Phage display of environmental protein toxins and virulence factors reveals the prevalence, persistence, and genetics of antibody responses.

Microbial exposures are crucial environmental factors that impact healthspan by sculpting the immune system and microbiota. Antibody profiling via Phage ImmunoPrecipitation Sequencing (PhIP-Seq) provides a high-throughput, cost-effective approach for detecting exposure and response to microbial protein products. We designed and constructed a library of 95,601 56-amino acid peptide tiles spanning 14,430 proteins with "toxin" or "virulence factor" keyword annotations. We used PhIP-Seq to profile the antibodies of ∼1,000 individuals against this "ToxScan" library. In addition to enumerating immunodominant antibody epitopes, we studied the age-dependent stability of the ToxScan profile and used a genome-wide association study to find that the MHC-II locus modulates bacterial epitope selection. We detected previously described anti-flagellin antibody responses in a Crohn's disease cohort and identified an association between anti-flagellin antibodies and juvenile dermatomyositis. PhIP-Seq with the ToxScan library is thus an effective tool for studying the environmental determinants of health and disease at cohort scale.

Multi-level profiling unravels mitochondrial dysfunction in myotonic dystrophy type 2.

Myotonic dystrophy type 2 (DM2) is an autosomal-dominant multisystemic disease with a core manifestation of proximal muscle weakness, muscle atrophy, myotonia, and myalgia. The disease-causing CCTG tetranucleotide expansion within the CNBP gene on chromosome 3 leads to an RNA-dominated spliceopathy, which is currently untreatable. Research exploring the pathophysiological mechanisms in myotonic dystrophy type 1 has resulted in new insights into disease mechanisms and identified mitochondrial dysfunction as a promising therapeutic target. It remains unclear whether similar mechanisms underlie DM2 and, if so, whether these might also serve as potential therapeutic targets. In this cross-sectional study, we studied DM2 skeletal muscle biopsy specimens on proteomic, molecular, and morphological, including ultrastructural levels in two separate patient cohorts consisting of 8 (explorative cohort) and 40 (confirmatory cohort) patients. Seven muscle biopsy specimens from four female and three male DM2 patients underwent proteomic analysis and respiratory chain enzymology. We performed bulk RNA sequencing, immunoblotting of respiratory chain complexes, mitochondrial DNA copy number determination, and long-range PCR (LR-PCR) to study mitochondrial DNA deletions on six biopsies. Proteomic and transcriptomic analyses revealed a downregulation of essential mitochondrial proteins and their respective RNA transcripts, namely of subunits of respiratory chain complexes I, III, and IV (e.g., mt-CO1, mt-ND1, mt-CYB, NDUFB6) and associated translation factors (TACO1). Light microscopy showed mitochondrial abnormalities (e.g., an age-inappropriate amount of COX-deficient fibers, subsarcolemmal accumulation) in most biopsy specimens. Electron microscopy revealed widespread ultrastructural mitochondrial abnormalities, including dysmorphic mitochondria with paracrystalline inclusions. Immunofluorescence studies with co-localization of autophagy (p62, LC-3) and mitochondrial marker proteins (TOM20, COX-IV), as well as immunohistochemistry for mitophagy marker BNIP3 indicated impaired mitophagic flux. Immunoblotting and LR-PCR did not reveal significant differences between patients and controls. In contrast, mtDNA copy number measurement showed a reduction of mtDNA copy numbers in the patient group compared to controls. This first multi-level study of DM2 unravels thus far undescribed functional and structural mitochondrial abnormalities. However, the molecular link between the tetranucleotide expansion and mitochondrial dysfunction needs to be further elucidated.

Agreement between local and central anti-synthetase antibodies detection: results from the Classification Criteria of Anti-Synthetase Syndrome project biobank.

OBJECTIVES: The CLASS (Classification Criteria of Anti-Synthetase Syndrome) project is a large international multicentre study that aims to create the first data-driven anti-synthetase syndrome (ASSD) classification criteria. Identifying anti-aminoacyl tRNA synthetase antibodies (anti-ARS) is crucial for diagnosis, and several commercial immunoassays are now available for this purpose. However, using these assays risks yielding false-positive or false-negative results, potentially leading to misdiagnosis. The established reference standard for detecting anti-ARS is immunoprecipitation (IP), typically employed in research rather than routine autoantibody testing. We gathered samples from participating centers and results from local anti-ARS testing. As an "ad-interim" study within the CLASS project, we aimed to assess how local immunoassays perform in real-world settings compared to our central definition of anti-ARS positivity. METHODS: We collected 787 serum samples from participating centres for the CLASS project and their local anti-ARS test results. These samples underwent initial central testing using RNA-IP. Following this, the specificity of ARS was reconfirmed centrally through ELISA, line-blot assay (LIA), and, in cases of conflicting results, protein-IP. The sensitivity, specificity, positive likelihood ratio and positive and negative predictive values were evaluated. We also calculated the inter-rater agreement between central and local results using a weighted κ co-efficient. RESULTS: Our analysis demonstrates that local, real-world detection of anti-Jo1 is reliable with high sensitivity and specificity with a very good level of agreement with our central definition of anti-Jo1 antibody positivity. However, the agreement between local immunoassay and central determination of anti-non-Jo1 antibodies varied, especially among results obtained using local LIA, ELISA and "other" methods. CONCLUSIONS: Our study evaluates the performance of real-world identification of anti-synthetase antibodies in a large cohort of multi-national patients with ASSD and controls. Our analysis reinforces the reliability of real-world anti-Jo1 detection methods. In contrast, challenges persist for anti-non-Jo1 identification, particularly anti-PL7 and rarer antibodies such as anti-OJ/KS. Clinicians should exercise caution when interpreting anti-synthetase antibodies, especially when commercial immunoassays test positive for non-anti-Jo1 antibodies.

The pattern of MHC class I expression in muscle biopsies from patients with myositis and other neuromuscular disorders.

OBJECTIVE: Diagnostic muscle biopsies are routinely immunostained for major histocompatibility complex class I (MHC-I) protein. In this study we analysed the prevalence and patterns of MHC-I immunostaining in biopsies from patients with different types of myopathies and neurogenic disorders. METHODS: All 357 diagnostic muscle biopsies processed at the Johns Hopkins Neuromuscular Pathology Laboratory from August 2013 to January 2017 were immunostained for MHC-I. The prevalence and patterns of MHC-I immunostaining were compared between patients with histologically normal muscle biopsies (n = 31), idiopathic inflammatory myopathies (IIMs; n = 170), non-inflammatory myopathies (n = 60) and neurogenic disorders (n = 96). RESULTS: MHC-I immunostaining was abnormal in most patients with DM (98%), sporadic IBM (sIBM; 100%), immune-mediated necrotizing myopathy (IMNM; 100%) and polymyositis (77%). In contrast, MHC-I immunostaining was less frequently present in non-inflammatory myopathies (32%) or neurogenic disorders (30%). Overall, abnormal MHC-I immunostaining had a sensitivity of 0.95 and a specificity of 0.82 for diagnosing IIMs. A focal MHC-I staining pattern was associated with IMNM, whereas a global pattern was more prevalent in sIBM and a perifascicular pattern was significantly more common in dermatomyositis. Among 18 DM biopsies without perifascicular atrophy, 50% had a perifascicular MHC-I staining pattern. Sarcoplasmic upregulation staining was more common than sarcolemmal staining across all groups. CONCLUSION: MHC-I immunostaining was useful to distinguish IIMs from non-inflammatory myopathies or neurogenic disorders. Of note, a perifascicular MHC-I staining pattern was present only in those with DM, including half of those without perifascicular atrophy; many of these biopsies may not otherwise have been diagnostic for DM.

Association of anti-TPM4 autoantibodies with vasculopathic cutaneous manifestations in juvenile dermatomyositis.

OBJECTIVES: AECAs are detected in multiple forms of vasculitis or vasculopathy, including JDM. High levels of tropomyosin alpha-4 chain (TPM4) gene expression in cutaneous lesions and TPM4 protein expression in some endothelial cells (ECs) have been proven. Furthermore, the presence of autoantibodies to tropomyosin proteins have been discovered in DM. We therefore investigated whether anti-TPM4 autoantibodies are an AECA in JDM and are correlated with clinical features of JDM. METHODS: The expression of TPM4 protein in cultured normal human dermal microvascular ECs was investigated by Western blotting. Plasma samples from 63 children with JDM, 50 children with polyarticular JIA (pJIA) and 40 healthy children (HC) were tested for the presence of anti-TPM4 autoantibodies using an ELISA. Clinical features were compared between JDM patients with and without anti-TPM4 autoantibodies. RESULTS: Autoantibodies to TPM4 were detected in the plasma of 30% of JDM, 2% of pJIA (P < 0.0001) and 0% of HC (P < 0.0001). In JDM, anti-TPM4 autoantibodies were associated with the presence of cutaneous ulcers (53%; P = 0.02), shawl sign rash (47%; P = 0.03), mucous membrane lesions (84%; P = 0.04) and subcutaneous edema (42%; P < 0.05). Anti-TPM4 autoantibodies significantly correlated with the use of intravenous steroids and IVIG therapy in JDM (both P = 0.01). The total number of medications received was higher in patients with anti-TPM4 autoantibodies (P = 0.02). CONCLUSION: Anti-TPM4 autoantibodies are detected frequently in children with JDM and are novel myositis-associated autoantibodies. Their presence correlates with vasculopathic and other cutaneous manifestations of JDM that may be indicative of more refractory disease.

Anti-FHL1 autoantibodies in juvenile myositis are associated with anti-Ro52 autoantibodies but not with severe disease features.

OBJECTIVES: Four-and-a-half LIM domains 1 (FHL1) is a muscle-specific protein. Autoantibodies against FHL1 were recently discovered in adults with idiopathic inflammatory myopathies (IIMs) and were found to be associated with clinical features and outcomes indicative of increased disease severity. Anti-FHL1 autoantibodies have not been described in children. Here, the prevalence and clinical features associated with anti-FHL1 autoantibodies were examined in a large North American cohort of juvenile patients with IIM. METHODS: Sera from 338 juvenile IIM patients and 91 juvenile healthy controls were screened for anti-FHL1 autoantibodies by ELISA. Clinical characteristics and HLA alleles of those with and without anti-FHL1 autoantibodies were compared among those with juvenile IIM. RESULTS: Anti-FHL1 autoantibodies were present in 10.9% of juvenile IIM patients and 1.1% of controls. The frequency of anti-FHL1 autoantibodies among clinical and serologic subgroups did not differ. A higher percentage of Asian patients had anti-FHL1 autoantibodies (11% vs 0.7%; P = 0.002). Myositis-associated autoantibodies (MAAs) [odds ratio (OR) 2.09 (CI 1.03, 4.32)], anti-Ro52 autoantibodies specifically [OR 4.17 (CI 1.83, 9.37)] and V-sign rash [OR 2.59 (CI 1.22, 5.40)] were associated with anti-FHL1 autoantibodies. There were no differences in other features or markers of disease severity. No HLA associations with anti-FHL1 autoantibodies in Caucasian myositis patients were identified. CONCLUSION: Anti-FHL1 autoantibodies are present in ∼11% of juvenile IIM patients and commonly co-occur with MAAs, including anti-Ro52 autoantibodies. In contrast to adult IIM, anti-FHL1 autoantibodies in juvenile myositis are associated with V-sign rash but not with other distinctive clinical features or worse outcomes.

Muscle Transcriptomics Shows Overexpression of Cadherin 1 in Inclusion Body Myositis.

OBJECTIVE: This study aimed to elucidate the molecular features of inclusion body myositis (IBM). METHODS: We performed RNA sequencing analysis of muscle biopsy samples from 67 participants, consisting of 58 myositis patients with the pathological finding of CD8-positive T cells invading non-necrotic muscle fibers expressing major histocompatibility complex class I (43 IBM, 6 polymyositis, and 9 unclassifiable myositis), and 9 controls. RESULTS: Cluster analysis, principal component analysis, and pathway analysis showed that differentially expressed genes and pathways identified in IBM and polymyositis were mostly comparable. However, pathways related to cell adhesion molecules were upregulated in IBM as compared with polymyositis and controls (p < 0.01). Notably, CDH1, which encodes the epidermal cell junction protein cadherin 1, was overexpressed in the muscles of IBM, which was validated by another RNA sequencing dataset from previous publications. Western blotting confirmed the presence of mature cadherin 1 protein in the muscles of IBM. Immunohistochemical staining confirmed the positivity for anti-cadherin 1 antibody in the muscles of IBM, whereas there was no muscle fiber positive for anti-cadherin 1 antibody in immune-mediated necrotizing myopathy, antisynthetase syndrome, and controls. The fibers stained with anti-cadherin 1 antibody did not have rimmed vacuoles or abnormal protein accumulation. Experimental skeletal muscle regeneration and differentiation systems showed that CDH1 is expressed during skeletal muscle regeneration and differentiation. INTERPRETATION: CDH1 was detected as a differentially expressed gene, and immunohistochemistry showed that cadherin 1 exists in the muscles of IBM, whereas it was rarely seen in those of other idiopathic inflammatory myopathies. Cadherin 1 upregulation in muscle could provide a valuable clue to the pathological mechanisms of IBM. ANN NEUROL 2022;91:317-328.

Variants in DTNA cause a mild, dominantly inherited muscular dystrophy.

DTNA encodes α-dystrobrevin, a component of the macromolecular dystrophin-glycoprotein complex (DGC) that binds to dystrophin/utrophin and α-syntrophin. Mice lacking α-dystrobrevin have a muscular dystrophy phenotype, but variants in DTNA have not previously been associated with human skeletal muscle disease. We present 12 individuals from four unrelated families with two different monoallelic DTNA variants affecting the coiled-coil domain of α-dystrobrevin. The five affected individuals from family A harbor a c.1585G > A; p.Glu529Lys variant, while the recurrent c.1567_1587del; p.Gln523_Glu529del DTNA variant was identified in the other three families (family B: four affected individuals, family C: one affected individual, and family D: two affected individuals). Myalgia and exercise intolerance, with variable ages of onset, were reported in 10 of 12 affected individuals. Proximal lower limb weakness with onset in the first decade of life was noted in three individuals. Persistent elevations of serum creatine kinase (CK) levels were detected in 11 of 12 affected individuals, 1 of whom had an episode of rhabdomyolysis at 20 years of age. Autism spectrum disorder or learning disabilities were reported in four individuals with the c.1567_1587 deletion. Muscle biopsies in eight affected individuals showed mixed myopathic and dystrophic findings, characterized by fiber size variability, internalized nuclei, and slightly increased extracellular connective tissue and inflammation. Immunofluorescence analysis of biopsies from five affected individuals showed reduced α-dystrobrevin immunoreactivity and variably reduced immunoreactivity of other DGC proteins: dystrophin, α, ß, δ and γ-sarcoglycans, and α and ß-dystroglycans. The DTNA deletion disrupted an interaction between α-dystrobrevin and syntrophin. Specific variants in the coiled-coil domain of DTNA cause skeletal muscle disease with variable penetrance. Affected individuals show a spectrum of clinical manifestations, with severity ranging from hyperCKemia, myalgias, and exercise intolerance to childhood-onset proximal muscle weakness. Our findings expand the molecular etiologies of both muscular dystrophy and paucisymptomatic hyperCKemia, to now include monoallelic DTNA variants as a novel cause of skeletal muscle disease in humans.

Anti-MDA5-positive dermatomyositis and remission in a single referral centre population.

OBJECTIVES: To describe a single-centre North American adult cohort of anti-MDA5-positive dermatomyositis patients, with emphasis on drug-free long-term remission. METHODS: We conducted an observational retrospective cohort study of anti-MDA5-positive DM patients. All consented patients seen in the Johns Hopkins Myositis Centre from 2003-2020 with suspected muscle disease were routinely screened for myositis-specific autoantibodies. All sera were screened for anti-MDA5 autoantibodies by line blot; positives were verified by enzyme-linked immunoassay. Patients whose sera were anti-MDA5 positive by both assays (n=52) were followed longitudinally. If clinical status was unavailable, structured telephone interviews were conducted. Clinical remission was defined as being off all immunosuppression >1 year while remaining asymptomatic. RESULTS: 38/52 (73%) of the patients were women with a median age at disease-onset of 47 (IQR 40-54). Twenty-five of the patients (48%) were White, 16 (30%) were Black and 3 (6%) were Asian. Most patients (42/52, 80%) had interstitial lung disease, defined by inflammatory or fibrotic changes on high resolution computed tomography (HRCT). 18/52 (35%) of patients required pulse-dose methylprednisolone, 4/52 (8%) experienced spontaneous pneumothorax/pneumomediastinum, 6/52 (12%) required intubation, and 5/52 (10%) died. Over longitudinal follow-up (median 3.5 years), 9 (18%) patients achieved clinical remission. The median time from symptom onset to clinical remission was 4 years, and the median duration of sustained remission was 3.5 years (range 1.4-7.8). No demographic or disease characteristics were significantly associated with remission. CONCLUSIONS: In this single centre, tertiary referral population of anti-MDA5-positive dermatomyositis, ~20% of patients experienced long-term drug-free remission after a median disease duration of 4 years. No clinical or biologic factors were associated with clinical remission.

Association of anti-HSC70 autoantibodies with cutaneous ulceration and severe disease in juvenile dermatomyositis.

OBJECTIVES: JDM is an inflammatory myopathy characterized by prominent vasculopathy. AECAs are frequently detected in inflammatory and autoimmune diseases. We sought to determine whether AECAs correlate with clinical features of JDM, and thus serve as biomarkers to guide therapy or predict outcome. METHODS: Plasma samples from 63 patients with JDM, 49 patients with polyarticular JIA and 40 juvenile healthy controls were used to detect anti-heat shock cognate 71 kDa protein (HSC70) autoantibodies, a newly identified AECA, in ELISA assays. Clinical features were compared between JDM patients with and without anti-HSC70 autoantibodies. RESULTS: Anti-HSC70 autoantibodies were detected in 35% of patients with JDM, in 0% of patients with JIA (P < 0.0001) and in 0% of healthy donors (P < 0.0001). Both the presence of cutaneous ulcers (59% vs 17%, P < 0.002) and the use of wheelchairs and/or assistive devices (64% vs 27%, P < 0.007) were strongly associated with anti-HSC70 autoantibodies in JDM. High scores on the severity of myositis damage measures at the time of measurement of anti-HSC70 autoantibodies and an increased number of hospitalizations were also associated with anti-HSC70 autoantibodies. Intravenous immunoglobulin therapy was used more often in anti-HSC70 autoantibody-positive patients. CONCLUSION: Anti-HCS70 autoantibodies are detected frequently in children with JDM and are novel myositis-associated autoantibodies correlating with disease severity.

Persistent upregulation of the ß-tubulin tubb6, linked to muscle regeneration, is a source of microtubule disorganization in dystrophic muscle.

In healthy adult skeletal muscle fibers microtubules form a three-dimensional grid-like network. In the mdx mouse, a model of Duchenne muscular dystrophy (DMD), microtubules are mostly disordered, without periodicity. These microtubule defects have been linked to the mdx mouse pathology. We now report that increased expression of the beta 6 class V ß-tubulin (tubb6) contributes to the microtubule changes of mdx muscles. Wild-type muscle fibers overexpressing green fluorescent protein (GFP)-tubb6 (but not GFP-tubb5) have disorganized microtubules whereas mdx muscle fibers depleted of tubb6 (but not of tubb5) normalize their microtubules, suggesting that increasing tubb6 is toxic. However, tubb6 increases spontaneously during differentiation of mouse and human muscle cultures. Furthermore, endogenous tubb6 is not uniformly expressed in mdx muscles but is selectively increased in fiber clusters, which we identify as regenerating. Similarly, mdx-based rescued transgenic mice that retain a higher than expected tubb6 level show focal expression of tubb6 in subsets of fibers. Tubb6 is also upregulated in cardiotoxin-induced mouse muscle regeneration, in human myositis and DMD biopsies, and the tubb6 level correlates with that of embryonic myosin heavy chain, a regeneration marker. In conclusion, modulation of a ß-tubulin isotype plays a role in muscle differentiation and regeneration. Increased tubb6 expression and microtubule reorganization are not pathological per se but reflect a return to an earlier developmental stage. However, chronic elevation of tubb6, as occurs in the mdx mouse, may contribute to the repeated cycles of regeneration and to the pathology of the disease.

A network of core and subtype-specific gene expression programs in myositis.

Myositis comprises a heterogeneous group of skeletal muscle disorders which converge on chronic muscle inflammation and weakness. Our understanding of myositis pathogenesis is limited, and many myositis patients lack effective therapies. Using muscle biopsy transcriptome profiles from 119 myositis patients (spanning major clinical and serological disease subtypes) and 20 normal controls, we generated a co-expression network of 8101 dynamically regulated transcripts. This network organized the myositis transcriptome into a map of gene expression modules representing interrelated biological processes and disease signatures. Universally myositis-upregulated network modules included muscle regeneration, specific cytokine signatures, the acute phase response, and neutrophil degranulation. Universally myositis-suppressed pathways included a specific subset of myofilaments, the mitochondrial envelope, and nuclear isoforms of the anti-apoptotic humanin protein. Myositis subtype-specific modules included type 1 interferon signaling and titin (dermatomyositis), RNA processing (antisynthetase syndrome), and vasculogenesis (inclusion body myositis). Importantly, therapies exist to target influential proteins in many myositis-dysregulated modules, and nearly all modules contained understudied proteins and non-coding RNAs - many of which were extraordinarily dysregulated in myositis and may represent novel therapeutic targets. Finally, we apply our network to patient classification, finding that a deep learning algorithm trained on patient-level network "images" successfully assigned patients to clinical groups and further into molecular subclusters. Altogether, we provide a global resource to probe and contextualize differential gene expression in myositis.

The indirect immunofluorescence assay autoantibody profiles of myositis patients without known myositis-specific autoantibodies.

OBJECTIVES: The indirect immunofluorescence assay (IIFA) is used to screen for the presence of autoantibodies. Our objective was to determine the prevalence and clinical features of IIFA positive myositis patients without known myositis-specific autoantibodies (MSA). METHODS: Sera from healthy comparators (HC) and patients with dermatomyositis (DM), inclusion body myositis (IBM), and polymyositis (PM) with no detectable MSA were tested by IIFA on HEp-2 cells. The pattern of positivity was classified according to the International Consensus on Antinuclear Antibody Patterns. The prevalence and frequency of each IIFA pattern were compared between the different groups. RESULTS: Sera from 100 HC, 71 DM, 53 IBM, and 69 PM subjects were included in the study. The IIFA was positive in 35% HC compared to 66% DM (p<0.001), 49% IBM, and 64% (p<0.001) PM sera. Among IIFA positive sera, the staining was moderate or intense in 43% HC compared to 79% DM (p<0.001) but just 54% IBM, and 52% PM sera. IIFA positivity was predominantly nuclear in all groups (all >69%). The most common pattern in myositis patients was fine speckled with no differences between groups. In general, IIFA positive and negative DM patients showed similar clinical features and disease activity. CONCLUSIONS: Half of MSA-negative DM patients have moderate/strong IIFA positivity, predominantly with a fine speckled pattern. In contrast, MSA-negative PM, IBM, and healthy comparators are more often weakly positive for IIFA. These findings suggest that unidentified autoantibodies are more likely to exist in DM patients than in the other myositis groups.

Accumulation of autophagosome cargo protein p62 is common in idiopathic inflammatory myopathies.

OBJECTIVES: The subsarcolemmal accumulation of p62 aggregates in myofibres has been proposed to be characteristic of sporadic inclusion body myositis (sIBM). The objective of this study was to analyse the patterns and prevalence of p62 immunostaining and to quantitate p62 gene expression in muscle biopsies from a large number of patients with different types of myopathic and neurogenic disorders. METHODS: For the p62 immunostaining analysis, all patients with a muscle biopsy immunostained for p62 at the Johns Hopkins Neuromuscular Pathology Laboratory from 2013 to 2017 were included (n=303). The prevalence and pattern of p62 immunostaining were compared between patients with histologically normal muscle (n=29), inflammatory myopathies (n=136), non-inflammatory myopathies (n=53), and neurogenic disorders (n=85). p62 expression levels were analysed using an existing RNAseq dataset including data from dermatomyositis (DM; n=39), immune-mediated necrotising myopathy (IMNM; n=49), antisynthetase syndrome (AS; n=18), and sIBM (n=23) patients as well as 20 histologically normal muscle biopsies. RESULTS: p62 staining was absent in normal biopsies, but present in biopsies from those with polymyositis (29%), non-inflammatory myopathies (all <31%), neurogenic disorders (31%), dermatomyositis (57%), sIBM (92%) and IMNM (87%). In all diseases studied, p62 accumulation was more prevalent in biopsies with more severe muscle damage. sIBM biopsies had decreased p62 expression levels compared to the other groups (corrected p<0.04). CONCLUSIONS: p62 accumulation is a general response to muscle injury and not a specific marker for sIBM. Also, in sIBM, p62 RNA levels are decreased, suggesting that, in this disease, p62 aggregation is not due to overexpression.

Machine learning algorithms reveal unique gene expression profiles in muscle biopsies from patients with different types of myositis.

OBJECTIVES: Myositis is a heterogeneous family of diseases that includes dermatomyositis (DM), antisynthetase syndrome (AS), immune-mediated necrotising myopathy (IMNM), inclusion body myositis (IBM), polymyositis and overlap myositis. Additional subtypes of myositis can be defined by the presence of myositis-specific autoantibodies (MSAs). The purpose of this study was to define unique gene expression profiles in muscle biopsies from patients with MSA-positive DM, AS and IMNM as well as IBM. METHODS: RNA-seq was performed on muscle biopsies from 119 myositis patients with IBM or defined MSAs and 20 controls. Machine learning algorithms were trained on transcriptomic data and recursive feature elimination was used to determine which genes were most useful for classifying muscle biopsies into each type and MSA-defined subtype of myositis. RESULTS: The support vector machine learning algorithm classified the muscle biopsies with >90% accuracy. Recursive feature elimination identified genes that are most useful to the machine learning algorithm and that are only overexpressed in one type of myositis. For example, CAMK1G (calcium/calmodulin-dependent protein kinase IG), EGR4 (early growth response protein 4) and CXCL8 (interleukin 8) are highly expressed in AS but not in DM or other types of myositis. Using the same computational approach, we also identified genes that are uniquely overexpressed in different MSA-defined subtypes. These included apolipoprotein A4 (APOA4), which is only expressed in anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) myopathy, and MADCAM1 (mucosal vascular addressin cell adhesion molecule 1), which is only expressed in anti-Mi2-positive DM. CONCLUSIONS: Unique gene expression profiles in muscle biopsies from patients with MSA-defined subtypes of myositis and IBM suggest that different pathological mechanisms underly muscle damage in each of these diseases.

Focused HLA analysis in Caucasians with myositis identifies significant associations with autoantibody subgroups.

OBJECTIVES: Idiopathic inflammatory myopathies (IIM) are a spectrum of rare autoimmune diseases characterised clinically by muscle weakness and heterogeneous systemic organ involvement. The strongest genetic risk is within the major histocompatibility complex (MHC). Since autoantibody presence defines specific clinical subgroups of IIM, we aimed to correlate serotype and genotype, to identify novel risk variants in the MHC region that co-occur with IIM autoantibodies. METHODS: We collected available autoantibody data in our cohort of 2582 Caucasian patients with IIM. High resolution human leucocyte antigen (HLA) alleles and corresponding amino acid sequences were imputed using SNP2HLA from existing genotyping data and tested for association with 12 autoantibody subgroups. RESULTS: We report associations with eight autoantibodies reaching our study-wide significance level of p<2.9×10-5. Associations with the 8.1 ancestral haplotype were found with anti-Jo-1 (HLA-B*08:01, p=2.28×10-53 and HLA-DRB1*03:01, p=3.25×10-9), anti-PM/Scl (HLA-DQB1*02:01, p=1.47×10-26) and anti-cN1A autoantibodies (HLA-DRB1*03:01, p=1.40×10-11). Associations independent of this haplotype were found with anti-Mi-2 (HLA-DRB1*07:01, p=4.92×10-13) and anti-HMGCR autoantibodies (HLA-DRB1*11, p=5.09×10-6). Amino acid positions may be more strongly associated than classical HLA associations; for example with anti-Jo-1 autoantibodies and position 74 of HLA-DRB1 (p=3.47×10-64) and position 9 of HLA-B (p=7.03×10-11). We report novel genetic associations with HLA-DQB1 anti-TIF1 autoantibodies and identify haplotypes that may differ between adult-onset and juvenile-onset patients with these autoantibodies. CONCLUSIONS: These findings provide new insights regarding the functional consequences of genetic polymorphisms within the MHC. As autoantibodies in IIM correlate with specific clinical features of disease, understanding genetic risk underlying development of autoantibody profiles has implications for future research.

Muscle endurance deficits in myositis patients despite normal manual muscle testing scores.

INTRODUCTION: It is unclear whether quantitating muscle endurance adds nonredundant information useful for the care of patients with muscular disease. METHODS: Records were retrospectively reviewed for all Johns Hopkins Myositis Center patients with a muscle endurance assessment (n = 128, 226 patient-visits). Muscle endurance and strength were quantitated with the Myositis Functional Index-2 (FI2) and manual muscle testing (MMT), respectively. RESULTS: Composite FI2 muscle endurance scores were comparable in inclusion body myositis (n = 58), dermatomyositis (n = 31), and polymyositis (n = 39). Overall, muscle endurance correlated with and evolved similarly to strength, inversely to serum creatine kinase. However, in patients with normal or near-normal strength (mean MMT > 9.75/10), muscle endurance was typically abnormal and highly variable (mean FI2, 5.6/10; interquartile range, 3.3-7.8/10). DISCUSSION: Muscle endurance testing may identify muscle impairment inadequately described by MMT, particularly in patients with high MMT scores. Muscle Nerve 59:70-75, 2019.

Efficacy and adverse effects of methotrexate compared with azathioprine in the antisynthetase syndrome.

OBJECTIVES: To study the efficacy in terms of muscle strength, and corticosteroid tapering as well as the prevalence of adverse effects in patients with the antisynthetase syndrome (ASyS) treated with azathioprine (AZA) compared to those treated with methotrexate (MTX). METHODS: We compared the clinical outcomes in ASyS patients treated with AZA versus MTX including change in corticosteroid dose, strength, and creatine kinase (CK) as well as the prevalence of adverse effects. RESULTS: Among 169 patients with ASyS, 102 were treated at some point exclusively with either AZA or MTX (± corticosteroids). There were no significant differences in the rate of muscle strength recovery, CK decrease or corticosteroid tapering between those ASyS patients treated with MTX versus AZA. The prevalence of adverse events in patients treated with AZA and MTX was similar (29% vs. 25%, p>0.05); elevated liver enzymes (17% AZA vs. 12% MTX) and gastrointestinal involvement (10% AZA vs. 8% MTX) were the most common adverse events. While no patients treated with AZA developed lung complications, two of the patients treated with MTX experienced reversible pneumonitis with MTX cessation. CONCLUSIONS: AZA and MTX showed similar efficacy and adverse events in patients with ASyS. Pneumonitis is a rare but important event in patients receiving MTX.

Dermatomyositis etiopathogenesis: a rebel soldier in the muscle.

PURPOSE OF REVIEW: The purpose of this article is to review the etiopathogenesis of dermatomyositis, including the predisposing factors, triggers, inflammatory cells, pathways and target antigens associated with dermatomyositis. RECENT FINDINGS: During the last few years, we have made considerable progress in unveiling the etiopathogenesis of dermatomyositis. In the first place, we have defined genes within the major histocompatibility complex locus as the greatest genetic risk factor for the disease. Second, we have demonstrated that certain environmental factors, as well as tumors, may trigger certain dermatomyositis subtypes. Moreover, we have established the importance of the interferon pathway in dermatomyositis pathogenesis compared with other myositis subtypes. But probably, the most remarkable advance has been the discovery of multiple autoantibodies that define groups of patients with characteristic clinical features, prognosis and response to treatment. SUMMARY: Dermatomyositis cause and pathogenesis have proven to be a complex and fascinating task for the scientific community and the last decade has been full of new findings on how the disease starts and how it causes damage to different organ systems. However, we have still more questions than answers in this topic, answers that will be critical to understanding autoimmunity and finding effective therapies to dermatomyositis.

Anti-NT5C1A autoantibodies are associated with more severe disease in patients with juvenile myositis.

OBJECTIVES: Autoantibodies recognising cytosolic 5'-nucleotidase 1A (NT5C1A) are found in adult patients with myositis and other autoimmune diseases. They are especially prevalent in adults with inclusion body myositis (IBM), in which they are associated with more severe weakness and higher mortality. This study was undertaken to define the prevalence and clinical features associated with anti-NT5C1A autoantibodies in juvenile myositis. METHODS: We screened sera from 380 patients with juvenile myositis, 30 patients with juvenile idiopathic arthritis (JIA) and 92 healthy control children for anti-NT5C1A autoantibodies. Clinical characteristics were compared between patients with myositis with and without anti-NT5C1A autoantibodies. RESULTS: Anti-NT5C1A autoantibodies were present in 102 of 380 (27%) patients with juvenile myositis and in 11 of 92 (12%) healthy control children (P=0.002) and 27% of children with JIA (P=0.05 vs controls). Sera of 83 of 307 (27%) patients with juvenile dermatomyositis and 16 of 46 (35%) patients with juvenile overlap myositis were anti-NT5C1A autoantibody-positive (P<0.01 vs healthy controls for each), but sera of only 3 of 27 (11%) patients with juvenile polymyositis were anti-NT5C1A-positive. Patients with juvenile myositis with and without anti-NT5C1A autoantibodies had similar clinical phenotypes. However, patients with anti-NT5C1A autoantibody-positive myositis had greater pulmonary symptoms at diagnosis (P=0.005), more frequent hospitalisations (P=0.01) and required a larger number of medications (P<0.001). CONCLUSION: Anti-NT5C1A autoantibodies are present in more than one-quarter of children with juvenile myositis and JIA compared with only 12% of healthy children, suggesting they are myositis-associated in children. As in adults with IBM, patients with juvenile myositis with anti-NT5C1A autoantibodies have more severe disease.