Neuromuscular Disease*

Date/Time: Tuesday, September 12, 2023 - 11:00 AM – 12:30 PM
Track: Special Interest Group (SIG) Session
Room: Franklin Hall 13 (4th Floor)
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Chair: Teerin Liewluck, MD

Co-Chair: Hani Kushlaf, MD, FAAN, FANA, FAANEM

There are 3 speakers for this session cover the following areas: 1) immune-mediated rippling muscle disease (iRMD), Charcot-Marie-Tooth type (CMT) 1A and myasthenia gravis (MG).iRMD is an emerging group of immune mediated myopathies that anti-cavin 4 antibody was recently discovered in most iRMD patients. CMT1A is the most common hereditary motor and sensory neuropathy that affect patients of all ages and there are recent developments regarding natural history study, outcome measures and gene therapy of this condition. As for MG, there are new therapeutic agents targeting complements and neonatal Fc receptors that have been approved by FDA in the recent years.

Learning Objectives:

  • Diagnose and manage patients with iRMD.
  • Diagnose and provide CMT1A patients with the current knowledge regarding the recent development of CMT1A therapies.
  • Select the appropriate therapeutic agents for individuals with myasthenia gravis.

CMT1A: Natural History, Outcome Measures and Therapeutic Development

Speaker: Michael E. Shy, MD, FANA

Charcot Marie Tooth Disease 1A (CMT1A), the most common inherited neuropathy, is a disease that slowly progresses over a person's lifetime. Because it is caused by a gene duplication of PMP22 it is susceptible to therapeutic approaches such as antisense oligonucleotides or RNAinterference designed to reduce transcription of PMP22. Clinical Outcome Measures (COA) and biomarkers have been developed to measure disease progression within a time frame suitable for clinical trials which are now being designed.

Novel therapies for myasthenia gravis

Speaker: Vera Bril, MD, FRCPC

The presentation will focus on newer therapies for MG including complement inhibitors and FcR inhibitors with the rationale for use and clinical trial evidence of efficacy.

Immune-mediated rippling muscle disease: from bench to bedside.

Speaker: Margherita Milone, MD, PhD, FANA

Rippling muscle disease (RMD) is a rare myopathy characterized by wave-like muscle contractions (rippling) and percussion/ stretch-induced muscle mounding. It can be inherited (hRMD) or immune-mediated (iRMD). iRMD responds to immunotherapy. We characterized clinical, electrophysiological, serological, and morphological features of a cohort of patients with iRMD and identified a novel disease biomarker. Rippling manifested in the lower limb or all limb muscles. Proximal muscle weakness occurred in approximately 50% of patients. Fatigue was common. All but one patient had elevated creatine kinase levels. Half of the tested patients had acetylcholine receptor binding antibodies with electrophysiological evidence of defect of neuromuscular transmission in some of them. One patient developed cancer 6 months after development of iRMD. Muscle biopsy showed a mosaic pattern of sarcolemmal caveolin-3 and cavin-4 immunoreactivity, in contrast to the uniform sarcolemmal dystrophin immunoreactivity. Sequential sections revealed that fibers with absent or reduced caveolin-3 immunoreactivity matched those with attenuated cavin-4 immunoreactivity. Contrary to what observed in iRMD, hRMD muscle biopsies showed severely reduced or absent caveolin-3 but normal cavin-4 immunoreactivity. Upregulation of MHC-I and C5b9 deposition on the sarcolemma of non-necrotic muscle fibers were observed in iRMD muscle biopsies but not in the hRMD biopsies. Anti-cavin-4 antibodies were identified in most patients with iRMD. These antibodies represent the first specific serological biomarker of iRMD. The combination of anti-cavin-4 antibodies and patchy loss of cavin-4 in muscle suggest a potential role of cavin-4 in disease mechanism. As cavin-4 plays a crucial role in the T-tubules maturation, the anti-cavin-4 antibodies could impair the T-tubule membrane stability and excitation-contraction coupling system. Transcriptomics profiling in iRMD muscle biopsies is currently ongoing to assess alterations in gene expression.

Early B Cell Tolerance Defects in Anti-Neurofascin-155-Mediated Autoimmune Nodopathy

Oral Abstract Presenter: Bhaskar Roy, MBBS, MHS

Background: In the majority of patients with chronic inflammatory demyelinating polyneuropathy (CIDP), no identifiable autoantibodies are present. However, autoantibodies against paranodal protein neurofascin 155 (NF-155) were recently identified in approximately 5% of patients fulfilling CIDP diagnostic criteria. Patients with anti-NF-155-mediated autoimmune nodopathy (AiN) present with a distinct clinical phenotype with predominantly distal involvement of weakness, sensory ataxia, and tremor, and they tend to respond better to B cell depletion therapy. NF-155 autoantibodies are predominantly of immunoglobulin G subclass 4 (IgG4). Our previous work in myasthenia gravis (MG) patients with muscle-specific tyrosine kinase (MuSK) autoantibody, another IgG4-mediated disease, showed that monoclonal antibodies (mAbs) bound to MuSK even when they were reverted to their unmutated common ancestors, suggesting a breach in early B cell tolerance and self-reactive autoantibody production. Impaired fidelity of B cell tolerance checkpoints has been implicated in the pathogenesis of other autoimmune neurological diseases but remains unexplored in anti-NF-155-mediated AiN. Methods: Recombinant MAbs from single B cells from the new emigrant (NE) and mature naïve (MN) compartments from patients with NF-155-mediated AiN were generated using a well-established protocol. The frequency of polyreactive clones in the NE compartment was measured by ELISA against dsDNA, insulin, and lipopolysaccharide, to assess the fidelity of the central B cell tolerance checkpoint. Furthermore, we performed gene expression profiling paired with full-length B cell receptor repertoire analysis at the single cell level from these early B cell populations between patients with NF-155-mediated AiN and healthy control. Results: 3 patients with NF-155-mediated AiN (age range 43-61 year) and one healthy young control were included in this study. A total of 60 and 58 unique clones of monoclonal antibodies were generated from the NE and MN B cells, respectively. Thirty-seven percent (± 11.5%) of clones from the NE B cells and 31.5% (± 2.5%) of clones from the MN B cells were polyreactive, compared to expected 5-11% polyreactivity from healthy controls (p-value 0.02 and 0.025 respectively). Phenotyping of the NE and MN B cells at the single-cell level by simultaneously profiling gene expression and full-length paired B-cell receptors from one healthy control and one patient with NF-155-mediated AiN patient showed aberrant expression of inflammatory markers including PI3K/Akt pathway, TXNDC5, MZB1. Conclusion: Our findings suggest that NF-155-mediated AiN is associated with early checkpoint tolerance defects. Further studies are warranted to understand the underlying pathomechanism driving such dysfunction.

Using Unsupervised Machine Learning to Identify Phenotypic Clusters of Small Fiber Neuropathy

Oral Abstract Presenter: Peyton Murin, MD

Introduction: Small fiber neuropathy (SFN) is a debilitating, neurodegenerative disease characterized by loss of peripheral small myelinated and unmyelinated axons. Patients with SFN suffer from various combinations of positive and negative symptoms, including burning pain, paresthesias, numbness, muscle cramps, autonomic dysfunction and fatigue. Although strength remains preserved throughout the course of the disease, the pain, paresthesias, and fatigue are frequently profoundly disabling. Frustratingly, no treatments are available to stop or reverse progression of SFN and while the few neuropathic pain medications may be helpful for some patients, they lack efficacy in others. A major impediment for developing effective SFN therapies is the significant between-patient heterogeneity of the disease. Here, we used unsupervised machine learning partitioning on a large cohort of well-characterized SFN patients to identify SFN subgroups. Methods: Demographic and detailed clinical information was collected from 94 patients with symptoms of SFN who had received skin biopsies at the ankle and thigh. Using pandas, data consisting of eight SFN symptoms graded by the patients from 0 (absent) to 10 (highest intensity) were imported and framed in the Anaconda platform. Using scikitlearn, matlab, and scikitlearn.cluster.extra, the silhouette method was used to determine the ideal number of clusters. Subsequently, K Mediods analysis was run, and an ANOVA performed to compare variance. A heat map was constructed based on cluster membership and symptom severity. Results: Three clusters were identified that were statistically significant different in all eight symptoms. Patients in cluster 1 (n=24) had many symptoms of high intensity (“severe”) whereas patients in cluster 2 (n=21) exhibited few symptoms, which were of low intensity (“mild”). Cluster 3 (n=49) was characterized by one or two intense symptoms plus a few symptoms at lower intensity (“intermediate”). Analyzing cluster 3 further revealed patients with severe muscle symptoms and numbness (“muscle group”), patients with moderate symptoms and severe pain and fatigue (“pain and fatigue”) and patients who had several symptoms at a moderate level of intensity. The age distribution of patients in the clusters was similar, but size and pattern of intra-epidermal nerve fiber loss and co-morbidities differed between the clusters. Conclusion: Using unsupervised machine learning, we identified three distinct clusters of SFN patients based on SFN symptom co-occurrence and intensity. The identification of SFN subtypes may lead to a deeper understanding of pathobiological mechanisms, improve clinical trial design, and advance the development of targeted, personalized treatments.

Alternative Polyadenylation in the Pathogenesis of Amyotrophic Lateral Sclerosis

Oral Abstract Presenter: Sebastian Michels, MD

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder characterized by progressive loss of upper and lower motor neurons. Dysregulation of RNA metabolism has emerged as a key driver of the underlying pathology of ALS. Indeed, almost all (>97%) ALS patients display proteinaceous cytoplasmic TDP-43 inclusions, which leads to widespread changes in post-transcriptional processing of various RNA species. Identifying key transcripts altered due to loss of nuclear TDP-43 could yield broadly applicable diagnostic and therapeutic targets for ALS. Recently, characterization of alternative splicing upon TDP-43 loss-of-function led to the development of a promising therapy approach by modulating the neuronal growth factor stathmin-2. However, other aspects of RNA metabolism regulated by TDP-43 remain unexplored. Thus, here we investigate changes in alternative polyadenylation (APA) upon TDP-43 mutation or depletion. APA is an RNA-processing mechanism that generates distinct 3’termini on mRNA transcripts. TDP-43 is known to regulate APA by binding target pre-mRNAs near a polyadenylation signal (PAS), thus creating mRNA isoforms with longer or shorter 3’ untranslated regions (3’UTR) harboring regulatory elements, such as binding sites for microRNAs and RNA-binding proteins. This can affect RNA stability, protein translation, and subcellular localization of a given transcript without altering steady-state transcript levels. APA events occurring in TDP-43 proteinopathy remain understudied, since conventional RNA-sequencing analysis do not fully capture APA changes. To address this gap, we recently applied the dynamic analysis of APA from RNA-seq (DaPars) tool to published RNA-seq datasets, finding previously unknown APA events that function in pathways implicated in ALS pathogenesis, (i.e. nucleocytoplasmic transport, oxidative stress response, and chromatin accessibility). Notably, in neuronal nuclei from the neocortex of postmortem ALS/FTD patients depleted of nuclear TDP-43, the most significant APA event occurred in MARK3 resulting in a longer 3’UTR upon depletion of TDP-43. We further confirmed that TDP-43 directly binds to the MARK3 transcript in the 3’UTR using eCLIP data generated by the ENCODE project. MARK3 is a tau kinase implicated in Alzheimer’s disease, reflecting a potentially novel mechanistic link between TDP-43 and tau pathology. Interestingly, shRNA-mediated knockdown of MARK3 in HEK293T cells leads to a substantial increase in the accumulation of the TDP-43 C-terminal fragment, while overexpression of MARK3 markedly reduced TDP-43 burden. Importantly, APA can be directly modulated by antisense oligonucleotides (ASOs); thus, newly identified APA genes may be candidates for rapid therapy development in ALS.