Human airway lineages derived from pluripotent stem cells reveal the epithelial responses to SARS-CoV-2 infection.

There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), and transmembrane protease serine 2 (TMPRSS2). Multiciliated cells are the primary initial target of SARS-CoV-2 infection. On infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses, and treatment with remdesivir or camostat mesylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia.

Facioscapulohumeral muscular dystrophy family studies of DUX4 expression: evidence for disease modifiers and a quantitative model of pathogenesis.

Facioscapulohumeral muscular dystrophy (FSHD), the most prevalent myopathy afflicting both children and adults, is predominantly associated with contractions in the 4q35-localized macrosatellite D4Z4 repeat array. Recent studies have proposed that FSHD pathology is caused by the misexpression of the DUX4 (double homeobox 4) gene resulting in production of a pathogenic protein, DUX4-FL, which has been detected in FSHD, but not in unaffected control myogenic cells and muscle tissue. Here, we report the analysis of DUX4 mRNA and protein expression in a much larger collection of myogenic cells and muscle biopsies derived from biceps and deltoid muscles of FSHD affected subjects and their unaffected first-degree relatives. We confirmed that stable DUX4-fl mRNA and protein were expressed in myogenic cells and muscle tissues derived from FSHD affected subjects, including several genetically diagnosed adult FSHD subjects yet to show clinical manifestations of the disease in the assayed muscles. In addition, we report DUX4-fl mRNA and protein expression in muscle biopsies and myogenic cells from genetically unaffected relatives of the FSHD subjects, although at a significantly lower frequency. These results establish that DUX4-fl expression per se is not sufficient for FSHD muscle pathology and indicate that quantitative modifiers of DUX4-fl expression and/or function and family genetic background are determinants of FSHD muscle disease progression.

Peripheral nerve pathology, including aberrant Schwann cell differentiation, is ameliorated by doxycycline in a laminin-α2-deficient mouse model of congenital muscular dystrophy.

The most common form of childhood congenital muscular dystrophy, Type 1A (MDC1A), is caused by mutations in the human LAMA2 gene that encodes the laminin-α2 subunit. In addition to skeletal muscle deficits, MDC1A patients typically show a loss of peripheral nerve function. To identify the mechanisms underlying this loss of nerve function, we have examined pathology and cell differentiation in sciatic nerves and ventral roots of the laminin-α2-deficient (Lama2(-/-)) mice, which are models for MDC1A. We found that, compared with wild-type, sciatic nerves of Lama2(-/-) mice had a significant increase in both proliferating (Ki67+) cells and premyelinating (Oct6+) Schwann cells, but also had a significant decrease in both immature/non-myelinating [glial fibrillary acidic protein (GFAP)(+)] and myelinating (Krox20+) Schwann cells. To extend our previous work in which we found that doxycycline, which has multiple effects on mammalian cells, improves motor behavior and more than doubles the median life-span of Lama2(-/-) mice, we also determined how nerve pathology was affected by doxycycline treatment. We found that myelinating (Krox20+) Schwann cells were significantly increased in doxycycline-treated compared with untreated sciatic nerves. In addition, doxycycline-treated peripheral nerves had significantly less pathology as measured by assays such as amount of unmyelinated or disorganized axons. This study thus identified aberrant proliferation and differentiation of Schwann cells as key components of pathogenesis in peripheral nerves and provided proof-of-concept that pharmaceutical therapy can be of potential benefit for peripheral nerve dysfunction in MDC1A.

Airway stem cell reconstitution by the transplantation of primary or pluripotent stem cell-derived basal cells.

Life-long reconstitution of a tissue's resident stem cell compartment with engrafted cells has the potential to durably replenish organ function. Here, we demonstrate the engraftment of the airway epithelial stem cell compartment via intra-airway transplantation of mouse or human primary and pluripotent stem cell (PSC)-derived airway basal cells (BCs). Murine primary or PSC-derived BCs transplanted into polidocanol-injured syngeneic recipients give rise for at least two years to progeny that stably display the morphologic, molecular, and functional phenotypes of airway epithelia. The engrafted basal-like cells retain extensive self-renewal potential, evident by the capacity to reconstitute the tracheal epithelium through seven generations of secondary transplantation. Using the same approach, human primary or PSC-derived BCs transplanted into NOD scid gamma (NSG) recipient mice similarly display multilineage airway epithelial differentiation in vivo. Our results may provide a step toward potential future syngeneic cell-based therapy for patients with diseases resulting from airway epithelial cell damage or dysfunction.

Proximity ligation assay to detect DUX4 protein in FSHD1 muscle: a pilot study.

OBJECTIVE: Aberrant expression in skeletal muscle of DUX4, a double homeobox transcription factor, underlies pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). Although previous studies of FSHD muscle biopsies detected mRNAs encoding DUX4 and its target genes, no studies had reported detection of DUX4 protein. Our objective was to develop a proximity ligation assay (PLA) for DUX4 and to determine if this assay could detect DUX4 protein in FSHD muscle sections. RESULTS: We developed a PLA protocol using two DUX4 antibodies previously reported by Stephen Tapscott's group: P2G4, a mouse mAb specific for an epitope in the N-terminal region, and E5-5, a rabbit mAb specific for an epitope in the C-terminal region, in combination with commercial PLA secondary reagents. We validated the DUX4 PLA using cultured human myogenic cells in which DUX4 was ectopically expressed in a small fraction of nuclei. Using this two primary mAb PLA on an FSHD1 biceps biopsy, we observed nuclei with apparent DUX4 PLA signals associated with a small subset of myofibers (~ 0.05-0.1%). Though a limited pilot study, these results suggest that the two primary mAb PLA protocol could be useful for detecting DUX4 protein in FSHD muscle biopsies.

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells.

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research. Induced pluripotent stem cells (iPSCs) are readily generated by "reprogramming" somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells. Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-ß and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects. Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.

A multimodal iPSC platform for cystic fibrosis drug testing.

Cystic fibrosis is a monogenic lung disease caused by dysfunction of the cystic fibrosis transmembrane conductance regulator anion channel, resulting in significant morbidity and mortality. The progress in elucidating the role of CFTR using established animal and cell-based models led to the recent discovery of effective modulators for most individuals with CF. However, a subset of individuals with CF do not respond to these modulators and there is an urgent need to develop novel therapeutic strategies. In this study, we generate a panel of airway epithelial cells using induced pluripotent stem cells from individuals with common or rare CFTR variants representative of three distinct classes of CFTR dysfunction. To measure CFTR function we adapt two established in vitro assays for use in induced pluripotent stem cell-derived airway cells. In both a 3-D spheroid assay using forskolin-induced swelling as well as planar cultures composed of polarized mucociliary airway epithelial cells, we detect genotype-specific differences in CFTR baseline function and response to CFTR modulators. These results demonstrate the potential of the human induced pluripotent stem cell platform as a research tool to study CF and in particular accelerate therapeutic development for CF caused by rare variants.

Differentiation of human pluripotent stem cells into functional airway basal stem cells.

Airway basal cells play an essential role in the maintenance of the airway epithelium. Here, we provide a detailed directed differentiation protocol to generate ''induced basal cells (iBCs)'' from human pluripotent stem cells. iBCs recapitulate biological and functional properties of airway basal cells including mucociliary differentiation in vitro or in vivo in tracheal xenografts, facilitating the study of inherited and acquired diseases of the airway, as well as potential use in regenerative medicine. For complete details on the use and execution of this protocol, please refer to Hawkins et al. (2021).

Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells.

The derivation of tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would have broad reaching implications for regenerative medicine. Here, we report the directed differentiation of human iPSCs into airway basal cells ("iBCs"), a population resembling the stem cell of the airway epithelium. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato), we track and purify these cells as they first emerge as developmentally immature NKX2-1GFP+ lung progenitors and subsequently augment a TP63 program during proximal airway epithelial patterning. In response to primary basal cell medium, NKX2-1GFP+/TP63tdTomato+ cells display the molecular and functional phenotype of airway basal cells, including the capacity to self-renew or undergo multi-lineage differentiation in vitro and in tracheal xenografts in vivo. iBCs and their differentiated progeny model perturbations that characterize acquired and genetic airway diseases, including the mucus metaplasia of asthma, chloride channel dysfunction of cystic fibrosis, and ciliary defects of primary ciliary dyskinesia.

Pathology is alleviated by doxycycline in a laminin-alpha2-null model of congenital muscular dystrophy.

OBJECTIVE: Congenital muscular dystrophy type 1A is an autosomal recessive disease that is caused by loss-of-function mutations in the laminin-alpha2 gene, and results in motor nerve and skeletal muscle dysfunction. In a previous study, we used genetic modifications to show that inappropriate induction of apoptosis was a significant contributor to pathogenesis in a laminin-alpha2-deficient mouse model of congenital muscular dystrophy type 1A. To identify a possible pharmacological therapy for laminin-alpha2 deficiency, we designed this study to determine whether treatment with minocycline or doxycycline, which are tetracycline derivatives reported to have antiapoptotic effects in mammals, would significantly increase lifespan and improve neuromuscular function in laminin-alpha2-deficient mice. METHODS: Mice that were homozygous for a targeted, inactivating mutation of the laminin-alpha2 gene were placed into control, minocycline-treated, or doxycycline-treated groups. Drug treatment began within 2 weeks of birth, and the progression of disease was followed over time using behavioral, growth, histological, and molecular assays. RESULTS: We found that treatment with either minocycline or doxycycline increased the median lifespan of laminin-alpha2-null mice from approximately 32 days to approximately 70 days. Furthermore, doxycycline improved postnatal growth rate and delayed the onset of hind-limb paralysis. Doxycycline-treated laminin-alpha2-deficient muscles had increased Akt phosphorylation, decreased inflammation, and decreased levels of Bax protein, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive myonuclei, and activated caspase-3. INTERPRETATION: Doxycycline or other drugs with similar functional profiles may be a possible route to improving neuromuscular dysfunction caused by laminin-alpha2-deficiency.

Human iPSC-derived alveolar and airway epithelial cells can be cultured at air-liquid interface and express SARS-CoV-2 host factors.

Development of an anti-SARS-CoV-2 therapeutic is hindered by the lack of physiologically relevant model systems that can recapitulate host-viral interactions in human cell types, specifically the epithelium of the lung. Here, we compare induced pluripotent stem cell (iPSC)-derived alveolar and airway epithelial cells to primary lung epithelial cell controls, focusing on expression levels of genes relevant for COVID-19 disease modeling. iPSC-derived alveolar epithelial type II-like cells (iAT2s) and iPSC-derived airway epithelial lineages express key transcripts associated with lung identity in the majority of cells produced in culture. They express ACE2 and TMPRSS2, transcripts encoding essential host factors required for SARS-CoV-2 infection, in a minor subset of each cell sub-lineage, similar to frequencies observed in primary cells. In order to prepare human culture systems that are amenable to modeling viral infection of both the proximal and distal lung epithelium, we adapt iPSC-derived alveolar and airway epithelial cells to two-dimensional air-liquid interface cultures. These engineered human lung cell systems represent sharable, physiologically relevant platforms for SARS-CoV-2 infection modeling and may therefore expedite the development of an effective pharmacologic intervention for COVID-19.

SARS-CoV-2 Infection of Pluripotent Stem Cell-Derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response.

A hallmark of severe COVID-19 pneumonia is SARS-CoV-2 infection of the facultative progenitors of lung alveoli, the alveolar epithelial type 2 cells (AT2s). However, inability to access these cells from patients, particularly at early stages of disease, limits an understanding of disease inception. Here, we present an in vitro human model that simulates the initial apical infection of alveolar epithelium with SARS-CoV-2 by using induced pluripotent stem cell-derived AT2s that have been adapted to air-liquid interface culture. We find a rapid transcriptomic change in infected cells, characterized by a shift to an inflammatory phenotype with upregulation of NF-κB signaling and loss of the mature alveolar program. Drug testing confirms the efficacy of remdesivir as well as TMPRSS2 protease inhibition, validating a putative mechanism used for viral entry in alveolar cells. Our model system reveals cell-intrinsic responses of a key lung target cell to SARS-CoV-2 infection and should facilitate drug development.

SARS-CoV-2 Infection of Pluripotent Stem Cell-derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response.

The most severe and fatal infections with SARS-CoV-2 result in the acute respiratory distress syndrome, a clinical phenotype of coronavirus disease 2019 (COVID-19) that is associated with virions targeting the epithelium of the distal lung, particularly the facultative progenitors of this tissue, alveolar epithelial type 2 cells (AT2s). Little is known about the initial responses of human lung alveoli to SARS-CoV-2 infection due in part to inability to access these cells from patients, particularly at early stages of disease. Here we present an in vitro human model that simulates the initial apical infection of the distal lung epithelium with SARS-CoV-2, using AT2s that have been adapted to air-liquid interface culture after their derivation from induced pluripotent stem cells (iAT2s). We find that SARS-CoV-2 induces a rapid global transcriptomic change in infected iAT2s characterized by a shift to an inflammatory phenotype predominated by the secretion of cytokines encoded by NF-kB target genes, delayed epithelial interferon responses, and rapid loss of the mature lung alveolar epithelial program. Over time, infected iAT2s exhibit cellular toxicity that can result in the death of these key alveolar facultative progenitors, as is observed in vivo in COVID-19 lung autopsies. Importantly, drug testing using iAT2s confirmed an antiviral dose-response to remdesivir and demonstrated the efficacy of TMPRSS2 protease inhibition, validating a putative mechanism used for viral entry in human alveolar cells. Our model system reveals the cell-intrinsic responses of a key lung target cell to infection, providing a physiologically relevant platform for further drug development and facilitating a deeper understanding of COVID-19 pathogenesis.

Efficient system for upstream mRNA trans-splicing to generate covalent, head-to-tail, protein multimers.

We present a plasmid-based system in which upstream trans-splicing efficiently generates mRNAs that encode head-to-tail protein multimers. In this system, trans-splicing occurs between one of two downstream splice donors in the sequence encoding a C-terminal V5 epitope tag and an upstream splice acceptor in the 5' region of the pCS2(+) host plasmid. Using deletion and fusion constructs of the DUX4 protein as an example, we found that this system produced trans-spliced mRNAs in which coding regions from independent transcripts were fused in phase such that covalent head-to-tail protein multimers were translated. For a cDNA of ~450 bp, about half of the expressed proteins were multimeric, with the efficiency of trans-splicing and extent of multimer expression decreasing as cDNA length increased. This system generated covalent heterodimeric proteins upon co-transfections of plasmids encoding separate proteins and did not require a long complementary binding domain to position mRNAs for trans-splicing. This plasmid-based trans-splicing system is adaptable to multiple gene delivery systems, and it presents new opportunities for investigating molecular mechanisms of trans-splicing, generating covalent protein multimers with novel functions within cells, and producing mRNAs encoding large proteins from split precursors.

Aberrant Caspase Activation in Laminin-α2-Deficient Human Myogenic Cells is Mediated by p53 and Sirtuin Activity.

BACKGROUND: Mutations in the LAMA2 gene encoding laminin-α2 cause congenital muscular dystrophy Type 1A (MDC1A), a severe recessive disease with no effective treatment. Previous studies have shown that aberrant activation of caspases and cell death through a pathway regulated by BAX and KU70 is a significant contributor to pathogenesis in laminin-α2-deficiency. OBJECTIVES: To identify mechanisms of pathogenesis in MDC1A. METHODS: We used immunocytochemical and molecular studies of human myogenic cells and mouse muscles-comparing laminin-α2-deficient vs. healthy controls-to identify mechanisms that regulate pathological activation of caspase in laminin-α2-deficiency. RESULTS: In cultures of myogenic cells from MDC1A donors, p53 accumulated in a subset of nuclei and aberrant caspase activation was inhibited by the p53 inhibitor pifithrin-alpha. Also, the p53 target BBC3 (PUMA) was upregulated in both MDC1A myogenic cells and Lama2-/- mouse muscles. In addition, studies with sirtuin inhibitors and SIRT1 overexpression showed that caspase activation in MDC1A myotubes was inversely related to sirtuin deacetylase activity. Caspase activation in laminin-α2-deficiency was, however, not associated with increased phosphorylation of p38 MAPK. CONCLUSIONS: Aberrant caspase activation in MDC1A cells was mediated both by sirtuin deacetylase activity and by p53. Interventions that inhibit aberrant caspase activation by targeting sirtuin or p53 function could potentially be useful in ameliorating MDC1A.

Functional domains of the FSHD-associated DUX4 protein.

Aberrant expression of the full-length isoform of DUX4 (DUX4-FL) appears to underlie pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). DUX4-FL is a transcription factor and ectopic expression of DUX4-FL is toxic to most cells. Previous studies showed that DUX4-FL-induced pathology requires intact homeodomains and that transcriptional activation required the C-terminal region. In this study, we further examined the functional domains of DUX4 by generating mutant, deletion, and fusion variants of DUX4. We compared each construct to DUX4-FL for (i) activation of a DUX4 promoter reporter, (ii) expression of the DUX4-FL target gene ZSCAN4, (iii) effect on cell viability, (iv) activation of endogenous caspases, and (v) level of protein ubiquitination. Each construct produced a similarly sized effect (or lack of effect) in each assay. Thus, the ability to activate transcription determined the extent of change in multiple molecular and cellular properties that may be relevant to FSHD pathology. Transcriptional activity was mediated by the C-terminal 80 amino acids of DUX4-FL, with most activity located in the C-terminal 20 amino acids. We also found that non-toxic constructs with both homeodomains intact could act as inhibitors of DUX4-FL transcriptional activation, likely due to competition for promoter sites.This article has an associated First Person interview with the first author of the paper.

Nuclear bodies reorganize during myogenesis in vitro and are differentially disrupted by expression of FSHD-associated DUX4.

BACKGROUND: Nuclear bodies, such as nucleoli, PML bodies, and SC35 speckles, are dynamic sub-nuclear structures that regulate multiple genetic and epigenetic processes. Additional regulation is provided by RNA/DNA handling proteins, notably TDP-43 and FUS, which have been linked to ALS pathology. Previous work showed that mouse cell line myotubes have fewer but larger nucleoli than myoblasts, and we had found that nuclear aggregation of TDP-43 in human myotubes was induced by expression of DUX4-FL, a transcription factor that is aberrantly expressed and causes pathology in facioscapulohumeral dystrophy (FSHD). However, questions remained about nuclear bodies in human myogenesis and in muscle disease. METHODS: We examined nucleoli, PML bodies, SC35 speckles, TDP-43, and FUS in myoblasts and myotubes derived from healthy donors and from patients with FSHD, laminin-alpha-2-deficiency (MDC1A), and alpha-sarcoglycan-deficiency (LGMD2D). We further examined how these nuclear bodies and proteins were affected by DUX4-FL expression. RESULTS: We found that nucleoli, PML bodies, and SC35 speckles reorganized during differentiation in vitro, with all three becoming less abundant in myotube vs. myoblast nuclei. In addition, though PML bodies did not change in size, both nucleoli and SC35 speckles were larger in myotube than myoblast nuclei. Similar patterns of nuclear body reorganization occurred in healthy control, MDC1A, and LGMD2D cultures, as well as in the large fraction of nuclei that did not show DUX4-FL expression in FSHD cultures. In contrast, nuclei that expressed endogenous or exogenous DUX4-FL, though retaining normal nucleoli, showed disrupted morphology of some PML bodies and most SC35 speckles and also co-aggregation of FUS with TDP-43. CONCLUSIONS: Nucleoli, PML bodies, and SC35 speckles reorganize during human myotube formation in vitro. These nuclear body reorganizations are likely needed to carry out the distinct gene transcription and splicing patterns that are induced upon myotube formation. DUX4-FL-induced disruption of some PML bodies and most SC35 speckles, along with co-aggregation of TDP-43 and FUS, could contribute to pathogenesis in FSHD, perhaps by locally interfering with genetic and epigenetic regulation of gene expression in the small subset of nuclei that express high levels of DUX4-FL at any one time.

Expression of FSHD-related DUX4-FL alters proteostasis and induces TDP-43 aggregation.

OBJECTIVE: Pathogenesis in facioscapulohumeral muscular dystrophy (FSHD) appears to be due to aberrant expression, particularly in skeletal muscle nuclei, of the full-length isoform of DUX4 (DUX4-FL). Expression of DUX4-FL is known to alter gene expression and to be cytotoxic, but cell responses to DUX4-FL are not fully understood. Our study was designed to identify cellular mechanisms of pathogenesis caused by DUX4-FL expression. METHODS: We used human myogenic cell cultures to analyze the effects of DUX4-FL when it was expressed either from its endogenous promoter in FSHD cells or by exogenous expression using BacMam vectors. We focused on determining the effects of DUX4-FL on protein ubiquitination and turnover and on aggregation of TDP-43. RESULTS: Human FSHD myotubes with endogenous DUX4-FL expression showed both altered nuclear and cytoplasmic distributions of ubiquitinated proteins and aggregation of TDP-43 in DUX4-FL-expressing nuclei. Similar changes were found upon exogenous expression of DUX4-FL, but were not seen upon expression of the non-toxic short isoform DUX4-S. DUX4-FL expression also inhibited protein turnover in a model system and increased the amounts of insoluble ubiquitinated proteins and insoluble TDP-43. Finally, inhibition of the ubiquitin-proteasome system with MG132 produced TDP-43 aggregation similar to DUX4-FL expression. INTERPRETATIONS: Our results identify DUX4-FL-induced inhibition of protein turnover and aggregation of TDP-43, which are pathological changes also found in diseases such as amyotrophic lateral sclerosis and inclusion body myopathy, as potential pathological mechanisms in FSHD.

Individual epigenetic status of the pathogenic D4Z4 macrosatellite correlates with disease in facioscapulohumeral muscular dystrophy.

BACKGROUND: Both forms of facioscapulohumeral muscular dystrophy (FSHD) are associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite. Chromatin changes due to large deletions of heterochromatin (FSHD1) or mutations in chromatin regulatory proteins (FSHD2) lead to relaxation of epigenetic repression and increased expression of the deleterious double homeobox 4 (DUX4) gene encoded within the distal D4Z4 repeat. However, many individuals with the genetic requirements for FSHD remain asymptomatic throughout their lives. Here we investigated family cohorts of FSHD1 individuals who were either affected (manifesting) or without any discernible weakness (nonmanifesting/asymptomatic) and their unaffected family members to determine if individual epigenetic status and stability of repression at the contracted 4q35 D4Z4 array in myocytes correlates with FSHD disease. RESULTS: Family cohorts were analyzed for DNA methylation on the distal pathogenic 4q35 D4Z4 repeat on permissive A-type subtelomeres. We found DNA hypomethylation in FSHD1-affected subjects, hypermethylation in healthy controls, and distinctly intermediate levels of methylation in nonmanifesting subjects. We next tested if these differences in DNA methylation had functional relevance by assaying DUX4-fl expression and the stability of epigenetic repression of DUX4-fl in myogenic cells. Treatment with drugs that alter epigenetic status revealed that healthy cells were refractory to treatment, maintaining stable repression of DUX4, while FSHD1-affected cells were highly responsive to treatment and thus epigenetically poised to express DUX4. Myocytes from nonmanifesting subjects had significantly higher levels of DNA methylation and were more resistant to DUX4 activation in response to epigenetic drug treatment than cells from FSHD1-affected first-degree relatives containing the same contraction, indicating that the epigenetic status of the contracted D4Z4 array is reflective of disease. CONCLUSIONS: The epigenetic status of the distal 4qA D4Z4 repeat correlates with FSHD disease; FSHD-affected subjects have hypomethylation, healthy unaffected subjects have hypermethylation, and nonmanifesting subjects have characteristically intermediate methylation. Thus, analysis of DNA methylation at the distal D4Z4 repeat could be used as a diagnostic indicator of developing clinical FSHD. In addition, the stability of epigenetic repression upstream of DUX4 expression is a key regulator of disease and a viable therapeutic target.