Emery-Dreifuss Muscular Dystrophy Research Articles

Generation of two iPSC lines (FAMRCi007-A and FAMRCi007-B) from patient with Emery-Dreifuss muscular dystrophy and heart rhythm abnormalities carrying genetic variant LMNA p.Arg249Gln.

Human iPSC lines were generated from peripheral blood mononuclear cells of patient carrying LMNA mutation associated with Emery-Dreifuss muscular dystrophy accompanied by atrioventricular block and paroxysmal atrial fibrillation. Reprogramming factors OCT4, KLF4, SOX2, CMYC were delivered using Sendai virus transduction. iPSCs were characterized in order to prove the pluripotency markers expression, normal karyotype, ability to differentiate into three embryonic germ layers. Generated iPSC lines would be useful model to investigate disease development associated with genetic variants in LMNA gene.

EDMD-Causing Emerin Mutant Myogenic Progenitors Exhibit Impaired Differentiation Using Similar Mechanisms.

Mutations in the gene encoding emerin (EMD) cause Emery–Dreifuss muscular dystrophy (EDMD1), an inherited disorder characterized by progressive skeletal muscle wasting, irregular heart rhythms and contractures of major tendons. The skeletal muscle defects seen in EDMD are caused by failure of muscle stem cells to differentiate and regenerate the damaged muscle. However, the underlying mechanisms remain poorly understood. Most EDMD1 patients harbor nonsense mutations and have no detectable emerin protein. There are three EDMD-causing emerin mutants (S54F, Q133H, and Δ95–99) that localize correctly to the nuclear envelope and are expressed at wildtype levels. We hypothesized these emerin mutants would share in the disruption of key molecular pathways involved in myogenic differentiation. We generated myogenic progenitors expressing wildtype emerin and each EDMD1-causing emerin mutation (S54F, Q133H, Δ95–99) in an emerin-null (EMD−/y) background. S54F, Q133H, and Δ95–99 failed to rescue EMD−/y myogenic differentiation, while wildtype emerin efficiently rescued differentiation. RNA sequencing was done to identify pathways and networks important for emerin regulation of myogenic differentiation. This analysis significantly reduced the number of pathways implicated in EDMD1 muscle pathogenesis.

An Omics View of Emery-Dreifuss Muscular Dystrophy.

Recent progress in Omics technologies has started to empower personalized healthcare development at a thorough biomolecular level. Omics have subsidized medical breakthroughs that have started to enter clinical proceedings. The use of this scientific know-how has surfaced as a way to provide a more far-reaching view of the biological mechanisms behind diseases. This review will focus on the discoveries made using Omics and the utility of these approaches for Emery–Dreifuss muscular dystrophy.

Emerin Phosphorylation during the Early Phase of the Oxidative Stress Response Influences Emerin-BAF Interaction and BAF Nuclear Localization.

Reactive Oxygen Species (ROS) are reactive molecules required for the maintenance of physiological functions. Oxidative stress arises when ROS production exceeds the cellular ability to eliminate such molecules. In this study, we showed that oxidative stress induces post-translational modification of the inner nuclear membrane protein emerin. In particular, emerin is phosphorylated at the early stages of the oxidative stress response, while protein phosphorylation is abolished upon recovery from stress. A finely tuned balance between emerin phosphorylation and O-GlcNAcylation seems to govern this dynamic and modulates emerin–BAF interaction and BAF nucleoplasmic localization during the oxidative stress response. Interestingly, emerin post-translational modifications, similar to those observed during the stress response, are detected in cells bearing LMNA gene mutations and are characterized by a free radical generating environment. On the other hand, under oxidative stress conditions, a delay in DNA damage repair and cell cycle progression is found in cells from Emery–Dreifuss Muscular Dystrophy type 1, which do not express emerin. These results suggest a role of the emerin–BAF protein platform in the DNA damage response aimed at counteracting the detrimental effects of elevated levels of ROS.

Histone acetyltransferase inhibition rescues differentiation of emerin-deficient myogenic progenitors.

Emery-Dreifuss muscular dystrophy (EDMD) is a disease characterized by skeletal muscle wasting, major tendon contractures, and cardiac conduction defects. Mutations in the gene encoding emerin cause EDMD1. Our previous studies suggested that emerin activation of histone deacetylase 3 (HDAC3) to reduce histone 4-lysine 5 (H4K5) acetylation (ac) is important for myogenic differentiation. Pharmacological inhibitors (Nu9056, L002) of histone acetyltransferases targeting acetylated H4K5 were used to test whether increased acetylated H4K5 was responsible for the impaired differentiation seen in emerin-deficient myogenic progenitors. Nu9056 and L002 rescued impaired differentiation in emerin deficiency. SRT1720, which inhibits the nicotinamide adenine dinucleotide (NAD)+ -dependent deacetylase sirtuin 1 (SIRT1), failed to rescue myotube formation. We conclude that emerin regulation of HDAC3 activity to affect H4K5 acetylation dynamics is important for myogenic differentiation. Targeting H4K5ac dynamics represents a potential new strategy for ameliorating the skeletal muscle wasting seen in EDMD1.

Dysfunctional polycomb transcriptional repression contributes to lamin A/C-dependent muscular dystrophy.

Lamin A is a component of the inner nuclear membrane that, together with epigenetic factors, organizes the genome in higher order structures required for transcriptional control. Mutations in the lamin A/C gene cause several diseases belonging to the class of laminopathies, including muscular dystrophies. Nevertheless, molecular mechanisms involved in the pathogenesis of lamin A-dependent dystrophies are still largely unknown. The polycomb group (PcG) of proteins are epigenetic repressors and lamin A interactors, primarily involved in the maintenance of cell identity. Using a murine model of Emery-Dreifuss muscular dystrophy (EDMD), we show here that lamin A loss deregulated PcG positioning in muscle satellite stem cells, leading to derepression of non-muscle-specific genes and p16INK4a, a senescence driver encoded in the Cdkn2a locus. This aberrant transcriptional program caused impairment in self-renewal, loss of cell identity, and premature exhaustion of the quiescent satellite cell pool. Genetic ablation of the Cdkn2a locus restored muscle stem cell properties in lamin A/C-null dystrophic mice. Our findings establish a direct link between lamin A and PcG epigenetic silencing and indicate that lamin A-dependent muscular dystrophy can be ascribed to intrinsic epigenetic dysfunctions of muscle stem cells.

Lamin A/C Assembly Defects in LMNA-Congenital Muscular Dystrophy Is Responsible for the Increased Severity of the Disease Compared with Emery–Dreifuss Muscular Dystrophy

LMNA encodes for Lamin A/C, type V intermediate filaments that polymerize under the inner nuclear membrane to form the nuclear lamina. A small fraction of Lamin A/C, less polymerized, is also found in the nucleoplasm. Lamin A/C functions include roles in nuclear resistance to mechanical stress and gene regulation. LMNA mutations are responsible for a wide variety of pathologies, including Emery–Dreifuss (EDMD) and LMNA-related congenital muscular dystrophies (L-CMD) without clear genotype–phenotype correlations. Both diseases presented with striated muscle disorders although L-CMD symptoms appear much earlier and are more severe. Seeking for pathomechanical differences to explain the severity of L-CMD mutations, we performed an in silico analysis of the UMD-LMNA database and found that L-CMD mutations mainly affect residues involved in Lamin dimer and tetramer stability. In line with this, we found increased nucleoplasmic Lamin A/C in L-CMD patient fibroblasts and mouse myoblasts compared to the control and EDMD. L-CMD myoblasts show differentiation defects linked to their inability to upregulate muscle specific nuclear envelope (NE) proteins expression. NE proteins were mislocalized, leading to misshapen nuclei. We conclude that these defects are due to both the absence of Lamin A/C from the nuclear lamina and its maintenance in the nucleoplasm of myotubes.

Heart Transplantation Mortality in Recipients with Muscular Dystrophy

We want to compare mortality in heart transplant (HTx) recipients with and without Muscular Dystrophy. Using the United Network of Organ Sharing (UNOS) Organ Registry we compared mortality rates for HTx recipients with a diagnosis of Muscular Dystrophy (MD), versus HTx recipients without a diagnosis of MD with a multivariate Cox proportional hazard regression model adjusting for age, sex, race, diabetes diagnosis, ischemic time, need for dialysis/life support, recipient wait-time, and HLA mismatch. MD was defined as Becker's Muscular Dystrophy, Emery-Dreifuss Muscular Dystrophy, Duchenne's Muscular Dystrophy, and Other Muscular Dystrophy. We excluded patients who were age <10, lost-to-follow-up, multi-organ transplant and redo transplant recipients. We censored data at 10 years. There were a total of 63,051 of non-MD HTx recipients versus 91 MD HTx patients. Non-MD HTx recipients were significantly older versus MD HTx recipients (46.7±19.2 years vs 27.5±14.3 years respectively, p<0.001), had a lower prevalence of diabetes (79.4% vs 97.8% respectively, p<0.001), were less likely to have had prior cardiac surgery (81.7% vs 94.5% respectively, p=0.002), had longer waitlist time (83.0 vs 61.0 days respectively, p =0.013), and were more likely to be a smoker (33.1% vs 11%, p,0.001). The mortality at 10 years was significantly higher for non-MD HTx recipients versus MD HTx recipients (45.9% vs 23.7% respectively, p = 0.0374). MD HTx patients have higher survival rates compared to NON-MD HTx patients, despite having a higher prevalence of diabetes and prior cardiac surgery.

Loss of an H3K9me anchor rescues laminopathy-linked changes in nuclear organization and muscle function in an Emery-Dreifuss muscular dystrophy model.

Mutations in the nuclear structural protein lamin A produce rare, tissue-specific diseases called laminopathies. The introduction of a human Emery-Dreifuss muscular dystrophy (EDMD)-inducing mutation into the C. elegans lamin (LMN-Y59C), recapitulates many muscular dystrophy phenotypes, and correlates with hyper-sequestration of a heterochromatic array at the nuclear periphery in muscle cells. Using muscle-specific emerin Dam-ID in worms, we monitored the effects of the mutation on endogenous chromatin. An increased contact with the nuclear periphery along chromosome arms, and an enhanced release of chromosomal centers, coincided with the disease phenotypes of reduced locomotion and compromised sarcomere integrity. The coupling of the LMN-Y59C mutation with the ablation of CEC-4, a chromodomain protein that anchors H3K9-methylated chromatin at the nuclear envelope (NE), suppressed the muscle-associated disease phenotypes. Deletion of cec-4 also rescued LMN-Y59C-linked alterations in chromatin organization and some changes in transcription. Sequences that changed position in the LMN-Y59C mutant, are enriched for E2F (EFL-2)-binding sites, consistent with previous studies suggesting that altered Rb-E2F interaction with lamin A may contribute to muscle dysfunction. In summary, we were able to counteract the dominant muscle-specific defects provoked by LMNA mutation by the ablation of a lamin-associated H3K9me anchor, suggesting a novel therapeutic pathway for EDMD.

Mapping the Emerin Interactome by Apex Proximity Labeling

Emerin is a highly conserved LEM-domain protein expressed mostly at the inner nuclear envelope of mammalian cells. Mutations in the EMD gene encoding for emerin cause X-linked Emery-Dreifuss muscular dystrophy (EDMD). Through its interaction with a variety of nuclear binding partners, emerin has a number of biological functions that include regulation of gene expression, nucleo-cytoskeleton mechanotransduction and maintenance of nuclear shape. The molecular pathogenesis of EDMD remains unclear and mapping the protein interaction network of emerin can potentially help elucidate the molecular mechanisms behind the disease. Here we used a proximity-dependent biotinylation method based on an engineered ascorbate peroxidase (APEX2) to obtain proteomic profiles of emerin interactions at the nuclear envelope and the endoplasmic reticulum in cells. We generated constructs of APEX2 fused to the N- or the C-terminus of emerin to study its interacting partners across the inner nuclear envelope, the outer nuclear envelope and the endoplasmic reticulum membrane. We also optimized proximity biotin labeling in both live and fixed cells, and showed that biotinylation patterns vary significantly between emerin fusions and for cells micropatterned on fibronectin strips of decreasing widths that we use to apply increasing mechanical perturbation to the cell nuclei. These data indicate that emerin explores different microenvironments and interacts with distinct partners as a function of nuclear strain. Using APEX2 quantitative proteomics, we also started to identify key interacting partners of emerin and their subcompartmental localizations for cells subjected to increasing mechanical challenges.

Relationship between infrared skin radiation and muscular strength tests in patients affected by Emery-Dreifuss muscular dystrophy

Considering that infrared thermography is presented as a diagnostic technique for non-invasive, non-ionizing, fast and easy to use imaging and Emery-Dreifuss muscular dystrophy is a clinical condition that seems to be related to changes in the emission of infrared radiation at the skin level due to its neurodegenerative character, we have conducted an investigation by infrared thermography and the use of functional strength tests in the lower limbs in a family of 4 affected members of Emery-Dreifuss muscular dystrophy to try to establish a relationship between the evolution of the disease and the emission of infrared radiation in this pathology at the lower limb level and provide a more general view of this disease for a better evaluation and monitoring of the disease.

Novel candidate alleles associated with gene regulation for Emery-Dreifuss muscular dystrophy.

Novel candidate alleles associated with gene regulation for Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy.

Emery‐Dreifuss muscular dystrophy (EDMD) is a rare muscular dystrophy, but is particularly important to diagnose due to frequent life‐threatening cardiac complications. EDMD classically presents with muscle weakness, early contractures, cardiac conduction abnormalities and cardiomyopathy, although the presence and severity of these manifestations vary by subtype and individual. Associated genes include EMD, LMNA, SYNE1, SYNE2, FHL1, TMEM43, SUN1, SUN2, and TTN, encoding emerin, lamin A/C, nesprin‐1, nesprin‐2, FHL1, LUMA, SUN1, SUN2, and titin, respectively. The Online Mendelian Inheritance in Man database recognizes subtypes 1 through 7, which captures most but not all of the associated genes. Genetic diagnosis is essential whenever available, but traditional diagnostic tools can help steer the evaluation toward EDMD and assist with interpretation of equivocal genetic test results. Management is primarily supportive, but it is important to monitor patients closely, especially for potential cardiac complications. There is a high potential for progress in the treatment of EDMD in the coming years.

A multistage sequencing strategy pinpoints novel candidate alleles for Emery-Dreifuss muscular dystrophy and supports gene misregulation as its pathomechanism.

BackgroundAs genome-wide approaches prove difficult with genetically heterogeneous orphan diseases, we developed a new approach to identify candidate genes. We applied this to Emery-Dreifuss muscular dystrophy (EDMD), characterised by early onset contractures, slowly progressive muscular wasting, and life-threatening heart conduction disturbances with wide intra- and inter-familial clinical variability. Roughly half of EDMD patients are linked to six genes encoding nuclear envelope proteins, but the disease mechanism remains unclear because the affected proteins function in both cell mechanics and genome regulation.MethodsA primer library was generated to test for mutations in 301 genes from four categories: (I) all known EDMD-linked genes; (II) genes mutated in related muscular dystrophies; (III) candidates generated by exome sequencing in five families; (IV) functional candidates — other muscle nuclear envelope proteins functioning in mechanical/genome processes affected in EDMD. This was used to sequence 56 unlinked patients with EDMD-like phenotype.FindingsTwenty-one patients could be clearly assigned: 18 with mutations in genes of similar muscular dystrophies; 3 with previously missed mutations in EDMD-linked genes. The other categories yielded novel candidate genes, most encoding nuclear envelope proteins with functions in gene regulation.InterpretationOur multi-pronged approach identified new disease alleles and many new candidate EDMD genes. Their known functions strongly argue the EDMD pathomechanism is from altered gene regulation and mechanotransduction due to connectivity of candidates from the nuclear envelope to the plasma membrane. This approach highlights the value of testing for related diseases using primer libraries and may be applied for other genetically heterogeneous orphan diseases.FundingThe Wellcome Trust, Muscular Dystrophy UK, Medical Research Council, European Community's Seventh Framework Programme “Integrated European –omics research project for diagnosis and therapy in rare neuromuscular and neurodegenerative diseases (NEUROMICS)”.

Mutant lamins cause nuclear envelope rupture and DNA damage in skeletal muscle cells.

Mutations in the LMNA gene, which encodes the nuclear envelope (NE) proteins lamins A/C, cause Emery-Dreifuss muscular dystrophy, congenital muscular dystrophy, and other diseases collectively known as laminopathies. The mechanisms responsible for these diseases remain incompletely understood. Using three mouse models of muscle laminopathies and muscle biopsies from individuals with LMNA-related muscular dystrophy, we found that Lmna mutations reduced nuclear stability and caused transient rupture of the NE in skeletal muscle cells, resulting in DNA damage, DNA damage response activation, and reduced cell viability. NE and DNA damage resulted from nuclear migration during skeletal muscle maturation and correlated with disease severity in the mouse models. Reducing cytoskeletal forces on the myonuclei prevented NE damage and rescued myofiber function and viability in Lmna mutant myofibers, indicating that myofiber dysfunction is the result of mechanically induced NE damage. Taken together, these findings implicate mechanically induced DNA damage as a pathogenic contributor for LMNA skeletal muscle diseases.

FHL1-related myopathy may not be classified by reducing bodies in muscle biopsy

FHL1-related myopathies, including reducing body myopathy (RBM), X-linked scapulo-axio-peroneal myopathy, rigid spine syndrome, X-linked myopathy with postural muscle atrophy (XMPMA), X-linked Emery–Dreifuss muscular dystrophy and hypertrophic cardiomyopathy, are clinically and pathologically heterogeneous disorders caused by FHL1 gene mutations. According to previous reports, the first three types are myopathies with reducing bodies observed in biopsies, and the last three are myopathies without reducing bodies. We report four FHL1-related myopathy patients, including an XMPMA patient and a RBM family with three patients. Clinical information, muscle biopsies, electromyograms and genetic testing were obtained. Muscle weakness and atrophy, spinal rigidity, and joint contracture were present in the RBM family. The XMPMA patient showed a pseudoathletic appearance with muscle weakness and atrophy, spinal rigidity and deformity. The index patient of the RBM family underwent two muscle biopsies to find reducing bodies. Interestingly, these muscle biopsies revealed reducing bodies and rimmed vacuoles not only in the RBM family but also in the XMPMA patient. Next-generation sequencing identified a reported single missense mutation c.448 C>T (p. C150R) in the RBM family and a novel mutation c.814T>C (p. S272P) in the XMPMA patient. Therefore, FHL1-related myopathies overlap substantially and may not be simply classified into subtypes depending on reducing bodies. Biopsies of additional affected muscles can aid in finding reducing bodies. We report the first XMPMA patient with a novel FHL1 mutation and reducing bodies in a muscle biopsy in China.

A Micropatterning Strategy to Study Nuclear Mechanotransduction in Cells.

Micropatterning techniques have been widely used in biology, particularly in studies involving cell adhesion and proliferation on different substrates. Cell micropatterning approaches are also increasingly employed as in vitro tools to investigate intracellular mechanotransduction processes. In this report, we examined how modulating cellular shapes on two-dimensional rectangular fibronectin micropatterns of different widths influences nuclear mechanotransduction mediated by emerin, a nuclear envelope protein implicated in Emery–Dreifuss muscular dystrophy (EDMD). Fibronectin microcontact printing was tested onto glass coverslips functionalized with three different silane reagents (hexamethyldisilazane (HMDS), (3-Aminopropyl)triethoxysilane (APTES) and (3-Glycidyloxypropyl)trimethoxysilane (GPTMS)) using a vapor-phase deposition method. We observed that HMDS provides the most reliable printing surface for cell micropatterning, notably because it forms a hydrophobic organosilane monolayer that favors the retainment of surface antifouling agents on the coverslips. We showed that, under specific mechanical cues, emerin-null human skin fibroblasts display a significantly more deformed nucleus than skin fibroblasts expressing wild type emerin, indicating that emerin plays a crucial role in nuclear adaptability to mechanical stresses. We further showed that proper nuclear responses to forces involve a significant relocation of emerin from the inner nuclear envelope towards the outer nuclear envelope and the endoplasmic reticulum membrane network. Cell micropatterning by fibronectin microcontact printing directly on HMDS-treated glass represents a simple approach to apply steady-state biophysical cues to cells and study their specific mechanobiology responses in vitro.

ESGCT 27th Annual Congress In collaboration with SETGyc Barcelona, Spain October 22–25, 2019 Abstracts

Xenotransplantation of transgenic porcine pancreatic islets offers a promising alternative source to circumvent current limitations posed by the scarcity of allogeneic donors. We have investigated the feasibility to generate tissue engineered SLA silenced islet cell clusters (ICC) to decrease xenogeneic immune responses Pancreatic islets single cell suspensions were generated by enzymatic digestion of porcine ICCs. Single cells were silenced for SLA expression by lentiviral vectors encoding for Nanoluciferase as reporter gene and for short hairpin RNAs targeting beta2-microglobulin or class II transactivator, respectively. SLA transcripts were evaluated by real-time PCR and protein levels by flow cytometry and fluorescence microscopy analyses. The effect of SLA silencing was evaluated in human T and NK cell cytotoxicity assays. SLA-silenced pancreatic beta-cells were then reassembled into ICCs in stirred bioreactors. SLA class I silencing reached a level of up to 84% and class II by up to 50% on pancreatic islet cell. Silencing SLA expression did not affect cell viability and the insulin-producing beta-cell phenotype. Xenogeneic T-cell immune responses (p < 0.05) as well as antibody-mediated cellular-dependent immune responses (p < 0.01) were significantly decreased. Silencing SLA class I expression did not increase susceptibility to NK-cell cytotoxicity. In stirred bioreactors, tissue engineered islets showing the typical 3D-structure and morphology of ICC were assembled from SLA-silenced pancreatic cell suspensions. These data shows the feasibility to generate low immunogenic porcine ICC from transgenic pigs after single cell engineering and post-transduction islet reassembling that might serve as a robust alternative to allogeneic pancreatic islet cell transplantation.

Drosophila emerins control LINC complex localization and transcription to regulate myonuclear position.

Mispositioned nuclei are a hallmark of skeletal muscle disease. Many of the genes that are linked to Emery-Dreifuss muscular dystrophy (EDMD) encode proteins that are critical for nuclear movement in various cells, suggesting that disruptions in nuclear movement and position may contribute to disease progression. However, how these genes are coordinated to move nuclei is not known. Here, we focussed on two different emerin proteins in Drosophila, Bocksbeutel and Otefin, and their effects on nuclear movement. Although nuclear position was dependent on both, elimination of either Bocksbeutel or Otefin produced distinct phenotypes that were based in differential effects on the KASH-domain protein Klarsicht. Specifically, loss of Bocksbeutel reduced Klarsicht localization to the nucleus and resulted in a disruption in nuclear separation. Loss of Otefin increased the transcription of Klarsicht and led to premature separation of nuclei and their positioning closer to the edge of the muscle. Consistent with opposing functions, nuclear position is normal in otefin; bocksbeutel double mutants. These data indicate emerin-dependent regulation of Klarsicht levels in the nuclear envelope is a critical determinant of nuclear position.

Emery-Dreifuss muscular dystrophy: focal point nuclear envelope.

Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in EMD encoding emerin and LMNA encoding A-type lamins, proteins of the nuclear envelope. In the past decade, there has been an extraordinary burst of research on the nuclear envelope. Discoveries resulting from this basic research have implications for better understanding the pathogenesis and developing treatments for EDMD. Recent clinical research has confirmed that EDMD is one of several overlapping skeletal muscle phenotypes that can result from mutations in EMD and LMNA with dilated cardiomyopathy as a common feature. Basic research on the nuclear envelope has provided new insights into how A-type lamins and emerin function in force transmission throughout the cell, which may be particularly important in striated muscle. Much of the recent research has focused on the heart and LMNA mutations. Prevalence and outcome studies have confirmed the relative severity of cardiac disease. Robust mouse models of EDMD caused by LMNA mutations has allowed for further insight into pathogenic mechanisms and potentially beneficial therapeutic approaches. Recent clinical and basic research on EDMD is gradually being translated to clinical practice and possibly novel therapies.