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Facio-Scapulo-Humeral Dystrophy (FSHD) is an enigmatic pathology. This autosomal dominant disorder is linked to deletion within a D4Z4 macrosatellite in the subtelomeric 4q35 region. The gene product leading to the disease has not been clearly identified and epigenetic changes are likely key players in the disease since beside reduction in the number of repeats, D4Z4 is hypomethylated in FSHD. Within D4Z4, DUX4 has been found upregulated in patients. Different DUX4 transcripts have been described Production of a long transcript encompassing the DUX4 sequence and a region distal to D4Z4 encoding a toxic protein has been proposed as the cause of disease. A causal link between DUX4 expression and D4Z4 hypomethylation subsequent to array shortening has been proposed but never firmly established. We analyzed DUX4 expression as well as the expression of 4q35 genes and muscular markers in FSHD and non-FSHD biopsies during fetal development and in adults in a large cohort of samples. We highlight several genes whose expression differs between control and FSHD samples. Furthermore, we detected DUX4 transcripts in both groups, in muscle but also in other tissues indicating that expression of the long transcript is not restricted to FSHD muscles. Using FSHD and control myoblasts, we showed that DUX4 expression is induced by hypomethylation after knock-down of DNA methyltransferases, independently of D4Z4 array shortening. Our result tends toward a stochastic activation DUX4 transcription rather than a muscle-specific expression pattern in FSHD patients. The mechanism precluding onset and progression of FSHD remains highly controversial and still debated. Our work is the first to uncover changes in gene expression in fetuses carrying a D4Z4-linked 4q35 defect. These results are important for understanding FSHD but also in general, to understand how epigenetic mechanism modulate the transcription of repetitive DNA sequences in the human genome especially in the human diseases. [Copyright &y& Elsevier]

Background: Approximately half of hypertrophic cardiomyopathy (HCM) patients lack a precise genetic diagnosis. The likelihood of identifying clinically relevant variants increased over time., Methods: In this study, we conducted a gene-centric reanalysis of exome data of 200 HCM cases 5 years after the initial analysis. This reanalysis prioritized genes with a matched HCM entry in the OMIM database and recently emerging HCM-associated genes gathered using a text mining-based literature review. Further classification of the identified genes and variants was performed using the Clinical Genome Resource (ClinGen) resource and American College of Medical Genetics and Genomics (ACMG) guidelines to assess the robustness of gene-disease association and the clinical actionability of the prioritized variants., Results: As expected, the majority of patients carried variants in MYBPC3 and M YH7 genes, 26% ( n  = 51) and 8% ( n  = 16), respectively, in accordance with the initial analysis. The vast majority of pathogenic (P) and likely pathogenic (LP) variants were found in MYBPC3 (22 out of 40 variants) and MYH7 (8 out of 16 variants) genes. Three genes-not included in the initial analysis-were identified: SVIL , FHOD3 , and TRIM63 . Considering only patients with unique variants in the last three genes, there was a 9% enhancement in variant identification. Importantly, SVIL variant carriers presented apical and septal HCM, aortopathies, and severe scoliosis for one patient. Ten patients (5%) carried variants in the FHOD3 gene, six in hotspot regions (exons 12 and 15). We identified seven variants within the TRIM63 gene in 12 patients (6%). Homozygous variants were detected in 2.5% of the cohort in MYBPC3 ( n  = 1), MYL3 ( n  = 1), and TRIM63 ( n  = 3) genes., Conclusion: Our study revealed that no variants were found in the ACTC1 , TPM1 , and TNNI3 genes in the HYPERGEN cohort. However, we identified variants in five out of the eight HCM core genes, with a high prevalence in young patients. We identified variants in three recent HCM-associated genes ( SVIL , FHOD3 , and TRIM63 ) in 35 patients, with 18 patients carrying unique variants (9%). Our results further emphasize the usefulness of exome data reanalysis, particularly in genotype-negative patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jaouadi, Morel, Martel, Lindenbaum, de la Chapelle, Herbane, Lucas, Magdinier, Gilbert, Schott, Zaffran and Nguyen.)

Rubinstein-Taybi syndrome (RTS) is a rare and severe genetic developmental disorder characterized by multiple congenital anomalies and intellectual disability. CREBBP and EP300, the two genes known to cause RTS encode transcriptional coactivators with a catalytic lysine acetyltransferase (KAT) activity. Loss of CBP or p300 function results in a deficit in protein acetylation, in particular at histones. In RTS, nothing is known on the consequences of the loss of histone acetylation on the transcriptomic profiles during neuronal differentiation. To address this question, we differentiated induced pluripotent stem cells from RTS patients carrying a recurrent CREBBP mutation that inactivates the KAT domain into cortical and pyramidal neurons. By comparing their acetylome and their transcriptome at different neuronal differentiation time points, we identified 25 specific acetylated histone residues altered in RTS. We also identified the transition between neural progenitors and immature neurons as a critical step of the differentiation process, with a delayed neuronal maturation in RTS. Overall, this study opens new perspectives in the definition of epigenetic biomarkers for RTS, whose methodology could be extended to other chromatinopathies., (© 2024. The Author(s).)

The gold standard for facioscapulohumeral muscular dystrophy (FSHD) genetic diagnostic procedures was published in 2012. With the increasing complexity of the genetics of FSHD1 and 2, the increase of genetic testing centers, and the start of clinical trials for FSHD, it is crucial to provide an update on our knowledge of the genetic features of the FSHD loci and renew the international consensus on the molecular testing recommendations. To this end, members of the FSHD European Trial Network summarized the evidence presented during the 2022 ENMC meeting on Genetic diagnosis, clinical outcome measures, and biomarkers. The working group additionally invited genetic and clinical experts from the USA, India, Japan, Australia, South-Africa, and Brazil to provide a global perspective. Six virtual meetings were organized to reach consensus on the minimal requirements for genetic confirmation of FSHD1 and FSHD2. Here, we present the clinical and genetic features of FSHD, specific features of FSHD1 and FSHD2, pros and cons of established and new technologies (Southern blot in combination with either linear or pulsed-field gel electrophoresis, molecular combing, optical genome mapping, FSHD2 methylation analysis and FSHD2 genotyping), the possibilities and challenges of prenatal testing, including pre-implantation genetic testing, and the minimal requirements and recommendations for genetic confirmation of FSHD1 and FSHD2. This consensus is expected to contribute to current clinical management and trial-readiness for FSHD., (© 2024 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)

Due to their biocompatibility and biodegradability and their unique structural and physicochemical properties, laser-synthesized silicon nanoparticles (Si-NPs) are one of the nanomaterials which have been most studied as potential theragnostic tools for non-invasive therapeutic modalities. However, their ability to modulate cell behavior and to promote proliferation and differentiation is still very little investigated or unknown. In this work, ultrapure ligand free Si-NPs of 50 ± 11.5 nm were prepared by femtosecond (fs) laser ablation in liquid. After showing the ability of Si-NPs to be internalized by murine C2C12 myoblasts, the cytotoxicity of the Si-NPs on these cells was evaluated at concentrations ranging from 14 to 224 μg mL -1 . Based on these findings, three concentrations of 14, 28 and 56 μg mL -1 were thus considered to study the effect on myoblast differentiation, proliferation and motility at the molecular and phenotypical levels. It was demonstrated that up to 28 μg mL -1 , the Si-NPs are able to promote the proliferation of myoblasts and their subsequent differentiation. Scratch tests were also performed revealing the positive Si-NP effect on cellular motility at 14 and 28 μg mL -1 . Finally, gene expression analysis confirmed the ability of Si-NPs to promote proliferation, differentiation and motility of myoblasts even at very low concentration. This work opens up novel exciting prospects for Si-NPs made by the laser process as innovative tools for skeletal muscle tissue engineering in view of developing novel therapeutic protocols for regenerative medicine., Competing Interests: The authors declare that they have no competing interest., (This journal is © The Royal Society of Chemistry.)

Many genetic syndromes are linked to mutations in genes encoding factors that guide chromatin organization. Among them, several distinct rare genetic diseases are linked to mutations in SMCHD1 that encodes the structural maintenance of chromosomes flexible hinge domain containing 1 chromatin-associated factor. In humans, its function as well as the impact of its mutations remains poorly defined. To fill this gap, we determined the episignature associated with heterozygous SMCHD1 variants in primary cells and cell lineages derived from induced pluripotent stem cells for Bosma arhinia and microphthalmia syndrome (BAMS) and type 2 facioscapulohumeral dystrophy (FSHD2). In human tissues, SMCHD1 regulates the distribution of methylated CpGs, H3K27 trimethylation and CTCF at repressed chromatin but also at euchromatin. Based on the exploration of tissues affected either in FSHD or in BAMS, i.e. skeletal muscle fibers and neural crest stem cells, respectively, our results emphasize multiple functions for SMCHD1, in chromatin compaction, chromatin insulation and gene regulation with variable targets or phenotypical outcomes. We concluded that in rare genetic diseases, SMCHD1 variants impact gene expression in two ways: (i) by changing the chromatin context at a number of euchromatin loci or (ii) by directly regulating some loci encoding master transcription factors required for cell fate determination and tissue differentiation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Telomeric repeat binding factor 2 (TRF2) binds to telomeres and protects chromosome ends against the DNA damage response and senescence. Although the expression of TRF2 is downregulated upon cellular senescence and in various aging tissues, including skeletal muscle tissues, very little is known about the contribution of this decline to aging. We previously showed that TRF2 loss in myofibers does not trigger telomere deprotection but mitochondrial dysfunction leading to an increased level of reactive oxygen species. We show here that this oxidative stress triggers the binding of FOXO3a to telomeres where it protects against ATM activation, revealing a previously unrecognized telomere protective function of FOXO3a, to the best of our knowledge. We further showed in transformed fibroblasts and myotubes that the telomere properties of FOXO3a are dependent on the C-terminal segment of its CR2 domain (CR2C) but independent of its Forkhead DNA binding domain and of its CR3 transactivation domain. We propose that these non-canonical properties of FOXO3a at telomeres play a role downstream of the mitochondrial signaling induced by TRF2 downregulation to regulate skeletal muscle homeostasis and aging., (© 2023. The Author(s).)

Background and Objectives: After clinical evaluation, the molecular diagnosis of type 1 facioscapulohumeral dystrophy (FSHD1) relies in most laboratories on the detection of a shortened D4Z4 array at the 4q35 locus by Southern blotting. In many instances, this molecular diagnosis remains inconclusive and requires additional experiments to determine the number of D4Z4 units or identify somatic mosaicism, 4q-10q translocations, and proximal p13E-11 deletions. These limitations highlight the need for alternative methodologies, illustrated by the recent emergence of novel technologies such as molecular combing (MC), single molecule optical mapping (SMOM), or Oxford Nanopore-based long-read sequencing providing a more comprehensive analysis of 4q and 10q loci. Over the last decade, MC revealed a further increasing complexity in the organization of the 4q and 10q distal regions in patients with FSHD with cis -duplication of D4Z4 arrays in approximately 1%-2% of cases., Methods: By using MC, we investigated in our center 2,363 cases for molecular diagnosis of FSHD. We also evaluated whether previously reported cis -duplications might be identified by SMOM using the Bionano EnFocus FSHD 1.0 algorithm., Results: In our cohort of 2,363 samples, we identified 147 individuals carrying an atypical organization of the 4q35 or 10q26 loci. Mosaicism is the most frequent category followed by cis -duplications of the D4Z4 array. We report here chromosomal abnormalities of the 4q35 or 10q26 loci in 54 patients clinically described as FSHD, which are not present in the healthy population. In one-third of the 54 patients, these rearrangements are the only genetic defect suggesting that they might be causative of the disease. By analyzing DNA samples from 3 patients carrying a complex rearrangement of the 4q35 region, we further showed that the SMOM direct assembly of the 4q and 10q alleles failed to reveal these abnormalities and lead to negative results for FSHD molecular diagnosis., Discussion: This work further highlights the complexity of the 4q and 10q subtelomeric regions and the need of in-depth analyses in a significant number of cases. This work also highlights the complexity of the 4q35 region and interpretation issues with consequences on the molecular diagnosis of patients or genetic counseling., Competing Interests: The authors report no relevant disclosures. Go to Neurology.org/NG for full disclosures., (Written work prepared by employees of the Federal Government as part of their official duties is, under the U.S. Copyright Act, a “work of the United States Government” for which copyright protection under Title 17 of the United States Code is not available. As such, copyright does not extend to the contributions of employees of the Federal Government.)

Neuromuscular disorders (NMDs) are a large group of diseases associated with either alterations of skeletal muscle fibers, motor neurons or neuromuscular junctions. Most of these diseases is characterized with muscle weakness or wasting and greatly alter the life of patients. Animal models do not always recapitulate the phenotype of patients. The development of innovative and representative human preclinical models is thus strongly needed for modeling the wide diversity of NMDs, characterization of disease-associated variants, investigation of novel genes function, or the development of therapies. Over the last decade, the use of patient's derived induced pluripotent stem cells (hiPSC) has resulted in tremendous progress in biomedical research, including for NMDs. Skeletal muscle is a complex tissue with multinucleated muscle fibers supported by a dense extracellular matrix and multiple cell types including motor neurons required for the contractile activity. Major challenges need now to be tackled by the scientific community to increase maturation of muscle fibers in vitro, in particular for modeling adult-onset diseases affecting this tissue (neuromuscular disorders, cachexia, sarcopenia) and the evaluation of therapeutic strategies. In the near future, rapidly evolving bioengineering approaches applied to hiPSC will undoubtedly become highly instrumental for investigating muscle pathophysiology and the development of therapeutic strategies.

McArdle disease is a rare autosomal recessive disorder caused by mutations in the PYGM gene. This gene encodes for the skeletal muscle isoform of glycogen phosphorylase (myophosphorylase), the first enzyme in glycogenolysis. Patients with this disorder are unable to obtain energy from their glycogen stored in skeletal muscle, prompting an exercise intolerance. Currently, there is no treatment for this disease, and the lack of suitable in vitro human models has prevented the search for therapies against it. In this article, we have established the first human iPSC-based model for McArdle disease. For the generation of this model, induced pluripotent stem cells (iPSCs) from a patient with McArdle disease (harbouring the homozygous mutation c.148C>T; p.R50* in the PYGM gene) were differentiated into myogenic cells able to contract spontaneously in the presence of motor neurons and generate calcium transients, a proof of their maturity and functionality. Additionally, an isogenic skeletal muscle model of McArdle disease was created. As a proof-of-concept, we have tested in this model the rescue of PYGM expression by two different read-through compounds (PTC124 and RTC13). The developed model will be very useful as a platform for testing drugs or compounds with potential pharmacological activity.

microRNAs (miRNAs) are small single strand non-coding RNAs and powerful gene expression regulators. They mainly bind to the 3'UTR sequence of targeted mRNA, leading to their degradation or translation inhibition. miR-140 gene encodes the pre-miR-140 that generates the two mature miRNAs miR-140-5p and miR-140-3p. miR-140-5p/-3p have been associated with the development and progression of cancers, but also non-neoplastic diseases. In aging-related diseases, miR-140-5p and miR-140-3p expressions are modulated. The seric levels of these two miRNAs are used as circulating biomarkers and may represent predictive tools. They are also considered key actors in the pathophysiology of aging-related diseases. miR-140-5p/-3p repress targets regulating cell proliferation, apoptosis, senescence, and inflammation. This work focuses on the roles of miR-140-3p and miR-140-5p in aging-related diseases, details their regulation (i.e., by long non-coding RNA), and reviews the molecular targets of theses miRNAs involved in aging pathophysiology.

Progeroid syndromes are rare genetic diseases with most of autosomal dominant transmission, the prevalence of which is less than 1/10,000,000. These syndromes caused by mutations in the <i>LMNA</i> gene encoding A-type lamins belong to a group of disorders called laminopathies. Lamins are implicated in the architecture and function of the nucleus and chromatin. Patients affected with progeroid laminopathies display accelerated aging of mesenchymal stem cells (MSCs)-derived tissues associated with nuclear morphological abnormalities. To identify pathways altered in progeroid patients' MSCs, we used induced pluripotent stem cells (hiPSCs) from patients affected with classical Hutchinson-Gilford progeria syndrome (HGPS, c.1824C>T-p.G608G), HGPS-like syndrome (HGPS-L; c.1868C>G-p.T623S) associated with farnesylated prelamin A accumulation, or atypical progeroid syndromes (APS; homozygous c.1583C> T-p.T528M; heterozygous c.1762T>C-p.C588R; compound heterozygous c.1583C>T and c.1619T>C-p.T528M and p.M540T) without progerin accumulation. By comparative analysis of the transcriptome and methylome of hiPSC-derived MSCs, we found that patient's MSCs display specific DNA methylation patterns and modulated transcription at early stages of differentiation. We further explored selected biological processes deregulated in the presence of <i>LMNA</i> variants and confirmed alterations of age-related pathways during MSC differentiation. In particular, we report the presence of an altered mitochondrial pattern; an increased response to double-strand DNA damage; and telomere erosion in HGPS, HGPS-L, and APS MSCs, suggesting converging pathways, independent of progerin accumulation, but a distinct DNA methylation profile in HGPS and HGPS-L compared with APS cells., (© 2022 Trani et al.)

The body plan of animals is laid out by an evolutionary-conserved HOX code which is colinearly transcribed after zygotic genome activation (ZGA). Here we report that SMCHD1, a chromatin-modifying enzyme needed for X-inactivation in mammals, is maternally required for timely HOX expression. Using zebrafish and mouse Smchd1 knockout animals, we demonstrate that Smchd1 haplo-insufficiency brings about precocious and ectopic HOX transcription during oogenesis and embryogenesis. Unexpectedly, wild-type offspring born to heterozygous knockout zebrafish smchd1 mothers exhibited patent vertebrate patterning defects. The loss of maternal Smchd1 was accompanied by HOX epi-mutations driven by aberrant DNA methylation. We further show that this regulation is mediated by Lrif1, a direct interacting partner of Smchd1, whose knockout in zebrafish phenocopies that of Smchd1. Rather than being a short-lived maternal effect, HOX mis-regulation is stably inherited through cell divisions and persists in cultured fibroblasts derived from FSHD2 patients haploinsufficient for SMCHD1. We conclude that maternal SMCHD1/LRIF1 sets up an epigenetic state in the HOX loci that can only be reset in the germline. Such an unusual inter-generational inheritance, whereby a phenotype can be one generation removed from its genotype, casts a new light on how unresolved Mendelian diseases may be interpreted., (© 2022. The Author(s).)

Germline mutations that activate genes in the canonical RAS/MAPK signaling pathway are responsible for rare human developmental disorders known as RASopathies. Here, we analyzed the molecular determinants of Costello syndrome (CS) using a mouse model expressing HRAS p.G12S, patient skin fibroblasts, hiPSC-derived human cardiomyocytes, a HRAS p.G12V zebrafish model, and human fibroblasts expressing lentiviral constructs carrying HRAS p.G12S or HRAS p.G12A mutations. The findings revealed alteration of mitochondrial proteostasis and defective oxidative phosphorylation in the heart and skeletal muscle of CS mice that were also found in the cell models of the disease. The underpinning mechanisms involved the inhibition of the AMPK signaling pathway by mutant forms of HRAS, leading to alteration of mitochondrial proteostasis and bioenergetics. Pharmacological activation of mitochondrial bioenergetics and quality control restored organelle function in HRAS p.G12A and p.G12S cell models, reduced left ventricle hypertrophy in CS mice, and diminished the occurrence of developmental defects in the CS zebrafish model. Collectively, these findings highlight the importance of mitochondrial proteostasis and bioenergetics in the pathophysiology of RASopathies and suggest that patients with CS may benefit from treatment with mitochondrial modulators.

Chromatin structure is an essential regulator of gene expression. Its state of compaction contributes to the regulation of genetic programs, in particular during differentiation. Epigenetic processes, which include post-translational modifications of histones, DNA methylation and implication of non-coding RNA, are powerful regulators of gene expression. Neurogenesis and neuronal differentiation are spatio-temporally regulated events that allow the formation of the central nervous system components. Here, we review the chromatin structure and post-translational histone modifications associated with neuronal differentiation. Studying the impact of histone modifications on neuronal differentiation improves our understanding of the pathophysiological mechanisms of chromatinopathies and opens up new therapeutic avenues. In addition, we will discuss techniques for the analysis of histone modifications on a genome-wide scale and the pathologies associated with the dysregulation of the epigenetic machinery.

Background: Facioscapulohumeral dystrophy (FSHD) is a late-onset autosomal dominant form of muscular dystrophy involving specific groups of muscles with variable weakness that precedes inflammatory response, fat infiltration, and muscle atrophy. As there is currently no cure for this disease, understanding and modelling the typical muscle weakness in FSHD remains a major milestone towards deciphering the disease pathogenesis as it will pave the way to therapeutic strategies aimed at correcting the functional muscular defect in patients., Methods: To gain further insights into the specificity of the muscle alteration in this disease, we derived induced pluripotent stem cells from patients affected with Types 1 and 2 FSHD but also from patients affected with Bosma arhinia and microphthalmia. We differentiated these cells into contractile innervated muscle fibres and analysed their transcriptome by RNA Seq in comparison with cells derived from healthy donors. To uncover biological pathways altered in the disease, we applied MOGAMUN, a multi-objective genetic algorithm that integrates multiplex complex networks of biological interactions (protein-protein interactions, co-expression, and biological pathways) and RNA Seq expression data to identify active modules., Results: We identified 132 differentially expressed genes that are specific to FSHD cells (false discovery rate < 0.05). In FSHD, the vast majority of active modules retrieved with MOGAMUN converges towards a decreased expression of genes encoding proteins involved in sarcomere organization (P value 2.63e -12 ), actin cytoskeleton (P value 9.4e -5 ), myofibril (P value 2.19e -12 ), actin-myosin sliding, and calcium handling (with P values ranging from 7.9e -35 to 7.9e -21 ). Combined with in vivo validations and functional investigations, our data emphasize a reduction in fibre contraction (P value < 0.0001) indicating that the muscle weakness that is typical of FSHD clinical spectrum might be associated with dysfunction of calcium release (P value < 0.0001), actin-myosin interactions, motor activity, mechano-transduction, and dysfunctional sarcomere contractility., Conclusions: Identification of biomarkers of FSHD muscle remain critical for understanding the process leading to the pathology but also for the definition of readouts to be used for drug design, outcome measures, and monitoring of therapies. The different pathways identified through a system biology approach have been largely overlooked in the disease. Overall, our work opens new perspectives in the definition of biomarkers able to define the muscle alteration but also in the development of novel strategies to improve muscle function as it provides functional parameters for active molecule screening., (© 2021 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder, in which an abnormal and toxic protein called progerin, accumulates in cell nuclei, leading to major cellular defects. Among them, chromatin remodeling drives gene expression changes, including miRNA dysregulation. In our study, we evaluated miRNA expression profiles in HGPS and control fibroblasts. We identified an enrichment of overexpressed miRNAs belonging to the 14q32.2-14q32.3 miRNA cluster. Using 3D FISH, we demonstrated that overexpression of these miRNAs is associated with chromatin remodeling at this specific locus in HGPS fibroblasts. We then focused on miR-376b-3p and miR-376a-3p, both overexpressed in HGPS fibroblasts. We demonstrated that their induced overexpression in control fibroblasts decreases cell proliferation and increases senescence, whereas their inhibition in HGPS fibroblasts rescues proliferation defects and senescence and decreases progerin accumulation. By targeting these major processes linked to premature aging, these two miRNAs may play a pivotal role in the pathophysiology of HGPS., Competing Interests: The authors have no conflicts of interest to declare., (© 2022 The Authors.)

Only a limited number of large-scale protocols describe the production of mature skeletal muscle fibers from human induced pluripotent stem cells (hiPSCs). Here we describe a novel procedure for simultaneous differentiation of hiPSC into muscle cells and motor neurons, that generates innervated and contractile multinucleated skeletal muscle fibers with sarcomeric organization. Our protocol permits the production of expandable skeletal muscle progenitor cells and mature skeletal muscle fibers that can be used for the exploration of skeletal muscle differentiation for basic research, disease modeling, and drug discovery., (© 2020. Springer Science+Business Media New York.)

Competing Interests: The authors have no conflict of interest to declare