Nintedanib decreases muscle fibrosis and improves muscle function in a murine model of dystrophinopathy

Duchenne muscle dystrophy (DMD) is a genetic disorder characterized by progressive skeletal muscle weakness. Dystrophin deficiency induces instability of the sarcolemma during muscle contraction that leads to muscle necrosis and replacement of muscle by fibro-adipose tissue. Several therapies have been developed to counteract the fibrotic process. We report the effects of nintedanib, a tyrosine kinase inhibitor, in the mdx murine model of DMD. Nintedanib reduced proliferation and migration of human fibroblasts in vitro and decreased the expression of fibrotic genes such as COL1A1, COL3A1, FN1, TGFB1, and PDGFA. We treated seven mdx mice with 60 mg/kg/day nintedanib for 1 month. Electrophysiological studies showed an increase in the amplitude of the motor action potentials and an improvement of the morphology of motor unit potentials in the animals treated. Histological studies demonstrated a significant reduction of the fibrotic areas present in the skeletal muscles. Analysis of mRNA expression from muscles of treated mice showed a reduction in Col1a1, Col3a1, Tgfb1, and Pdgfa. Western blot showed a reduction in the expression of collagen I in skeletal muscles. In conclusion, nintedanib reduced the fibrotic process in a murine model of dystrophinopathy after 1 month of treatment, suggesting its potential use as a therapeutic drug in DMD patients.España, Ministerio de Economía y Competitividad BFU2016-74975-PEspaña, Instituto Ramón y Cajal PI13/0134

Survey on the management of Pompe disease in routine clinical practice in Spain

Antibodies; Diagnosis; Pompe diseaseAnticuerpos; Diagnóstico; Enfermedad de pompeAnticossos; Diagnòstic; Malaltia de pompeBackground Despite the availability of several clinical guidelines, not all health professionals use their recommendations to manage patients with Pompe disease, a rare genetic disorder involving high-impact therapy. Through several discussion meetings and a survey, the present study aimed to learn about the management of Pompe disease in routine clinical practice in Spain, to improve clinical care in a real-life situation. Results The survey was sent to 42 healthcare professionals who manage patients with Pompe disease in their clinical practice. Although most respondents followed the clinical guidelines, clinical practice differed from the expert recommendations in many cases. Approximately 7% did not request a genetic study to confirm the diagnosis before starting treatment, and 21% considered that only two dried blood spot determinations suffice to establish the diagnosis. About 76% requested anti-GAA antibodies when there is a suspicion of lack of treatment efficacy, though a significant percentage of respondents have never requested such antibodies. According to 31% of the respondents, significant impairment of motor function and/or respiratory insufficiency is a requirement for authorizing medication at their hospital. Up to 26% waited for improvements over the clinical follow-up to maintain treatment and withdrew it in the absence of improvement since they did not consider disease stabilization to be a satisfactory outcome. Conclusions The results highlight the lack of experience and/or knowledge of some professionals caring for patients with Pompe disease. It is necessary to develop and disseminate simple guidelines that help to apply the expert recommendations better or centralize patient follow-up in highly specialized centers.Sanofi has sponsored this project without participating in the article’s design, data analysis, or writing

Comprehensive functional characterization of SGCB coding variants predicts pathogenicity in limb-girdle muscular dystrophy type R4/2E

Genetic testing is essential for patients with a suspected hereditary myopathy. More than 50% of patients clinically diagnosed with a myopathy carry a variant of unknown significance in a myopathy gene, often leaving them without a genetic diagnosis. Limb-girdle muscular dystrophy (LGMD) type R4/2E is caused by mutations in β-sarcoglycan (SGCB). Together, β-, α-, γ-, and δ-sarcoglycan form a 4-protein transmembrane complex (SGC) that localizes to the sarcolemma. Biallelic loss-of-function mutations in any subunit can lead to LGMD. To provide functional evidence for the pathogenicity of missense variants, we performed deep mutational scanning of SGCB and assessed SGC cell surface localization for all 6,340 possible amino acid changes. Variant functional scores were bimodally distributed and perfectly predicted pathogenicity of known variants. Variants with less severe functional scores more often appeared in patients with slower disease progression, implying a relationship between variant function and disease severity. Amino acid positions intolerant to variation mapped to points of predicted SGC interactions, validated in silico structural models, and enabled accurate prediction of pathogenic variants in other SGC genes. These results will be useful for clinical interpretation of SGCB variants and improving diagnosis of LGMD; we hope they enable wider use of potentially life-saving gene therapy

Hypoxia triggers IFN-I production in muscle: Implications in dermatomyositis

Dermatomyositis is an inflammatory myopathy characterized by symmetrical proximal muscle weakness and skin changes. Muscle biopsy hallmarks include perifascicular atrophy, loss of intramuscular capillaries, perivascular and perimysial inflammation and the overexpression of IFN-inducible genes. Among them, the retinoic-acid inducible gene 1 (RIG-I) is specifically overexpressed in perifascicular areas of dermatomyositis muscle. The aim of this work was to study if RIG-I expression may be modulated by hypoxia using an in vitro approach. We identified putative hypoxia response elements (HRE) in RIG-I regulatory regions and luciferase assays confirmed that RIG-I is a new HIF-inducible gene. We observed an increase expression of RIG-I both by Real time PCR and Western blot in hypoxic conditions in human muscle cells. Cell transfection with a constitutive RIG-I expression vector increased levels of phospho-IRF-3, indicating that RIG-I promotes binding of transcription factors to the enhancer sequence of IFN. Moreover, release of IFN-beta was observed in hypoxic conditions. Finally, HIF-1 alpha overexpression was confirmed in the muscle biopsies and in some RIG-I positive perifascicular muscle fibres but not in controls. Our results indicate that hypoxia triggers the production of IFN-I in vitro, and may contribute to the pathogenesis of DM together with other inflammatory factors

Applying the win ratio method in clinical trials of orphan drugs: an analysis of data from the COMET trial of avalglucosidase alfa in patients with late-onset Pompe disease

Background: Clinical trials for rare diseases often include multiple endpoints that capture the effects of treatment on different disease domains. In many rare diseases, the primary endpoint is not standardized across trials. The win ratio approach was designed to analyze multiple endpoints of interest in clinical trials and has mostly been applied in cardiovascular trials. Here, we applied the win ratio approach to data from COMET, a phase 3 trial in late-onset Pompe disease, to illustrate how this approach can be used to analyze multiple endpoints in the orphan drug context. Methods: All possible participant pairings from both arms of COMET were compared sequentially on changes at week 49 in upright forced vital capacity (FVC) % predicted and six-minute walk test (6MWT). Each participant’s response for the two endpoints was first classified as a meaningful improvement, no meaningful change, or a meaningful decline using thresholds based on published minimal clinically important differences (FVC ± 4% predicted, 6MWT ± 39 m). Each comparison assessed whether the outcome with avalglucosidase alfa (AVA) was better than (win), worse than (loss), or equivalent to (tie) the outcome with alglucosidase alfa (ALG). If tied on FVC, 6MWT was compared. In this approach, the treatment effect is the ratio of wins to losses (“win ratio”), with ties excluded. Results: In the 2499 possible pairings (51 receiving AVA × 49 receiving ALG), the win ratio was 2.37 (95% confidence interval [CI], 1.30–4.29, p = 0.005) when FVC was compared before 6MWT. When the order was reversed, the win ratio was 2.02 (95% CI, 1.13–3.62, p = 0.018). Conclusion: The win ratio approach can be used in clinical trials of rare diseases to provide meaningful insight on treatment benefits from multiple endpoints and across disease domains

Different Approaches to Analyze Muscle Fat Replacement With Dixon MRI in Pompe Disease

Altres ajuts: Asociación Española de Enfermos de Glucogenosis (AEEG)Quantitative MRI is an increasingly used method to monitor disease progression in muscular disorders due to its ability to measure changes in muscle fat content (reported as fat fraction) over a short period. Being able to objectively measure such changes is crucial for the development of new treatments in clinical trials. However, the analysis of the images involved continues to be a daunting task because of the time needed. Whether a more specific analysis selecting individual muscles or a global one analyzing the whole thigh or compartments could be a suitable alternative has only been marginally studied. In our study we compare three methods of analysis of 2-point-dixon images in a cohort of 34 patients with late onset Pompe disease followed over a period of one year. We measured fat fraction on MRIs obtained at baseline and at year 1, and we calculated the increment of fat fraction. We correlated the results obtained with the results of muscle function tests to investigate whether the three methods of analysis were equivalent or not. We observed significant differences between the three methods in the estimation of the fat fraction at both baseline and year 1, but no difference was found in the increment in fat fraction between baseline and year 1. When we correlated the fat fraction obtained with each method and the muscle function tests, we found a significant correlation with most tests in all three methods, although in most comparisons the highest correlation coefficient was found with the analysis of individual muscles. We conclude that the fastest strategy of analysis assessing compartments or the whole thigh could be reliable for certain cohorts of patients where the variable to study is the fat increment. In other sorts of studies, an individual muscle approach seems the most reliable technique

Quantitative muscle MRI to follow up late onset Pompe patients : a prospective study

Late onset Pompe disease (LOPD) is a slow, progressive disorder characterized by skeletal and respiratory muscle weakness. Enzyme replacement therapy (ERT) slows down the progression of muscle symptoms. Reliable biomarkers are needed to follow up ERT-treated and asymptomatic LOPD patients in clinical practice. In this study, 32 LOPD patients (22 symptomatic and 10 asymptomatic) underwent muscle MRI using 3-point Dixon and were evaluated at the time of the MRI with several motor function tests and patient-reported outcome measures, and again after one year. Muscle MRI showed a significant increase of 1.7% in the fat content of the thigh muscles in symptomatic LOPD patients. In contrast, there were no noteworthy differences between muscle function tests in the same period of time. We did not observe any significant changes either in muscle MRI or in muscle function tests in asymptomatic patients over the year. We conclude that 3-point Dixon muscle MRI is a useful tool for detecting changes in muscle structure in symptomatic LOPD patients and could become part of the current follow-up protocol in daily clinics

Differences In Adipose Tissue And Lean Mass Distribution In Patients With Collagen Vi Related Myopathies Are Associated With Disease Severity And Physical Ability

Mutations in human collagen VI genes cause a spectrum of musculoskeletal conditions in children and adults collectively termed collagen VI-related myopathies (COL6-RM) characterized by a varying degree of muscle weakness and joint contractures and which include Ullrich Congenital Muscular Dystrophy (UCMD) and Bethlem Myopathy (BM). Given that collagen VI is one of the most abundant extracellular matrix proteins in adipose tissue and its emerging role in energy metabolism we hypothesized that collagen VI deficiency might be associated with alterations in adipose tissue distribution and adipokines serum profile. We analyzed body composition by means of dual-energy X-ray absorptiometry in 30 pediatric and adult COL6-RM myopathy patients representing a range of severities (UCMD, intermediate-COL6-RM, and BM). We found a distinctive pattern of regional adipose tissue accumulation which was more evident in children at the most severe end of the spectrum. In particular, the accumulation of fat in the android region was a distinguishing feature of UCMD patients. In parallel, there was a decrease in lean mass compatible with a state of sarcopenia, particularly in ambulant children with an intermediate phenotype. All children and adult patients that were sarcopenic were also obese. These changes were significantly more pronounced in children with collagen VI deficiency than in children with Duchenne Muscular Dystrophy of the same ambulatory status. High molecular weight adiponectin and leptin were significantly increased in sera from children in the intermediate and BM group. Correlation analysis showed that the parameters of fat mass were negatively associated with motor function according to several validated outcome measures. In contrast, lean mass parameters correlated positively with physical performance and quality of life. Leptin and adiponectin circulating levels correlated positively with fat mass parameters and negatively with lean mass and thus may be relevant to the disease pathogenesis and as circulating markers. Taken together our results indicate that COL6-RM are characterized by specific changes in total fat mass and distribution which associate with disease severity, motor function, and quality of life and which are clinically meaningful and thus should be taken into consideration in the management of these patients

Isolation of human fibroadipogenic progenitors and satellite cells from frozen muscle biopsies

Altres ajuts: Association Française contre les Myopathies (22525)Altres ajuts: Fundación Isabel GemioSkeletal muscle contains multiple cell types that work together to maintain tissue homeostasis. Among these, satellite cells (SC) and fibroadipogenic progenitors cells (FAPs) are the two main stem cell pools. Studies of these cells using animal models have shown the importance of interactions between these cells in repair of healthy muscle, and degeneration of dystrophic muscle. Due to the unavailability of fresh patient muscle biopsies, similar analysis of interactions between human FAPs and SCs is limited especially among the muscular dystrophy patients. To address this issue here we describe a method that allows the use of frozen human skeletal muscle biopsies to simultaneously isolate and grow SCs and FAPs from healthy or dystrophic patients. We show that while the purified SCs differentiate into mature myotubes, purified FAPs can differentiate into adipocytes or fibroblasts demonstrating their multipotency. We find that these FAPs can be immortalized and the immortalized FAPs (iFAPs) retain their multipotency. These approaches open the door for carrying out personalized analysis of patient FAPs and interactions with the SCs that lead to muscle loss