SMCHD1 is involved in de novo methylation of the DUX4-encoding D4Z4 macrosatellite.

The DNA methylation epigenetic signature is a key determinant during development. Rules governing its establishment and maintenance remain elusive especially at repetitive sequences, which account for the majority of methylated CGs. DNA methylation is altered in a number of diseases including those linked to mutations in factors that modify chromatin. Among them, SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain Containing 1) has been of major interest following identification of germline mutations in Facio-Scapulo-Humeral Dystrophy (FSHD) and in an unrelated developmental disorder, Bosma Arhinia Microphthalmia Syndrome (BAMS). By investigating why germline SMCHD1 mutations lead to these two different diseases, we uncovered a role for this factor in de novo methylation at the pluripotent stage. SMCHD1 is required for the dynamic methylation of the D4Z4 macrosatellite upon reprogramming but seems dispensable for methylation maintenance. We find that FSHD and BAMS patient's cells carrying SMCHD1 mutations are both permissive for DUX4 expression, a transcription factor whose regulation has been proposed as the main trigger for FSHD. These findings open new questions as to what is the true aetiology for FSHD, the epigenetic events associated with the disease thus calling the current model into question and opening new perspectives for understanding repetitive DNA sequences regulation.

One-hour universal protocol for mouse genotyping.

BACKGROUND: Transgenic animals are widely used for research and for most of them, genotyping is unavoidable. Published protocols may be powerful but may also present disadvantages such as their cost or the requirement of additional steps/equipment. Moreover, if more than one strain must be genotyped, several protocols may need to be developed. METHODS: we adapted the existing amplification-resistant mutation protocol to develop the 1-h universal genotyping protocol (1-HUG), which allows the robust genotyping of genetically modified mice in 1 h from sample isolation to polymerase chain reaction gel running. RESULTS: This protocol allows the genotyping of different mouse models including mdx mouse, and FLExDUX4 and HSA-MerCreMer alone or in combination. It can be applied to different types of genomic modifications and to sexing. CONCLUSIONS: The 1-HUG protocol can be used routinely in any laboratory using mouse models for neuromuscular diseases.

Genetic Evidence That Captured Retroviral Envelope syncytins Contribute to Myoblast Fusion and Muscle Sexual Dimorphism in Mice.

Syncytins are envelope genes from endogenous retroviruses, "captured" for a role in placentation. They mediate cell-cell fusion, resulting in the formation of a syncytium (the syncytiotrophoblast) at the fetomaternal interface. These genes have been found in all placental mammals in which they have been searched for. Cell-cell fusion is also pivotal for muscle fiber formation and repair, where the myotubes are formed from the fusion of mononucleated myoblasts into large multinucleated structures. Here we show, taking advantage of mice knocked out for syncytins, that these captured genes contribute to myoblast fusion, with a >20% reduction in muscle mass, mean muscle fiber area and number of nuclei per fiber in knocked out mice for one of the two murine syncytin genes. Remarkably, this reduction is only observed in males, which subsequently show muscle quantitative traits more similar to those of females. In addition, we show that syncytins also contribute to muscle repair after cardiotoxin-induced injury, with again a male-specific effect on the rate and extent of regeneration. Finally, ex vivo experiments carried out on murine myoblasts demonstrate the direct involvement of syncytins in fusion, with a >40% reduction in fusion index upon addition of siRNA against both syncytins. Importantly, similar effects are observed with primary myoblasts from sheep, dog and human, with a 20-40% reduction upon addition of siRNA against the corresponding syncytins. Altogether, these results show a direct contribution of the fusogenic syncytins to myogenesis, with a demonstrated male-dependence of the effect in mice, suggesting that these captured genes could be responsible for the muscle sexual dimorphism observed in placental mammals.

Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: a new gene-silencing approach.

Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy.

Targeting the Polyadenylation Signal of Pre-mRNA: A New Gene Silencing Approach for Facioscapulohumeral Dystrophy.

Facioscapulohumeral dystrophy (FSHD) is characterized by the contraction of the D4Z4 array located in the sub-telomeric region of the chromosome 4, leading to the aberrant expression of the DUX4 transcription factor and the mis-regulation of hundreds of genes. Several therapeutic strategies have been proposed among which the possibility to target the polyadenylation signal to silence the causative gene of the disease. Indeed, defects in mRNA polyadenylation leads to an alteration of the transcription termination, a disruption of mRNA transport from the nucleus to the cytoplasm decreasing the mRNA stability and translation efficiency. This review discusses the polyadenylation mechanisms, why alternative polyadenylation impacts gene expression, and how targeting polyadenylation signal may be a potential therapeutic approach for FSHD.

Nuclear protein spreading: implication for pathophysiology of neuromuscular diseases.

While transfer of a protein encoded by a single nucleus to nearby nuclei in multinucleated cells has been known for almost 25 years, the biological consequences for gain-of-function diseases have not been considered. Here, we have investigated nuclear protein spreading and its potential consequences in two of the three most prevalent neuromuscular diseases. By performing co-cultures between diseased or control human myoblasts and murine C2C12 myoblasts, we demonstrate that in facioscapulohumeral dystrophy, although the transcription of the toxic protein DUX4 occurs in only a limited number of nuclei, the resulting protein diffuses into nearby nuclei within the myotubes, thus spreading aberrant gene expression. In myotonic dystrophy type 1, we observed that in human-mouse heterokaryons, the expression of a mutated DMPK from human nuclei titrates splicing factors produced by neighboring nuclei, inducing the mis-splicing of several pre-mRNAs in murine nuclei. In both cases, the spreading of the pathological phenotypes from one nucleus to another is observed, highlighting an additional mechanism that contributes to the dissemination and worsening of the muscle pathogenesis. These results indicate that nuclear protein spreading may be an important component of pathophysiology of gain of function muscular diseases which should be taken into consideration in the design of new therapeutic approaches.

DUX4 and DUX4 downstream target genes are expressed in fetal FSHD muscles.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent adult muscular dystrophies. The common clinical signs usually appear during the second decade of life but when the first molecular dysregulations occur is still unknown. Our aim was to determine whether molecular dysregulations can be identified during FSHD fetal muscle development. We compared muscle biopsies derived from FSHD1 fetuses and the cells derived from some of these biopsies with biopsies and cells derived from control fetuses. We mainly focus on DUX4 isoform expression because the expression of DUX4 has been confirmed in both FSHD cells and biopsies by several laboratories. We measured DUX4 isoform expression by using qRT-PCR in fetal FSHD1 myotubes treated or not with an shRNA directed against DUX4 mRNA. We also analyzed DUX4 downstream target gene expression in myotubes and fetal or adult FSHD1 and control quadriceps biopsies. We show that both DUX4-FL isoforms are already expressed in FSHD1 myotubes. Interestingly, DUX4-FL expression level is much lower in trapezius than in quadriceps myotubes, which is confirmed by the level of expression of DUX4 downstream genes. We observed that TRIM43 and MBD3L2 are already overexpressed in FSHD1 fetal quadriceps biopsies, at similar levels to those observed in adult FSHD1 quadriceps biopsies. These results indicate that molecular markers of the disease are already expressed during fetal life, thus opening a new field of investigation for mechanisms leading to FSHD.

Segregation between SMCHD1 mutation, D4Z4 hypomethylation and Facio-Scapulo-Humeral Dystrophy: a case report.

BACKGROUND: The main form of Facio-Scapulo-Humeral muscular Dystrophy is linked to copy number reduction of the 4q D4Z4 macrosatellite (FSHD1). In 5 % of cases, FSHD phenotype appears in the absence of D4Z4 reduction (FSHD2). In 70-80 % of these patients, variants of the SMCHD1 gene segregate with 4qA haplotypes and D4Z4 hypomethylation. CASE PRESENTATION: We report a family presenting with neuromuscular symptoms reminiscent of FSHD but without D4Z4 copy reduction. We characterized the 4q35 region using molecular combing, searched for mutation in the SMCHD1 gene and determined D4Z4 methylation level by sodium bisulfite sequencing. We further investigated the impact of the SMCHD1 mutation at the protein level and on the NMD-dependent degradation of transcript. In muscle, we observe moderate but significant reduction in D4Z4 methylation, not correlated with DUX4-fl expression. Exome sequencing revealed a heterozygous insertion of 7 bp in exon 37 of the SMCHD1 gene producing a loss of frame with premature stop codon 4 amino acids after the insertion (c.4614-4615insTATAATA). Both wild-type and mutated transcripts are detected. CONCLUSION: The truncated protein is absent and the full-length protein level is similar in patients and controls indicating that in this family, FSHD is not associated with SMCHD1 haploinsufficiency.

Correlation between low FAT1 expression and early affected muscle in facioscapulohumeral muscular dystrophy.

OBJECTIVE: Facioscapulohumeral muscular dystrophy (FSHD) is linked to either contraction of D4Z4 repeats on chromosome 4 or to mutations in the SMCHD1 gene, both of which result in the aberrant expression of the transcription factor DUX4. However, it is still difficult to correlate these genotypes with the phenotypes observed in patients. Because we have recently shown that mice with disrupted Fat1 functions exhibit FSHD-like phenotypes, we have investigated the expression of the human FAT1 gene in FSHD. METHODS: We first analyzed FAT1 expression in FSHD adult muscles and determined whether FAT1 expression was driven by DUX4. We next determined FAT1 expression levels in 64 muscles isolated from 16 control fetuses. These data were further complemented with analysis of Fat1 expression in developing mouse embryos. RESULTS: We demonstrated that FAT1 expression is independent of DUX4. Moreover, we observed that (1) in control fetal human biopsies or in developing mouse embryos, FAT1 is expressed at lower levels in muscles that are affected at early stages of FSHD progression than in muscles that are affected later or are nonaffected; and (2) in adult muscle biopsies, FAT1 expression is lower in FSHD muscles compared to control muscles. INTERPRETATION: We propose a revised model for FSHD in which FAT1 levels might play a role in determining which muscles will exhibit early and late disease onset, whereas DUX4 may worsen the muscle phenotype.

Generation of isogenic D4Z4 contracted and noncontracted immortal muscle cell clones from a mosaic patient: a cellular model for FSHD.

In most cases facioscapulohumeral muscular dystrophy (FSHD) is caused by contraction of the D4Z4 repeat in the 4q subtelomere. This contraction is associated with local chromatin decondensation and derepression of the DUX4 retrogene. Its complex genetic and epigenetic cause and high clinical variability in disease severity complicate investigations on the pathogenic mechanism underlying FSHD. A validated cellular model bypassing the considerable heterogeneity would facilitate mechanistic and therapeutic studies of FSHD. Taking advantage of the high incidence of somatic mosaicism for D4Z4 repeat contraction in de novo FSHD, we have established a clonal myogenic cell model from a mosaic patient. Individual clones are genetically identical except for the size of the D4Z4 repeat array, being either normal or FSHD sized. These clones retain their myogenic characteristics, and D4Z4 contracted clones differ from the noncontracted clones by the bursts of expression of DUX4 in sporadic nuclei, showing that this burst-like phenomenon is a locus-intrinsic feature. Consequently, downstream effects of DUX4 expression can be observed in D4Z4 contracted clones, like differential expression of DUX4 target genes. We also show their participation to in vivo regeneration with immunodeficient mice, further expanding the potential of these clones for mechanistic and therapeutic studies. These cell lines will facilitate pairwise comparisons to identify FSHD-specific differences and are expected to create new opportunities for high-throughput drug screens.

Quantification of the methylation at the GNAS locus identifies subtypes of sporadic pseudohypoparathyroidism type Ib.

BACKGROUND: Pseudohypoparathyroidism type Ib (PHP-Ib) is due to epigenetic changes at the imprinted GNAS locus, including loss of methylation at the A/B differentially methylated region (DMR) and sometimes at the XL and AS DMRs and gain of methylation at the NESP DMR. OBJECTIVE: To investigate if quantitative measurement of the methylation at the GNAS DMRs identifies subtypes of PHP-Ib. DESIGN AND METHODS: In 19 patients with PHP-Ib and 7 controls, methylation was characterised at the four GNAS DMRs through combined bisulfite restriction analysis and quantified through cytosine specific real-time PCR in blood lymphocyte DNA. RESULTS: A principal component analysis using the per cent of methylation at seven cytosines of the GNAS locus provided three clusters of subjects (controls n=7, autosomal dominant PHP-Ib with loss of methylation restricted to the A/B DMR n=3, and sporadic PHP-Ib with broad GNAS methylation changes n=16) that matched perfectly the combined bisulfite restriction analysis classification. Furthermore, three sub-clusters of patients with sporadic PHP-Ib, that displayed different patterns of methylation, were identified: incomplete changes at all DMRs compatible with somatic mosaicism (n=5), profound epigenetic changes at all DMRs (n=8), and unmodified methylation at XL in contrast with the other DMRs (n=3). Interestingly, parathyroid hormone concentration at the time of diagnosis correlated with the per cent of methylation at the A/B DMR. CONCLUSION: Quantitative assessment of the methylation in blood lymphocyte DNA is of clinical relevance, allows the diagnosis of PHP-Ib, and identifies subtypes of PHP-Ib. These epigenetic findings suggest mosaicism at least in some patients.

Gene Editing to Tackle Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral dystrophy (FSHD) is a skeletal muscle disease caused by the aberrant expression of the DUX4 gene in the muscle tissue. To date, different therapeutic approaches have been proposed, targeting DUX4 at the DNA, RNA or protein levels. The recent development of the clustered regularly interspaced short-palindromic repeat (CRISPR) based technology opened new avenues of research, and FSHD is no exception. For the first time, a cure for genetic muscular diseases can be considered. Here, we describe CRISPR-based strategies that are currently being investigated for FSHD. The different approaches include the epigenome editing targeting the DUX4 gene and its promoter, gene editing targeting the polyadenylation of DUX4 using TALEN, CRISPR/cas9 or adenine base editing and the CRISPR-Cas9 genome editing for SMCHD1. We also discuss challenges facing the development of these gene editing based therapeutics.

Transiently expressed CRISPR/Cas9 induces wild-type dystrophin in vitro in DMD patient myoblasts carrying duplications.

Among the mutations arising in the DMD gene and causing Duchenne Muscular Dystrophy (DMD), 10-15% are multi-exon duplications. There are no current therapeutic approaches with the ability to excise large multi-exon duplications, leaving this patient cohort without mutation-specific treatment. Using CRISPR/Cas9 could provide a valid alternative to achieve targeted excision of genomic duplications of any size. Here we show that the expression of a single CRISPR/Cas9 nuclease targeting a genomic region within a DMD duplication can restore the production of wild-type dystrophin in vitro. We assessed the extent of dystrophin repair following both constitutive and transient nuclease expression by either transducing DMD patient-derived myoblasts with integrating lentiviral vectors or electroporating them with CRISPR/Cas9 expressing plasmids. Comparing genomic, transcript and protein data, we observed that both continuous and transient nuclease expression resulted in approximately 50% dystrophin protein restoration in treated myoblasts. Our data demonstrate that a high transient expression profile of Cas9 circumvents its requirement of continuous expression within the cell for targeting DMD duplications. This proof-of-concept study therefore helps progress towards a clinically relevant gene editing strategy for in vivo dystrophin restoration, by highlighting important considerations for optimizing future therapeutic approaches.

Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles.

BACKGROUND: The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology. METHODS: Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged-matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT-qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls. RESULTS: An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti-CD63 immuno-blocking of vesicle uptake. CONCLUSIONS: ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS.

RIPK3-mediated cell death is involved in DUX4-mediated toxicity in facioscapulohumeral dystrophy.

BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is caused by mutations leading to the aberrant expression of the DUX4 transcription factor in muscles. DUX4 was proposed to induce cell death, but the involvement of different death pathways is still discussed. A possible pro-apoptotic role of DUX4 was proposed, but as FSHD muscles are characterized by necrosis and inflammatory infiltrates, non-apoptotic pathways may be also involved. METHODS: We explored DUX4-mediated cell death by focusing on the role of one regulated necrosis pathway called necroptosis, which is regulated by RIPK3. We investigated the effect of necroptosis on cell death in vitro and in vivo experiments using RIPK3 inhibitors and a RIPK3-deficient transgenic mouse model. RESULTS: We showed in vitro that DUX4 expression causes a caspase-independent and RIPK3-mediated cell death in both myoblasts and myotubes. In vivo, RIPK3-deficient animals present improved body and muscle weights, a reduction of the aberrant activation of the DUX4 network genes, and an improvement of muscle histology. CONCLUSIONS: These results provide evidence for a role of RIPK3 in DUX4-mediated cell death and open new avenues of research.

Chitosan-Shelled Nanobubbles Irreversibly Encapsulate Morpholino Conjugate Antisense Oligonucleotides and Are Ineffective for Phosphorodiamidate Morpholino-Mediated Gene Silencing of DUX4.

Orphan drugs, including antisense oligonucleotides (AONs), siRNAs/miRNAs, Cas9 nuclease, and recombinant genes, have recently been made available for rare diseases. However, the main bottleneck for these new therapies is delivery. Drugs/synthetic genes need to reach the affected tissues with minimal off-target effects and immune reactions. AON molecules are currently delivered as backboned naked compounds or via viral vectors. Nanocarriers are considered promising vehicles, able to improve drug distribution by organ targeting and limiting safety issues. We tested perfluoropentane-based nanobubbles (NBs) as vehicles for loading phosphorodiamidate morpholino (PMO) AON to suppress DUX4 expression in a facioscapulohumeral muscular dystrophy cell model. In vitro cell-free analysis demonstrated a good loading capacity of PMO into NBs, while experiments in cell cultures showed lack of therapeutic effect since expression of DUX4 and its targets remained unmodified. We conclude that these types of chitosan-shelled NBs do not release PMO-AON and are therefore not ideal for PMO AON-related therapies.

Therapeutic Strategies Targeting DUX4 in FSHD.

Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle dystrophy typically affecting patients within their second decade. Patients initially exhibit asymmetric facial and humeral muscle damage, followed by lower body muscle involvement. FSHD is associated with a derepression of DUX4 gene encoded by the D4Z4 macrosatellite located on the subtelomeric part of chromosome 4. DUX4 is a highly regulated transcription factor and its expression in skeletal muscle contributes to multiple cellular toxicities and pathologies ultimately leading to muscle weakness and atrophy. Since the discovery of the FSHD candidate gene DUX4, many cell and animal models have been designed for therapeutic approaches and clinical trials. Today there is no treatment available for FSHD patients and therapeutic strategies targeting DUX4 toxicity in skeletal muscle are being actively investigated. In this review, we will discuss different research areas that are currently being considered to alter DUX4 expression and toxicity in muscle tissue and the cell and animal models designed to date.

DUX4 Expression in FSHD Muscles: Focus on Its mRNA Regulation.

Facioscapulohumeral dystrophy (FSHD) is the most frequent muscular disease in adults. FSHD is characterized by a weakness and atrophy of a specific set of muscles located in the face, the shoulder, and the upper arms. FSHD patients may present different genetic defects, but they all present epigenetic alterations of the D4Z4 array located on the subtelomeric part of chromosome 4, leading to chromatin relaxation and, ultimately, to the aberrant expression of one gene called DUX4. Once expressed, DUX4 triggers a cascade of deleterious events, eventually leading to muscle dysfunction and cell death. Here, we review studies on DUX4 expression in skeletal muscle to determine the genetic/epigenetic factors and regulatory proteins governing DUX4 expression, with particular attention to the different transcripts and their very low expression in muscle.

A Deoxyribonucleic Acid Decoy Trapping DUX4 for the Treatment of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral dystrophy (FSHD) is characterized by a loss of repressive epigenetic marks leading to the aberrant expression of the DUX4 transcription factor. In muscle, DUX4 acts as a poison protein though the induction of multiple downstream genes. So far, there is no therapeutic solution for FSHD. Because DUX4 is a transcription factor, we developed an original therapeutic approach, based on a DNA decoy trapping the DUX4 protein, preventing its binding to genomic DNA and thereby blocking the aberrant activation of DUX4's transcriptional network. In vitro, transfection of a DUX4 decoy into FSHD myotubes reduced the expression of the DUX4 network genes. In vivo, both double-stand DNA DUX4 decoys and adeno-associated viruses (AAVs) carrying DUX4 binding sites reduced transcriptional activation of genes downstream of DUX4 in a DUX4-expressing mouse model. Our study demonstrates, both in vitro and in vivo, the feasibility of the decoy strategy and opens new avenues of research.

Gene Editing Targeting the DUX4 Polyadenylation Signal: A Therapy for FSHD?

Facioscapulohumeral dystrophy (FSHD, OMIM: 158900, 158901) is the most common dystrophy in adults and so far, there is no treatment. Different loci of the disease have been characterized and they all lead to the aberrant expression of the DUX4 protein, which impairs the function of the muscle, ultimately leading to cell death. Here, we used gene editing to try to permanently shut down DUX4 expression by targeting its poly(A) sequence. We used transcription activator-like effector nucleases (TALEN) and CRISPR-Cas9 nucleases in vitro on FSHD myoblasts. More than 150 TOPO clones were sequenced and only indels were observed in 4%. Importantly, in 2 of them, the DUX4 poly(A) signal was eliminated at the genomic level but DUX4 mRNA was still produced thanks to the use of a non-canonical upstream poly(A) signal sequence. These experiments show that targeting DUX4 PAS at the genomic level might not be an appropriate gene editing strategy for FSHD therapy.