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.

Meta-analysis towards FSHD reveals misregulation of neuromuscular junction, nuclear envelope, and spliceosome.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common autosomal dominant muscle disorders, yet no cure or amelioration exists. The clinical presentation is diverse, making it difficult to identify the actual driving pathomechanism among many downstream events. To unravel this complexity, we performed a meta-analysis of 13 original omics datasets (in total 171 FSHD and 129 control samples). Our approach confirmed previous findings about the disease pathology and specified them further. We confirmed increased expression of former proposed DUX4 biomarkers, and furthermore impairment of the respiratory chain. Notably, the meta-analysis provides insights about so far not reported pathways, including misregulation of neuromuscular junction protein encoding genes, downregulation of the spliceosome, and extensive alterations of nuclear envelope protein expression. Finally, we developed a publicly available shiny app to provide a platform for researchers who want to search our analysis for genes of interest in the future.

Clinical Application of Optical Genome Mapping for Molecular Diagnosis of Facioscapulohumeral Muscular Dystrophy.

Background: Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy that mainly affects skeletal muscle. FSHD1 accounts for 95% of all FSHD cases and can be diagnosed based on the pathogenic contraction of the D4Z4-repeat array on chromosome 4q35. Genetic diagnosis of FSHD1 is challenging because of the large size and repetitive nature of the D4Z4 region. We evaluated the clinical applicability of optical genome mapping (OGM) for the genetic diagnosis of FSHD1. Methods: We included 25 individuals with clinically confirmed or suspected/probable FSHD and their families. Ultra-high-molecular-weight DNA from peripheral blood was labeled, stained, and imaged using a single-molecule OGM platform (Bionano Genomics Saphyr system). D4Z4 repeat size and haplotype information were analyzed using the manufacturer's dedicated pipeline. We also compared the workflow and test time between Southern blot analysis and OGM. Results: We obtained concordant OGM and Southern blot results with 10 samples from patients with clinically confirmed FSHD. The D4Z4 repeat size differed within 1 unit between the Southern blot analysis and OGM. Among nine patients with clinically suspected or probable FSHD, six patients were confirmed to have pathogenic contractions by OGM. In our cohort, one de novo mosaic FSHD1 patient was successfully diagnosed with OGM. Moreover, OGM has a more straightforward and less time-consuming workflow than Southern blot analysis. Conclusions: OGM enables accurate and reliable detection of pathogenic contraction of the D4Z4-repeat array and is a valuable tool for the genetic diagnosis of FSHD1.

Quality of life and support needs in children, adolescents, and young adults with facioscapulohumeral dystrophy, a mixed-method study.

BACKGROUND AND OBJECTIVES: Quality of life (QoL) in children with facioscapulohumeral dystrophy (FSHD) seems plausible decreased. Little is known about factors influencing QoL in children with FSHD. Our objective is to explore factors contributing to the QoL of children, adolescents, and young adults with FSHD, to describe how they experience life with FSHD, and to report their support needs. METHODS: We performed a mixed-method study with individual age-appropriate semi-structured interviews assessing QoL in children, adolescents, and young adults with FSHD and their parents. To characterize the sample, quantitative data on QoL, pain, fatigue, and participation were collected. Interview data was analyzed using a thematic analysis. RESULTS: Fourteen patients participated (age between 9 and 26 years old, eight males and six females). The degree of FSHD severity, as indicated by the FSHD-score, did not correlate with QoL. Older children had a lower QoL than younger children. Children and adolescents strived for normality regardless of physical discomfort. Phenotypical features of FSHD led to insecurity aggravated by hurtful comments of others. The unpredictability of disease progression and its implications for career and parenthood choices led to a generalized feeling of uncertainty about the future. Support was found within family and friends. Participants expressed a need for peer support and psychological support as well as recommending it to others. DISCUSSION: Quality of life in childhood FSHD is diminished caused by their physical limitations, altered appearance, fear of social rejection, and uncertainty of the disease progression in the future. A fear of social rejection most likely contributes to striving for normality regardless of physical discomfort. Support should be focused on acceptance and coping with hurtful comments. It should preferably be individualized, easily accessible and not offered as therapy but rather as tutoring for children.

DNMT3B splicing dysregulation mediated by SMCHD1 loss contributes to DUX4 overexpression and FSHD pathogenesis.

Structural maintenance of chromosomes flexible hinge domain-containing 1 (SMCHD1) is a noncanonical SMC protein and an epigenetic regulator. Mutations in SMCHD1 cause facioscapulohumeral muscular dystrophy (FSHD), by overexpressing DUX4 in muscle cells. Here, we demonstrate that SMCHD1 is a key regulator of alternative splicing in various cell types. We show how SMCHD1 loss causes splicing alterations of DNMT3B, which can lead to hypomethylation and DUX4 overexpression. Analyzing RNA sequencing data from muscle biopsies of patients with FSHD and Smchd1 knocked out cells, we found mis-splicing of hundreds of genes upon SMCHD1 loss. We conducted a high-throughput screen of splicing factors, revealing the involvement of the splicing factor RBM5 in the mis-splicing of DNMT3B. Subsequent RNA immunoprecipitation experiments confirmed that SMCHD1 is required for RBM5 recruitment. Last, we show that mis-splicing of DNMT3B leads to hypomethylation of the D4Z4 region and to DUX4 overexpression. These results suggest that DNMT3B mis-splicing due to SMCHD1 loss plays a major role in FSHD pathogenesis.

Single-cell spatial transcriptomics reveals a dystrophic trajectory following a developmental bifurcation of myoblast cell fates in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to abnormal derepression of the transcription activator DUX4. This effect is localized to a low percentage of cells, requiring single-cell analysis. However, single-cell/nucleus RNA-seq cannot fully capture the transcriptome of multinucleated large myotubes. To circumvent these issues, we use multiplexed error-robust fluorescent in situ hybridization (MERFISH) spatial transcriptomics that allows profiling of RNA transcripts at a subcellular resolution. We simultaneously examined spatial distributions of 140 genes, including 24 direct DUX4 targets, in in vitro differentiated myotubes and unfused mononuclear cells (MNCs) of control, isogenic D4Z4 contraction mutant and FSHD patient samples, as well as the individual nuclei within them. We find myocyte nuclei segregate into two clusters defined by the expression of DUX4 target genes, which is exclusively found in patient/mutant nuclei, whereas MNCs cluster based on developmental states. Patient/mutant myotubes are found in "FSHD-hi" and "FSHD-lo" states with the former signified by high DUX4 target expression and decreased muscle gene expression. Pseudotime analyses reveal a clear bifurcation of myoblast differentiation into control and FSHD-hi myotube branches, with variable numbers of DUX4 target-expressing nuclei found in multinucleated FSHD-hi myotubes. Gene coexpression modules related to extracellular matrix and stress gene ontologies are significantly altered in patient/mutant myotubes compared with the control. We also identify distinct subpathways within the DUX4 gene network that may differentially contribute to the disease transcriptomic phenotype. Taken together, our MERFISH-based study provides effective gene network profiling of multinucleated cells and identifies FSHD-induced transcriptomic alterations during myoblast differentiation.

Exchange of subtelomeric regions between chromosomes 4q and 10q reverts the FSHD genotype and phenotype.

The most common form of facioscapulohumeral dystrophy (FSHD1) is caused by a partial loss of the D4Z4 macrosatellite repeat array in the subtelomeric region of chromosome 4. Patients with FSHD1 typically carry 1 to 10 D4Z4 repeats, whereas nonaffected individuals have 11 to 150 repeats. The ~150-kilobyte subtelomeric region of the chromosome 10q exhibits a ~99% sequence identity to the 4q, including the D4Z4 array. Nevertheless, contractions of the chr10 array do not cause FSHD or any known disease, as in most people D4Z4 array on chr10 is flanked by the nonfunctional polyadenylation signal, not permitting the DUX4 expression. Here, we attempted to correct the FSHD genotype by a CRISPR-Cas9-induced exchange of the chr4 and chr10 subtelomeric regions. We demonstrated that the induced t(4;10) translocation can generate recombinant genotypes translated into improved FSHD phenotype. FSHD myoblasts with the t(4;10) exhibited reduced expression of the DUX4 targets, restored PAX7 target expression, reduced sensitivity to oxidative stress, and improved differentiation capacity.

Comprehensive genetic analysis of facioscapulohumeral muscular dystrophy by Nanopore long-read whole-genome sequencing.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is a high-prevalence autosomal dominant neuromuscular disease characterized by significant clinical and genetic heterogeneity. Genetic diagnosis of FSHD remains a challenge because it cannot be detected by standard sequencing methods and requires a complex diagnosis workflow. METHODS: We developed a comprehensive genetic FSHD detection method based on Oxford Nanopore Technologies (ONT) whole-genome sequencing. Using a case-control design, we applied this procedure to 29 samples and compared the results with those from optical genome mapping (OGM), bisulfite sequencing (BSS), and whole-exome sequencing (WES). RESULTS: Using our ONT-based method, we identified 59 haplotypes (35 4qA and 24 4qB) among the 29 samples (including a mosaic sample), as well as the number of D4Z4 repeat units (RUs). The pathogenetic D4Z4 RU contraction identified by our ONT-based method showed 100% concordance with OGM results. The methylation levels of the most distal D4Z4 RU and the double homeobox 4 gene (DUX4) detected by ONT sequencing are highly consistent with the BSS results and showed excellent diagnostic efficiency. Additionally, our ONT-based method provided an independent methylation profile analysis of two permissive 4qA alleles, reflecting a more accurate scenario than traditional BSS. The ONT-based method detected 17 variations in three FSHD2-related genes from nine samples, showing 100% concordance with WES. CONCLUSIONS: Our ONT-based FSHD detection method is a comprehensive method for identifying pathogenetic D4Z4 RU contractions, methylation level alterations, allele-specific methylation of two 4qA haplotypes, and variations in FSHD2-related genes, which will all greatly improve genetic testing for FSHD.

Safety and efficacy of losmapimod in facioscapulohumeral muscular dystrophy (ReDUX4): a randomised, double-blind, placebo-controlled phase 2b trial.

BACKGROUND: Facioscapulohumeral muscular dystrophy is a hereditary progressive myopathy caused by aberrant expression of the transcription factor DUX4 in skeletal muscle. No approved disease-modifying treatments are available for this disorder. We aimed to assess the safety and efficacy of losmapimod (a small molecule that inhibits p38α MAPK, a regulator of DUX4 expression, and p38ß MAPK) for the treatment of facioscapulohumeral muscular dystrophy. METHODS: We did a randomised, double-blind, placebo-controlled phase 2b trial at 17 neurology centres in Canada, France, Spain, and the USA. We included adults aged 18-65 years with type 1 facioscapulohumeral muscular dystrophy (ie, with loss of repression of DUX4 expression, as ascertained by genotyping), a Ricci clinical severity score of 2-4, and at least one skeletal muscle judged using MRI to be suitable for biopsy. Participants were randomly allocated (1:1) to either oral losmapimod (15 mg twice a day) or matching placebo for 48 weeks, via an interactive response technology system. The investigator, study staff, participants, sponsor, primary outcome assessors, and study monitor were masked to the treatment allocation until study closure. The primary endpoint was change from baseline to either week 16 or 36 in DUX4-driven gene expression in skeletal muscle biopsy samples, as measured by quantitative RT-PCR. The primary efficacy analysis was done in all participants who were randomly assigned and who had available data for assessment, according to the modified intention-to-treat principle. Safety and tolerability were assessed as secondary endpoints. This study is registered at ClinicalTrials.gov, number NCT04003974. The phase 2b trial is complete; an open-label extension is ongoing. FINDINGS: Between Aug 27, 2019, and Feb 27, 2020, 80 people were enrolled. 40 were randomly allocated to losmapimod and 40 to placebo. 54 (68%) participants were male and 26 (33%) were female, 70 (88%) were White, and mean age was 45·7 (SD 12·5) years. Least squares mean changes from baseline in DUX4-driven gene expression did not differ significantly between the losmapimod (0·83 [SE 0·61]) and placebo (0·40 [0·65]) groups (difference 0·43 [SE 0·56; 95% CI -1·04 to 1·89]; p=0·56). Losmapimod was well tolerated. 29 treatment-emergent adverse events (nine drug-related) were reported in the losmapimod group compared with 23 (two drug-related) in the placebo group. Two participants in the losmapimod group had serious adverse events that were deemed unrelated to losmapimod by the investigators (alcohol poisoning and suicide attempt; postoperative wound infection) compared with none in the placebo group. No treatment discontinuations due to adverse events occurred and no participants died during the study. INTERPRETATION: Although losmapimod did not significantly change DUX4-driven gene expression, it was associated with potential improvements in prespecified structural outcomes (muscle fat infiltration), functional outcomes (reachable workspace, a measure of shoulder girdle function), and patient-reported global impression of change compared with placebo. These findings have informed the design and choice of efficacy endpoints for a phase 3 study of losmapimod in adults with facioscapulohumeral muscular dystrophy. FUNDING: Fulcrum Therapeutics.

Best practice guidelines on genetic diagnostics of facioscapulohumeral muscular dystrophy: Update of the 2012 guidelines.

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.

Muscle strength, quantity and quality and muscle fat quantity and their association with oxidative stress in patients with facioscapulohumeral muscular dystrophy: Effect of antioxidant supplementation.

The purpose of this study was to identify causes of quadriceps muscle weakness in facioscapulohumeral muscular dystrophy (FSHD). To this aim, we evaluated quadriceps muscle and fat volumes by magnetic resonance imaging and their relationships with muscle strength and oxidative stress markers in adult patients with FSHD (n = 32) and healthy controls (n = 7), and the effect of antioxidant supplementation in 20 of the 32 patients with FSHD (n = 10 supplementation and n = 10 placebo) (NCT01596803). Compared with healthy controls, the dominant quadriceps strength and quality (muscle strength per unit of muscle volume) were decreased in patients with FSHD. In addition, fat volume was increased, without changes in total muscle volume. Moreover, in patients with FSHD, the lower strength of the non-dominant quadriceps was associated with lower muscle quality compared with the dominant muscle. Antioxidant supplementation significantly changed muscle and fat volumes in the non-dominant quadriceps, and muscle quality in the dominant quadriceps. This was associated with improved muscle strength (both quadriceps) and antioxidant response. These findings suggest that quadriceps muscle strength decline may not be simply explained by atrophy and may be influenced also by the muscle intrinsic characteristics. As FSHD is associated with increased oxidative stress, supplementation might reduce oxidative stress and increase antioxidant defenses, promoting changes in muscle function.

Optimization of Xenografting Methods for Generating Human Skeletal Muscle in Mice.

Xenografts of human skeletal muscle generated in mice can be used to study muscle pathology and to test drugs designed to treat myopathies and muscular dystrophies for their efficacy and specificity in human tissue. We previously developed methods to generate mature human skeletal muscles in immunocompromised mice starting with human myogenic precursor cells (hMPCs) from healthy individuals and individuals with facioscapulohumeral muscular dystrophy (FSHD). Here, we examine a series of alternative treatments at each stage in order to optimize engraftment. We show that (i) X-irradiation at 25Gy is optimal in preventing regeneration of murine muscle while supporting robust engraftment and the formation of human fibers without significant murine contamination; (ii) hMPC lines differ in their capacity to engraft; (iii) some hMPC lines yield grafts that respond better to intermittent neuromuscular electrical stimulation (iNMES) than others; (iv) some lines engraft better in male than in female mice; (v) coinjection of hMPCs with laminin, gelatin, Matrigel, or Growdex does not improve engraftment; (vi) BaCl2 is an acceptable replacement for cardiotoxin, but other snake venom preparations and toxins, including the major component of cardiotoxin, cytotoxin 5, are not; and (vii) generating grafts in both hindlimbs followed by iNMES of each limb yields more robust grafts than housing mice in cages with running wheels. Our results suggest that replacing cardiotoxin with BaCl2 and engrafting both tibialis anterior muscles generates robust grafts of adult human muscle tissue in mice.

Treatment Approaches for Altered Facial Expression: A Systematic Review in Facioscapulohumeral Muscular Dystrophy and Other Neurological Diseases.

Background: Facial weakness is a key feature of facioscapulohumeral muscular dystrophy (FSHD) and may lead to altered facial expression and subsequent psychosocial impairment. There is no cure and supportive treatments focus on optimizing physical fitness and compensation of functional disabilities. Objective: We hypothesize that symptomatic treatment options and psychosocial interventions for other neurological diseases with altered facial expression could be applicable to FSHD. Therefore, the aim of this review is to collect symptomatic treatment approaches that target facial muscle function and psychosocial interventions in various neurological diseases with altered facial expression in order to discuss the applicability to FSHD. Methods: A systematic search was performed. Selected studies had to include FSHD, Bell's palsy, Moebius syndrome, myotonic dystrophy type 1, or Parkinson's disease and treatment options which target altered facial expression. Data was extracted for study and patients' characteristics, outcome assessment tools, treatment, outcome of facial expression and or psychosocial functioning. Results: Forty studies met the inclusion criteria, of which only three studies included FSHD patients exclusively. Most, twenty-one, studies were performed in patients with Bell's palsy. Studies included twelve different therapy categories and results were assessed with different outcomes measures. Conclusions: Five therapy categories were considered applicable to FSHD: training of (non-verbal) communication compensation strategies, speech training, physical therapy, conference attendance, and smile restoration surgery. Further research is needed to establish the effect of these therapies in FSHD. We recommend to include outcome measures in these studies that cover at least cosmetic, functional, communication, and quality of life domains.

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.

First-trimester noninvasive prenatal diagnosis of seven facioscapulohumeral muscular dystrophy type 1 families using SNP-based amplicon sequencing: An earlier, rapid and safer way.

The study is to explore the feasibility and value of SNP-based noninvasive prenatal diagnosis (NIPD) for facioscapulohumeral muscular dystrophy type 1 (FSHD1) in early pregnancy weeks. We prospectively collected seven FSHD1 families, with an average gestational age of 8+6. Among these seven couples, there were three affected FSHD1 mothers and four affected fathers. A multiplex-PCR panel comprising 402 amplicons was designed to selective enrich for highly heterozygous SNPs upstream of the DUX4 gene. Risk haplotype was constructed based on familial linkage analysis. Fetal genotypes were accurately inferred through relative haplotype dosage analysis using Bayes Factor. All tests were successfully completed in a single attempt, and no recombination events were detected. NIPD results were provided within a week, which is 4 weeks earlier than karyomapping and 7 weeks earlier than Bionano single-molecule optical mapping (BOM). Ultimately, five FSHD1 fetuses and two normal fetuses were successfully identified, with a 100% concordance rate with karyomapping and BOM. Therefore, SNP-based NIPD for FSHD1 was demonstrated to be feasible and accurate in early weeks of gestation, although the risk of recombination events cannot be completely eliminated. In the future, testing of more cases is still necessary to fully determine the clinical utility.

An up-to-date myopathologic characterisation of facioscapulohumeral muscular dystrophy type 1 muscle biopsies shows sarcolemmal complement membrane attack complex deposits and increased skeletal muscle regeneration.

The aim of this study was to identify key routinely used myopathologic biomarkers of FSHD1. Needle muscle biopsies were taken in 34 affected muscles (m. quadriceps femoris (QF), n = 20, m. tibialis anterior (TA), n = 13, m. biceps brachii, n = 1) from 22 patients (age, 53.5 (10) years; M = 12, F = 10). Eleven patients had more than one biopsy (2xQF, n = 1; QF+TA, n = 9; 2xQF+TA, n = 1). Histochemistry, immunoperoxidase, and immunofluorescence stainings were performed and compared to age and muscle type matched muscle specimens of 11 healthy controls. Myopathologic features observed in our FSHD1 cohort were internalized nuclei, type 1 fibre hypertrophy and NADH central clearances/cores. We observed a prominent inflammatory response with MAC deposits, MHC I expression, and muscle regeneration that correlated with the inflammatory score. Our up-to-date characterization of FSHD1 points towards MHC I, MAC, and embryonic Myosin Heavy Chain/muscle regeneration as useful myopathologic readouts of FSHD1.

Fitness and walking outcomes following aerobic and lower extremity strength training in facioscapulohumeral dystrophy: a case series.

Facioscapulohumeral muscular dystrophy (FSHD) is a common form of adult muscular dystrophy often resulting also in cardiorespiratory deconditioning and weakness of the lower limbs. Although previous studies examined outcomes of interventions aimed at improving either cardiorespiratory fitness or muscle strength, the potential benefits of a rehabilitation program targeting both remain unexplored. Thus, the aim of this study was to evaluate changes following participation in a rehabilitation program combining aerobic and strength exercises. We conducted a retrospective analysis of 10 subjects with FSHD who participated in our rehabilitation program during 2018 and 2019. Each of the 20 sessions consisted of aerobic training on a cycloergometer and a moderate lower limb strength exercises on an isokinetic machine in combination with conventional therapy. The primary outcomes were walking speed, aerobic performance and isokinetic strength of the knee extensors and flexors. The secondary outcomes were fatigue, insomnia. VO2max and walking speed increased significantly by 2.125 ml·kg-1·min-1 [95% confidence interval (CI): 0.75-3.62, P = 0.022] and 0.28 m/s (95% CI: 0.16-0.4, P = 0.002), respectively. The effect size was small for V02max (Hedge's g, 0.44; 95% CI: -0.5 to 1.37) and large for walking speed (Hedge's g, 0.99; 95% CI: 0.06-1.92). The knee flexor strength significantly increased at rehabilitation discharge (repeated measures analysis of variance P = 0.004). Positive changes in fatigue and insomnia were also observed. Our preliminary results provide evidence that a relatively short course of a comprehensive rehabilitation program targeting both cardiorespiratory fitness and knee muscle strength can be beneficial for people with FSHD, which warrants further prospective studies.

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.