Association with HLA-DRß1 position 37 distinguishes juvenile dermatomyositis from adult-onset myositis.

Juvenile dermatomyositis (JDM) is a rare, severe autoimmune disease and the most common idiopathic inflammatory myopathy of children. JDM and adult-onset dermatomyositis (DM) have similar clinical, biological and serological features, although these features differ in prevalence between childhood-onset and adult-onset disease, suggesting that age of disease onset may influence pathogenesis. Therefore, a JDM-focused genetic analysis was performed using the largest collection of JDM samples to date. Caucasian JDM samples (n = 952) obtained via international collaboration were genotyped using the Illumina HumanCoreExome chip. Additional non-assayed human leukocyte antigen (HLA) loci and genome-wide single-nucleotide polymorphisms (SNPs) were imputed. HLA-DRB1*03:01 was confirmed as the classical HLA allele most strongly associated with JDM [odds ratio (OR) 1.66; 95% confidence interval (CI) 1.46, 1.89; P = 1.4 × 10-14], with an independent association at HLA-C*02:02 (OR = 1.74; 95% CI 1.42, 2.13, P = 7.13 × 10-8). Analyses of amino acid positions within HLA-DRB1 indicated that the strongest association was at position 37 (omnibus P = 3.3 × 10-19), with suggestive evidence this association was independent of position 74 (omnibus P = 5.1 × 10-5), the position most strongly associated with adult-onset DM. Conditional analyses also suggested that the association at position 37 of HLA-DRB1 was independent of some alleles of the Caucasian HLA 8.1 ancestral haplotype (AH8.1) such as HLA-DQB1*02:01 (OR = 1.62; 95% CI 1.36, 1.93; P = 8.70 × 10-8), but not HLA-DRB1*03:01 (OR = 1.49; 95% CR 1.24, 1.80; P = 2.24 × 10-5). No associations outside the HLA region were identified. Our findings confirm previous associations with AH8.1 and HLA-DRB1*03:01, HLA-C*02:02 and identify a novel association with amino acid position 37 within HLA-DRB1, which may distinguish JDM from adult DM.

Comprehensive transcriptomic analysis and machine learning reveal unique gene expression profiles in patients with immune-mediated necrotizing myopathy.

BACKGROUND: Immune-mediated necrotizing myopathy (IMNM) is an autoimmune myopathy characterized by severe proximal weakness and muscle fiber necrosis, yet its pathogenesis remains unclear. So far, there are few bioinformatics studies on underlying pathogenic genes and infiltrating immune cell profiles of IMNM. Therefore, we aimed to characterize differentially expressed genes (DEGs) and infiltrating cells in IMNM muscle biopsy specimens, which may be useful for elucidating the pathogenesis of IMNM. METHODS: Three datasets (GSE39454, GSE48280 and GSE128470) of gene expression profiling related to IMNM were obtained from the Gene Expression Omnibus database. Data were normalized, and DEG analysis was performed using the limma package. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs were performed using clusterProfiler. The CIBERSORT algorithm was performed to identify infiltrating cells. Machine learning algorithm and gene set enrichment analysis (GSEA) were performed to find distinctive gene signatures and the underlying signaling pathways of IMNM. RESULTS: DEG analysis identified upregulated and downregulated in IMNM muscle compared to the gene expression levels of other groups. GO and KEGG analysis showed that the pathogenesis of IMNM was notable for the under-representation of pathways that were important in dermatomyositis and inclusion body myositis. Three immune cells (M2 macrophages, resting dendritic cells and resting natural killer cells) with differential infiltration and five key genes (NDUFAF7, POLR2J, CD99, ARF5 and SKAP2) in patients with IMNM were identified through the CIBERSORT and machine learning algorithm. The GSEA results revealed that the key genes were remarkably enriched in diverse immunological and muscle metabolism-related pathways. CONCLUSIONS: We comprehensively explored immunological landscape of IMNM, which is indicative for the research of IMNM pathogenesis.

Characterised intron retention profiles in muscle tissue of idiopathic inflammatory myopathy subtypes.

OBJECTIVES: Idiopathic inflammatory myopathies (IIMs) are a group of heterogeneous autoimmune diseases. Intron retention (IR) serves as an important post-transcriptional and translational regulatory mechanism. This study aims to identify changes in IR profiles in IIM subtypes, investigating their influence on proteins and their correlations with clinical features. METHODS: RNA sequencing and liquid chromatography-tandem mass spectrometry were performed on muscle tissues obtained from 174 patients with IIM and 19 controls, following QC procedures. GTFtools and iREAD software were used for IR identification. An analysis of differentially expressed IRs (DEIs), exons and proteins was carried out using edgeR or DEP. Functional analysis was performed with clusterProfiler, and SPIRON was used to assess splicing factors. RESULTS: A total of 6783 IRs located in 3111 unique genes were identified in all IIM subtypes compared with controls. IIM subtype-specific DEIs were associated with the pathogenesis of respective IIM subtypes. Splicing factors YBX1 and HSPA2 exhibited the most changes in dermatomyositis and immune-mediated necrotising myopathy. Increased IR was associated with reduced protein expression. Some of the IIM-specific DEIs were correlated with clinical parameters (skin rash, MMT-8 scores and muscle enzymes) and muscle histopathological features (myofiber necrosis, regeneration and inflammation). IRs in IFIH1 and TRIM21 were strongly correlated with anti-MDA5+ antibody, while IRs in SRP14 were associated with anti-SRP+ antibody. CONCLUSION: This study revealed distinct IRs and specific splicing factors associated with IIM subtypes, which might be contributing to the pathogenesis of IIM. We also emphasised the potential impact of IR on protein expression in IIM muscles.

Novel endotypes of antisynthetase syndrome identified independent of anti-aminoacyl transfer RNA synthetase antibody specificity that improve prognostic stratification.

OBJECTIVES: To systemically analyse the heterogeneity in the clinical manifestations and prognoses of patients with antisynthetase syndrome (ASS) and evaluate the transcriptional signatures related to different clinical phenotypes. METHODS: A total of 701 patients with ASS were retrospectively enrolled. The clinical presentation and prognosis were assessed in association with four anti-aminoacyl transfer RNA synthetase (ARS) antibodies: anti-Jo1, anti-PL7, anti-PL12 and anti-EJ. Unsupervised machine learning was performed for patient clustering independent of anti-ARS antibodies. Transcriptome sequencing was conducted in clustered ASS patients and healthy controls. RESULTS: Patients with four different anti-ARS antibody subtypes demonstrated no significant differences in the incidence of rapidly progressive interstitial lung disease (RP-ILD) or prognoses. Unsupervised machine learning, independent of anti-ARS specificity, identified three endotypes with distinct clinical features and outcomes. Endotype 1 (RP-ILD cluster, 23.7%) was characterised by a high incidence of RP-ILD and a high mortality rate. Endotype 2 (dermatomyositis (DM)-like cluster, 14.5%) corresponded to patients with DM-like skin and muscle symptoms with an intermediate prognosis. Endotype 3 (arthritis cluster, 61.8%) was characterised by arthritis and mechanic's hands, with a good prognosis. Transcriptome sequencing revealed that the different endotypes had distinct gene signatures and biological processes. CONCLUSIONS: Anti-ARS antibodies were not significant in stratifying ASS patients into subgroups with greater homogeneity in RP-ILD and prognoses. Novel ASS endotypes were identified independent of anti-ARS specificity and differed in clinical outcomes and transcriptional signatures, providing new insights into the pathogenesis of ASS.

K2P2.1 is a regulator of inflammatory cell responses in idiopathic inflammatory myopathies.

K2P2.1 (TREK1), a two-pore domain potassium channel, has emerged as regulator of leukocyte transmigration into the central nervous system. In the context of skeletal muscle, immune cell infiltration constitutes the pathogenic hallmark of idiopathic inflammatory myopathies (IIMs). However, the underlying mechanisms remain to be elucidated. In this study, we investigated the role of K2P2.1 in the autoimmune response of IIMs. We detected K2P2.1 expression in primary skeletal muscle and endothelial cells of murine and human origin. We observed an increased pro-inflammatory cell response, adhesion and transmigration by pharmacological blockade or genetic deletion of K2P2.1 in vitro and in in vivo myositis mouse models. Of note, our findings were not restricted to endothelial cells as skeletal muscle cells with impaired K2P2.1 function also demonstrated a strong pro-inflammatory response. Conversely, these features were abrogated by activation of K2P2.1 and improved the disease course of a myositis mouse model. In humans, K2P2.1 expression was diminished in IIM patients compared to non-diseased controls arguing for the translatability of our findings. In summary, K2P2.1 may regulate the inflammatory response of skeletal muscle. Further research is required to understand whether K2P2.1 could serve as novel therapeutic target.

Causality Assessment Between Idiopathic Inflammatory Myopathies and Lung Cancer: A Bidirectional 2-Sample Mendelian Randomization.

BACKGROUND: Although observational studies have revealed associations between idiopathic inflammatory myopathies (IIMs) and lung cancer (LC), they have not established a causal relationship between these 2 conditions. METHODS: We used a 2-sample Mendelian randomization approach to examine the bidirectional causal associations between IIMs and LC, using single-nucleotide polymorphisms selected from high-quality genome-wide association studies in the FinnGen database. Sensitivity analyses were conducted to assess potential heterogeneity and pleiotropy impacts on the Mendelian randomization results. RESULTS: Our analysis demonstrated a positive causal effect of genetically increased IIM risk on LC (odds ratio, 1.114; 95% confidence interval, 1.057-1.173; p = 5.63 × 10 -5 ), particularly on the lung squamous cell carcinoma subtype (odds ratio, 1.168, 95% confidence interval, 1.049-1.300, p = 0.00451), but not on lung adenocarcinoma or small cell lung cancer. No causal effect of LC on IIMs was identified. Sensitivity analyses indicated that horizontal pleiotropy was unlikely to influence causality, and leave-one-out analysis confirmed that the observed associations were not driven by a single-nucleotide polymorphism. CONCLUSION: Our findings offer compelling evidence of a positive causal relationship between IIMs and LC, particularly with regard to lung squamous cell carcinoma, in the European population. Conversely, there is no evidence of LC causing IIMs. We recommend that LC diagnosis consider the specific characteristics of IIMs.

Familial autoimmunity in patients with idiopathic inflammatory myopathies.

BACKGROUND: Familial associations can be indicators of shared genetic susceptibility between two diseases. Previous data on familial autoimmunity in patients with idiopathic inflammatory myopathies (IIM) are scarce and inconsistent. OBJECTIVES: To investigate which autoimmune diseases (ADs) may share genetic susceptibility with IIM, we examined the familial associations between IIM and different ADs. METHODS: In this Swedish population-based family study, we assembled 7615 first-degree relatives (FDRs) of 1620 patients with IIM and 37,309 relatives of 7797 matched individuals without IIM. Via register linkages, we ascertained rheumatoid arthritis, other rheumatic inflammatory diseases (RIDs), multiple sclerosis, inflammatory bowel diseases (IBD), type 1 diabetes mellitus, autoimmune thyroid diseases (AITD), coeliac disease (CeD) and myasthenia gravis among the FDRs. We estimated the familial association between IIM and each AD using conditional logistic regression and performed subgroup analyses by kinship. RESULTS: Patients with IIM had significantly higher odds of having ≥1 FDR affected by other RIDs (adjusted odds ratio [aOR] = 1.40, 95% confidence interval [CI] 1.11-1.78) and greater odds of having ≥2 FDRs affected by CeD (aOR = 3.57, 95% CI 1.28-9.92) compared to the individuals without IIM. In the analyses of any FDR pairs, we observed familial associations for other RIDs (aOR = 1.34, 95% CI 1.14-1.56), IBD (aOR = 1.20, 95% CI 1.02-1.41), AITD (aOR = 1.10, 95% CI 1.02-1.19) and CeD (aOR = 1.37, 95% CI 1.08-1.74) while associations for other ADs were not statistically significant. CONCLUSION: The observed familial associations may suggest that IIM shares genetic susceptibility with various ADs, information that may be useful for clinical counselling and guiding future genetic studies of IIM.

Transcriptomic profiling reveals distinct subsets of immune checkpoint inhibitor induced myositis.

OBJECTIVES: Inflammatory myopathy or myositis is a heterogeneous family of immune-mediated diseases including dermatomyositis (DM), antisynthetase syndrome (AS), immune-mediated necrotising myopathy (IMNM) and inclusion body myositis (IBM). Immune checkpoint inhibitors (ICIs) can also cause myositis (ICI-myositis). This study was designed to define gene expression patterns in muscle biopsies from patients with ICI-myositis. METHODS: Bulk RNA sequencing was performed on 200 muscle biopsies (35 ICI-myositis, 44 DM, 18 AS, 54 IMNM, 16 IBM and 33 normal muscle biopsies) and single nuclei RNA sequencing was performed on 22 muscle biopsies (seven ICI-myositis, four DM, three AS, six IMNM and two IBM). RESULTS: Unsupervised clustering defined three distinct transcriptomic subsets of ICI-myositis: ICI-DM, ICI-MYO1 and ICI-MYO2. ICI-DM included patients with DM and anti-TIF1γ autoantibodies who, like DM patients, overexpressed type 1 interferon-inducible genes. ICI-MYO1 patients had highly inflammatory muscle biopsies and included all patients that developed coexisting myocarditis. ICI-MYO2 was composed of patients with predominant necrotising pathology and low levels of muscle inflammation. The type 2 interferon pathway was activated both in ICI-DM and ICI-MYO1. Unlike the other types of myositis, all three subsets of ICI-myositis patients overexpressed genes involved in the IL6 pathway. CONCLUSIONS: We identified three distinct types of ICI-myositis based on transcriptomic analyses. The IL6 pathway was overexpressed in all groups, the type I interferon pathway activation was specific for ICI-DM, the type 2 IFN pathway was overexpressed in both ICI-DM and ICI-MYO1 and only ICI-MYO1 patients developed myocarditis.

Molecular signature of neutrophil extracellular trap mediating disease module in idiopathic inflammatory myopathy.

The rarity and heterogeneity of idiopathic inflammatory myopathy (IIM) pose challenges for researching IIM in affected individuals. We analyzed integrated transcriptomic datasets obtained using muscle tissues from patients with five distinct IIM subtypes to investigate the shared and distinctive cellular and molecular characteristics. A transcriptomic dataset of muscle tissues from normal controls (n = 105) and patients with dermatomyositis (n = 89), polymyositis (n = 33), inclusion body myositis (n = 121), immune-mediated necrotizing myositis (n = 75), and anti-synthetase syndrome (n = 18) was used for differential gene-expression analysis, functional-enrichment analysis, gene set-enrichment analysis, disease-module identification, and kernel-based diffusion scoring. Damage-associated molecular pattern-associated pathways and neutrophil-mediated immunity were significantly enriched across different IIM subtypes, although their activities varied. Interferons-signaling pathways were differentially activated across all five IIM subtypes. In particular, neutrophil extracellular trap (NET) formation was significantly activated and correlated with Fcγ R-mediated signaling pathways. NET formation-associated genes were key for establishing disease modules, and FCGRs, C1QA, and SERPINE1 markedly perturbed the disease modules. Integrated transcriptomic analysis of muscle tissues identified NETs as key components of neutrophil-mediated immunity involved in the pathogenesis of IIM subtypes and, thus, has therapeutically targetable value.

Transcriptomic profiles of muscular dystrophy with myositis (mdm) in extensor digitorum longus, psoas, and soleus muscles from mice.

BACKGROUND: Titinopathies are inherited muscular diseases triggered by genetic mutations in the titin gene. Muscular dystrophy with myositis (mdm) is one such disease caused by a LINE repeat insertion, leading to exon skipping and an 83-amino acid residue deletion in the N2A-PEVK region of mouse titin. This region has been implicated in a number of titin-titin ligand interactions, hence are important for myocyte signaling and health. Mice with this mdm mutation develop a severe and progressive muscle degeneration. The range of phenotypic differences observed in mdm mice shows that the deletion of this region induces a cascade of transcriptional changes extending to numerous signaling pathways affected by the titin filament. Previous research has focused on correlating phenotypic differences with muscle function in mdm mice. These studies have provided understanding of the downstream physiological effects resulting from the mdm mutation but only provide insights on processes that can be physiologically observed and measured. We used differential gene expression (DGE) to compare the transcriptomes of extensor digitorum longus (EDL), psoas and soleus muscles from wild-type and mdm mice to develop a deeper understand of these tissue-specific responses. RESULTS: The overall expression pattern observed shows a well-differentiated transcriptional signature in mdm muscles compared to wild type. Muscle-specific clusters observed within the mdm transcriptome highlight the level of variability of each muscle to the deletion. Differential gene expression and weighted gene co-expression network analysis showed a strong directional response in oxidative respiration-associated mitochondrial genes, which aligns with the poor shivering and non-shivering thermogenesis previously observed. Sln, which is a marker associated with shivering and non-shivering thermogenesis, showed the strongest expression change in fast-fibered muscles. No drastic changes in MYH expression levels were reported, which indicated an absence of major fiber-type switching events. Overall expression shifts in MYH isoforms, MARPs, and extracellular matrix associated genes demonstrated the transcriptional complexity associated with mdm mutation. The expression alterations in mitochondrial respiration and metabolism related genes in the mdm muscle dominated over other transcriptomic changes, and likely account for the late stage cellular responses in the mdm muscles. CONCLUSIONS: We were able to demonstrate that the complex nature of mdm mutation extends beyond a simple rearrangement in titin gene. EDL, psoas and soleus exemplify unique response modes observed in skeletal muscles with mdm mutation. Our data also raises the possibility that failure to maintain proper energy homeostasis in mdm muscles may contribute to the pathogenesis of the degenerative phenotype in mdm mice. Understanding the full disease-causing molecular cascade is difficult using bulk RNA sequencing techniques due to intricate nature of the disease. The development of the mdm phenotype is temporally and spatially regulated, hence future studies should focus on single fiber level investigations.

Abnormal paraoxonase-1 (PON1) enzyme activity in idiopathic inflammatory myopathies.

OBJECTIVES: Patients with idiopathic inflammatory myopathies (IIM) have severe vascular involvement, which contributes to disease morbidity and mortality. Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL) associated protein that protects the vascular endothelium from oxidative injury and damage. The current work assessed the functional and genetic determinants of PON1 activity in IIM patients. METHODS: A total of 184 IIM patients and 112 healthy controls (HC) were included. PON1 enzyme activity was assessed by paraoxonase, arylesterase and lactonase assays, and the Q192R PON1 single nucleotide polymorphism (SNP) was analysed. Multivariate regression models examined associations of PON1 activity with IIM diagnosis and myositis disease outcomes. RESULTS: The arylesterase and lactonase activities of PON1 were significantly lower in IIM patients compared with HC. Higher myositis disease activity, the presence of severe IIM-associated interstitial lung disease (ILD), and the presence of MDA5 or anti-synthetase antibodies were significantly associated with lower PON1 activity. The PON1 Q192R polymorphism was strongly linked to the paraoxonase activity of PON1 in IIM, and patients with the PON1 QQ genotype had better IIM disease outcomes compared with patients with the QR or RR genotypes. CONCLUSIONS: The arylesterase and lactonase activities of PON1 are significantly impaired in IIM patients compared with HC, and inversely associate with IIM disease activity and the presence of severe ILD. The PON1 QQ genotype associates with more favourable disease outcomes in IIM patients. Large prospective studies are needed to further evaluate the role of PON1 and PON1 genetic polymorphisms in the development and propagation of IIM and IIM-ILD.

Integrating 16S RRNA gene sequencing and metabolomics to evaluate the association between gut microbiota and serum metabolites in patients with myositis.

AIMS: Gut microbiota and metabolites have a profound impact on the maintenance of body health. In this study, we assessed the association between gut microbiota and serum metabolite changes in myositis using 16S rRNA gene sequencing and metabolomics to provide new ideas for screening and treating myositis. METHODS AND RESULTS: Blood and faecal samples were collected from 20 myositis patients and 20 healthy control subjects. Then, 16S rRNA gene sequencing, enzyme-linked immunosorbent assays and untargeted metabolomics study were performed to evaluate the relationship between gut microbiota and serum metabolites in patients with myositis. Compared to healthy control subjects, the blood samples from the patients with myositis had elevated levels of interleukin-4 (IL-4), tumour necrosis factor-α (TNF-α), and malondialdehyde (MDA) and decreased superoxide dismutase (SOD) levels. The increase in Bacteroidota (including Bacteroides and Parabacteroides, but not Prevotella) and the decrease in Firmicutes in the patients were accompanied by functional changes in amino acid and lipid metabolism. The gut microbiota (Bacteroides and Parabacteroides) were negatively correlated with the differential serum metabolites (glutamate and taurine). The differential serum metabolites (glutamate, pyrrolidonecarboxylic acid, and taurine) were also correlated with inflammatory factors (IL-4 and TNF-α) and oxidative stress indexes (MDA and SOD). CONCLUSION: Dysbiosis of gut microbiota in patients with myositis was accompanied by changes in inflammatory factors, oxidative stress indexes, and small molecule metabolites in serum. SIGNIFICANCE AND IMPACT OF STUDY: Blood and faecal biomarkers could be used for screening myositis.

Recurrent FOS rearrangement in proliferative fasciitis/proliferative myositis.

Proliferative fasciitis (PF) and proliferative myositis (PM) are rare benign soft tissue lesions, usually affecting the extremities of middle-aged or older adults. Presenting as poorly circumscribed masses, they histologically show bland spindle cell proliferation in a myxoid to fibrous background and a hallmark component of large epithelioid "ganglion-like" cells in various numbers, which may lead to their misdiagnosis as sarcoma. PF/PM has been long considered as reactive, akin to nodular fasciitis; however, its pathogenesis has remained unknown. In this study, we analyzed the FOS status in 6 PF/PMs (5 PFs and 1 PM). Five PF/PMs occurred in adults, all showing diffuse strong expression of c-FOS primarily in the epithelioid cells, whereas spindle cell components were largely negative. Using fluorescence in situ hybridization (FISH), all 5 c-FOS-immunopositive tumors showed evidence of FOS gene rearrangement in the epithelioid cells. RNA sequencing in 1 case detected a FOS-VIM fusion transcript, which was subsequently validated by reverse transcriptase-polymerase chain reaction, Sanger sequencing, and VIM FISH. The one pediatric PF case lacked c-FOS expression and FOS rearrangement. c-FOS immunohistochemistry was negative in 45 cases of selected mesenchymal tumor types with epithelioid components that may histologically mimic PF/PM, including pleomorphic sarcoma with epithelioid features and epithelioid sarcoma. Recurrent FOS rearrangement and c-FOS overexpression in PF/PM suggested these lesions to be neoplastic. FOS abnormality was largely restricted to the epithelioid cell population, clarifying the histological composition of at least 2 different cell types. c-FOS immunohistochemistry may serve as a useful adjunct to accurately distinguish PF/PM from mimics.

Familial aggregation and heritability: a nationwide family-based study of idiopathic inflammatory myopathies.

OBJECTIVES: The magnitude of the genetic contribution to idiopathic inflammatory myopathies (IIMs) is unknown. In this project, we aimed to investigate the familial aggregation and heritability of IIM. METHODS: This is a family-based study using nationwide healthcare register data in Sweden. We matched each patient with IIM to individuals without IIM, identified their first-degree relatives and determined the IIM status among all first-degree relatives. We estimated the adjusted ORs (aORs) of familial aggregation of IIM using conditional logistic regression. In addition, we used tetrachoric correlation to estimate the heritability of IIM. RESULTS: We included 7615 first-degree relatives of 1620 patients with IIM diagnosed between 1997 and 2016 and 37 309 first-degree relatives of 7797 individuals without IIM. Compared with individuals without IIM, patients with IIM were more likely to have ≥1 first-degree relative affected by IIM (aOR=4.32, 95% CI 2.00 to 9.34). Furthermore, the aOR of familial aggregation of IIM in full siblings was 2.53 (95% CI 1.62 to 3.96). The heritability of IIM was 22% (95% CI 12% to 31%) among any first-degree relatives and 24% (95% CI 12% to 37%) among full siblings. CONCLUSIONS: IIM has a familial component with a risk of aggregation among first-degree relatives and a heritability of about 20%. This information is of importance for future aetiological studies and in clinical counselling.

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.

Clinical exome sequencing in the diagnosis of pediatric neuromuscular disease.

BACKGROUND: The diagnosis of uncommon pediatric neuromuscular disease (NMD) is challenging due to genetic and phenotypic heterogeneity, yet is important to guide treatment, prognosis, and recurrence risk. Patients with diagnostically challenging presentations typically undergo extensive testing with variable molecular diagnostic yield. Given the advancement in next generation sequencing (NGS), we investigated the value of clinical whole exome sequencing (ES) in uncommon pediatric NMD. METHODS: A retrospective cohort study of 106 pediatric NMD patients with a combination of ES, chromosomal microarray (CMA), and candidate gene testing was completed at a large tertiary referral center. RESULTS: A molecular diagnosis was achieved in 37/79 (46%) patients with ES, 4/44 (9%) patients with CMA, and 15/74 (20%) patients with candidate gene testing. In 2/79 (3%) patients, a dual molecular diagnosis explaining the neuromuscular disease process was identified. A total of 42 patients (53%) who received ES remained without a molecular diagnosis at the conclusion of the study. CONCLUSIONS: Due to NGS, molecular diagnostic yield of rare neurological diseases is at an all-time high. We show that ES has a higher diagnostic rate compared to other genetic tests in a complex pediatric neuromuscular disease cohort and should be considered early in the diagnostic journey for select NMD patients with challenging presentations in which a clinical diagnosis is not evident.

Muscle regeneration in adiponectin knockout mice showed early activation of anti-inflammatory response with perturbations in myogenesis.

Activation, proliferation, and differentiation of satellite cells can be influenced by extracellular factors, such as adiponectin. This adipokine has been proposed as a regulator of in vitro myogenesis, but its action on in vivo regeneration is not still elucidated. We used C57BL/6 (wild-type [WT]) and adiponectin knockout (AdKO) mice injured with barium chloride at periods of 3, 7, and 14 days after injury. The AdKO presented a higher number of centralized nuclei after 7 days, and a reduction in myogenic genes was observed after 3 days. Moreover, these mice presented an increase in anti-inflammatory cytokines after 3 and 7 days, and an increase in the M2 gene marker and proinflammatory cytokines after 7 days. The WT demonstrated an increase in adiponectin messenger RNA after 7 days. These results demonstrate that adiponectin is important in tissue remodeling during regeneration and that its deficiency does not compromise the maturation of muscle fibers, due to an increase in anti-inflammatory response; however, there is a possible impairment in proinflammatory response and an increase in centralized myonuclei.

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.

Myositis with sarcoplasmic inclusions in Nakajo-Nishimura syndrome: a genetic inflammatory myopathy.

AIMS: Nakajo-Nishimura syndrome (NNS) is an autosomal recessive disease caused by biallelic mutations in the PSMB8 gene that encodes the immunoproteasome subunit ß5i. There have been only a limited number of reports on the clinicopathological features of the disease in genetically confirmed cases. METHODS: We studied clinical and pathological features of three NNS patients who all carry the homozygous p.G201V mutations in PSMB8. Patients' muscle specimens were analysed with histology and immunohistochemistry. RESULTS: All patients had episodes of typical periodic fever and skin rash, and later developed progressive muscle weakness and atrophy, similar to previous reports. Oral corticosteroid was used for treatment but showed no obvious efficacy. On muscle pathology, lymphocytes were present in the endomysium surrounding non-necrotic fibres, as well as in the perimysium perivascular area. Nearly all fibres strongly expressed MHC-I in the sarcolemma. In the eldest patient, there were abnormal protein aggregates in the sarcoplasm, immunoreactive to p62, TDP-43 and ubiquitin antibodies. CONCLUSIONS: These results suggest that inflammation, inclusion pathology and aggregation of abnormal proteins underlie the progressive clinical course of the NNS pathomechanism.

Insights into pathogenesis and clinical implications in myositis-associated interstitial lung diseases.

PURPOSE OF REVIEW: Interstitial lung diseases (ILDs) have been reported to be associated with myositis (including polymyositis and dermatomyositis). These myositis-associated ILDs carry significant morbidity and mortality. This review summarizes recent findings on myositis-associated ILD with a focus on pathogenesis and emerging treatment. RECENT FINDINGS: Recent advances in genetics have revealed 22 myositis-associated genome-wide loci, which were significantly enriched in regulatory regions in immune cells. An analysis of such disease-associated loci elucidated potential drug targets (e.g., TYK2 targeted by tofacitinib). In another study, an intronic variant in WDFY4 in association with clinically amyopathic dermatomyositis (CADM) had an effect for higher expression of a truncated WDFY4 isoform. Truncated WDFY4 markedly enhanced the MDA5-mediated NF-κB activation and cell apoptosis, indicating the dysregulated WDFY4-MDA5 pathway as a novel pathogenesis of CADM. As a novel strategy, tofacitinib treatment showed a promising improvement in survival and clinical features of CADM-associated ILD. SUMMARY: The genetic differences in the myositis-susceptible loci may explain the heterogeneous phenotypes and treatment responses in myositis-associated ILD. The understanding of pathogenesis with the genetic background as well as autoantibodies will enable the practice of personalized treatment in the management of the disease.