Professional Session Abstracts - 2023 MDF Annual Conference
We are excited to bring together leading scientists, clinicians, and experts in the field to present the latest findings, cutting edge research, and innovative approaches to understanding and treating myotonic dystrophy. This track was designed with expert guidance from members of MDF’s Scientific Advisory Committee. Our DM Professional Topics & Talks serve as a crucial platform for knowledge exchange, collaboration, and collective efforts to advance research efforts, all with the shared goal of accelerating progress towards effective treatments and ultimately, a cure for myotonic dystrophy. Click here to learn more about the 2023 MDF Annual Conference.
Friday, September 8th
9:00 - 10:00 AM | Professional Session #1
Involvement of Aberrant Splicing of the NFIX Transcription Factor in Transcriptome Changes in Muscles of DM Patients
Krzysztof Sobczak, PhD
Instytut Biologii Molekularnej i Biotechnologii, Adam Mickiewicz University, Poznań, Poland
Myotonic dystrophy type 1 (DM1) is an hereditary disease caused by expansion of trinucleotide CTG repeats in the 3’UTR of DMPK gene. Expanded CUG repeats (CUGexp) in mutant mRNA is a major toxic product of mutant gene. It sequester MBNL splicing factors and due to functional insufficiency of these proteins trigger global changes in alternative splicing in skeletal muscles, heart and brain, which result in symptoms characteristic for DM1. One of the MBNL-dependent exons, which inclusion is significantly higher in skeletal muscles of DM, is exon 7 of NFIX, a gene encoding for transcription factor essential for muscle development. The aim of this study was to understand the contribution of two NFIX splicing isoforms on transcriptional activity in a few model systems and in DM pathology. To answer this question two alternative approaches were used. First of them was generation of two cell lines with stable and inducible overexpression of NFIX (NFIX-OE) based on a HEK cells with functional knockout of endogenous NFIX (NFIX-KO). The second approach based on manipulation of exon 7 inclusion of endogenous NFIX mRNA using the antisense oligonucleotide (AON) targeting exon 7/intron 7 boundary. In human skeletal muscle cells, in which NFIX+ex7 isoform predominates, this AON induces efficient exon 7 skipping and production of NFIX-ex7 isoform. The results of RNA-seq revealed hundreds of genes which expression was significantly changed after AON treatment, suggesting that both splicing isoforms, NFIX+ex7 and NFIX-ex7, differ in transcriptional activity. Gene ontology analysis showed significant enrichment of groups of genes that are sensitive to NFIX splicing isoforms and abnormally expressed in DM1 muscles. They include the extracellular matrix structural constituent, and collagen binding. Taken together, these results showed that abnormal distribution of NFIX exon 7 caused by sequestration of MBNL proteins on CUGexp triggers a subset of gene expression changes occurring in DM1 skeletal muscles.
Toxic RNA Selective Screening to Identify New Drugs, Drug Targets and Genetic Modifiers for Myotonic Dystrophy
Kaalak Reddy, PhD
State University of New York at Albany’s RNA Institute, New York, United States
Myotonic dystrophy (DM) is caused by DNA repeat expansions that produce toxic repeat expansion RNA. These toxic RNA molecules gain a number of aberrant functions in the cell leading to disease. One major consequence is the sequestration of the MBNL family of RNA processing proteins. By acting as a molecular sponge to the MBNL proteins, toxic RNA production causes widespread RNA processing defects as a result. Thus, when considering development of therapeutic approaches, a cellular system where the toxic RNA abundance can be sensitively and rapidly measured on a large scale can permit screening of large numbers of chemical and biological agents with therapeutic potential. We previously developed a HeLa DM cell line that enables us to simultaneously measure the levels of a toxic RNA and a non-toxic control RNA to rapidly identify treatments that selectively affect the toxic RNA. We previously used this cellular screening system to identify new candidate small molecule therapeutics for DM. We are now expanding our approach to screen the human genome for genetic modifiers of toxic RNA levels in DM. Through genetic screening, we identified new splicing factors that modulate toxic RNA effects in DM patient cells. Identifying genetic modifiers of toxic RNA in DM provides new biological and therapeutic insights and may shed light on new disease modifiers for DM that can help to explain the considerable disease heterogeneity in patients.
10:00 - 11:00 AM | Professional Session #2
Use of Human Pluripotent Stem Cells for Deciphering Myotonic Dystrophy Type 1
Cécile Martinat, PhD
I-Stem - Institut des cellules Souches pour le Traitement et l'Etude des maladies Monogéniques, Corbeil-Essonnes, France
Authors: Sandrine Baghdoyan, Azania Abatan, Noémie Berenger-Currias, Morgan Gazzola, Cécile Martinat
Understanding the mechanisms by which a genetic variation contributes to diseases is a central aim of human genetics and should greatly facilitate the development of preventive strategies and treatments. Implementing this approach to understand the cellular and molecular basis of neuromuscular disorders (NMDs) is particularly challenging due to the inherent inaccessibility of the affected cell types from patients. Despite the wealth of existing cellular and animal models, progresses towards identification of new treatments have been hampered by the incomplete understanding of the pathogenic mechanisms involved in these diseases as well as the availability of relevant screening tools. The development of convenient Human models that even more closely replicate the disease will undoubtedly improve pathological modeling of neuromuscular disorders as well as more adapted therapeutics. In this context, my group is interested in developing an in vitro human “toolbox” to establish pathological models of representative NMDs based on the use of human pluripotent stem cells. Validating this concept, we demonstrated that human pluripotent stem cells, expressing the causal mutation implicated in Myotonic Dystrophy type 1 (DM1), offer pertinent disease-cell models, applicable for a wide systemic analysis ranging from mechanistic studies to therapeutic screening. Thus, we identified developmental molecular defects involved in myogenesis as well as in neurite formation and establishment of neuromuscular connections. In parallel to these mechanistic studies, we are also interested in using these new disease-specific cell models to identify new therapeutic strategies.
Timing is Everything: Understanding Sleep Dysregulation in Myotonic Dystrophy
Belinda Pinto, PhD
University of Florida, Gainesville, Florida, United States
Authors: Belinda Pinto, Fangke Xu, Miguel Gutierrez, Andrew Morris, Andrew Liu, Karyn Esser, Ravi Allada, Eric Wang
While a lot of attention has been given to understanding the basis of muscle pathology in Myotonic Dystrophy (DM), very little is known about the basis of the CNS symptoms in this disease, including excessive daytime sleepiness and sleep dysregulation. Sleep timing and structure are regulated by communication between the circadian network and the sleep-arousal pathways. Here, we describe our efforts to examine how circadian disruption contributes to sleep dysregulation in DM through studies in Drosophila and mouse models. Circadian activity analysis of a CTG250 expressing Drosophila model displays lengthening of the circadian period caused by perturbations to core clock proteins. To determine whether expanded CTG repeat expression perturbs circadian rhythms in mammals, we studied the DmpkCTG480 knock-in mouse model. Interestingly, we find that these mice display changes in circadian activity behavior with a shortening of the circadian period to ~23.5 hours. We are investigating the basis for this circadian disruption by transcriptomic analysis of central and peripheral clock tissues and assessment of molecular rhythms of clock proteins via bioluminescence imaging of clock gene-luciferase fusion reporters. We are also extending these studies to examine circadian biomarkers in the DM patient population through urine-based ELISA assays. Taken together, our data show that circadian rhythms are disrupted in DM. Future studies will provide key insights into how circadian disruption contributes to excessive daytime sleepiness in DM.
12:00 - 1:00 PM | Professional Session #3
Age-related Corneal Disease Mediated by Expanded CUG Repeat RNA
Vinod Mootha, MD
University of Texas Southwestern Medical Center, Dallas, Texas, United States
Fuchs endothelial corneal dystrophy (FECD) is an age-related degenerative eye disease that impacts up to 4% of United States population. Seventy percent of cases are caused by an intronic trinucleotide repeat expansion in the TCF4 gene. While corneal transplants are a useful treatment, pharmacological interventions would likely have an important impact on patient care. Successful drugs will require a better understanding of the molecular, cellular, and phenotypic consequences of the disease. Here we examine those consequences in adjacent eye tissues beyond the corneal endothelium: lens epithelium, corneal epithelium, corneal stroma, and trabecular meshwork. Expanded CUG repeat RNA nuclear foci, the hallmark of FECD in corneal endothelium, are rare in corneal epithelium and stroma, and absent in lens epithelium. By contrast, we observe higher numbers of foci in the trabecular meshwork, a key tissue involved in glaucoma. With few exceptions including mis-splicing in trabecular meshwork, differential gene expression and splicing changes associated with the expanded repeat in corneal endothelial cells are not observed in other cell types. Expression of the TCF4 transcripts including full length isoforms containing the repeat sequence at the 5’ end is much higher in corneal endothelium or trabecular meshwork than in corneal stroma or corneal epithelium. Expression of the CUG repeat containingTCF4 transcripts is higher in corneal endothelium, likely contributing to foci formation and the large molecular and pathologic impact on those cells.
Genetic Modifiers of Huntington Disease: Biological Insights and Therapeutic Opportunities
Darren Monckton, PhD
University of Glasgow, Scotland, United Kingdom
Huntington disease (HD) is a typically late onset neurodegenerative condition characterized by loss of motor control, cognitive decline and behavioral and psychiatric changes. HD is caused by the expansion of a polyglutamine encoding CAG repeat in the HTT gene. Inherited repeat length is inversely associated with age at onset. The repeat tract is genetically unstable in the germline and is biased toward expansion, explaining the anticipation observed in affected families. The repeat is also somatically unstable in a process that is expansion-biased, cell type-specific, allele-length and age-dependent. Notably, very large expansions up to 1,000s of repeats are observed in striatal neurons. High-throughput ultra-deep sequencing of the HTT repeat has revealed rare synonymous CAA variants in the polyglutamine encoding CAG/CAA array that are associated with decreased somatic instability and a later age at onset. Bafflingly, rare synonymous CCA/CCG variants in the downstream polyproline encoding region also have dramatic impacts on HD severity. Contrary to expectations, large scale genome-wide associations studies for modifiers of disease severity in HD have revealed multiple hits in DNA repair genes, most of which have established roles in somatic expansion. Similarly, genome-wide association studies of the modifiers of somatic expansion have confirmed a major role for largely overlapping components of the DNA mismatch repair pathway in humans. Multiple lines of evidence have converged on somatic expansion as a key driver of HD pathology and multiple academic and commercial entities are developing strategies to suppress somatic expansion, primarily via targeting of components of the DNA mismatch repair pathway.
5:00 - 6:00 PM | Build Connections for a Cure - Networking & Social Hour
Join the Myotonic Dystrophy Foundation's professional networking session. Connect with researchers, scientists, pharmaceutical representatives, government officials, and funders dedicated to advancing myotonic dystrophy research. Forge partnerships, share insights, and accelerate progress towards treatments and a cure. Don't miss this unique opportunity to collaborate and make a difference.
Saturday, September 9th
9:00- 9:30 AM | Professional Session #4
Brain Disease Mechanisms in Myotonic Dystrophy and Why Neurons Aren't the Whole Story
Mario Gomes-Pereira, PhD
Sorbonne Université, Inserm, Association Institut de Myologie, Paris, France
Brain function relies on the complex interplay between highly specialised and ramified neuronal and glia cells, which together regulate cognition, emotion and sleep/wake cycles. All these are profoundly affected in myotonic dystrophy type 1 (DM1). While it is established that mutant RNA accumulates in the nucleus of DM1 cells and perturbs the activity of key RNA-binding proteins, our understanding of how different cell types in the central nervous system drive brain pathology remains incomplete. To bridge this knowledge gap, we sought to investigate the impact of DM1 RNA toxicity on different brain cells, taking advantage of a unique transgenic mouse model that preserves the spatiotemporal expression of an expanded DMPK transgene. We found a marked deleterious effect of toxic RNA on glial cells, characterized by impaired astrocyte ramification and delayed myelination in vivo, as well as defective morphology, adhesion and migration in primary cultures. Glial phenotypes were associated with pronounced spliceopathy of cytoskeleton-related transcripts in both astrocytes and oligodendrocytes, which recreated a molecular signature of impaired differentiation. We suggest that RNA toxicity in glia cells disrupts the intricate neuron-glia crosstalk and impacts neuronal physiology. By focusing our investigation on glial cells, which are often overshadowed by neurons, our research sheds light on the underlying mechanisms of DM1 in the brain. Importantly, our data strengthen the need to target both neuronal and non-neuronal cells in future therapeutic strategies designed to alleviate the neuropsychological symptoms of the disease.
9:30- 11:00 AM | Professional Session #5 - Accelerating Knowledge
Moderator: Tom Cooper, MD
Baylor College of Medicine, Houston, Texas, United States
Experience rapid-fire talks in our Lightning Round featuring top poster submissions on myotonic dystrophy. Predoctoral, postdoctoral, and early career scholars present their groundbreaking research, showcasing the most promising findings. Witness the future of myotonic dystrophy research condensed into concise, dynamic talks. Engage with rising stars in the field and gain valuable insights.
Lightning Round Speakers:
REACH DM1- Remote assessments and genetic analysis in patients with Myotonic Dystrophy Type 1 (Poster ID # 45)
Johanna Hamel, MD
University of Rochester Medical Center, Department of Neurology, Rochester, New York, United States
The role of MBNL in smooth muscle function and DM1 gastrointestinal pathologies (Poster ID # 13)
Department of Pathology & Immunology, Baylor College of Medicine, Houston, Texas, United States
Individual transcriptomic response to strength training for patients with myotonic dystrophy type 1 (Poster ID # 29)
The RNA Institute, College of Arts and Sciences, State University of New York at Albany, New York, United States
Supervised strength training in women with myotonic dystrophy type 1 (Poster ID # 40)
Université du Québec à Chicoutimi (UQAC), Saguenay, Québec, Canada, Interdisciplinary Research Group on Neuromuscular Disorders, Centre intégré universitaire de santé et de services sociaux du Saguenay-Lac-St-Jean, Jonquière, Canada
Extracellular RNA splice events in cerebrospinal fluid as candidate biomarkers of myotonic dystrophy type 1 (Poster ID # 39)
Preeti Kumari, PhD
Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States
Investigating global and cell type-specific transcriptomic dysregulation in the DM1 brain (Poster ID # 43)
Department of Molecular Genetics & Microbiology, Center for NeuroGenetics, Genetics Institute, University of Florida, Gainesville, Florida, United States
Choroid plexus mis-splicing and altered cerebrospinal fluid composition in myotonic dystrophy type 1 (Poster ID # 23)
Curtis Nutter, PhD
Department of Molecular Genetics and Microbiology, Center for NeuroGenetics and the Genetics Institute, University of Florida, Gainesville, Florida, United States
Quercetin selectively reduces expanded repeat RNA levels in models of myotonic dystrophy (Poster ID # 34)
Subodh Mishra, PhD
The RNA Institute, State University of New York at Albany, New York, United States
Benefit of amlodipine or ranolazine on myotonic dystrophy bi-channelopathy (Poster ID # 21)
University of Rochester, New York, United States
11:00 AM - 12:10 PM | Professional Session #6
Disease Severity and Progression in Myotonic Dystrophy Type 2
Johanna Hamel, MD
University of Rochester Medical Center, Rochester, New York, United States
Authors: Johanna Hamel, Katy Eichinger, Jeanne Dekdebrun, James Hilbert, Chad Heatwole, Richard Moxley, Michael McDermott, Charles Thornton
Myotonic dystrophy type 2 (DM2) causes progressive muscle weakness, myotonia, variable muscle pain, cardiac conduction block, cataracts, and GI dysmotility. Here we utilize the National Registry and a longitudinal prospective study to characterize disease burden and progression in DM2. The National Registry provides up to 20 years of patient-reported follow-up on important disease milestones, such as the use of assistive devices, non-invasive ventilation, or implantable cardiac devices. The natural history study spans 3 years and provides comprehensive and quantified information on strength and function in people with DM2. To date, 39 participants with DM2 enrolled. Preliminary data on strength, function, and effects on the transcriptome will be presented.
Insights Into Muscle Pathology: Imaging Analysis and Clinical Endpoints in Myotonic Dystrophy Type 2
Araya Puwanant, MD, MS
Wake Forest University School of Medicine, Winston Salem, North Carolina, United States
Myotonic dystrophy type 2 (DM2), an autosomal dominant muscular dystrophy, is characterized by late-onset progressive proximal muscle weakness, myotonia, and multisystem features. DM2 results from a CCTG repeat expansion in the cellular nucleic acid binding protein (CNBP) gene, where the RNA gain-of-function is considered the primary mechanism that leads to myopathy. Although muscle structure measures from magnetic resonance imaging (MRI) have been used to assess disease severity in other muscular dystrophies, relatively little is known about how these measures are affected in DM2. While major progress has been made in drug development in myotonic dystrophy type 1 (DM1), identifying sensitive biomarkers of disease severity is essential to inform future clinical trial design in DM2. This presentation will review various imaging modalities employed in studies of DM2, from muscle ultrasound and DXA regional body composition to advanced MRI. We will discuss the strengths and limitations of each imaging technique in capturing abnormalities of muscle structure and function. The talk will focus on the latest research findings on muscle MRI in patients with DM2 compared to the control and DM1 groups, how these findings correlate with clinical endpoints, and whether MRI measures could serve as sensitive biomarkers of disease progression in DM2. Finally, we will discuss our pilot data from the DM2 brain study and the role of white matter abnormalities facilitating motor dysfunction, which is already compromised by dystrophic muscle pathology in DM2.
Generation and Characterization of a DM2 Mouse Model
Center for NeuroGenetics, Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, United States
Authors: Avery Engelbrecht, Tala Ortiz, Lisa E.L. Romano, Kiruphagaran Thangaraju, S. Elaine Ames, John Cleary, Tao Zu, Laura P.W. Ranum
Objective: Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are multisystemic diseases caused by CTG or CCTG repeat expansions located in the DMPK or CNBP genes, respectively. RNA gain of function effects, bidirectional transcription and repeat associated non-ATG (RAN) translation are found in DM1 and DM2. In DM2, RAN translation of sense (CCUG) and antisense (CAGG) expansion transcripts produce (LPAC) and (QAGR) RAN proteins. LPAC and QAGR proteins are toxic to cells and found in brain regions with neuropathological changes and white matter loss. Understanding the role of RAN proteins in DM2 and developing therapeutic approaches requires animal models that mirror DM2 patient disease features. Methodology: Using a bacterial artificial chromosome (BAC) approach we generated two independent lines of DM2 BAC transgenic mice. We are characterizing these mice for RNA foci using HCR FISH, repeat instability by long-range PCR, histopathological and behavioral features. Results: Both DM2 mouse lines contain the entire CNBP gene with substantial flanking sequence and unstable expanded repeats ranging from ~470 to over 2000 CCTGs. HCR-FISH detects signal in transgenic mice for CCUG repeats in skeletal muscle and brain. Both sense LPAC and antisense QAGR RAN proteins accumulate in brain tissue. These mice demonstrate substantial repeat instability in both germline and somatic tissues. Conclusions: We have generated a novel DM2 BAC transgenic mouse model that shows a number of disease relevant molecular phenotypes. We are continuing to characterize these mice and hope that this model will provide a useful tool for better understanding the molecular mechanisms of DM2 and therapy development.
12:30-1:30 PM | Discover Breakthroughs in Myotonic Dystrophy Research: Poster Showcase
Engage with cutting-edge research at our Poster Showcase. Pre-doctoral, post-doctoral, and early career scholars, alongside industry experts, present their innovative findings on myotonic dystrophy. Explore the latest advancements, network with leading researchers, and witness the potential for new treatments. Don't miss this vibrant display of scientific excellence.
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