Muscle eosinophilia is a hallmark of chronic disease in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive myopathy caused by the aberrant increased expression of the DUX4 retrogene in skeletal muscle cells. The DUX4 gene encodes a transcription factor that functions in zygotic genome activation and then is silenced in most adult somatic tissues. DUX4 expression in FSHD disrupts normal muscle cell function; however, the downstream pathogenic mechanisms are still unclear. Histologically, FSHD affected muscles show a characteristic dystrophic phenotype that is often accompanied by a pronounced immune cell infiltration, but the role of the immune system in FSHD is not understood. Previously, we used ACTA1;FLExDUX4 FSHD-like mouse models varying in severity as discovery tools to identify increased Interleukin 6 and microRNA-206 levels as serum biomarkers for FSHD disease severity. In this study, we use the ACTA1;FLExDUX4 chronic FSHD-like mouse model to provide insight into the immune response to DUX4 expression in skeletal muscles. We demonstrate that these FSHD-like muscles are enriched with the chemoattractant eotaxin and the cytotoxic eosinophil peroxidase, and exhibit muscle eosinophilia. We further identified muscle fibers with positive staining for eosinophil peroxidase in human FSHD muscle. Our data supports that skeletal muscle eosinophilia is a hallmark of FSHD pathology.

snRNA-seq analysis in multinucleated myogenic FSHD cells identifies heterogeneous FSHD transcriptome signatures associated with embryonic-like program activation and oxidative stress-induced apoptosis.

The sporadic nature of DUX4 expression in FSHD muscle challenges comparative transcriptome analyses between FSHD and control samples. A variety of DUX4 and FSHD-associated transcriptional changes have been identified, but bulk RNA-seq strategies prohibit comprehensive analysis of their spatiotemporal relation, interdependence and role in the disease process. In this study, we used single-nucleus RNA-sequencing of nuclei isolated from patient- and control-derived multinucleated primary myotubes to investigate the cellular heterogeneity in FSHD. Taking advantage of the increased resolution in snRNA-sequencing of fully differentiated myotubes, two distinct populations of DUX4-affected nuclei could be defined by their transcriptional profiles. Our data provides insights into the differences between these two populations and suggests heterogeneity in two well-known FSHD-associated transcriptional aberrations: increased oxidative stress and inhibition of myogenic differentiation. Additionally, we provide evidence that DUX4-affected nuclei share transcriptome features with early embryonic cells beyond the well-described cleavage stage, progressing into the 8-cell and blastocyst stages. Altogether, our data suggests that the FSHD transcriptional profile is defined by a mixture of individual and sometimes mutually exclusive DUX4-induced responses and cellular state-dependent downstream effects.

FSHD muscle shows perturbation in fibroadipogenic progenitor cells, mitochondrial function and alternative splicing independently of inflammation.

Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable myopathy. FSHD is highly heterogeneous, with patients following a variety of clinical trajectories, complicating clinical trials. Skeletal muscle in FSHD undergoes fibrosis and fatty replacement that can be accelerated by inflammation, adding to heterogeneity. Well controlled molecular studies are thus essential to both categorize FSHD patients into distinct subtypes and understand pathomechanisms. Here, we further analyzed RNA-sequencing data from 24 FSHD patients, each of whom donated a biopsy from both a non-inflamed (TIRM-) and inflamed (TIRM+) muscle, and 15 FSHD patients who donated peripheral blood mononucleated cells (PBMCs), alongside non-affected control individuals. Differential gene expression analysis identified suppression of mitochondrial biogenesis and up-regulation of fibroadipogenic progenitor (FAP) gene expression in FSHD muscle, which was particularly marked on inflamed samples. PBMCs demonstrated suppression of antigen presentation in FSHD. Gene expression deconvolution revealed FAP expansion as a consistent feature of FSHD muscle, via meta-analysis of 7 independent transcriptomic datasets. Clustering of muscle biopsies separated patients in an unbiased manner into clinically mild and severe subtypes, independently of known disease modifiers (age, sex, D4Z4 repeat length). Lastly, the first genome-wide analysis of alternative splicing in FSHD muscle revealed perturbation of autophagy, BMP2 and HMGB1 signalling. Overall, our findings reveal molecular subtypes of FSHD with clinical relevance and identify novel pathomechanisms for this highly heterogeneous condition.

Autosomal dominant in cis D4Z4 repeat array duplication alleles in facioscapulohumeral dystrophy.

Facioscapulohumeral dystrophy (FSHD) has a unique genetic aetiology resulting in partial chromatin relaxation of the D4Z4 macrosatellite repeat array on 4qter. This D4Z4 chromatin relaxation facilitates inappropriate expression of the transcription factor DUX4 in skeletal muscle. DUX4 is encoded by a retrogene that is embedded within the distal region of the D4Z4 repeat array. In the European population, the D4Z4 repeat array is usually organized in a single array that ranges between 8 and 100 units. D4Z4 chromatin relaxation and DUX4 derepression in FSHD is most often caused by repeat array contraction to 1-10 units (FSHD1) or by a digenic mechanism requiring pathogenic variants in a D4Z4 chromatin repressor like SMCHD1, combined with a repeat array between 8 and 20 units (FSHD2). With a prevalence of 1.5% in the European population, in cis duplications of the D4Z4 repeat array, where two adjacent D4Z4 arrays are interrupted by a spacer sequence, are relatively common but their relationship to FSHD is not well understood. In cis duplication alleles were shown to be pathogenic in FSHD2 patients; however, there is inconsistent evidence for the necessity of an SMCHD1 mutation for disease development. To explore the pathogenic nature of these alleles we compared in cis duplication alleles in FSHD patients with or without pathogenic SMCHD1 variant. For both groups we showed duplication-allele-specific DUX4 expression. We studied these alleles in detail using pulsed-field gel electrophoresis-based Southern blotting and molecular combing, emphasizing the challenges in the characterization of these rearrangements. Nanopore sequencing was instrumental to study the composition and methylation of the duplicated D4Z4 repeat arrays and to identify the breakpoints and the spacer sequence between the arrays. By comparing the composition of the D4Z4 repeat array of in cis duplication alleles in both groups, we found that specific combinations of proximal and distal repeat array sizes determine their pathogenicity. Supported by our algorithm to predict pathogenicity, diagnostic laboratories should now be furnished to accurately interpret these in cis D4Z4 repeat array duplications, alleles that can easily be missed in routine settings.

Facioscapulohumeral Muscular Dystrophy is Associated With Altered Myoblast Proteome Dynamics.

Proteomic studies in facioscapulohumeral muscular dystrophy (FSHD) could offer new insight into disease mechanisms underpinned by post-transcriptional processes. We used stable isotope (deuterium oxide; D2O) labeling and peptide mass spectrometry to investigate the abundance and turnover rates of proteins in cultured muscle cells from two individuals affected by FSHD and their unaffected siblings (UASb). We measured the abundance of 4420 proteins and the turnover rate of 2324 proteins in each (n = 4) myoblast sample. FSHD myoblasts exhibited a greater abundance but slower turnover rate of subunits of mitochondrial respiratory complexes and mitochondrial ribosomal proteins, which may indicate an accumulation of "older" less viable mitochondrial proteins in myoblasts from individuals affected by FSHD. Treatment with a 2'-O-methoxyethyl modified antisense oligonucleotide targeting exon 3 of the double homeobox 4 (DUX4) transcript tended to reverse mitochondrial protein dysregulation in FSHD myoblasts, indicating the effect on mitochondrial proteins may be a DUX4-dependent mechanism. Our results highlight the importance of post-transcriptional processes and protein turnover in FSHD pathology and provide a resource for the FSHD research community to explore this burgeoning aspect of FSHD.

Characterization of D4Z4 alleles and assessment of de novo cases in Facioscapulohumeral dystrophy (FSHD) in a cohort of Italian families.

Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant disease, although 10%-30% of cases are sporadic. However, this percentage may include truly de novo patients (carrying a reduced D4Z4 allele that is not present in either of the parents) and patients with apparently sporadic disease resulting from mosaicism, non-penetrance, or complex genetic situations in either patients or parents. In this study, we characterized the D4Z4 Reduced Alleles (DRA) and evaluated the frequency of truly de novo cases in FSHD1 in a cohort of DNA samples received consecutively for FSHD-diagnostic from 100 Italian families. The D4Z4 testing revealed that 60 families reported a DRA compatible with FSHD1 (1-10 RU). The DRA co-segregated with the disease in most cases. Five families with truly de novo cases were identified, suggesting that this condition may be slightly lower (8%) than previously reported. In addition, D4Z4 characterization in the investigated families showed 4% of mosaic cases and 2% with translocations. This study further highlighted the importance of performing family studies for clarifying apparently sporadic FSHD cases, with significant implications for genetic counseling, diagnosis, clinical management, and procreative choices for patients and families.

A longitudinal study of disease progression in facioscapulohumeral muscular dystrophy (FSHD).

INTRODUCTION/AIMS: In preparation for clinical trials, it is important to better understand how disease burden changes over time in facioscapulohumeral muscular dystrophy (FSHD) and to assess the capability of select metrics to detect these changes. This study aims to evaluate FSHD disease progression over 1 year and to examine the sensitivity of several outcome measures in detecting changes during this interval. METHODS: We conducted a 12-month prospective observational study of 41 participants with FSHD. Participants were evaluated at baseline, 6 months, and 12 months with serial strength testing (manual muscle testing or MMT and maximum voluntary isometric contraction testing or MVICT), functional testing (FSHD-Composite Outcome Measure or FSHD-COM, FSHD Clinical Severity Score or CSS, and FSHD Evaluation Score or FES), sleep and fatigue assessments, lean body mass measurements, respiratory testing, and the FSHD-Health Index patient-reported outcome. Changes in these outcome measures were assessed over the 12-month period. Associations between changes in outcome measures and both age and sex were also examined. RESULTS: In a 12-month period, FSHD participant function remained largely stable with a mild worsening of strength, measured by MMT and standardized MVICT scores, and a mild loss in lean body mass. DISCUSSION: The abilities and disease burden of adults with FSHD are largely static over a 12-month period with participants demonstrating a mild average reduction in some measures of strength. Selection of patients, outcome measures, and trial duration should be carefully considered during the design and implementation of future clinical studies involving FSHD patients.

Muscle diffusion tensor imaging in facioscapulohumeral muscular dystrophy.

INTRODUCTION/AIMS: Muscle diffusion tensor imaging has not yet been explored in facioscapulohumeral muscular dystrophy (FSHD). We assessed diffusivity parameters in FSHD subjects compared with healthy controls (HCs), with regard to their ability to precede any fat replacement or edema. METHODS: Fat fraction (FF), water T2 (wT2), mean, radial, axial diffusivity (MD, RD, AD), and fractional anisotropy (FA) of thigh muscles were calculated in 10 FSHD subjects and 15 HCs. All parameters were compared between FSHD and controls, also exploring their gradient along the main axis of the muscle. Diffusivity parameters were tested in a subgroup analysis as predictors of disease involvement in muscle compartments with different degrees of FF and wT2 and were also correlated with clinical severity scores. RESULTS: We found that MD, RD, and AD were significantly lower in FSHD subjects than in controls, whereas we failed to find a difference for FA. In contrast, we found a significant positive correlation between FF and FA and a negative correlation between MD, RD, and AD and FF. No correlation was found with wT2. In our subgroup analysis we found that muscle compartments with no significant fat replacement or edema (FF < 10% and wT2 < 41 ms) showed a reduced AD and FA compared with controls. Less involved compartments showed different diffusivity parameters than more involved compartments. DISCUSSION: Our exploratory study was able to demonstrate diffusivity parameter abnormalities even in muscles with no significant fat replacement or edema. Larger cohorts are needed to confirm these preliminary findings.

Methylation of the 4q35 D4Z4 repeat defines disease status in facioscapulohumeral muscular dystrophy.

Genetic diagnosis of facioscapulohumeral muscular dystrophy (FSHD) remains a challenge in clinical practice as it cannot be detected by standard sequencing methods despite being the third most common muscular dystrophy. The conventional diagnostic strategy addresses the known genetic parameters of FSHD: the required presence of a permissive haplotype, a size reduction of the D4Z4 repeat of chromosome 4q35 (defining FSHD1) or a pathogenic variant in an epigenetic suppressor gene (consistent with FSHD2). Incomplete penetrance and epistatic effects of the underlying genetic parameters as well as epigenetic parameters (D4Z4 methylation) pose challenges to diagnostic accuracy and hinder prediction of clinical severity. In order to circumvent the known limitations of conventional diagnostics and to complement genetic parameters with epigenetic ones, we developed and validated a multistage diagnostic workflow that consists of a haplotype analysis and a high-throughput methylation profile analysis (FSHD-MPA). FSHD-MPA determines the average global methylation level of the D4Z4 repeat array as well as the regional methylation of the most distal repeat unit by combining bisulphite conversion with next-generation sequencing and a bioinformatics pipeline and uses these as diagnostic parameters. We applied the diagnostic workflow to a cohort of 148 patients and compared the epigenetic parameters based on FSHD-MPA to genetic parameters of conventional genetic testing. In addition, we studied the correlation of repeat length and methylation level within the most distal repeat unit with age-corrected clinical severity and age at disease onset in FSHD patients. The results of our study show that FSHD-MPA is a powerful tool to accurately determine the epigenetic parameters of FSHD, allowing discrimination between FSHD patients and healthy individuals, while simultaneously distinguishing FSHD1 and FSHD2. The strong correlation between methylation level and clinical severity indicates that the methylation level determined by FSHD-MPA accounts for differences in disease severity among individuals with similar genetic parameters. Thus, our findings further confirm that epigenetic parameters rather than genetic parameters represent FSHD disease status and may serve as a valuable biomarker for disease status.

Physical activity practiced at a young age is associated with a less severe subsequent clinical presentation in facioscapulohumeral muscular dystrophy.

BACKGROUND: In facioscapulohumeral muscular dystrophy (FSHD), it is not known whether physical activity (PA) practiced at young age is associated with the clinical presentation of disease. To assess this issue, we performed a retrospective cohort study concerning the previous practice of sports and, among them, those with medium-high cardiovascular commitment in clinically categorized carriers of a D4Z4 reduced allele (DRA). METHODS: People aged between 18 and 60 were recruited as being DRA carriers. Subcategory (classical phenotype, A; incomplete phenotype, B; asymptomatic carriers, C; complex phenotype, D) and FSHD score, which measures muscle functional impairment, were assessed for all participants. Information on PAs was retrieved by using an online survey dealing with the practice of sports at a young age. RESULTS: 368 participants were included in the study, average age 36.6 years (SD = 9.4), 47.6% male. The FSHD subcategory A was observed in 157 (42.7%) participants with average (± SD) FSHD score of 5.8 ± 3.0; the incomplete phenotype (category B) in 46 (12.5%) participants (average score 2.2 ± 1.7) and the D phenotype in 61 (16.6%, average score 6.5 ± 3.8). Asymptomatic carriers were 104 (subcategory C, 28.3%, score 0.0 ± 0.2). Time from symptoms onset was higher for patients with A (15.8 ± 11.1 years) and D phenotype (13.3 ± 11.9) than for patients with B phenotype (7.3 ± 9.0). The practice of sports was associated with lower FSHD score (-17%) in participants with A phenotype (MR = 0.83, 95% CI = 0.73-0.95, p = 0.007) and by 33% in participants with D phenotype (MR = 0.67, 95% CI = 0.51-0.89, p = 0.006). Conversely, no improvement was observed in participants with incomplete phenotype with mild severity (B). CONCLUSIONS: PAs at a young age are associated with a lower clinical score in the adult A and D FSHD subcategories. These results corroborate the need to consider PAs at the young age as a fundamental indicator for the correct clinical stratification of the disease and its possible evolution.

The DUX4-HIF1α Axis in Murine and Human Muscle Cells: A Link More Complex Than Expected.

FacioScapuloHumeral muscular Dystrophy (FSHD) is one of the most prevalent inherited muscle disorders and is linked to the inappropriate expression of the DUX4 transcription factor in skeletal muscles. The deregulated molecular network causing FSHD muscle dysfunction and pathology is not well understood. It has been shown that the hypoxia response factor HIF1α is critically disturbed in FSHD and has a major role in DUX4-induced cell death. In this study, we further explored the relationship between DUX4 and HIF1α. We found that the DUX4 and HIF1α link differed according to the stage of myogenic differentiation and was conserved between human and mouse muscle. Furthermore, we found that HIF1α knockdown in a mouse model of DUX4 local expression exacerbated DUX4-mediated muscle fibrosis. Our data indicate that the suggested role of HIF1α in DUX4 toxicity is complex and that targeting HIF1α might be challenging in the context of FSHD therapeutic approaches.

Systemic Pharmacotherapeutic Treatment of the ACTA1-MCM/FLExDUX4 Preclinical Mouse Model of FSHD.

Aberrant expression of the double homeobox 4 (DUX4) gene in skeletal muscle predominantly drives the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD). We recently demonstrated that berberine, an herbal extract known for its ability to stabilize guanine-quadruplex structures, effectively downregulates DUX4 expression in FSHD patient-derived myoblasts and in mice overexpressing exogenous DUX4 after viral vector-based treatment. Here, we sought to confirm berberine's inhibitory efficacy on DUX4 in the widely used FSHD-like transgenic mouse model, ACTA1-MCM/FLExDUX4, where DUX4 is induced at pathogenic levels using tamoxifen. Animals repeatedly treated with berberine via intraperitoneal injections for 4 weeks exhibited significant reductions in both mRNA and protein levels of DUX4, and in mRNA expression of murine DUX4-related genes. This inhibition translated into improved forelimb muscle strength and positive alterations in important FSHD-relevant cellular pathways, although its impact on muscle mass and histopathology was less pronounced. Collectively, our data confirm the efficacy of berberine in downregulating DUX4 expression in the most relevant FSHD mouse model. However, further optimization of dosing regimens and new studies to enhance the bioavailability of berberine in skeletal muscle are warranted to fully leverage its therapeutic potential for FSHD treatment.

Epigenetic profiling of the D4Z4 locus: Optimization of the protocol for studying DNA methylation at single CpG site level.

The alteration of epigenetic modifications, including DNA methylation, can contribute to the etiopathogenesis and progression of many diseases. Among them, facioscapulohumeral dystrophy (FSHD) is a muscular disorder characterized by the loss of repressive epigenetic features affecting the D4Z4 locus (4q35). As a consequence, these alterations are responsible for DNA hypomethylation and a transcriptional-active chromatin conformation change that, in turn, lead to the aberrant expression of DUX4 in muscle cells. In the present study, methylation levels of 29 CpG sites of the DR1 region (within each repeat unit of the D4Z4 macrosatellite) were assessed on 335 subjects by employing primers designed for enhancing the performance of the assay. First, the DR1 original primers were optimized by adding M13 oligonucleotide tails. Moreover, the DR1 reverse primer was replaced with a degenerate one. As a result, the protocol optimization allowed a better sequencing resolution and a more accurate evaluation of DR1 methylation levels. Moreover, the assessment of the repeatability of measurements proved the reliability and robustness of the assay. The optimized protocol emerges as an excellent method to detect methylation levels compatible with FSHD.

Three-dimensional quantitative muscle ultrasound in patients with facioscapulohumeral dystrophy and myotonic dystrophy.

INTRODUCTION/AIMS: Ultrasound imaging of muscle tissue conventionally results in two-dimensional sampling of tissue. For heterogeneously affected muscles, a sampling error using two-dimensional (2D) ultrasound can therefore be expected. In this study, we aimed to quantify and extend ultrasound imaging findings in neuromuscular disorders by using three-dimensional quantitative muscle ultrasound (3D QMUS). METHODS: Patients with facioscapulohumeral dystrophy (n = 31) and myotonic dystrophy type 1 (n = 16) were included in this study. After physical examination, including Medical Research Council (MRC) scores, the tibialis anterior muscle was scanned with automated ultrasound. QMUS parameters were calculated over 15 cm of the length of the tibialis anterior muscle and were compared with a healthy reference data set. RESULTS: With 3D QMUS local deviations from the healthy reference could be detected. Significant Pearson correlations (P < .01) between MRC score and QMUS parameters in male patients (n = 23) included the mean echo intensity (EI) (0.684), the standard deviation of EI (0.737), and the residual attenuation (0.841). In 91% of all patients, mean EI deviated by more than 1 standard deviation from the healthy reference. In general, the proportion of muscle tissue with a Z score >1 was about 50%. DISCUSSION: In addition to mean EI, multiple QMUS parameters reported in this study are potential biomarkers for pathology. Besides a moderate correlation of mean EI with muscle weakness, two other parameters showed strong correlations: standard deviation of EI and residual attenuation. Local detection of abnormalities makes 3D QMUS a promising method that can be used in research and potentially for clinical evaluation.

Outcomes of scapulothoracic fusion in patients with facioscapulohumeral dystrophy: a comparison of allograft versus autograft bone grafting.

BACKGROUND: Scapulothoracic (ST) fusion for facioscapulohumeral muscular dystrophy (FSHD) is an established treatment that corrects scapular instability, although it has high reported complication rates. The purpose of our study was to characterize the outcomes of ST fusion for FSHD in a large patient cohort and compare the outcomes based on bone graft type and fixation technique. Our hypothesis was that union rates would not differ by bone graft type during ST fusion. METHODS: A retrospective chart review was undertaken to identify patients who underwent ST fusion at multiple institutions performed by a single surgeon between 2013 and 2019 with minimum 2-year follow-up. Patient demographic characteristics, surgical technique, time to union, complications, and clinical outcomes including patient-reported outcome measures were recorded. Univariate and multivariate statistical analyses including regression analyses were performed to compare preoperative and postoperative outcomes. RESULTS: A total of 50 patients with 54 ST fusions (bilateral in 4 patients) and an average follow-up period of 5.8 years (standard deviation, 1.6 years) were included for analysis. Active forward elevation (77° vs. 124°, P < .00001) and abduction (60° vs. 90°, P < .00001) both improved significantly after fusion. Average internal rotation after fusion was at spinal level L3-L4. The visual analog scale pain score (2.6 vs. 1.2, P < .00001), Subjective Shoulder Value score (33 vs. 76, P < .00001), and American Shoulder and Elbow Surgeons (ASES) score (41.8 vs. 76.1, P < .00001) all improved significantly postoperatively. Of the shoulders, 50% (27 of 54) received treatment with cerclage wires and 50% (27 of 54) received treatment with Luque wires. Femoral head allograft was used in 53.7% of shoulders (29 of 54), whereas iliac crest autograft was used in 46.3% (25 of 54). Average radiographic time to healing was 11.1 weeks (standard deviation, 3.2 weeks), with no incidence of nonunion, and did not significantly differ by bone graft type (P = .26) or technique (P = .20). The complication rate was 24.1%, including seroma (n = 3), superficial infection (n = 2), transient neurologic injury (n = 2), hemothorax (n = 1), rib fracture (n = 1), pneumothorax (n = 1), and shortness of breath (n = 1), although none requiring reoperation. There was no significant difference in the rate of postoperative complications when compared by surgical technique (P = .81) and bone graft type (P = .93). There were no independently predictive factors influencing the rate of postoperative complications by multivariate regression. Regression analysis showed that the postoperative ASES score was independently associated with the preoperative ASES score (P < .0001), use of iliac crest autograft (P < .011), and presence of complications (P < .043). CONCLUSION: Patients receiving ST fusion for FSHD demonstrate globally improved active motion and patient-reported outcome measures. Fusion construct or type of bone graft does not affect time to union or complication rates. Surgeons should be aware of a relatively high complication rate in the early postoperative period.

Interaction between mesenchymal stem cells and myoblasts in the context of facioscapulohumeral muscular dystrophy contributes to the disease phenotype.

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic disease associated with ectopic expression of the DUX4 gene in skeletal muscle. Muscle degeneration in FSHD is accompanied by muscle tissue replacement with fat and connective tissue. Expression of DUX4 in myoblasts stimulates mesenchymal stem cells (MSC) migration via the CXCR4-CXCL12 axis. MSCs participate in adipose and connective tissue formation and can contribute to fibrosis. Here we studied the interaction between myoblasts and MSCs and the consequences of this interaction in the FSHD context. We used cell motility assays and coculture of MSCs with myoblasts to study their mutual effects on cell migration, differentiation, proliferation, and extracellular matrix formation. The growth medium conditioned by FSHD myoblasts stimulated MSCs migration 1.6-fold (p < 0.04) compared to nonconditioned medium. Blocking the CXCL12-CXCR4 axis with the CXCR4 inhibitor (AMD3100) or neutralizing antibodies to CXCL12 abolished this effect. FSHD myoblasts stimulated MSC proliferation 1.5-2 times (p < 0.05) compared to control myoblasts, while the presence of MSCs impaired myoblast differentiation. Under inflammatory conditions, medium conditioned by FSHD myoblasts stimulated collagen secretion by MSCs 2.2-fold as compared to the nonconditioned medium, p < 0.03. FSHD myoblasts attract MSCs via the CXCL12-CXCR4 axis, stimulate MSC proliferation and collagen secretion by MSCs. Interaction between MSCs and FSHD myoblasts accounts for several important aspects of FSHD pathophysiology. The CXCL12-CXCR4 axis may serve as a potential target to improve the state of the diseased muscles.

The Genetics and Epigenetics of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD), a progressive myopathy that afflicts individuals of all ages, provides a powerful model of the complex interplay between genetic and epigenetic mechanisms of chromatin regulation. FSHD is caused by dysregulation of a macrosatellite repeat, either by contraction of the repeat or by mutations in silencing proteins. Both cases lead to chromatin relaxation and, in the context of a permissive allele, aberrant expression of the DUX4 gene in skeletal muscle. DUX4 is a pioneer transcription factor that activates a program of gene expression during early human development, after which its expression is silenced in most somatic cells. When misexpressed in FSHD skeletal muscle, the DUX4 program leads to accumulated muscle pathology. Epigenetic regulators of the disease locus represent particularly attractive therapeutic targets for FSHD, as many are not global modifiers of the genome, and altering their expression or activity should allow correction of the underlying defect.

Randomized phase 2 study of ACE-083, a muscle-promoting agent, in facioscapulohumeral muscular dystrophy.

INTRODUCTION/AIMS: Facioscapulohumeral muscular dystrophy (FSHD) is a slowly progressive muscular dystrophy without approved therapies. In this study we evaluated whether locally acting ACE-083 could safely increase muscle volume and improve functional outcomes in adults with FSHD. METHODS: Participants were at least 18 years old and had FSHD1/FSHD2. Part 1 was open label, ascending dose, assessing safety and tolerability (primary objective). Part 2 was randomized, double-blind for 6 months, evaluating ACE-083240 mg/muscle vs placebo injected bilaterally every 3 weeks in the biceps brachii (BB) or tibialis anterior (TA) muscles, followed by 6 months of open label. Magnetic resonance imaging measures included total muscle volume (TMV; primary objective), fat fraction (FF), and contractile muscle volume (CMV). Functional measures included 6-minute walk test, 10-meter walk/run, and 4-stair climb (TA group), and performance of upper limb midlevel/elbow score (BB group). Strength, patient-reported outcomes (PROs), and safety were also evaluated. RESULTS: Parts 1 and 2 enrolled 37 and 58 participants, respectively. Among 55 participants evaluable in Part 2, the least-squares mean (90% confidence interval, analysis of covariance) treatment difference for TMV was 16.4% (9.8%-23.0%) in the BB group (P < .0001) and 9.5% (3.2%-15.9%) in the TA group (P = .01). CMV increased significantly in the BB and TA groups and FF decreased in the TA group. There were no consistent improvements in functional or PRO measures in either group. The most common adverse events were mild or moderate injection-site reactions. DISCUSSION: Significant increases in TMV with ACE-083 vs placebo did not result in consistent functional or PRO improvements with up to 12 months of treatment.

DUX4 Transcript Knockdown with Antisense 2'-O-Methoxyethyl Gapmers for the Treatment of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder characterized by a progressive, asymmetric weakening of muscles, starting with those in the upper body. It is caused by aberrant expression of the double homeobox protein 4 gene (DUX4) in skeletal muscle. FSHD is currently incurable. We propose to develop a therapy for FSHD using antisense 2'-O-methoxyethyl (2'-MOE) gapmers, to knock down DUX4 mRNA expression. Using immortalized patient-derived muscle cells and local intramuscular injections in the FLExDUX4 FSHD mouse model, we showed that our designed 2'-MOE gapmers significantly reduced DUX4 transcript levels in vitro and in vivo, respectively. Furthermore, in vitro, we observed significantly reduced expression of DUX4-activated downstream targets, restoration of FSHD signature genes by RNA sequencing, significant improvements in myotube morphology, and minimal off-target activity. This work facilitates the development of a promising candidate therapy for FSHD and lays down the foundation for in vivo systemic treatment studies.

Relationship of DUX4 and target gene expression in FSHD myocytes.

Facioscapulohumeral dystrophy (FSHD) is associated with the upregulation of the DUX4 transcription factor and its target genes. However, low-frequency DUX4 upregulation in patient myocytes is difficult to detect and examining the relationship and dynamics of DUX4 and target gene expression has been challenging. Using RNAScope in situ hybridization with highly specific probes, we detect the endogenous DUX4 and target gene transcripts in situ in patient skeletal myotubes during 13-day differentiation in vitro. We found that the endogenous DUX4 transcripts primarily localize as foci in one or two nuclei as compared with the accumulation of the recombinant DUX4 transcripts in the cytoplasm. We also found the continuous increase of DUX4 and target gene-positive myotubes after Day 3, arguing against its expected immediate cytotoxicity. Interestingly, DUX4 and target gene expression become discordant later in differentiation with the increase of DUX4-positive/target gene-negative as well as DUX4-negative/target gene-positive myotubes. Depletion of DUX4-activated transcription factors, DUXA and LEUTX, specifically repressed a DUX4-target gene, KDM4E, later in differentiation, suggesting that after the initial activation by DUX4, target genes themselves contribute to the maintenance of downstream gene expression. Together, the study provides important new insights into the dynamics of the DUX4 transcriptional network in FSHD patient myocytes.