0.0017) and decreases in the percentage of lymphocytes that were CD3(-) CD16(+) and/or CD56(+) (NK cells; P = 0. 01) and CD3(+) CD8(+) (T suppressor/cytotoxic cells; P = 0.02). There were no significant differences in any of the B-cell activation markers assessed. Of note, the percentage of CD54(+) non-B lymphocytes (i.e., T cells and NK cells expressing CD54) was significantly lower in the JDM patients (25% +/- 5%) than in the "age-related" healthy control group (43% +/- 4%; P = 0.013). These results suggest the following for untreated children with active JDM: (i) the increase in the percentage of peripheral blood B cells is not due to intrinsic B-cell activation, and (ii) CD54/ICAM-1(+) non-B cells, CD8(+) T cells, and NK cells are being removed from circulation and may be participating in the pathophysiology of the disease.

Autoantibodies to DEK oncoprotein in human inflammatory disease.

OBJECTIVE: To evaluate the specificity of anti-DEK antibodies for juvenile rheumatoid arthritis (JRA). METHODS: Anti-DEK autoantibodies were measured by enzyme-linked immunosorbent assay (ELISA) using affinity-purified his6-DEK fusion protein. Sera from 639 subjects (417 patients with systemic autoimmune disease, 13 with sarcoidosis, 44 with pulmonary tuberculosis, 125 with uveitis, and 6 with scleritis, and 34 healthy control subjects) were screened. Reactivity was verified by immunoblotting and immunoprecipitation studies using baculovirus-expressed human DEK. RESULTS: Anti-DEK activity was found at the following frequencies: JRA 39.4% (n = 71), systemic lupus erythematosus (SLE) 25.1% (n = 216), sarcoidosis 46.2% (n = 13), rheumatoid arthritis 15.5% (n = 71), systemic sclerosis 36.0% (n = 22), polymyositis 6.2% (n = 16), and adult Still's disease 0% (n = 21). Autoantibodies also were detected in 9.1% of tuberculosis sera (n = 44), but were undetectable in sera from the 34 healthy controls. Western blot and immunoprecipitation assay results correlated well with the ELISA findings. In general, levels of anti-DEK autoantibodies were higher in SLE than in other patient subsets, including JRA. CONCLUSION: Anti-DEK autoantibodies are less specific for JRA than previously believed. They are produced in association with a variety of inflammatory conditions, many of which are associated with granuloma formation and/or predominant Thl cytokine production. Anti-DEK antibodies may be a marker for a subset of autoimmunity associated with interferon-gamma production rather than a particular disease subset.

The economic impact of intermittent high-dose intravenous versus oral corticosteroid treatment of juvenile dermatomyositis.

OBJECTIVE: To perform a cost-identification and cost-effectiveness analysis comparing oral corticosteroids (OCS) with high-dose intermittent intravenous corticosteroid (IVCS) regimens in the treatment of juvenile dermatomyositis (JDM). METHODS: Children previously diagnosed and treated for JDM (without myositis-specific or myositis-associated autoantibodies) at a single medical center by a single provider were identified. Two treatment protocols were compared: OCS and IVCS. Data on initial disease severity, time to remission, resource use, and costs generated were collected from patient records. Incremental cost-effectiveness ratios (ICE) were constructed. RESULTS: Patients treated with IVCS achieved median remission 2 years earlier at median increased cost of $13,736. The ICE ratio comparing IVCS to OCS is $6,868 per year of disease avoided. CONCLUSION: This study suggests that, although IVCS treatments are costly, they are cost-effective.

Development of validated disease activity and damage indices for the juvenile idiopathic inflammatory myopathies. II. The Childhood Myositis Assessment Scale (CMAS): a quantitative tool for the evaluation of muscle function. The Juvenile Dermatomyositis Disease Activity Collaborative Study Group.

OBJECTIVE: To develop, validate, and determine the measurement characteristics of a quantitative tool for assessing the severity of muscle involvement in children with idiopathic inflammatory myopathies. METHODS: The Childhood Myositis Assessment Scale (CMAS) was developed from 2 existing observational functional assessment tools to assess muscle function in the areas of strength and endurance across a wide range of ability and ages. The 14 ordinal items included were chosen to assess primarily axial and proximal muscle groups and are ranked with standard performance and scoring methods. Following the development of the CMAS, a training video and written instructions were developed and reviewed by the physicians participating in this study. Subsequently, utilizing a randomized block design, 12 physicians independently scored 10 children (9 with dermatomyositis, 1 with polymyositis; ages 4-15 years) twice in one day (morning and afternoon) on the CMAS. A pediatric physical therapist performed quantitative manual muscle strength testing (MMT) twice on each child (morning and afternoon), including the neck, trunk, and proximal and distal extremity muscle groups. RESULTS: The CMAS has a potential range of 0-51, with higher scores indicating greater muscle strength and endurance. The observed mean for the 10 patients was 36.4 (median 44, SD 14.1, observed range 5-51). The total score for the CMAS correlated with the physician's global assessment (by visual analog scale) of disease activity, the MMT score, serum creatine kinase level, and the Juvenile Arthritis Functional Assessment Report score. The score on the CMAS was not correlated with patient age. Interrater reliability (Kendall's coefficient of concordance) ranged from 0.77 to 1.0 for individual items (all P < 0.001), and overall, it was 0.95 (P < 0.001). Intrarater reliability for the individual physicians was measured by correlation of the CMAS scores for each patient on 2 separate evaluations and ranged from 0.97 to 0.99, with an overall correlation for all physicians of 0.98 (all P < 0.001). CONCLUSION: The CMAS demonstrated an acceptable range of observed scores, excellent convergent validity, and excellent inter- and intrarater reliability. The CMAS is validated to quantitatively assess muscle function in the areas of strength and endurance in children with idiopathic inflammatory myopathies. It can be used in routine clinical care as well as therapeutic trials.

Association among somatic HPRT mutant frequency, peripheral blood T-lymphocyte clonality, and serologic parameters of disease activity in children with juvenile onset dermatomyositis.

Somatic mutant frequencies (Mf) were determined using the HPRT T-cell cloning assay of peripheral blood T-lymphocytes from 14 children with juvenile onset dermatomyositis (JDM). Serologic parameters, specifically muscle enzyme determinations in JDM subjects, were correlated with residual lnMf (delta) in these patients to compare T-cell activation with clinical parameters associated with JDM. In addition TCR analysis was performed to determine T-cell proliferation and clonality on 12 HPRT mutant isolates from two individuals with JDM. Statistically significant correlations were found between residual lnMf and the following serologic parameters: aldolase (r = 0.771, P = 0.015); CPK (r = 0.602, P = 0.023); and SGOT (r = 0.656, P = 0.011) in children with JDM. In addition, identical TCR gene rearrangements were identified in 86 and 40% of the HPRT mutant isolates from the two patient samples analyzed, which is a significantly higher level of clonality than the 10-15% expected in normal individuals. These data suggest that determining HPRT Mf can be a useful antigen-independent method of selecting clonally expanding T-lymphocytes in autoimmune disease where relevant antigens are unknown. Future analysis of HPRT mutant isolates from children with active myositis may increase our understand of the activated T-cells involved in this disease.

Torg osteolysis syndrome.

We describe a 9-year-old girl who initially presented at age 4 with evidence of arthritis in her hands, feet, and large joints. Although she had a partial response to anti-inflammatory medications and had some laboratory results consistent with inflammatory disease, radiographs showed carpal and tarsal osteolysis associated with interphalangeal joint erosions. There was also widening of the shafts of the metacarpals and metatarsals with thinning of the cortices. Based on both the clinical progression of her illness and the radiologic characteristics, this child most likely has the Torg syndrome.

Intravenous corticosteroids: adverse reactions are more variable than expected in children.

OBJECTIVE: To determine the frequency and severity of adverse reactions associated with high dose intermittent intravenous corticosteroids (IVCS) in children with rheumatic disease. METHODS: Prospective documentation of adverse reactions associated with IVCS given to 213 pediatric rheumatology patients over a 4 year period. RESULTS: Forty-six of the 213 children (22%) reported an adverse reaction. The 46 patients received 2622 doses of IVCS. Twenty-one patients (10% of all patients studied) had behavioral changes, including altered mood (14), hyperactivity (4), psychosis (2), disorientation (1), and sleep disturbances (3). Nonbehavioral adverse reactions included headache (5.2%), abdominal complaints (4.7%), pruritus (4.2%), vomiting (3.8%), hives (2.3%), hypertension (2.3%), bone pain (1.5%), dizziness (1.5%), fatigue (1%), lethargy (1%), hypotension (1%), tachycardia (1%), hyperglycemia (1%), fracture (1%), tremor (0.5%), anaphylaxis (0.5%), ulcer (0.5%), and "gray appearance" (0.5%). Using chi-squared analysis, there were no statistical differences in ethnicity (p = 0.54) or diagnosis (p = 0.46) between patient groups, with or without adverse reactions. There was a significant statistical association between history of drug induced cutaneous reaction and adverse reactions to IVCS (p < 0.01). CONCLUSION: IVCS are associated with a spectrum of adverse reactions in children with rheumatic disease, of which volatile behavior is the most frequent. Children with a history of drug induced cutaneous reaction are more likely to have an adverse reaction to IVCS.

Juvenile dermatomyositis at diagnosis: clinical characteristics of 79 children.

OBJECTIVE: To evaluate demographic and clinical characteristics, duration of time between disease onset (date of first rash and/or weakness), and diagnosis/therapy, as well as socioeconomic status, of children with newly diagnosed juvenile dermatomyositis (JDM). METHODS: Structured telephone interview of families of a cohort of 79 children with JDM: interval between onset of symptoms to diagnosis, median of 3 months (range 0.5-20.0). RESULTS: At diagnosis, all the children had rash (100%) and proximal muscle weakness (100%); 58 (73%) had muscle pain; 51 (65%) fever; 35 (44%) dysphagia; 34 (43%) hoarseness; 29 (37%) abdominal pain; 28 (35%) arthritis; 18 (23%) calcinosis, and 10 (13%) melena. Muscle derived enzymes were normal in 10% of the children. Of the 43 children who had an electromyogram (EMG), 8 (19%) had normal results. Fifty-one children had a muscle biopsy; the results were normal/nondiagnostic in 10 (20%). Median time from disease onset to diagnosis was different between racial groups: Caucasians (n=59) 2.0 months: for minorities (n=20), 6.5 months, (p=0.0008). The median time from disease onset to therapy was: Caucasians. 3.0 months; minorities, 7.2 months (p=0.002). Report of calcinosis was associated with increased time to diagnosis and therapy (p=0.04). In the 33 children whose first symptom occurred in June-September, rash preceded or accompanied onset of muscle weakness in 83% (n=27). Ninety-one percent of the children were given steroid therapy and 9% received methotrexate as well. CONCLUSION: The results of an undirected site for muscle biopsy or EMG may not be diagnostic. Minority children had a longer interval between first JDM symptom and diagnosis/therapy than Caucasian children. Delay in diagnosis/therapy was associated with calcinosis.

Immunogenetic studies in families of children with juvenile dermatomyositis.

OBJECTIVE: We reported an association between juvenile dermatomyositis (JDMS) and the HLA-DQA1*0501 allele. The purpose of this study was to determine whether there is evidence for linkage between JDMS and the DQA1*0501 allele in JDMS families. METHODS: The study population included 18 unrelated patients with JDMS, their parents, and 49 unaffected siblings. Using molecular genetic techniques, we studied the HLA genes, DRB1, DQA1, and tumor necrosis factor-alpha. RESULTS: Using the transmission disequilibrium test, we confirmed our earlier observations that the HLA-DQA1*0501 allele confers primary susceptibility to JDMS. CONCLUSION: DQA1*0501 confers genetic risk for JDMS; we cannot exclude the effects of alleles at other linked loci that were not studied or interactive effects between DQA1 alleles and alleles at other loci.

Atypical Pneumocystis carinii pneumonia in a child with hyper-IgM syndrome.

Children with hyper-immunoglobulin M (hyper-IgM) syndrome are at increased risk for Pneumocystis carinii pneumonia (PCP), an opportunistic infection often found in immunodeficient hosts. PCP can present with increasing hypoxia, fever, cough, and respiratory distress. We describe a child with hyper-IgM syndrome in whom bronchoalveolar washings were negative for PCP. However, there was an atypical lung response in which caseating granulomas predominated. The histopathology, resembling that found in tuberculosis, stresses the importance of a high index of clinical suspicion and histologic confirmation for early intervention and treatment. Immunocompromised children with rapidly progressive pulmonary disease may require lung biopsy and stains such as GMS to identify PCP.

Correction of neutropenia and hypogammaglobulinemia in X-linked hyper-IgM syndrome by allogeneic bone marrow transplantation.

X-linked hyper-IgM (X-HIM) syndrome is a primary immunodeficiency disease characterized by defects in both cellular and humoral immunity. X-HIM is caused by mutations in the gene for CD40 ligand (CD40L), a T cell membrane protein that mediates T cell-dependent immune functions. We report the case of a 6-year-old male with X-HIM due to an intronic mutation resulting in aberrant CD40L RNA splicing and absence of detectable CD40L protein. The patient had a history of multiple infectious complications and chronic neutropenia requiring treatment with recombinant granulocyte colony-stimulating factor, and underwent allogeneic bone marrow transplantation from an HLA-matched sibling donor. Following successful engraftment, T cell CD40L expression and immunoglobulin isotype switching were reconstituted and neutropenia resolved. Allogeneic bone marrow transplantation can correct neutropenia and reconstitute immune function in X-HIM.

Development of a rapid whole blood flow cytometry procedure for the diagnosis of X-linked hyper-IgM syndrome patients and carriers.

The CD40 ligand expressed on activated T cells plays a pivotal role in B cell proliferation and differentiation. Mutations in the CD40 ligand gene, which alter its expression on the surface of activated T cells, are associated with the X-linked form of Hyper-IgM syndrome (XHIM). A rapid and simple, three-color whole blood flow cytometry procedure was developed for maximal expression and detection of the CD40L on the surface of in vitro activated CD4+ T cells. Approximately 90% of in vitro activated CD4+ T cells obtained from healthy controls expressed the CD40L compared to only 5% of in vitro activated CD4+ T cells obtained from the XHIM patients. The CD40L was expressed on approximately 50% of the in vitro activated CD4+ T cells obtained from the mothers of XHIM patients, consistent with a diagnosis of their carrier status. This is the first report of a whole blood procedure adapted for routine clinical use which is able to detect abnormal CD40L expression in XHIM patients and carriers.

Correlations between change in disease activity and changes in peripheral blood lymphocyte subsets in patients with juvenile dermatomyositis.

OBJECTIVE: To investigate whether changes in peripheral blood lymphocytes correlate with changes in disease activity in juvenile dermatomyositis (JDM). METHODS: Clinical changes in disease expression measured by a disease activity score were correlated with changes in percentages of peripheral blood lymphocyte subsets. RESULTS: Changes in the percentage of peripheral blood CD19 positive lymphocytes (B cells) correlated with changes in disease activity (Spearman rank coefficients = 0.47, p = 0.02). There were no significant correlations in disease activity with changes of T cell subsets or the T cell activation markers CD25 or DR. CONCLUSION: Change in the percentage of peripheral blood B cells correlates with change in disease activity in patients with JDM. This variable may be of use as an indicator of immunologic activity and response to therapy.