Autophagy in non-immune-mediated rhabdomyolysis: Assessment of p62 immunohistochemistry.

INTRODUCTION/AIMS: p62 immunochemistry (IHC) has been shown to aid diagnosis with distinct patterns of muscle fiber staining observed in some inflammatory, hereditary, and degenerative myopathies, such as immune-mediated necrotizing myopathy (IMNM). The pattern of p62 staining may help narrow the pathological differential diagnosis of rhabdomyolysis. However, there is a lack of information on the pattern of p62 IHC in non-immune-mediated rhabdomyolysis. In this study we aim to describe histopathological findings in non-immune-mediated rhabdomyolysis, with particular emphasis on the pattern of p62 IHC. METHODS: We retrospectively reviewed the histopathological features of patients with a confirmed diagnoses of non-immune-mediated rhabdomyolysis referred to our center. RESULTS: Five patients were identified. Rhabdomyolysis was determined to be due to statin-associated toxicity in three patients, alcohol overuse in one patient, and intensive exercise in one patient. All patients showed increased numbers of necrotic and regenerating muscle fibers. Diffuse and finely granular sarcoplasmic positive p62 staining was present in scattered non-necrotic muscle fibers in all patients. DISCUSSION: Disturbance of autophagy appears to be a common mechanism in non-immune-mediated rhabdomyolysis. Our results show p62 IHC is sensitive but lacks specificity. Therefore, the pattern of p62 staining does not distinguish non-immune-mediated rhabdomyolysis from histopathologically similar IMNM.

Factors associated with the severity of COVID-19 outcomes in people with neuromuscular diseases: Data from the International Neuromuscular COVID-19 Registry.

BACKGROUND AND PURPOSE: Clinical outcome information on patients with neuromuscular diseases (NMDs) who have been infected with SARS-CoV-2 is limited. The aim of this study was to determine factors associated with the severity of COVID-19 outcomes in people with NMDs. METHODS: Cases of NMD, of any age, and confirmed/presumptive COVID-19, submitted to the International Neuromuscular COVID-19 Registry up to 31 December 2021, were included. A mutually exclusive ordinal COVID-19 severity scale was defined as follows: (1) no hospitalization; (2) hospitalization without oxygenation; (3) hospitalization with ventilation/oxygenation; and (4) death. Multivariable ordinal logistic regression analyses were used to estimate odds ratios (ORs) for severe outcome, adjusting for age, sex, race/ethnicity, NMD, comorbidities, baseline functional status (modified Rankin scale [mRS]), use of immunosuppressive/immunomodulatory medication, and pandemic calendar period. RESULTS: Of 315 patients from 13 countries (mean age 50.3 [±17.7] years, 154 [48.9%] female), 175 (55.5%) were not hospitalized, 27 (8.6%) were hospitalized without supplemental oxygen, 91 (28.9%) were hospitalized with ventilation/supplemental oxygen, and 22 (7%) died. Higher odds of severe COVID-19 outcomes were observed for: age ≥50 years (50-64 years: OR 2.4, 95% confidence interval [CI] 1.33-4.31; >64 years: OR 4.16, 95% CI 2.12-8.15; both vs. <50 years); non-White race/ethnicity (OR 1.81, 95% CI 1.07-3.06; vs. White); mRS moderately severe/severe disability (OR 3.02, 95% CI 1.6-5.69; vs. no/slight/moderate disability); history of respiratory dysfunction (OR 3.16, 95% CI 1.79-5.58); obesity (OR 2.24, 95% CI 1.18-4.25); ≥3 comorbidities (OR 3.2, 95% CI 1.76-5.83; vs. ≤2; if comorbidity count used instead of specific comorbidities); glucocorticoid treatment (OR 2.33, 95% CI 1.14-4.78); and Guillain-Barré syndrome (OR 3.1, 95% CI 1.35-7.13; vs. mitochondrial disease). CONCLUSIONS: Among people with NMDs, there is a differential risk of COVID-19 outcomes according to demographic and clinical characteristics. These findings could be used to develop tailored management strategies and evidence-based recommendations for NMD patients.

Bi-allelic loss-of-function OBSCN variants predispose individuals to severe recurrent rhabdomyolysis.

Rhabdomyolysis is the acute breakdown of skeletal myofibres in response to an initiating factor, most commonly toxins and over exertion. A variety of genetic disorders predispose to rhabdomyolysis through different pathogenic mechanisms, particularly in patients with recurrent episodes. However, most cases remain without a genetic diagnosis. Here we present six patients who presented with severe and recurrent rhabdomyolysis, usually with onset in the teenage years; other features included a history of myalgia and muscle cramps. We identified 10 bi-allelic loss-of-function variants in the gene encoding obscurin (OBSCN) predisposing individuals to recurrent rhabdomyolysis. We show reduced expression of OBSCN and loss of obscurin protein in patient muscle. Obscurin is proposed to be involved in sarcoplasmic reticulum function and Ca2+ handling. Patient cultured myoblasts appear more susceptible to starvation as evidenced by a greater decreased in sarcoplasmic reticulum Ca2+ content compared to control myoblasts. This likely reflects a lower efficiency when pumping Ca2+ back into the sarcoplasmic reticulum and/or a decrease in Ca2+ sarcoplasmic reticulum storage ability when metabolism is diminished. OSBCN variants have previously been associated with cardiomyopathies. None of the patients presented with a cardiomyopathy and cardiac examinations were normal in all cases in which cardiac function was assessed. There was also no history of cardiomyopathy in first degree relatives, in particular in any of the carrier parents. This cohort is relatively young, thus follow-up studies and the identification of additional cases with bi-allelic null OBSCN variants will further delineate OBSCN-related disease and the clinical course of disease.

Challenges in the diagnosis and management of immune-mediated necrotising myopathy (IMNM) in a patient on long-term statins.

Immune-mediated necrotising myopathy (IMNM) is a severe and poorly understood complication of statin use. Prompt management with immunosuppressive treatment is often needed to control the condition, which differs from the management of the more commonly recognised statin-induced myopathy. We present a case report and brief review of the literature regarding the pathogenesis, diagnosis, and management of anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) positive IMNM (HMGCR IMNM). There are no randomised clinical trials, but several smaller studies and cases suggest a triple therapy of corticosteroids, IVIG, and a corticosteroid-sparing immunosuppressant appears efficacious in patients with IMNM and proximal weakness. The mechanism of statin-induced IMNM is uncertain, and this is further complicated by the reports of HMGCR IMNM in statin-naïve patients, including children. We present a case of biopsy-confirmed HMGCR IMNM in a woman taking daily statins for treatment of hypercholesterolaemia for 4 years. She presented with symptoms consistent with a urinary tract infection (UTI), including muscle weakness. She was treated as an isolated case of UTI. One month later, she presented again with worsening weakness in her shoulders and hips. Creatine kinase was elevated, and MRI showed increased signal with STIR sequences in both thighs. Anti-HMGCR was positive and leg biopsy-confirmed necrotising changes. Stopping her statin prescription and a short course of prednisolone did not improve her muscle weakness. Adding methotrexate resulted in eventual resolution of her symptoms. IMNM should be considered as a differential in any patient taking statins presenting with muscle weakness, and this case suggests that immunosuppressant therapy in addition to cessation of statins is effective at treating IMNM. Clinical trials are needed to further investigate the efficacy of different combinations of immunosuppressants.

Genotype-phenotype correlations in valosin-containing protein disease: a retrospective muticentre study.

BACKGROUND: Valosin-containing protein (VCP) disease, caused by mutations in the VCP gene, results in myopathy, Paget's disease of bone (PBD) and frontotemporal dementia (FTD). Natural history and genotype-phenotype correlation data are limited. This study characterises patients with mutations in VCP gene and investigates genotype-phenotype correlations. METHODS: Descriptive retrospective international study collecting clinical and genetic data of patients with mutations in the VCP gene. RESULTS: Two hundred and fifty-five patients (70.0% males) were included in the study. Mean age was 56.8±9.6 years and mean age of onset 45.6±9.3 years. Mean diagnostic delay was 7.7±6 years. Symmetric lower limb weakness was reported in 50% at onset progressing to generalised muscle weakness. Other common symptoms were ventilatory insufficiency 40.3%, PDB 28.2%, dysautonomia 21.4% and FTD 14.3%. Fifty-seven genetic variants were identified, 18 of these no previously reported. c.464G>A (p.Arg155His) was the most frequent variant, identified in the 28%. Full time wheelchair users accounted for 19.1% with a median time from disease onset to been wheelchair user of 8.5 years. Variant c.463C>T (p.Arg155Cys) showed an earlier onset (37.8±7.6 year) and a higher frequency of axial and upper limb weakness, scapular winging and cognitive impairment. Forced vital capacity (FVC) below 50% was as risk factor for being full-time wheelchair user, while FVC <70% and being a full-time wheelchair user were associated with death. CONCLUSION: This study expands the knowledge on the phenotypic presentation, natural history, genotype-phenotype correlations and risk factors for disease progression of VCP disease and is useful to improve the care provided to patient with this complex disease.

Acute small fiber neuropathy after Oxford-AstraZeneca ChAdOx1-S vaccination: A report of three cases and review of the literature.

Small fiber neuropathy usually presents with gradual and progressive chronic length-dependent pain. Acute small fiber neuropathy is rarely reported. Three patients with acute onset neuropathic pain after Oxford-AstraZeneca ChAdOx1-S vaccination are described. Two patients were identified at the Oxford University NHS Foundation Trust, Oxford, UK and one patient in Red de Salud UC Christus, Santiago, Chile. All patients underwent a clinical assessment that included a detailed neurological examination, laboratory investigations, nerve conduction studies, thermal threshold testing, and skin biopsy for intra-epidermal nerve fiber density. Patients seen in Oxford underwent MRI of the brain and spinal cord. Cerebrospinal analysis was not performed. Neuropathic symptoms (burning pain, dysaesthesias) developed in the hands and feet within 2 weeks of vaccination. On clinical examination, there was pinprick and thermal hyposensitivity in the area of neuropathic pain. Laboratory investigation, nerve conduction tests, sympathetic skin responses, and MRI showed no relevant abnormalities. Thermal thresholds were abnormal and intra-epidermal nerve fiber density in the lower leg was reduced. In two cases symptoms persist after several months. Three cases of definite acute small fiber neuropathy after Oxford-AstraZeneca ChAdOx1-S vaccination are described. At follow up, neuropathic pain was present in two of the patients.

Proteomics of rimmed vacuoles define new risk allele in inclusion body myositis.

OBJECTIVE: Sporadic inclusion body myositis (sIBM) pathogenesis is unknown; however, rimmed vacuoles (RVs) are a constant feature. We propose to identify proteins that accumulate within RVs. METHODS: RVs and intact myofibers were laser microdissected from skeletal muscle of 18 sIBM patients and analyzed by a sensitive mass spectrometry approach using label-free spectral count-based relative protein quantification. Whole exome sequencing was performed on 62 sIBM patients. Immunofluorescence was performed on patient and mouse skeletal muscle. RESULTS: A total of 213 proteins were enriched by >1.5 -fold in RVs compared to controls and included proteins previously reported to accumulate in sIBM tissue or when mutated cause myopathies with RVs. Proteins associated with protein folding and autophagy were the largest group represented. One autophagic adaptor protein not previously identified in sIBM was FYCO1. Rare missense coding FYCO1 variants were present in 11.3% of sIBM patients compared with 2.6% of controls (p = 0.003). FYCO1 colocalized at RVs with autophagic proteins such as MAP1LC3 and SQSTM1 in sIBM and other RV myopathies. One FYCO1 variant protein had reduced colocalization with MAP1LC3 when expressed in mouse muscle. INTERPRETATION: This study used an unbiased proteomic approach to identify RV proteins in sIBM that included a novel protein involved in sIBM pathogenesis. FYCO1 accumulates at RVs, and rare missense variants in FYCO1 are overrepresented in sIBM patients. These FYCO1 variants may impair autophagic function, leading to RV formation in sIBM patient muscle. FYCO1 functionally connects autophagic and endocytic pathways, supporting the hypothesis that impaired endolysosomal degradation underlies the pathogenesis of sIBM. Ann Neurol 2017;81:227-239.

A novel quantitative assay of mitophagy: Combining high content fluorescence microscopy and mitochondrial DNA load to quantify mitophagy and identify novel pharmacological tools against pathogenic heteroplasmic mtDNA.

Mitophagy is a cellular mechanism for the recycling of mitochondrial fragments. This process is able to improve mitochondrial DNA (mtDNA) quality in heteroplasmic mtDNA disease, in which mutant mtDNA co-exists with normal mtDNA. In disorders where the load of mutant mtDNA determines disease severity it is likely to be an important determinant of disease progression. Measuring mitophagy is technically demanding. We used pharmacological modulators of autophagy to validate two techniques for quantifying mitophagy. First we used the IN Cell 1000 analyzer to quantify mitochondrial co-localisation with LC3-II positive autophagosomes. Unlike conventional fluorescence and electron microscopy, this high-throughput system is sufficiently sensitive to detect transient low frequency autophagosomes. Secondly, because mitophagy preferentially removes pathogenic heteroplasmic mtDNA mutants, we developed a heteroplasmy assay based on loss of m.3243A>G mtDNA, during culture conditions requiring oxidative metabolism ("energetic stress"). The effects of the pharmacological modulators on these two measures were consistent, confirming that the high throughput imaging output (autophagosomes co-localising with mitochondria) reflects mitochondrial quality control. To further validate these methods, we performed a more detailed study using metformin, the most commonly prescribed antidiabetic drug that is still sometimes used in Maternally Inherited Diabetes and Deafness (MIDD). This confirmed our initial findings and revealed that metformin inhibits mitophagy at clinically relevant concentrations, suggesting that it may have novel therapeutic uses.

Ongoing developments in sporadic inclusion body myositis.

Sporadic inclusion body myositis (IBM) is an acquired muscle disorder associated with ageing, for which there is no effective treatment. Ongoing developments include: genetic studies that may provide insights regarding the pathogenesis of IBM, improved histopathological markers, the description of a new IBM autoantibody, scrutiny of the diagnostic utility of clinical features and biomarkers, the refinement of diagnostic criteria, the emerging use of MRI as a diagnostic and monitoring tool, and new pathogenic insights that have led to novel therapeutic approaches being trialled for IBM, including treatments with the objective of restoring protein homeostasis and myostatin blockers. The effect of exercise in IBM continues to be investigated. However, despite these ongoing developments, the aetiopathogenesis of IBM remains uncertain. A translational and multidisciplinary collaborative approach is critical to improve the diagnosis, treatment, and care of patients with IBM.

Update in inclusion body myositis.

PURPOSE OF REVIEW: The purpose of this study is to review recent scientific advances relating to the natural history, cause, treatment and serum and imaging biomarkers of inclusion body myositis (IBM). RECENT FINDINGS: Several theories regarding the aetiopathogenesis of IBM are being explored and new therapeutic approaches are being investigated. New diagnostic criteria have been proposed, reflecting the knowledge that the diagnostic pathological findings may be absent in patients with clinically typical IBM. The role of MRI in IBM is expanding and knowledge about pathological biomarkers is increasing. The recent description of autoantibodies to cytosolic 5' nucleotidase 1A in patients with IBM is a potentially important advance that may aid early diagnosis and provides new evidence regarding the role of autoimmunity in IBM. SUMMARY: IBM remains an enigmatic and often misdiagnosed disease. The pathogenesis of the disease is still not fully understood. To date, pharmacological treatment trials have failed to show clear efficacy. Future research should continue to focus on improving understanding of the pathophysiological mechanisms of the disease and on the identification of reliable and sensitive outcome measures for clinical trials. IBM is a rare disease and international multicentre collaboration for trials is important to translate research advances into improved patient outcomes.

Clinical assessment determines the diagnosis of inclusion body myositis independently of pathological features.

BACKGROUND AND OBJECTIVE: Historically, the diagnosis of sporadic inclusion body myositis (IBM) has required the demonstration of the presence of a number of histopathological findings on muscle biopsy--namely, rimmed vacuoles, an inflammatory infiltrate with invasion of non-necrotic muscle fibres (partial invasion) and amyloid or 15-18 nm tubulofilamentous inclusions (Griggs criteria). However, biopsies of many patients with clinically typical IBM do not show all of these histopathological findings, at least at presentation. We compared the clinical features at presentation and during the course of disease in 67 patients with histopathologically diagnosed IBM and clinically diagnosed IBM seen within a single UK specialist muscle centre. METHODS AND RESULTS: At presentation, using clinically focused diagnostic criteria (European Neuromuscular Centre (ENMC) 2011), a diagnosis of IBM was made in 88% of patients whereas 76% fulfilled the 1997 ENMC criteria and only 27% satisfied the histopathologically focused Griggs criteria. There were no differences in clinical features or outcomes between clinically and histopathologically diagnosed patients, but patients lacking the classical histopathological finding of rimmed vacuoles were younger, suggesting that rimmed vacuoles may be a later feature of the disease. CONCLUSIONS: These findings have important implications for diagnosis and future studies or trials in IBM as adherence to histopathologically focused diagnostic criteria will exclude large numbers of patients with IBM. Importantly, those excluded may be at an earlier stage of the disease and more amenable to treatment.

Long-term observational study of sporadic inclusion body myositis.

We describe a long-term observational study of a large cohort of patients with sporadic inclusion body myositis and propose a sporadic inclusion body myositis weakness composite index that is easy to perform during a clinic. Data collection from two groups of patients (Paris and Oxford) was completed either during a clinic visit (52%), or by extraction from previous medical records (48%). One hundred and thirty-six patients [57% males, 61 (interquartile range 55-69) years at onset] were included. At the last visit all patients had muscle weakness (proximal British Medical Research Council scale <3/5 in 48%, distal British Medical Research Council scale <3/5 in 40%, swallowing problems in 46%). During their follow-up, 75% of patients had significant walking difficulties and 37% used a wheelchair (after a median duration from onset of 14 years). The sporadic inclusion body myositis weakness composite index, which correlated with grip strength (correlation coefficient: 0.47; P < 0.001) and Rivermead Mobility Index (correlation coefficient: 0.85; P < 0.001), decreased significantly with disease duration (correlation coefficient: -0.47; P < 0.001). The risk of death was only influenced by older age at onset of first symptoms. Seventy-one (52%) patients received immunosuppressive treatments [prednisone in 91.5%, associated (in 64.8%) with other immunomodulatory drugs (intravenous immunoglobulins, methotrexate or azathioprine) for a median duration of 40.8 months]. At the last assessment, patients who had been treated were more severely affected on disability scales (Walton P = 0.007, Rivermead Mobility Index P = 0.004) and on the sporadic inclusion body myositis weakness composite index (P = 0.04). The first stage of disease progression towards handicap for walking was more rapid among patients receiving immunosuppressive treatments (hazard ratio = 2.0, P = 0.002). This study confirms that sporadic inclusion body myositis is slowly progressive but not lethal and that immunosuppressive treatments do not ameliorate its natural course, thus confirming findings from smaller studies. Furthermore, our findings suggest that immunosuppressant drug therapy could have modestly exacerbated progression of disability. The sporadic inclusion body myositis weakness composite index might be a valuable outcome measure for future clinical trials, but requires further assessment and validation.

Mitochondrial dysfunction in anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) immune-mediated necrotising myopathy.

Mitochondrial dysfunction is a plausible cause of muscle fibre damage in a number of myopathies including immune-mediated necrotising myopathy. However, histopathological evidence of mitochondrial dysfunction is not often described in immune-mediated necrotising myopathy and, when present, it is often attributed to patient age. The purpose of this study was to describe features of mitochondrial dysfunction on muscle biopsy in anti-3­hydroxy-3-methylglutaryl-CoA reductase immune-mediated necrotising myopathy and explore whether these features are age-related. In this observational case control study, a statistically significant increase in the number of muscle fibres with increased lipid content (p = 0.004) and cytochrome c oxidase-negative/succinate dehydrogenase-positive fibres (p = 0.037) in anti-3­hydroxy-3-methylglutaryl-coenzyme immune-mediated necrotising myopathy was found compared to age-matched controls. Therefore, histopathological features of mitochondrial dysfunction are more frequent in anti-3­hydroxy-3-methylglutaryl-coenzyme immune-mediated necrotising myopathy than aged-matched controls and therefore, may be contributing to the pathogenesis.

Genetic defects are common in myopathies with tubular aggregates.

OBJECTIVE: A group of genes have been reported to be associated with myopathies with tubular aggregates (TAs). Many cases with TAs still lack of genetic clarification. This study aims to explore the genetic background of cases with TAs in order to improve our knowledge of the pathogenesis of these rare pathological structures. METHODS: Thirty-three patients including two family members with biopsy confirmed TAs were collected. Whole-exome sequencing was performed on 31 unrelated index patients and a candidate gene search strategy was conducted. The identified variants were confirmed by Sanger sequencing. The wild-type and the mutant p.Ala11Thr of ALG14 were transfected into human embryonic kidney 293 cells (HEK293), and western blot analysis was performed to quantify protein expression levels. RESULTS: Eleven index cases (33%) were found to have pathogenic variant or likely pathogenic variants in STIM1, ORAI1, PGAM2, SCN4A, CASQ1 and ALG14. Among them, the c.764A>T (p.Glu255Val) in STIM1 and the c.1333G>C (p.Val445Leu) in SCN4A were novel. Western blot analysis showed that the expression of ALG14 protein was severely reduced in the mutant ALG14 HEK293 cells (p.Ala11Thr) compared with wild type. The ALG14 variants might be associated with TAs in patients with complex multisystem disorders. INTERPRETATION: This study expands the phenotypic and genotypic spectrums of myopathies with TAs. Our findings further confirm previous hypothesis that genes related with calcium signalling pathway and N-linked glycosylation pathway are the main genetic causes of myopathies with TAs.

A rare case of adult onset LPIN1 associated rhabdomyolysis.

Pathogenic variants in LPIN1 are a recognised cause of severe and often fatal rhabdomyolysis in childhood. We present a rare case of adult onset recurrent rhabdomyolysis due to compound heterozygous variants in LPIN1. Despite first presenting with rhabdomyolysis in his twenties and having undergone extensive investigations, the patient did not receive a diagnosis until he was 46 years of age. DNA sequencing revealed a pathogenic deletion involving exon 18 of LPIN1 in conjunction with a c.2410G>A missense variant in exon 19. Whilst LPIN1 variants are a noteworthy cause of severe recurrent rhabdomyolysis in childhood, this is the first detailed description and only the second reported case of adult onset rhabdomyolysis. Variants in LPIN1 should be considered as a cause of recurrent severe rhabdomyolysis in adults when other more common causes have been excluded.

Tubular Aggregates and Cylindrical Spirals Have Distinct Immunohistochemical Signatures.

Tubular aggregates and cylindrical spirals are 2 distinct ultrastructural abnormalities observed in muscle biopsies that have similar histochemical staining characteristics on light microscopy. Both are found in a wide range of disorders. Recently, a number of genetic mutations have been reported in conditions with tubular aggregates in skeletal muscle. It is widely accepted that tubular aggregates arise from the sarcoplasmic reticulum, but the origin of cylindrical spirals has been less clearly defined. We describe the histopathological features of myopathies with tubular aggregates, including a detailed immunohistochemical analysis of congenital myasthenic syndromes with tubular aggregates due to mutations in GFPT1 and DPAGT1, and myopathies with cylindrical spirals. Our findings support the notion that cylindrical spirals, like tubular aggregates, derive primarily from the sarcoplasmic reticulum; however, immunohistochemistry indicates that different molecular components of the sarcoplasmic reticulum may be involved and can be used to distinguish between these different inclusions. The immunohistochemical differences may also help to guide genetic testing.