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.

Regional and bilateral MRI and gene signatures in facioscapulohumeral dystrophy: implications for clinical trial design and mechanisms of disease progression.

Identifying the aberrant expression of DUX4 in skeletal muscle as the cause of facioscapulohumeral dystrophy (FSHD) has led to rational therapeutic development and clinical trials. Several studies support the use of MRI characteristics and the expression of DUX4-regulated genes in muscle biopsies as biomarkers of FSHD disease activity and progression. We performed lower-extremity MRI and muscle biopsies in the mid-portion of the tibialis anterior (TA) muscles bilaterally in FSHD subjects and validated our prior reports of the strong association between MRI characteristics and expression of genes regulated by DUX4 and other gene categories associated with FSHD disease activity. We further show that measurements of normalized fat content in the entire TA muscle strongly predict molecular signatures in the mid-portion of the TA, indicating that regional biopsies can accurately measure progression in the whole muscle and providing a strong basis for inclusion of MRI and molecular biomarkers in clinical trial design. An unanticipated finding was the strong correlations of molecular signatures in the bilateral comparisons, including markers of B-cells and other immune cell populations, suggesting that a systemic immune cell infiltration of skeletal muscle might have a role in disease progression.

Validation of the association between MRI and gene signatures in facioscapulohumeral dystrophy muscle: implications for clinical trial design.

Identifying the aberrant expression of DUX4 in skeletal muscle as the cause of facioscapulohumeral dystrophy (FSHD) has led to rational therapeutic development and clinical trials. Several studies support the use of MRI characteristics and the expression of DUX4-regulated genes in muscle biopsies as biomarkers of FSHD disease activity and progression, but reproducibility across studies needs further validation. We performed lower-extremity MRI and muscle biopsies in the mid-portion of the tibialis anterior (TA) muscles bilaterally in FSHD subjects and validated our prior reports of the strong association between MRI characteristics and expression of genes regulated by DUX4 and other gene categories associated with FSHD disease activity. We further show that measurements of normalized fat content in the entire TA muscle strongly predict molecular signatures in the mid-portion of the TA. Together with moderate-to-strong correlations of gene signatures and MRI characteristics between the TA muscles bilaterally, these results suggest a whole muscle model of disease progression and provide a strong basis for inclusion of MRI and molecular biomarkers in clinical trial design.

Human DUX4 and porcine DUXC activate similar early embryonic programs in pig muscle cells: implications for preclinical models of FSHD.

Human DUX4 and its mouse ortholog Dux are normally expressed in the early embryo-the 4-cell or 2-cell cleavage stage embryo, respectively-and activate a portion of the first wave of zygotic gene expression. DUX4 is epigenetically suppressed in nearly all somatic tissue, whereas facioscapulohumeral dystrophy (FSHD)-causing mutations result in its aberrant expression in skeletal muscle, transcriptional activation of the early embryonic program and subsequent muscle pathology. Although DUX4 and Dux both activate an early totipotent transcriptional program, divergence of their DNA binding domains limits the use of DUX4 expressed in mice as a preclinical model for FSHD. In this study, we identify the porcine DUXC messenger ribonucleic acid expressed in early development and show that both pig DUXC and human DUX4 robustly activate a highly similar early embryonic program in pig muscle cells. These results support further investigation of pig preclinical models for FSHD.

FSHD Therapeutic Strategies: What Will It Take to Get to Clinic?

Facioscapulohumeral muscular dystrophy (FSHD) is arguably one of the most challenging genetic diseases to understand and treat. The disease is caused by epigenetic 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, pathogenic misexpression of DUX4 in skeletal muscle. The complex nature of the locus and the fact that FSHD is a toxic, gain-of-function disease present unique challenges for the design of therapeutic strategies. There are three major DUX4-targeting avenues of therapy for FSHD: small molecules, oligonucleotide therapeutics, and CRISPR-based approaches. Here, we evaluate the preclinical progress of each avenue, and discuss efforts being made to overcome major hurdles to translation.

Cross-sectional Neuromuscular Phenotyping Study of Patients With Arhinia With SMCHD1 Variants.

BACKGROUND AND OBJECTIVES: Facioscapulohumeral muscular dystrophy type 2 (FSHD2) and arhinia are 2 distinct disorders caused by pathogenic variants in the same gene: SMCHD1. The mechanism underlying this phenotypic divergence remains unclear. In this study, we characterize the neuromuscular phenotype of individuals with arhinia caused by SMCHD1 variants and analyze their complex genetic and epigenetic criteria to assess their risk for FSHD2. METHODS: Eleven individuals with congenital nasal anomalies, including arhinia, nasal hypoplasia, or anosmia, underwent a neuromuscular examination, genetic testing, muscle ultrasound, and muscle MRI. Risk for FSHD2 was determined by combined genetic and epigenetic analysis of 4q35 haplotype, D4Z4 repeat length, and methylation profile. We also compared expression levels of pathogenic DUX4 mRNA in primary myoblasts or dermal fibroblasts (upon myogenic differentiation or epigenetic transdifferentiation, respectively) in these individuals vs those with confirmed FSHD2. RESULTS: Among the 11 individuals with rare, pathogenic, heterozygous missense variants in exons 3-11 of SMCHD1, only a subset (n = 3/11; 1 male, 2 female; age 25-51 years) met the strict genetic and epigenetic criteria for FSHD2 (D4Z4 repeat unit length <21 in cis with a 4qA haplotype and D4Z4 methylation <30%). None of the 3 individuals had typical clinical manifestations or muscle imaging findings consistent with FSHD2. However, the patients with arhinia meeting the permissive genetic and epigenetic criteria for FSHD2 displayed some DUX4 expression in dermal fibroblasts under the epigenetic de-repression by drug treatment and in the primary myoblasts undergoing myogenic differentiation. DISCUSSION: In this cross-sectional study, we identified patients with arhinia who meet the full genetic and epigenetic criteria for FSHD2 and display the molecular hallmark of FSHD-DUX4 de-repression and expression in vitro-but who do not manifest with the typical clinicopathologic phenotype of FSHD2. The distinct dichotomy between FSHD2 and arhinia phenotypes despite an otherwise poised DUX4 locus implies the presence of novel disease-modifying factors that seem to operate as a switch, resulting in one phenotype and not the other. Identification and further understanding of these disease-modifying factors will provide valuable insight with therapeutic implications for both diseases.

Identification of Serum Interleukin 6 Levels as a Disease Severity Biomarker in Facioscapulohumeral Muscular Dystrophy.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common myopathies in adults, displaying a progressive, frequently asymmetric involvement of a typical muscles' pattern. FSHD is associated with epigenetic derepression of the polymorphic D4Z4 repeat on chromosome 4q, leading to DUX4 retrogene toxic expression in skeletal muscles. Identifying biomarkers that correlate with disease severity would facilitate clinical management and assess potential FSHD therapeutics' efficacy. OBJECTIVES: This study purpose was to analyze serum cytokines to identify potential biomarkers in a large cohort of adult patients with FSHD. METHODS: We retrospectively measured the levels of 20 pro-inflammatory and regulatory cytokines in sera from 100 genetically confirmed adult FSHD1 patients. Associations between cytokine concentrations and various clinical scores were investigated. We then measured serum and muscle interleukin 6 (IL-6) levels in a validated FSHD-like mouse model, ranging in severity and DUX4 expression. RESULTS: IL-6 was identified as the only cytokine with a concentration correlating with several clinical severity and functional scores, including Clinical Severity Score, Manual Muscle Testing sum score, Brooke and Vignos scores. Further, FSHD patients displayed overall IL-6 levels more than twice high as control, and patients with milder phenotypes exhibited lower IL-6 serum concentration than those with severe muscular weakness. Lastly, an FSHD-like mouse model analysis confirmed that IL-6 levels positively correlate with disease severity and DUX4 expression. CONCLUSIONS: Serum IL-6, therefore, shows promise as a serum biomarker of FSHD severity in a large cohort of FSHD1 adult patients.

Identification of candidate miRNA biomarkers for facioscapulohumeral muscular dystrophy using DUX4-based mouse models.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by misexpression of DUX4 in skeletal myocytes. As DUX4 is the key therapeutic target in FSHD, surrogate biomarkers of DUX4 expression in skeletal muscle are critically needed for clinical trials. Although no natural animal models of FSHD exist, transgenic mice with inducible DUX4 expression in skeletal muscles rapidly develop myopathic phenotypes consistent with FSHD. Here, we established a new, more-accurate FSHD-like mouse model based on chronic DUX4 expression in a small fraction of skeletal myonuclei that develops pathology mimicking key aspects of FSHD across its lifespan. Utilizing this new aged mouse model and DUX4-inducible mouse models, we characterized the DUX4-related microRNA signatures in skeletal muscles, which represent potential biomarkers for FSHD. We found increased expression of miR-31-5p and miR-206 in muscles expressing different levels of DUX4 and displaying varying degrees of pathology. Importantly, miR-206 expression is significantly increased in serum samples from FSHD patients compared with healthy controls. Our data support miR-31-5p and miR-206 as new potential regulators of muscle pathology and miR-206 as a potential circulating biomarker for FSHD. This article has an associated First Person interview with the first author of the paper.

Precise Epigenetic Analysis Using Targeted Bisulfite Genomic Sequencing Distinguishes FSHD1, FSHD2, and Healthy Subjects.

The true prevalence of facioscapulohumeral muscular dystrophy (FSHD) is unknown due to difficulties with accurate clinical evaluation and the complexities of current genetic diagnostics. Interestingly, all forms of FSHD are linked to epigenetic changes in the chromosome 4q35 D4Z4 macrosatellite, suggesting that epigenetic analysis could provide an avenue for sequence-based FSHD diagnostics. However, studies assessing DNA methylation at the FSHD locus have produced conflicting results; thus, the utility of this technique as an FSHD diagnostic remains controversial. Here, we critically compared two protocols for epigenetic analysis of the FSHD region using bisulfite genomic sequencing: Jones et al., that contends to be individually diagnostic for FSHD1 and FSHD2, and Gaillard et al., that can identify some changes in DNA methylation levels between groups of clinically affected FSHD and healthy subjects, but is not individually diagnostic for any form of FSHD. We performed both sets of assays on the same genetically confirmed samples and showed that this discrepancy was due strictly to differences in amplicon specificity. We propose that the epigenetic status of the FSHD-associated D4Z4 arrays, when accurately assessed, is a diagnostic for genetic FSHD and can readily distinguish between healthy, FSHD1 and FSHD2. Thus, epigenetic diagnosis of FSHD, which can be performed on saliva DNA, will greatly increase accessibility to FSHD diagnostics for populations around the world.

Targeted epigenetic repression by CRISPR/dSaCas9 suppresses pathogenic DUX4-fl expression in FSHD.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete silencing of the disease locus, leading to pathogenic misexpression of DUX4 in skeletal muscle. Previously, we showed that CRISPR inhibition could successfully target and repress DUX4 in FSHD myocytes. However, an effective therapy will require both efficient delivery of therapeutic components to skeletal muscles and long-term repression of the disease locus. Thus, we re-engineered our platform to allow in vivo delivery of more potent epigenetic repressors. We designed an FSHD-optimized regulatory cassette to drive skeletal muscle-specific expression of dCas9 from Staphylococcus aureus fused to HP1α, HP1γ, the MeCP2 transcriptional repression domain, or the SUV39H1 SET domain. Targeting each regulator to the DUX4 promoter/exon 1 increased chromatin repression at the locus, specifically suppressing DUX4 and its target genes in FSHD myocytes and in a mouse model of the disease. Importantly, minimizing the regulatory cassette and using the smaller Cas9 ortholog allowed our therapeutic cassettes to be effectively packaged into adeno-associated virus (AAV) vectors for in vivo delivery. By engineering a muscle-specific epigenetic CRISPR platform compatible with AAV vectors for gene therapy, we have laid the groundwork for clinical use of dCas9-based chromatin effectors in skeletal muscle disorders.

Interpretation of the Epigenetic Signature of Facioscapulohumeral Muscular Dystrophy in Light of Genotype-Phenotype Studies.

Facioscapulohumeral muscular dystrophy (FSHD) is characterized by incomplete penetrance and intra-familial clinical variability. The disease has been associated with the genetic and epigenetic features of the D4Z4 repetitive elements at 4q35. Recently, D4Z4 hypomethylation has been proposed as a reliable marker in the FSHD diagnosis. We exploited the Italian Registry for FSHD, in which FSHD families are classified using the Clinical Comprehensive Evaluation Form (CCEF). A total of 122 index cases showing a classical FSHD phenotype (CCEF, category A) and 110 relatives were selected to test with the receiver operating characteristic (ROC) curve, the diagnostic and predictive value of D4Z4 methylation. Moreover, we performed DNA methylation analysis in selected large families with reduced penetrance characterized by the co-presence of subjects carriers of one D4Z4 reduced allele with no signs of disease or presenting the classic FSHD clinical phenotype. We observed a wide variability in the D4Z4 methylation levels among index cases revealing no association with clinical manifestation or disease severity. By extending the analysis to family members, we revealed the low predictive value of D4Z4 methylation in detecting the affected condition. In view of the variability in D4Z4 methylation profiles observed in our large cohort, we conclude that D4Z4 methylation does not mirror the clinical expression of FSHD. We recommend that measurement of this epigenetic mark must be interpreted with caution in clinical practice.

Transgenic mice expressing tunable levels of DUX4 develop characteristic facioscapulohumeral muscular dystrophy-like pathophysiology ranging in severity.

BACKGROUND: All types of facioscapulohumeral muscular dystrophy (FSHD) are caused by the aberrant activation of the somatically silent DUX4 gene, the expression of which initiates a cascade of cellular events ultimately leading to FSHD pathophysiology. Typically, progressive skeletal muscle weakness becomes noticeable in the second or third decade of life, yet there are many individuals who are genetically FSHD but develop symptoms much later in life or remain relatively asymptomatic throughout their lives. Conversely, FSHD may clinically present prior to 5-10 years of age, ultimately manifesting as a severe early-onset form of the disease. These phenotypic differences are thought to be due to the timing and levels of DUX4 misexpression. METHODS: FSHD is a dominant gain-of-function disease that is amenable to modeling by DUX4 overexpression. We have recently created a line of conditional DUX4 transgenic mice, FLExDUX4, that develop a myopathy upon induction of human DUX4-fl expression in skeletal muscle. Here, we use the FLExDUX4 mouse crossed with the skeletal muscle-specific and tamoxifen-inducible line ACTA1-MerCreMer to generate a highly versatile bi-transgenic mouse model with chronic, low-level DUX4-fl expression and cumulative mild FSHD-like pathology that can be reproducibly induced to develop more severe pathology via tamoxifen induction of DUX4-fl in skeletal muscles. RESULTS: We identified conditions to generate FSHD-like models exhibiting reproducibly mild, moderate, or severe DUX4-dependent pathophysiology and characterized progression of pathology. We assayed DUX4-fl mRNA and protein levels, fitness, strength, global gene expression, and histopathology, all of which are consistent with an FSHD-like myopathic phenotype. Importantly, we identified sex-specific and muscle-specific differences that should be considered when using these models for preclinical studies. CONCLUSIONS: The ACTA1-MCM;FLExDUX4 bi-transgenic mouse model has mild FSHD-like pathology and detectable muscle weakness. The onset and progression of more severe DUX4-dependent pathologies can be controlled via tamoxifen injection to increase the levels of mosaic DUX4-fl expression, providing consistent and readily screenable phenotypes for assessing therapies targeting DUX4-fl mRNA and/or protein and are useful to investigate certain conserved downstream FSHD-like pathophysiology. Overall, this model supports that DUX4 expression levels in skeletal muscle directly correlate with FSHD-like pathology by numerous metrics.

Applying genome-wide CRISPR-Cas9 screens for therapeutic discovery in facioscapulohumeral muscular dystrophy.

The emergence of CRISPR-Cas9 gene-editing technologies and genome-wide CRISPR-Cas9 libraries enables efficient unbiased genetic screening that can accelerate the process of therapeutic discovery for genetic disorders. Here, we demonstrate the utility of a genome-wide CRISPR-Cas9 loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex type of muscular dystrophy for which there is currently no treatment. In FSHD, both genetic and epigenetic changes lead to misexpression of DUX4, the FSHD causal gene that encodes the highly cytotoxic DUX4 protein. We performed a genome-wide CRISPR-Cas9 screen to identify genes whose loss-of-function conferred survival when DUX4 was expressed in muscle cells. Genes emerging from our screen illuminated a pathogenic link to the cellular hypoxia response, which was revealed to be the main driver of DUX4-induced cell death. Application of hypoxia signaling inhibitors resulted in increased DUX4 protein turnover and subsequent reduction of the cellular hypoxia response and cell death. In addition, these compounds proved successful in reducing FSHD disease biomarkers in patient myogenic lines, as well as improving structural and functional properties in two zebrafish models of FSHD. Our genome-wide perturbation of pathways affecting DUX4 expression has provided insight into key drivers of DUX4-induced pathogenesis and has identified existing compounds with potential therapeutic benefit for FSHD. Our experimental approach presents an accelerated paradigm toward mechanistic understanding and therapeutic discovery of a complex genetic disease, which may be translatable to other diseases with well-established phenotypic selection assays.

The Good, The Bad, and The Unexpected: Roles of DUX4 in Health and Disease.

In this issue of Developmental Cell, Chew et al. (2019) show that the pioneer factor DUX4 is misexpressed in tumors, where it suppresses anti-tumor immune activity. Their findings provide a new mechanism for immune evasion in cancer and highlight the pathogenic effects of re-expressing an embryonic program in adult cells.

Muscle xenografts reproduce key molecular features of facioscapulohumeral muscular dystrophy.

Aberrant expression of DUX4, a gene unique to humans and primates, causes Facioscapulohumeral Muscular Dystrophy-1 (FSHD), yet the pathogenic mechanism is unknown. As transgenic overexpression models have largely failed to replicate the genetic changes seen in FSHD, many studies of endogenously expressed DUX4 have been limited to patient biopsies and myogenic cell cultures, which never fully differentiate into mature muscle fibers. We have developed a method to xenograft immortalized human muscle precursor cells from patients with FSHD and first-degree relative controls into the tibialis anterior muscle compartment of immunodeficient mice, generating human muscle xenografts. We report that FSHD cells mature into organized and innervated human muscle fibers with minimal contamination of murine myonuclei. They also reconstitute the satellite cell niche within the xenografts. FSHD xenografts express DUX4 and DUX4 downstream targets, retain the 4q35 epigenetic signature of their original donors, and express a novel protein biomarker of FSHD, SLC34A2. Ours is the first scalable, mature in vivo human model of FSHD. It should be useful for studies of the pathogenic mechanism of the disease as well as for testing therapeutic strategies targeting DUX4 expression.

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.

Identification of Epigenetic Regulators of DUX4-fl for Targeted Therapy of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by epigenetic de-repression of the disease locus, leading to pathogenic misexpression of the DUX4 gene in skeletal muscle. While the factors and pathways involved in normal repression of the FSHD locus in healthy cells have been well characterized, very little is known about those responsible for the aberrant activation of DUX4-fl in FSHD myocytes. Reasoning that DUX4-fl activators might represent useful targets for small molecule inhibition, we performed a highly targeted, candidate-based screen of epigenetic regulators in primary FSHD myocytes. We confirmed several of the strongest and most specific candidates (ASH1L, BRD2, KDM4C, and SMARCA5) in skeletal myocytes from two other unrelated FSHD1 patients, and we showed that knockdown led to reduced levels of DUX4-fl and DUX4-FL target genes, as well as altered chromatin at the D4Z4 locus. As a second mode of validation, targeting the CRISPR/dCas9-KRAB transcriptional repressor to the promoters of several candidates also led to reduced levels of DUX4-fl. Furthermore, these candidates can be repressed by different methods in skeletal myocytes without major effects on certain critical muscle genes. Our results demonstrate that expression of DUX4-fl is regulated by multiple epigenetic pathways, and they indicate viable, druggable candidates for therapeutic target development.

A cre-inducible DUX4 transgenic mouse model for investigating facioscapulohumeral muscular dystrophy.

The Double homeobox 4 (DUX4) gene is an important regulator of early human development and its aberrant expression is causal for facioscapulohumeral muscular dystrophy (FSHD). The DUX4-full length (DUX4-fl) mRNA splice isoform encodes a transcriptional activator; however, DUX4 and its unique DNA binding preferences are specific to old-world primates. Regardless, the somatic cytotoxicity caused by DUX4 expression is conserved when expressed in cells and animals ranging from fly to mouse. Thus, viable animal models based on DUX4-fl expression have been difficult to generate due in large part to overt developmental toxicity of low DUX4-fl expression from leaky transgenes. We have overcome this obstacle and here we report the generation and initial characterization of a line of conditional floxed DUX4-fl transgenic mice, FLExDUX4, that is viable and fertile. In the absence of cre, these mice express a very low level of DUX4-fl mRNA from the transgene, resulting in mild phenotypes. However, when crossed with appropriate cre-driver lines of mice, the double transgenic offspring readily express DUX4-fl mRNA, protein, and target genes with the spatiotemporal pattern of nuclear cre expression dictated by the chosen system. When cre is expressed from the ACTA1 skeletal muscle-specific promoter, the double transgenic animals exhibit a developmental myopathy. When crossed with tamoxifen-inducible cre lines, DUX4-mediated pathology can be induced in adult animals. Thus, the appearance and progression of pathology can be controlled to provide readily screenable phenotypes useful for assessing therapeutic approaches targeting DUX4-fl mRNA and protein. Overall, the FLExDUX4 line of mice is quite versatile and will allow new investigations into mechanisms of DUX4-mediated pathophysiology as well as much-needed pre-clinical testing of DUX4-targeted FSHD interventions in vivo.

Large family cohorts of lymphoblastoid cells provide a new cellular model for investigating facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite repeat. The resulting DNA hypomethylation and relaxation of epigenetic repression leads to increased expression of the deleterious DUX4-fl mRNA encoded within the distal D4Z4 repeat. With the typical late onset of muscle weakness, prevalence of asymptomatic individuals, and an autosomal dominant mode of inheritance, FSHD is often passed on from one generation to the next and affects multiple individuals within a family. Here we have characterized unique collections of 114 lymphoblastoid cell lines (LCLs) generated from 12 multigenerational FSHD families, including 56 LCLs from large, genetically homogeneous families in Utah. We found robust expression of DUX4-fl in most FSHD LCLs and a good correlation between DNA hypomethylation and repeat length. In addition, DUX4-fl levels can be manipulated using epigenetic drugs as in myocytes, suggesting that some epigenetic pathways regulating DUX4-fl in myocytes are maintained in LCLs. Overall, these FSHD LCLs provide an alternative cellular model in which to study many aspects of D4Z4, DUX4, and FSHD gene regulation in a background of low genetic variation. Significantly, these non-adherent immortal LCLs are amenable for high-throughput screening of potential therapeutics targeting DUX4-fl mRNA or protein expression.

Transgenic Drosophila for Investigating DUX4 and FRG1, Two Genes Associated with Facioscapulohumeral Muscular Dystrophy (FSHD).

Facioscapulohumeral muscular dystrophy (FSHD) is typically an adult onset dominant myopathy. Epigenetic changes in the chromosome 4q35 region linked to both forms of FSHD lead to a relaxation of repression and increased somatic expression of DUX4-fl (DUX4-full length), the pathogenic alternative splicing isoform of the DUX4 gene. DUX4-fl encodes a transcription factor expressed in healthy testis and pluripotent stem cells; however, in FSHD, increased levels of DUX4-fl in myogenic cells lead to aberrant regulation of target genes. DUX4-fl has proven difficult to study in vivo; thus, little is known about its normal and pathogenic roles. The endogenous expression of DUX4-fl in FSHD-derived human muscle and myogenic cells is extremely low, exogenous expression of DUX4-fl in somatic cells rapidly induces cytotoxicity, and, due in part to the lack of conservation beyond primate lineages, viable animal models based on DUX4-fl have been difficult to generate. By contrast, the FRG1 (FSHD region gene 1), which is linked to FSHD, is evolutionarily conserved from invertebrates to humans, and has been studied in several model organisms. FRG1 expression is critical for the development of musculature and vasculature, and overexpression of FRG1 produces a myopathic phenotype, yet the normal and pathological functions of FRG1 are not well understood. Interestingly, DUX4 and FRG1 were recently linked when the latter was identified as a direct transcriptional target of DUX4-FL. To better understand the pathways affected in FSHD by DUX4-fl and FRG1, we generated transgenic lines of Drosophila expressing either gene under control of the UAS/GAL4 binary system. Utilizing these lines, we generated screenable phenotypes recapitulating certain known consequences of DUX4-fl or FRG1 overexpression. These transgenic Drosophila lines provide resources to dissect the pathways affected by DUX4-fl or FRG1 in a genetically tractable organism and may provide insight into both muscle development and pathogenic mechanisms in FSHD.