Guanylate Kinase 1 Deficiency: A Novel and Potentially Treatable Mitochondrial DNA Depletion/Deletions Disease.

OBJECTIVE: Mitochondrial DNA (mtDNA) depletion/deletions syndrome (MDDS) comprises a group of diseases caused by primary autosomal defects of mtDNA maintenance. Our objective was to study the etiology of MDDS in 4 patients who lack pathogenic variants in known genetic causes. METHODS: Whole exome sequencing of the probands was performed to identify pathogenic variants. We validated the mitochondrial defect by analyzing mtDNA, mitochondrial dNTP pools, respiratory chain activities, and GUK1 activity. To confirm pathogenicity of GUK1 deficiency, we expressed 2 GUK1 isoforms in patient cells. RESULTS: We identified biallelic GUK1 pathogenic variants in all 4 probands who presented with ptosis, ophthalmoparesis, and myopathic proximal limb weakness, as well as variable hepatopathy and altered T-lymphocyte profiles. Muscle biopsies from all probands showed mtDNA depletion, deletions, or both, as well as reduced activities of mitochondrial respiratory chain enzymes. GUK1 encodes guanylate kinase, originally identified as a cytosolic enzyme. Long and short isoforms of GUK1 exist. We observed that the long isoform is intramitochondrial and the short is cytosolic. In probands' fibroblasts, we noted decreased GUK1 activity causing unbalanced mitochondrial dNTP pools and mtDNA depletion in both replicating and quiescent fibroblasts indicating that GUK1 deficiency impairs de novo and salvage nucleotide pathways. Proband fibroblasts treated with deoxyguanosine and/or forodesine, a purine phosphatase inhibitor, ameliorated mtDNA depletion, indicating potential pharmacological therapies. INTERPRETATION: Primary GUK1 deficiency is a new and potentially treatable cause of MDDS. The cytosolic isoform of GUK1 may contribute to the T-lymphocyte abnormality, which has not been observed in other MDDS disorders. ANN NEUROL 2024.

Pathological autoantibody internalisation in myositis.

OBJECTIVES: Autoantibodies targeting intracellular proteins are common in various autoimmune diseases. In the context of myositis, the pathologic significance of these autoantibodies has been questioned due to the assumption that autoantibodies cannot enter living muscle cells. This study aims to investigate the validity of this assumption. METHODS: Confocal immunofluorescence microscopy was employed to localise antibodies and other proteins of interest in myositis muscle biopsies. Bulk RNA sequencing was used to examine the transcriptomic profiles of 669 samples, including those from patients with myositis, disease controls and healthy controls. Additionally, antibodies from myositis patients were introduced into cultured myoblasts through electroporation, and their transcriptomic profiles were analysed using RNA sequencing. RESULTS: In patients with myositis autoantibodies, antibodies accumulated inside myofibres in the same subcellular compartment as the autoantigen. Bulk RNA sequencing revealed that muscle biopsies from patients with autoantibodies targeting transcriptional regulators exhibited transcriptomic patterns consistent with dysfunction of the autoantigen. For instance, in muscle biopsies from patients with anti-PM/Scl autoantibodies recognising components of the nuclear RNA exosome complex, an accumulation of divergent transcripts and long non-coding RNAs was observed; these RNA forms are typically degraded by the nuclear RNA exosome complex. Introducing patient antibodies into cultured muscle cells recapitulated the transcriptomic effects observed in human disease. Further supporting evidence suggested that myositis autoantibodies recognising other autoantigens may also disrupt the function of their targets. CONCLUSIONS: This study demonstrates that, in myositis, autoantibodies are internalised into living cells, causing biological effects consistent with the disrupted function of their autoantigen.

Transcriptional derepression of CHD4/NuRD-regulated genes in the muscle of patients with dermatomyositis and anti-Mi2 autoantibodies.

OBJECTIVES: Myositis is a heterogeneous family of diseases including dermatomyositis (DM), immune-mediated necrotising myopathy (IMNM), antisynthetase syndrome (AS) and inclusion body myositis (IBM). Myositis-specific autoantibodies define different subtypes of myositis. For example, patients with anti-Mi2 autoantibodies targeting the chromodomain helicase DNA-binding protein 4 (CHD4)/NuRD complex (a transcriptional repressor) have more severe muscle disease than other DM patients. This study aimed to define the transcriptional profile of muscle biopsies from anti-Mi2-positive DM patients. METHODS: RNA sequencing was performed on muscle biopsies (n=171) from patients with anti-Mi2-positive DM (n=18), DM without anti-Mi2 autoantibodies (n=32), AS (n=18), IMNM (n=54) and IBM (n=16) as well as 33 normal muscle biopsies. Genes specifically upregulated in anti-Mi2-positive DM were identified. Muscle biopsies were stained for human immunoglobulin and protein products corresponding to genes specifically upregulated in anti-Mi2-positive muscle biopsies. RESULTS: A set of 135 genes, including SCRT1 and MADCAM1, was specifically overexpressed in anti-Mi2-positive DM muscle. This set was enriched for CHD4/NuRD-regulated genes and included genes that are not otherwise expressed in skeletal muscle. The expression levels of these genes correlated with anti-Mi2 autoantibody titres, markers of disease activity and with the other members of the gene set. In anti-Mi2-positive muscle biopsies, immunoglobulin was localised to the myonuclei, MAdCAM-1 protein was present in the cytoplasm of perifascicular fibres, and SCRT1 protein was localised to myofibre nuclei. CONCLUSIONS: Based on these findings, we hypothesise that anti-Mi2 autoantibodies could exert a pathogenic effect by entering damaged myofibres, inhibiting the CHD4/NuRD complex, and subsequently derepressing the unique set of genes defined in this study.

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.

Clinico-pathological phenotypes of systemic sclerosis-associated myopathy: analysis of a large multicentre cohort.

OBJECTIVE: The objective of this study was to analyse the clinico-serological and histological phenotypes of patients with SSc with associated myopathy. METHODS: From November 2002 to September 2020, 52 patients with SSc underwent a muscle biopsy for suspected myopathy. We established two subgroups according to the histological findings based on the presence of isolated fibrosis or fibrosis together with significant inflammation. These patterns were designated as fibrosing and inflammatory, respectively. Clinical data, antibody profile, electrophysiologic studies, muscle biopsy findings and data regarding treatment, mortality and survival were compared between the two groups. RESULTS: Fourteen biopsies had a fibrosing pattern, whereas 26 showed an inflammatory pattern that could be classified (according to the predominant pattern) into DM (n = 7), necrotizing myopathy (n = 4) and non-specific myositis (n = 15). Additionally, 12 muscle biopsies were reported as neurogenic atrophy (n = 2), or normal muscle or minimal changes (n = 10). Compared with the inflammatory group, SSc patients with the fibrosing pattern presented a higher prevalence of ischaemic heart disease (38.5% vs 3.8%, P = 0.011), conduction abnormalities or arrhythmias (61.5% vs 26.9%, P = 0.036), anti-topo I antibodies (42.9% vs 11.5%, P = 0.044), greater median ESR (53.5 mm/h vs 32.5 mm/h, P = 0.013), with poor response to treatment and a higher mortality (42.9% vs 3.8%, P = 0.004) and lower cumulative survival (P = 0.035). CONCLUSIONS: Patients with SSc-associated myopathy require a comprehensive approach that encompasses clinical, serological and histopathological aspects, given their outcome predictive capacity. At least two different phenotypes can be drawn, considering clinico-pathological features. Significant differences are delineated between both a fibrotic and an inflammatory phenotype.

Anoctamin 5 (ANO5) muscular dystrophy-three different phenotypes and a new histological pattern.

OBJECTIVE: Anoctamin 5 (ANO5) is a putative intracellular calcium-activated chloride channel. Recessive mutations in ANO5 may present from asymptomatic hyperCKemia and exercise-induced myalgia to proximal and/or distal muscle weakness. Here we describe the clinical, pathological, and molecular findings of three unrelated patients with ANO5-related muscular dystrophy. METHODS: In this retrospective study, we analyzed our database which includes 1700 muscle biopsies performed for diagnostic purposes from October 2004 to February 2019. Patients were attended by two myology experts, who performed and analyzed the muscle biopsies. Muscle biopsies were frozen in cooled isopenthane, cryostat sectioned, and routinely stained and reacted (minimum 16 stainings). A custom panel, including 115 genes (Nextera Rapid Capture, Illumina) and whole-exome sequencing analysis, was used for next-generation sequencing in cases without a definite pathological diagnosis. RESULTS: Three patients were diagnosed with ANO5-related muscular dystrophy, with all presenting the common exon 5 mutation c.191dup plus a compound heterozygous missense mutation. They showed three different phenotypes (distal myopathy, LGMD2L, and asymptomatic hyperCKemia). Curiously, all three muscle biopsies showed different patterns, but numerous ragged-red fibers with little endomysial inflammation and partial invasion cell by T lymphocytes were observed in one. CONCLUSION: ANO5-related muscular dystrophy is a heterogeneous disease with different clinical phenotypes as well as different histological patterns, which may even mimic a mitochondrial myopathy. The results of this study provide further knowledge of the clinical, histological, and pathological features related to ANO5 mutations.

Pathogenic autoantibody internalization in myositis.

Objectives: Myositis is a heterogeneous family of autoimmune muscle diseases. As myositis autoantibodies recognize intracellular proteins, their role in disease pathogenesis has been unclear. This study aimed to determine whether myositis autoantibodies reach their autoantigen targets within muscle cells and disrupt the normal function of these proteins. Methods: Confocal immunofluorescence microscopy was used to localize antibodies and other proteins of interest in myositis muscle biopsies. Bulk RNA sequencing was used to study the transcriptomic profiles of 668 samples from patients with myositis, disease controls, and healthy controls. Antibodies from myositis patients were introduced into cultured myoblasts by electroporation and the transcriptomic profiles of the treated myoblasts were studied by bulk RNA sequencing. Results: In patients with myositis autoantibodies, antibodies accumulated inside myofibers in the same subcellular compartment as the autoantigen. Each autoantibody was associated with effects consistent with dysfunction of its autoantigen, such as the derepression of genes normally repressed by Mi2/NuRD in patients with anti-Mi2 autoantibodies, the accumulation of RNAs degraded by the nuclear RNA exosome complex in patients with anti-PM/Scl autoantibodies targeting this complex, and the accumulation of lipids within myofibers of anti-HMGCR-positive patients. Internalization of patient immunoglobulin into cultured myoblasts recapitulated the transcriptomic phenotypes observed in human disease, including the derepression of Mi2/NuRD-regulated genes in anti-Mi2-positive dermatomyositis and the increased expression of genes normally degraded by the nuclear RNA exosome complex in anti-PM/Scl-positive myositis. Conclusions: In myositis, autoantibodies are internalized into muscle fibers, disrupt the biological function of their autoantigen, and mediate the pathophysiology of the disease.

Clinical Outcomes of Patients with Chronic Neuropathic Form of Gaucher Disease in the Spanish Real-World Setting: A Retrospective Study.

This was a retrospective, multicenter study that aimed to report the characteristics of type 3 Gaucher disease (GD3) patients in Spain, including the genotype, phenotype, therapeutic options, and treatment responses. A total of 19 patients with GD3 from 10 Spanish hospitals were enrolled in the study (14 men, 5 women). The median age at disease onset and diagnosis was 1 and 1.2 years, respectively, and the mean age at follow-up completion was 12.37 years (range: 1-25 years). Most patients exhibited splenomegaly (18/19) and hepatomegaly (17/19) at the time of diagnosis. The most frequent neurological abnormalities at onset were psychomotor retardation (14/19) and extrinsic muscle disorders (11/19), including oculomotor apraxia, supranuclear palsy, and strabismus. The L444P (c.1448T>C) allele was predominant, with the L444P (c.1448T>C) homozygous genotype mainly associated with visceral manifestations like hepatosplenomegaly, anemia, and thrombocytopenia. All patients received enzyme replacement therapy (ERT); other treatments included miglustat and the chaperone (ambroxol). Visceral manifestations, including hepatosplenomegaly and hematological and bone manifestations, were mostly controlled with ERT, except for kyphosis. The data from this study may help to increase the evidence base on this rare disease and contribute to improving the clinical management of GD3 patients.

Coordinated local RNA overexpression of complement induced by interferon gamma in myositis.

Complement proteins are deposited in the muscles of patients with myositis. However, the local expression and regulation of complement genes within myositis muscle have not been well characterized. In this study, bulk RNA sequencing (RNAseq) analyses of muscle biopsy specimens revealed that complement genes are locally overexpressed and correlate with markers of myositis disease activity, including the expression of interferon-gamma (IFNγ)-induced genes. Single cell and single nuclei RNAseq analyses showed that most local expression of complement genes occurs in macrophages, fibroblasts, and satellite cells, with each cell type expressing different sets of complement genes. Biopsies from immune-mediated necrotizing myopathy patients, who have the lowest levels of IFNγ-induced genes, also had the lowest complement gene expression levels. Furthermore, data from cultured human cells showed that IFNγ upregulates complement expression in macrophages, fibroblasts, and muscle cells. Taken together, our results suggest that in myositis muscle, IFNγ coordinates the local overexpression of complement genes that occurs in several cell types.

Dermatomyositis unleashed by immune checkpoint inhibitors. Three additional cases and a review of the literature.

OBJECTIVES: Immune checkpoint inhibitors (ICI) have revolutionized the treatment of several locally advanced and metastatic tumors. They enhance the effector function of the immune system, consequently leading to different immune-related adverse events. The aim of the present study was to describe three cases of dermatomyositis (DM) triggered by ICI diagnosed at our institution and to perform a review of the literature. METHODS: We performed a retrospective clinical, laboratory, and pathological evaluation of three cases of DM triggered by ICI belonging to a cohort of 187 DM patients from the Clinic Hospital Muscle Research Group of Barcelona from January 2009 to July 2022. Moreover, we undertook a narrative review of the literature from January 1990 to June 2022. RESULTS: Cases from our institution were triggered by avelumab, an anti-PD-1 ligand (PD-L1), nivolumab, and pembrolizumab, both anti-programmed death-1 (PD-1). One of these patients had locally advanced melanoma, and two had urothelial carcinoma. The severity and response to treatment were heterogeneous among the different cases. All were positive at high titers for anti-TIF1γ autoantibodies; in one of them, serum before the onset of ICI was available, and anti-TIF1γ autoantibodies were already present. RNA expression of IFNB1, IFNG and genes stimulated by these cytokines were markedly elevated in these patients. CONCLUSIONS: In conclusion, data from our patients and the narrative review suggest that early positivity to anti-TIF1γ unleashed by ICI may play a role in the development of full-blown DM, at least in some cases.

Gastrointestinal Involvement in Dermatomyositis.

Dermatomyositis is a systemic vasculopathy mainly affecting skin, muscle and lung, but may affect the gastrointestinal tract. We aim to describe clinical characteristics of patients with severe gastrointestinal involvement related to dermatomyositis in our center and medical literature. We retrospectively analysed these patients in our center, including cases of erosions/ulcers, perforation or digestive bleeding. Reported cases from April 1990 to April 2021 were reviewed through PubMed and Cochrane. From our cohort (n = 188), only 3 presented gastrointestinal compromise. All were women (10, 46 and 68 years). The initial symptom was abdominal pain and all had ≥2 episodes of digestive bleeding. All died due to complications of gastrointestinal involvement. Available pathological samples showed vascular ectasia. From the literature review (n = 50), 77% were women with a mean age of 49 years and the main symptom was abdominal pain (65%). All presented active muscular and cutaneous involvement at complication diagnosis. Mortality was 41.7%. The underlying lesion was perforation or ulcer (n = 22), intestinal wall thickening (n = 2), macroscopic inflammation (n = 2) or intestinal pneumatosis (n = 15). In 13 cases, vasculitis was described. Gastrointestinal involvement in dermatomyositis denotes severity, so an early intensive treatment is recommended. Pathological findings suggest that the underlying pathophysiological mechanism is a vasculopathy and not a true vasculitis.

Emerging PD-1 and PD-1L inhibitors-associated myopathy with a characteristic histopathological pattern.

BACKGROUND AND OBJECTIVE: Drug-induced myopathy is among the most common causes of muscle disease. An association has recently been described between programmed death-1 (PD-1)/PD-1 ligand (PD-L1) inhibitors and immune-related adverse events (irAE) affecting the muscle. Here, we report the clinical and pathological findings of nine unrelated patients with PD-1 and PD-L1 inhibitors-associated myopathy. METHODS: We retrospectively analyzed 317 muscle biopsies performed for diagnostic purposes from January 2017 to June 2019. Patients were attended in two tertiary centers and muscle biopsies were performed and analyzed by two myology experts. Muscle biopsies were frozen in cooled isopenthane, cryostat sectioned and stained. Immunohistochemistry studies were also performed as a routine procedure in our lab. RESULTS: We identified 9 patients receiving anti-PD-1 or PD-L1 inhibitors consulting for either muscle weakness, asthenia, myasthenic-like syndrome or other muscle related-symptoms, along with biopsy-proven inflammatory myopathy. One had concomitant myocarditis. In most of the cases muscle biopsy showed a marked phenomenon of necrosis, macrophagy and muscle regeneration with perivascular inflammatory infiltrates with a large component of macrophagic cells. A tendency to perifascicular atrophy was also noticed. The expression of MHC class I antigens predominated in the perifascicular zones. Raised muscle enzymes were detected in 7 patients. CONCLUSION: A characteristic clinic-pathological pattern, including a myasthenia gravis-like syndrome plus myositis was found in patients receiving PD-1 and PD-1 L inhibitors. A large component of macrophages resembling granulomas seems to be the pathological hallmark of the syndrome. Further information is required to understand the wide spectrum of immune-related adverse events involving the muscle during or after treatment with anti-PD-1 inhibitors, but the pathological picture seems to be characteristic.

Antiphospholipase 2 receptor antibody levels to predict complete spontaneous remission in primary membranous nephropathy.

BACKGROUND: M-type phospholipase A2 receptor (APLA2R) is considered the major antigen involved in the pathogenesis of adult primary membranous nephropathy (MN), which is the leading cause of non-diabetic nephrotic syndrome. Antibodies to this antigen have been proved to be an excellent biomarker of disease activity in primary MN. In fact, preliminary data suggest that the higher the antibody level the more proteinuria, and that a decrease in antibody level precedes the remission of proteinuria, but more solid evidence is needed. METHODS: The present work aims to characterize the predictive value of the level of antibodies against PLA2R as a biomarker of disease course and treatment response in a well-defined cohort of 62 patients from University Hospitals Clinic of Barcelona and Josep Trueta in Girona. The primary outcome was the appearance of a spontaneous complete remission (CR), defined as induction of a CR without the use of immunosuppressive agents. RESULTS: In common with other reports, this work confirms that spontaneous CR is more frequent in patients with low titre of APLA2R at diagnosis, but strikingly, in this cohort we found that spontaneous CR was achieved in patients with APLA2R levels <40 UI/mL. Furthermore, spontaneous CR were less frequently observed in patients with proteinuria >8 g/day. CONCLUSIONS: In conclusion, these findings point out the important role of APLA2R as a tool to predict the disease course and establish personalized therapeutic options at the moment of diagnosis of primary MN. Specifically, patients with low titre of APLA2R (<40 UI/mL) and proteinuria <4/day could obtain benefit of a longer period of follow-up with conservative treatment after diagnosis.

Sporadic inclusion body myositis: Diagnostic value of p62 immunostaining / Miositis con cuerpos de inclusión: valor de la inmunohistoquímica para p62 en su diagnóstico

Background and objectives: Sporadic inclusion body myositis (sIBM) diagnosis is frequently delayed or confused with another class of disorders, and misdiagnosis is common. Sometimes, we have problems diagnosing an sIBM in the early stages or predicting when a PM is going to become an sIBM. In this sense, we believe that p62 immunostaining could help clinicians. Case report: We report the case of a 61-year-old patient with sIBM who six years earlier had been diagnosed with polymyositis (PM). After muscle biopsies analyses, we showed the natural history of sIBM by p62 expression. Results: When we looked for p62 aggregates retrospectively we could see small dotted p62 aggregates in the muscle fibres of the first muscle biopsy. Six years later, the patient presented with the typical clinical picture of sIBM, also the muscle biopsy was characteristic, with large p62 aggregates. Conclusions: Probably p62 immunostaining could help to distinguish PM patients that are going to become sIBM, but to date there has been no systematic study to clarify p62 utility in myositis

Fundamento y objetivo: El diagnóstico de la miositis por cuerpos de inclusión en su forma esporádica (MCI) con frecuencia se retrasa o se confunde con otra clase de trastornos. En ocasiones, surgen problemas para diagnosticar una MCI en estadios iniciales. En este sentido, pensamos que la inmunohistoquímica para p62 podría ser una herramienta de gran utilidad para el diagnóstico. Observación clínica: Presentamos el caso de un paciente de 61 años con MCI que seis años antes recibió el diagnóstico de polimiositis (PM). Después de analizar las biopsias musculares realizadas, mostramos la historia natural de la MCI a través de la expresión de p62. Resultados: Cuando buscamos los agregados de p62 retrospectivamente pudimos ver pequeños agregados de p62 en las fibras musculares de la primera biopsia. Seis años más tarde, coincidiendo con el cuadro de presentación clínica, típico de MCI, la biopsia muscular resultó característica, mostrando grandes agregados de p62. Conclusiones: Probablemente la inmunotinción para p62 ayudaría a distinguir los pacientes con diagnóstico de PM que posteriormente van a convertirse en MCI, pero hasta ahora no hay un estudio sistemático para dilucidar la utilidad de p62 en miositis