Cellular and molecular mechanisms underlying muscular dystrophy

The muscular dystrophies are a group of heterogeneous genetic diseases characterized by progressive degeneration and weakness of skeletal muscle. Since the discovery of the first muscular dystrophy gene encoding dystrophin, a large number of genes have been identified that are involved in various muscle-wasting and neuromuscular disorders. Human genetic studies complemented by animal model systems have substantially contributed to our understanding of the molecular pathomechanisms underlying muscle degeneration. Moreover, these studies have revealed distinct molecular and cellular mechanisms that link genetic mutations to diverse muscle wasting phenotypes

Telomere Position Effect (TPE) Regulates DUX4 in Human Facioscapulohumeral Muscular Dystrophy (FSHD)

Telomeres may regulate human disease by at least two independent mechanisms. 1) Replicative senescence occurs once short telomeres generate DNA damage signals that produce a barrier to tumor progression. 2) Telomere Position Effect (TPE) can change gene expression at intermediate telomere lengths in cultured human cells. We here report a human disease, facioscapulohumeral muscular dystrophy (FSHD) where telomere length may well contribute to its pathogenesis. FSHD is age-related and genetically only 25-60 kb from the end of chromosome 4q. We used a floxable telomerase to generate isogenic clones with different telomere lengths from patients and their unaffected siblings. DUX4, the primary candidate for FSHD pathogenesis, is upregulated >10-fold in FSHD myoblasts-myotubes with short versus long telomeres, and its expression is inversely proportional to telomere length. FSHD may represent a human disease in which TPE contributes to its age-related phenotype

Analysis of Myogenic and Candidate Disease Biomarkers in FSHD Muscle Cells

The UMMS Wellstone Program is a foundation and NIH-funded cooperative research center focusing on identifying biomarkers for facioscapulohumeral muscular dystrophy (FSHD) to gain insight into the molecular pathology of the disease and to develop potential therapies. FSHD is characterized by progressive wasting of skeletal muscles, with weakness often initiating in facial muscles and muscles supporting the scapula and upper arms. While the genetics associated with FSHD are complex, the major form of the disease, FSHD1, is linked to contraction of the D4Z4 repeat region located at chromosome 4q. Recently, a transcript encoded at the distal end of the repeat region, Dux4-fl, normally expressed in embryonic stem cells and germ cells, was also detected in differentiated muscle cells and biopsies from FSHD subjects, giving rise to the hypothesis that DUX4-FL function contributes to muscle weakness. We established a repository of high quality, well-characterized primary and immortalized muscle cell strains from FSHD and control subjects in affected families to provide biomaterials for cell and molecular studies to the FSHD research community. qPCR and immunostaining analyses demonstrate similar growth and differentiation characteristics in cells from FSHD and control subjects within families. We detected Dux4-fl transcript and protein in FSHD cells as recently described; interestingly, we also detected Dux4-fl in muscle cells from a subset of control individuals, suggesting that any Dux4-fl-mediated myopathy would require additional modifying elements. Microarray analysis of FSHD and control muscle cells demonstrated that several genes were upregulated in FSHD cells, including genes that were concurrently identified as downstream targets of Dux4-fl and as candidate FSHD disease genes. Future studies will further characterize the RNA and protein expression of candidate disease genes in cells from FSHD and control subjects, including nonmanifesting subjects with the D4Z4 lesion but no muscle weakness, and utilizing whole transcriptome sequencing (RNAseq) to identify additional candidates

Medical Sequencing of Candidate Genes for Nonsyndromic Cleft Lip and Palate

Nonsyndromic or isolated cleft lip with or without cleft palate (CL/P) occurs in wide geographic distribution with an average birth prevalence of 1/700. We used direct sequencing as an approach to study candidate genes for CL/P. We report here the results of sequencing on 20 candidate genes for clefts in 184 cases with CL/P selected with an emphasis on severity and positive family history. Genes were selected based on expression patterns, animal models, and/or role in known human clefting syndromes. For seven genes with identified coding mutations that are potentially etiologic, we performed linkage disequilibrium studies as well in 501 family triads (affected child/mother/father). The recently reported MSX1 P147Q mutation was also studied in an additional 1,098 cleft cases. Selected missense mutations were screened in 1,064 controls from unrelated individuals on the Centre d'Étude du Polymorphisme Humain (CEPH) diversity cell line panel. Our aggregate data suggest that point mutations in these candidate genes are likely to contribute to 6% of isolated clefts, particularly those with more severe phenotypes (bilateral cleft of the lip with cleft palate). Additional cases, possibly due to microdeletions or isodisomy, were also detected and may contribute to clefts as well. Sequence analysis alone suggests that point mutations in FOXE1, GLI2, JAG2, LHX8, MSX1, MSX2, SATB2, SKI, SPRY2, and TBX10 may be rare causes of isolated cleft lip with or without cleft palate, and the linkage disequilibrium data support a larger, as yet unspecified, role for variants in or near MSX2, JAG2, and SKI. This study also illustrates the need to test large numbers of controls to distinguish rare polymorphic variants and prioritize functional studies for rare point mutations

Emerging preclinical animal models for FSHD

Facioscapulohumeral dystrophy (FSHD) is a unique and complex genetic disease that is not entirely solved. Recent advances in the field have led to a consensus genetic premise for the disorder, enabling researchers to now pursue the design of preclinical models. In this review we explore all available FSHD models (DUX4-dependent and -independent) for their utility in therapeutic discovery and potential to yield novel disease insights. Owing to the complex nature of FSHD, there is currently no single model that accurately recapitulates the genetic and pathophysiological spectrum of the disorder. Existing models emphasize only specific aspects of the disease, highlighting the need for more collaborative research and novel paradigms to advance the translational research space of FSHD

Transcriptional profiling in facioscapulohumeral muscular dystrophy to identify candidate biomarkers

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive neuromuscular disorder caused by contractions of repetitive elements within the macrosatellite D4Z4 on chromosome 4q35. The pathophysiology of FSHD is unknown and, as a result, there is currently no effective treatment available for this disease. To better understand the pathophysiology of FSHD and develop mRNA-based biomarkers of affected muscles, we compared global analysis of gene expression in two distinct muscles obtained from a large number of FSHD subjects and their unaffected first-degree relatives. Gene expression in two muscle types was analyzed using GeneChip Gene 1.0 ST arrays: biceps, which typically shows an early and severe disease involvement; and deltoid, which is relatively uninvolved. For both muscle types, the expression differences were mild: using relaxed cutoffs for differential expression (fold change \u3e/=1.2; nominal P value \u3c0.01), we identified 191 and 110 genes differentially expressed between affected and control samples of biceps and deltoid muscle tissues, respectively, with 29 genes in common. Controlling for a false-discovery rate of \u3c0.25 reduced the number of differentially expressed genes in biceps to 188 and in deltoid to 7. Expression levels of 15 genes altered in this study were used as a molecular signature in a validation study of an additional 26 subjects and predicted them as FSHD or control with 90% accuracy based on biceps and 80% accuracy based on deltoids

Gene expression profiling of skeletal muscles treated with a soluble activin type IIB receptor

Inhibition of the myostatin signaling pathway is emerging as a promising therapeutic means to treat muscle wasting and degenerative disorders. Activin type IIB receptor (ActRIIB) is the putative myostatin receptor, and a soluble activin receptor (ActRIIB-Fc) has been demonstrated to potently inhibit a subset of transforming growth factor (TGF)-β family members including myostatin. To determine reliable and valid biomarkers for ActRIIB-Fc treatment, we assessed gene expression profiles for quadriceps muscles from mice treated with ActRIIB-Fc compared with mice genetically lacking myostatin and control mice. Expression of 134 genes was significantly altered in mice treated with ActRIIB-Fc over a 2-wk period relative to control mice (fold change > 1.5, P < 0.001), whereas the number of significantly altered genes in mice treated for 2 days was 38, demonstrating a time-dependent response to ActRIIB-Fc in overall muscle gene expression. The number of significantly altered genes in Mstn−/− mice relative to control mice was substantially higher (360), but for most of these genes the expression levels in the 2-wk treated mice were closer to the levels in the Mstn−/− mice than in control mice (P < 10−30). Expression levels of 30 selected genes were further validated with quantitative real-time polymerase chain reaction (qPCR), and a correlation of ≥0.89 was observed between the fold changes from the microarray analysis and the qPCR analysis. These data suggest that treatment with ActRIIB-Fc results in overlapping but distinct gene expression signatures compared with myostatin genetic mutation. Differentially expressed genes identified in this study can be used as potential biomarkers for ActRIIB-Fc treatment, which is currently in clinical trials as a therapeutic agent for muscle wasting and degenerative disorders

Telomere position effect regulates DUX4 in human facioscapulohumeral muscular dystrophy

Telomeres may regulate human disease by at least two independent mechanisms. First, replicative senescence occurs once short telomeres generate DNA-damage signals that produce a barrier to tumor progression. Second, telomere position effects (TPE) could change gene expression at intermediate telomere lengths in cultured human cells. Here we report that telomere length may contribute to the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD). FSHD is a late-onset disease genetically residing only 25-60 kilobases from the end of chromosome 4q. We used a floxable telomerase to generate isogenic clones with different telomere lengths from affected patients and their unaffected siblings. DUX4, the primary candidate for FSHD pathogenesis, is upregulated over ten-fold in FSHD myoblasts and myotubes with short telomeres, and its expression is inversely proportional to telomere length. FSHD may be the first known human disease in which TPE contributes to age-related phenotype

A unique library of myogenic cells from facioscapulohumeral muscular dystrophy subjects and unaffected relatives: family, disease and cell function

To explore possible mechanisms of pathology in facioscapulohumeral muscular dystrophy (FSHD), we generated a novel library of myogenic cells composed of paired cultures derived from FSHD subjects and unaffected first-degree relatives. We prepared cells from biopsies of both biceps and deltoid muscles obtained from each of 10 FSHD and 9 unaffected donors. We used this new collection to determine how family background and disease affected patterns of growth and differentiation, expression of a panel of candidate, and muscle-specific genes, and responses to exogenous stressors. We found that FSHD and unaffected cells had, on average, indistinguishable patterns of differentiation, gene expression, and dose-response curves to staurosporine, paraquat, hydrogen peroxide, and glutathione depletion. Differentiated FSHD and unaffected cultures were both more sensitive to glutathione depletion than proliferating cultures, but showed similar responses to paraquat, staurosporine, and peroxide. For stress responses, the sample size was sufficient to detect a 10% change in effect at the observed variability with a power of \u3e99%. In contrast, for each of these properties, we found significant differences among cells from different cohorts, and these differences were independent of disease status, gender, or muscle biopsied. Thus, though none of the properties we examined could be used to reliably distinguish between FSHD and unaffected cells, family of origin was an important contributor to gene-expression patterns and stressor responses in cultures of both FSHD and unaffected myogenic cells

Human skeletal muscle xenograft as a new preclinical model for muscle disorders

Development of novel therapeutics requires good animal models of disease. Disorders for which good animal models do not exist have very few drugs in development or clinical trial. Even where there are accepted, albeit imperfect models, the leap from promising preclinical drug results to positive clinical trials commonly fails, including in disorders of skeletal muscle. The main alternative model for early drug development, tissue culture, lacks both the architecture and, usually, the metabolic fidelity of the normal tissue in vivo. Herein, we demonstrate the feasibility and validity of human to mouse xenografts as a preclinical model of myopathy. Human skeletal muscle biopsies transplanted into the anterior tibial compartment of the hindlimbs of NOD-Rag1null IL2rgammanull immunodeficient host mice regenerate new vascularized and innervated myofibers from human myogenic precursor cells. The grafts exhibit contractile and calcium release behavior, characteristic of functional muscle tissue. The validity of the human graft as a model of facioscapulohumeral muscular dystrophy is demonstrated in disease biomarker studies, showing that gene expression profiles of xenografts mirror those of the fresh donor biopsies. These findings illustrate the value of a new experimental model of muscle disease, the human muscle xenograft in mice, as a feasible and valid preclinical tool to better investigate the pathogenesis of human genetic myopathies and to more accurately predict their response to novel therapeutics