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FSH Society Research Fellows and Grantees
For a list of current FSH Society Research Fellows and Grantees, granted in the August 2012, February 2012, August 2011 and February 2011 grant cycles and a description of the research projects, please click here for PDF or MS-Word For a list of FSH Society Research Fellows and Grantees, granted in the August 2010, February 2010, August 2009 grant cycles and a description of the research projects, please click here for PDF or MS-Word For a list of past FSH Society Research Fellows and Grantees, and a description of the research projects, please click here for PDF or MS-Word The FSH Society Scientific Advisory Board (SAB) met in December 2012 to review grant applications received for the August 2012 round of FSH Society grants funding. Below is a list of the funded projects, including project descriptions as submitted by grant applicant(s). 1. Role of Polycomb Group Proteins in Facioscapulohumeral dystrophy Summary (Provided by Applicant): Facioscapulohumeral muscular dystrophy (FSHD), the third most common myopathy, is an autosomal dominant neuromuscular disorder characterized by progressive weakness and atrophy affecting specific muscle groups. FSHD is not due to a mutation within a protein-coding gene, but is caused by contraction of the 3.3 kb macrosatellite repeat D4Z4 in the subtelomeric region of chromosome 4q35. While there is general agreement that D4Z4 deletion leads to over-expression of 4q35 genes, the molecular mechanism through which D4Z4 regulates chromatin structure and gene expression is poorly understood. Consequently, no therapeutic tool to control the aberrant 4q35 gene expression in FSHD is currently available. Polycomb (PcG) and Trithorax (TrxG) group proteins act antagonistically in the epigenetic regulation of gene expression and they play crucial roles in many biological aspects such as development, cell proliferation and cancer. In Drosophila, PcG and TrxG proteins bind to specific DNA regions termed Polycomb/Trithorax Response Elements (PREs/TREs), constituting a regulated switchable element that influences chromatin architecture and expression of nearby genes. D4Z4 shares several features with PREs/TREs. Indeed, my previous results (Cell 2012 149:819-31). showed that Polycomb group of epigenetic repressors targets D4Z4 in healthy subjects. Furthermore, I found that Polycomb proteins are required to maintain 4q35 genes repressed and that D4Z4 deletion is associated with reduced Polycomb silencing in FSHD patients (Cell 2012 149:819-31). My preliminary results strongly suggest that D4Z4 could be the first PRE involved in a human genetic disease. An attractive hypothesis would be that a D4Z4 copy number above the threshold of 11 repeats is able to stably substain a Polycomb-mediated repression of 4q35 genes, while few copies of the repeat fail to do this efficiently. Here, I propose to rigorously investigate the PRE activity of D4Z4. These studies will allow a deep understanding of the D4Z4 mechanism of action and will lay the basis to develop therapeutic approaches aimed at normalizing aberrant 4q35 gene expression in FSHD. My specific aims are: 1.) To understand the mechanism through which the deletion of D4Z4 repeats below a threshold copy number is affecting 4q35 gene expression in FSHD; 2. Derivation of human induced pluripotent stem cells from FSH patient fibroblasts Summary (Provided by Applicant): The genetic and biological events that result in Facioscapulohumeral muscular dystrophy (FSHD) pathogenesis are complex and the link between the genetic aberration and manifestation of symptoms is still elusive. We hypothesize that there might be cellular and genetic alteration in the early stage of myogenesis in FSHD patients. The establishment of human induced pluripotent stem cells (hiPSCs) ushered a new era in biomedicine and can be useful for modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening. Our lab has been studied human genetic disorders by using induced pluripotent stem cells (hiPSCs) that is a new type of stem cells without destruction of any embryonic tissues or embryos. In addition, we already built a novel methodology in highly innovative manner to directly derive and prospectively isolate skeletal muscle from the hiPSCs. Here we propose to establish hiPSC lines from FSHD patient’s somatic cells. Our proposed study will enable us to isolate FSHD-specific skeletal muscle cells for better understanding of FSHD pathogenesis in human system as well as potential autologous cellular therapy accompanying with genetic correction in near future. 3. Autophagy defects in FSHD Summary (Provided by Applicant): Our previous study showed that DUX4 was up-regulated in patient’s muscles of FSHD and transcriptionally regulated paired-like homeodomain transcription factor 1 (PITX1). The muscle-specific expression of Pitx1 in transgenic mouse model showed muscular dystrophy phenotype similar to FSHD [Pandey et al., 2012]. Expression profiling data of Pitx1 transgenic mice showed that 16 major autophagy genes, including damage-regulated autophagy modulator (Dram1) were mis-regulated in the muscle over-expressing PITX1. To determine whether the autophagy pathways were also affected in FSHD, we investigated the autophagy state in FSHD myoblasts as well as patients’ muscle biopsies. Our data showed disease-specific up-regulation of a master autophagy regulator, DRAM, in FSHD muscle biopsies but not DMD or controls. To further characterize the autophagy state in FSHD myoblasts we cultured the myoblast in differentiation media and we found that DRAM was up-regulated in FSHD myoblasts compared to the control myoblasts. We then examined two proteins critical to autophagy activities, p62 and LC3B. The p62 protein binds both ubiquitinated substrates and LC3B [Pankiv et al., 2007], and has been used as an indicator of autophagic flux. In addition, the accumulating of p62 has been used as an indicator of defective autophagy [Settembre et al., 2008; Ju et al., 2010]. In our study, instead of down regulation when autophagy is activated, p62 showed up-regulation in FSHD myoblasts suggesting a defect in autophagy activation. We further checked the LC3B-II to LCB3-I ratio (LC3B-II/I) which is a commonly used marker for autophagy activation. Because LC3B-II is formed only when autophagosomes are generated, the LC3B-II/LC3B-I ratio represents the density of autophagosomes in cells. The significantly lower LC3B-II/LC3B-I ratio in the FSHD myoblasts indicated again a suppression of autophagy in the myoblast. The suppression of autophagy is also supported by accumulation of ubiquitinated protein in the FSHD cells. While the activation of DRAM should activate the downstream autophagy pathways, we observed a defect in autophagosome formation. Interestingly, the up-regulation of LAMP1 and 2 at mRNA level in muscle biopsy of patients with FSHD suggests that the lysosomal system is activated and ready for the later steps of forming autophagolysosomes. However, the autophagy process is somehow disrupted in FSHD myoblast.In addition, the aberrant expression of DUX4 is a cause of FSHD so we would like determine whether defect in autophagy process is directly linked with expression of DUX4. On the basis of our preliminary result we hypothesize that defect in autophagy causes differentiation defect in myotubes formation in FSHD. In addition, autophagy defect is directly induced by aberrant expression of DUX4. In proposed study, we will examine the expression changes of the key regulators of autophagy and further investigate the mechanisms involved in autophagy defects in FSHD. In addition, we will knock-down the DUX4 expression in the FSHD myoblasts to determine whether the autophagy defects are directly induced by the aberrant expression of DUX4 in the cells.The goal of this current proposal is to understand the mechanism and to identify molecular pathways for treatment development. The aim for this study as follows: Aim 1: To determine the expression of DRAM, p62, Autophagy related gene 5 (ATG5), Autophagy related gene 4B (ATG4B), LC3B, and LAMP1 in patients with FSHD. We anticipate that DRAM, p62 and LAMP1 will show higher expression whereas LC3B-II/LC3B-I ratio will be low in FSHD. Aim 1A: To determine the expression of DRAM, p62, ATG5, ATG4B, LC3B, and LAMP1 in FSHD myoblasts with and without autophagy induction. Aim 1B: To determine the expression of DRAM, p62, ATG5, ATG4B, LC3B, and LAMP1 in muscle biopsies of patients with FSHD. Aim 2: To determine whether the defects in autophagy are due to inhibition of fusion between the autophagosomes and lysosomes. We anticipate a reduction in fusion of lysosome to autophagosomes will be observed in FSHD myoblasts but not in the control myoblasts. Aim 2A: To determine the inhibition of fusion efficiency between the autophagosomes and lysosomes in immortalized FSHD myoblasts. Aim 2B: To determine the expression of ATG4B, a key regulator of LC3B conversion, in immortalized FSHD myoblasts. Aim 3: To determine whether the defects are induced by DUX4 by knocking down DUX4 in the immortalized human myoblasts using antisense oligonucleotides against DUX4. Aim 3A: To determine the expression of DRAM, p62, ATG5, ATG4B, LC3B in proliferating and differentiating myoblasts after knocking-down DUX4. Aim 3B: To determine whether DUX4 knock-down can correct the inhibition of fusion between the autophagosomes and lysosomes in FSHD myoblasts. 4. Evaluation of an FSHD-specific patient reported outcome measure and a disease specific functional rating scale Summary (Provided by Applicant): Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common forms of muscular dystrophy with an estimated prevalence between 1: 15,000 and 1 :20,000. The clinical spectrum of disease severity is wide, and the regional distribution of muscle weakness, as well as the pattern of progression, is unique. The molecular defect in FSHD on chromosome 4q35 was described in 1992 but the molecular pathophysiology remained unknown until recently. A unifying model has now emerged proposing the aberrant reactivation of the DUX4 gene - resulting in a toxic gain of function- in the pathophysiology of FSHD. This FSHD model has provided, for the first time, therapeutic targets for FSHD, and it is expected that several potential therapeutic interventions will emerge in the coming years. Because of these recent discoveries, there is an urgent need to develop the tools necessary to effectively and efficiently conduct therapeutic trials in FSHD. Existing validated outcome measures in FSHD are neither sensitive to change nor intuitively patient-relevant. More sensitive outcome measures are needed for a more efficient drug development process. The need for patient-relevant outcome measures was emphasized in the proceedings of the 2010 FSHD European Neuromuscular Centre meeting. Moreover, there is increasing emphasis by the FDA on the development of outcome measures that are clinically meaningful and based on the patient's perspective. There are currently two validated, commonly utilized outcome measures in FSHD (manual muscle testing [MMT] and quantitative myometry) both of which are based on direct strength testing. Although direct measurement of muscle strength makes intuitive sense in a myopathy, what minimum change in such a measure can be considered clinically relevant is not clear. There are, additionally, two FSHD-specific clinical severity scales that have been validated in cross-sectional studies; however, neither the responsiveness to change over time nor the direct relevance to patients has been demonstrated. Moreover, as 10 and 15 point ordinal scales, they are not likely to be highly sensitive to change. Here we plan to test the reliability, validity and responsiveness to change of two FSHD-specific outcomes: the FSHD Health Inventory (FSHDHI) and the FSHD Functional Outcome (FSHD-FO). Both of these instruments were developed based on direct patient input to reflect the most prevalent and important physical limitations of FSHD. We will recruit 35 participants with FSHD for 4 visits over 1 year of follow up. Outcomes will be compared at baseline and longitudinally to traditional measurements such as the composite MMT score, existing FSHD clinical rating scales, and SF-36 health survey. Additionally an anchoring technique will be used to determine the minimally clinically important change. We expect that this proposal will provide preliminary data on the utility, ease of administration, reliability and validity, and responsiveness to change over 1 year of two novel and clinically relevant FSHD-specific outcome measures. We have designed our budget so that reliability and convergent validity are tested in year 1; and responsiveness in year 2, months 12-18. It is of vital importance for the FSHD research community that development of outcome measures parallels advancements in molecular pathophysiology and drug development. The scales presented here both represent valuable, patient-relevant tools for the FSHD clinical trial toolkit. 5. Specific Silencing of FAT1: Role in Pathogenesis of FSHD Summary (Provided by Applicant): This proposal outlines a postdoctoral fellowship project, which I (AK Zimmermann) am planning to conduct in the laboratory of Francoise Helmbacher at IBDML in Marseille, France over the next 2 years. I arrived in the lab 5 months ago and have already initiated the research project described below (see preliminary data). The Helmbacher laboratory studies mechanisms that contribute to assembly of neuromuscular circuits during development and associated pathologies. Recently the lab has begun work on facioscapulohumeral muscular dystrophy (FSHD), a devastating human disease characterized by degeneration of muscles in the face and shoulder area. Mechanisms contributing to FSHD remain elusive although pathogenic deletions within a D4Z4 macrosatellite array on chromosome 4 have been identified in the majority of clinical cases. Several studies investigating how this genetic alteration exerts its pathogenic effect have led to propose a contribution of deregulated expression of genes proximal to the deleted region (FRG1 & 2, ANT1), as well as a toxic effect of DUX4, a transcription factor whose transcription is enabled in the pathogenic context. However, a role for these genes in the selective muscle deficits associated with FSHD has not been irrevocably established and therefore additional candidates remain to be identified. The Helmbacher laboratory has shown that FAT1, a protocadherin gene also located near the D4Z4 locus, regulates muscle development and may play a role in physiology of mature skeletal muscles as well. In two mouse mutant models, FAT1-deficient mice were found to develop an FSHD-like phenotype, including both muscle and non-muscle defects. Notably, the developmental abnormalities of muscle shape appear to prefigure the map of muscles affected in FSHD patients. In addition, analyses of human foetal biopsies suggested that tissue-specific silencing of FAT1 might be a causal mechanism in FSHD. This was supported by the finding that several FSHD patients without the classical D4Z4 abnormality carried a deletion of a cis-regulatory enhancer of the FAT1 gene. Here, I propose to study the role of FAT1 in FSHD pathogenesis, and specifically to answer the following questions: 1. Owing to a conditional allele of FAT1 developed in the Helmbacher lab, I will use a tissue-specific ablation approach to ask in which tissues FAT1 expression is relevant for proper muscle migration (candidates include muscle, nerve, vascular and connective tissues) 2. Using primary culture assays on myoblasts and myotubes isolated from Fat1 mutant embryos, I will ask whether FAT1 also play a role in muscle function, as suggested by its subcellular association to the t-tubule excitation-contraction coupling system. Ultimately, alterations of these functions in FSHD patients might be accessible to preventive therapies. 3. Finally, I will ask how FAT1 silencing contributes to dysregulation of retina vasculature, a symptom of FSHD that may provide clues about the molecular mechanisms associated with both muscle and non-muscle phenotypes of disease? Ultimately we hope these strategies will contribute to the development of therapeutic targets aimed at bypassing FAT1 silencing in FSHD and maintaining functional Fat1 levels in muscle prior to worsening of the muscle degeneration symptom. Awards for February 2012 Cycle The FSH Society Scientific Advisory Board (SAB) met in June 2012 to review grant applications received for the February 2012 round of FSH Society grants funding. Below is a list of the funded projects, including project descriptions as submitted by grant applicant. 1. A transgenic model of DUX4-mediated FSHD Summary (Provided by Applicant): The most critical need in the FSHD field is a reliable and faithful mouse model of FSHD. This has been inhibited in the past by lack of a consistent and consensus understanding of the gene misregulation in the human condition that leads to FSHD pathology. Now that there is wide spread agreement about the involvement of DUX4-fl in FSHD pathology there are different barriers; the severe cytotoxicity of DUX4 and its lack of conservation in mammals. As such, the field has so far failed to generate a genetic mouse model based on DUX4 expression that recapitulates the DUX4-fl expression profile and FSHD-like pathophysiology. This project proposes to generate a regulable and tunable strain of D4Z4/DUX4 transgenic mice using the Cre/lox system and targeted transgenesis into the Rosa26 locus. Importantly, this model incorporates the downstream cis regulatory elements and DUX4 splicing and polyadenylation of the FSHD-associated 4q35 locus. This is different from any of the mouse models discussed at meetings (none are published) that fail to show any phenotype. The targeting construct has already been generated and shown to function properly in human and mouse myogenic cell culture and myotubes. With this construct we believe we can manipulate DUX4-expression in mice 1) to a range of cells in a population (1:50 down to 1:5000) in the developmental profile of DUX4 expression and/or 2) in any select tissue or spacio-temporal pattern desired. These mice will prove invaluable for therapeutic screening and understanding DUX4 function. As such, once generated and initially characterized we will make these mice available to the FSH community at large in a timely manner for those with therapeutic approaches. 2. Expression of Human DUX4 in Zebrafish Development Summary (Provided by Applicant): FSHD is characterized by an asymmetric progressive weakness and wasting of the facial, shoulder and upper arm muscles. Hearing loss and retinal vasculopathy are frequently accompanied. Accumulating evidence supports the hypothesis that derepression of DUX4, a double homeobox gene located within D4Z4 unit in chromosome 4q35, play a role in the pathogenesis of FSHD. However, a major problem with this hypothesis is the extremely low abundance of DUX4 expression in FSHD muscle. It has been shown that approximately one cell per 1000 expresses DUX4 protein in cultured FSHD muscle cells. How this sporadic burst of DUX4 expression can cause a chronic and progressive myopathy is largely unknown. To address this question, generation of DUX4 animal model is essential. However, there is currently no good animal model due to the toxic nature of DUX4 where overexpression of DUX4 induces apoptosis to many types of cells, resulting in embryonic lethality. This has hampered the further understanding of FSHD pathogenesis and the development of therapeutic approaches. To generate DUX4 animal model, we injected extremely small amount of human DUX4 mRNA into zebrafish embryos. Microinjection of 0.2 or 0.1 pg of DUX4 mRNA (≈ 1 x 105 copies) caused asymmetric abnormalities on their eyes and fins. Injected embryos also showed affected muscle birefringence and slow swimming, suggesting muscle degeneration and weakness. These phenotypes are very similar to those observed in FSHD patients. We believe that DUX4 injected in small amounts into zebrafish is a good animal model for investigating the pathogenesis of FSHD and the impact of DUX4 on development. In this proposal, we plan to define the phenotype of the DUX4 mRNA injected zebrafish model to determine how much and where in development expression of DUX4 can cause the human FSHD-like phenotype in zebrafish. Furthermore, we plan to develop a conditional DUX4-transgenic zebrafish to create stable model. These zebrafish models will help us to understand DUX4-mediated pathogenesis in vivo, and provide us a platform to screen a number of small molecules for therapeutic approach. 3. FAT1 roles in muscular physiology and FSHD onset Summary (Provided by Applicant): FSHD is an autosomal dominant pathology recently ranked to the most prevalent muscular dystrophy. The genetic locus of the FSHD pathology has been identified 20 years ago, but the molecular mechanisms leading from this genotype to FSHD are still not clearly understood. Indeed, despite recent findings which highlighted the notion of permissive chromosome for FSHD, and the fact that Dux4 is always expressed in FSHD biopsies from these permissive chromosomes, the consequence of this expression on muscle development and function is not well established and the link between Dux4 expression and the development of FSHD pathology is not clearly understood. This reinforces the complexity of FSHD and emphasizes the need to identify other genetic elements putatively involved. 4. A humanized mouse model for investigations of FSHD pathology and therapeutic development Summary (Provided by Applicant): Facioscapulohumeral muscular dystrophy (FSHD) is genetically caused by the contraction of D4Z4 DNA repeats located on chromosome 4 in 4q35. Although the genetic defect was identified 20 years ago, the exact molecular mechanism causing the disease is unknown. Because of the unique nature of human D4Z4 repeats, there is currently no mouse disease model. To provide such a valuable tool, we have developed a humanized mouse model for FSHD. This model was obtained by the engraftment of FSHD patient derived myoblasts into mouse muscle. Because of the dominant nature of the disorder, we hypothesized that the FSHD engrafted fibers will display a disease phenotype and recapitulate pathological molecular mechanisms associated with FSHD that will allow us to study the development of the disease. 5. Tri-dimensional organization of the FSHD locus during prolifération and différentiation of muscle Cells in FSHD patients and controls Summary (Provided by Applicant): FSHD is an autosomal dominant neuromuscular disorder, with an incidence of 7: 100,000 recently ranked as the most common and prevalent rare muscular dystrophy of the adult. The number of D4Z4 is a critical determinant of the age of onset and clinical severity of the disease. A region distal to D4Z4 generates two allelic forms, 4qA and 4qB both equally common in the general population but FSHD is mainly associated with the 4qA allele. In addition, other polymorphisms, qualified as permissive alleles, exist; the pathology being often associated with the 4qA161 allele. In 5-10 % of families with a typical FSHD phenotype, there is no linkage to 4q35 and this type is referred to as type 2. Over the last decade, major advances have occurred in the understanding of the genetics of this disorder however the exact patho-mechanisms secondary to the genetic defect are still not understood. The DUX4 ORF is localized within D4Z4 and since at least one repeat is necessary to generate a pathogenic phenotype it has been hypothesized that DUX4 overexpression contributes to the pathology by leading to the production of a toxic protein in 1 in 1000 muscle nucleus. While several groups are actively seeking targets of the DUX4 protein, a technically challenging approach, our goal is to understand what leads to DUX4 expression. The deletion of repetitive elements and changes in epigenetic marks across the D4Z4 array such as DNA hypomethylation or decrease in H3K9 trimethylation also indicates that FSHD involves chromatin changes and epigenetic alterations. Interestingly, a localization of the 4q telomere at the nuclear periphery has been reported including by our group (Ottaviani et al., 2009; Arnoult et al., 2010), in close proximity to heterochromatin suggesting that subnuclear positioning contributes to this peculiar pathology. However, the links between epigenetic changes, nature of the 4q35 sequences, DUX4 expression and muscle phenotype have never been fully demonstrated. • Within the frame of this project, a cohort of samples from patients is available including accompanying detailed patient histories and genotyping. The epigenetic mechanisms regulating the 4q35 locus remain poorly understood, including how DNA methylation is controlled in the pathology and whether hypomethylation is a cause (increased instability) or a consequence of D4Z4 array shortening. We aim at understanding whether D4Z4 hypomethylation is an early event that precedes shortening of the repeated array or simply the consequence of the loss of a certain number of repeats by comparing methylation profiles in normal and diseased cells, and investigate the intergenerational transmission of DNA methylation in FSHD1 and 2 patients and correlate this methylation level with disease penetrance, D4Z4 array compaction and DUX4 expression. We have already shown that is it possible to visualize methylated regions through the detection of methylcytosines on combed DNA molecules using anti-m5C antibodies. We wish to use MC combined with DNA methylation detection with the probes designed for genomic analysis in order to evaluate the DNA methylation pattern of the D4Z4 array and flanking sequences for each individual allele on the slide and all D4Z4 copies. The sensitivity and stringency of the method requires further development and tests of different experimental conditions are in progress.
The FSH Society Scientific Advisory Board (SAB) met in October 2011 to review grant applications received for the August 2011 round of FSH Society grants funding. Below is a list of the funded projects, including project descriptions as submitted by grant applicant. 1. Identification of the epigenetic mechanisms that regulate DUX4 activity in skeletal muscle Summary (Provided by Applicant): D4Z4 repeat array chromatin relaxation and transcriptional de-repression of the non-polyadenylated double homeobox 4 (DUX4) gene unifies D4Z4 contraction-dependent FSHD1 and contraction-independent FSHD2. Only from FSHD-permissive genetic backgrounds the DUX4 transcript originating from the most telomeric unit of the array can be stabilized by a polyadenylation (polyA) signal outside the array. Non-permissive chromosomes fail to stabilize DUX4 in the absence of this polyA signal. Somatic DUX4 derepression in FSHD1 and FSHD2 leads to bursts of DUX4 protein in sporadic nuclei of cultured FSHD myotubes. 2. Resonance Imaging and Spectroscopy Biomarkers in FSHD Summary (Provided by Applicant): Facioscapulohumeral muscular dystrophy (FSHD) is the most prevalent hereditary progressive muscle disorder in humans. It is an autosomal dominant disease that causes wasting and weakness in multiple muscle groups (face, shoulders, and upper arms initially, and legs later in the disease) as well as significant disability in affected individuals. Recent advances in understanding the pathophysiology of FSHD have led to the identification of therapeutic targets. However, we lack appropriate biomarkers that reflect the degree of muscle degeneration and regeneration in these patients. Such biomarkers will be necessary for the successful completion of clinical trials. The proposed study will test the hypothesis that proton magnetic resonance spectroscopy (MRS) and quantitative magnetic resonance imaging (MRI) can be used to define unique metabolic profiles in the skeletal muscle of patients with FSHD and controls with normal muscles. These profiles can then be used as biomarkers of disease severity and surrogate outcome measures in therapeutic clinical trials in FSHD. Summary (Provided by Applicant): This $12,500 per year grant is supplementary support linked to an NIH R21 1R21NS076671-01 (2-year) application directed towards identifying chemical inhibitors of DUX4. We have previously screened 200,000 compounds and identified approximately 600 inhibitors of cell death induced by DUX4, and current work is directed towards identifying the most promising leads within this set of 600. The NIH application was recently selected for funding and the grant was initiated. This support from the FSH Society will be used to supplement the NIH project, principally by providing additional medicinal chemistry support (allowing us to increase the number of compounds that we follow-up by purchase/synthesis. These compounds help us to understand pathways that can indirectly inhibit toxicity associated with DUX4. See 1R21NS076671-01 [ http://projectreporter.nih.gov/project_info_description.cfm?aid=8225734&icde=10980193 ] for more information on the R21. Awards for February 2011 Cycle The FSH Society Scientific Advisory Board (SAB) met in June 2011 to review grant applications received for the February 2011 round of FSH Society grants funding. Below is a list of the funded projects, including project descriptions as submitted by grant applicants. 1. Antisense strategies against DUX4 as therapeutic approaches for FSHD Summary (Provided by Applicant): FSHD is a muscle degeneration disease genetically linked to contractions of the D4Z4 repeat array on the 4q35 subtelomeric region. Our group has identified the double homeobox 4 (DUX4) gene within each unit of the D4Z4 repeat array and shown that the encoded protein was expressed in primary myoblasts and biopsies of patients with FSHD but not in non-affected individuals. We found that the only stable DUX4 messenger RNAs derive from the last unit and extend to the flanking pLAM sequence that provides a polyA addition signal. This signal is required to develop FSHD as independently confirmed by an eight-laboratory consortium which studied genetic polymorphisms in hundreds of patients and thousands of healthy individuals. In aggregate our collaborative studies with four different groups have shown that the DUX4 protein is a transcription factor that targets a large set of genes, some of which encode other transcription factors that in turn target additional genes. Globally, DUX4 activation at the FSHD locus initiates a transcription cascade leading to muscle atrophy, inflammation, decreased differentiation potential and oxidative stress, the key features of the disease. By differential protein, RNA and gene studies we keep identifying additional FSHD biomarkers and define whether they are direct or indirect DUX4 targets. 2. Humanized mouse model for the study of Facioscapulohumeral Dystrophy FSHD is genetically caused by the contraction of D4Z4 DNA repeats located on chromosome 4 in 4q35. Although the genetic defect was identified 20 years ago, the exact molecular mechanism causing the disease is unknown, and there is currently no mouse disease model. To provide such a valuable tool, we will develop a humanized mouse model for FSHD, obtained by the engraftment of FSHD patient-derived myoblasts into mouse muscle. Engrafted human cells are able to form muscle fibers in the host mouse muscle, thus allowing pioneering studies in an in vivo context. Because of the dominant nature of FSHD, we hypothesize that the engrafted fibers will display a disease phenotype and recapitulate pathological molecular mechanisms associated with FSHD that will allow us to study the development of the disease. Our preliminary studies have already established the feasibility of this project. Through the cell repository of the Boston Biomedical Research Institute (BBRI) Wellstone Center, we have the unique opportunity to access early passage myoblast cells from cohorts of FSHD probands and their appropriate controls, i.e., a first degree relative. We will graft these standardized cultured cells into mouse muscle to obtain the FSHD humanized mouse model, thereby generating a well-controlled in vivo model for the study of FSHD. The very pressing issue in the field today is the verification of the current DUX4 model. The humanized mice produced will be used to investigate the hypothesis that DUX4 gene expression is a major cause of FSHD pathogenesis. In the obtained model, DUX4 expression will be evaluated during in vivo regeneration, and the consequence of its expression on fiber turnover and satellite cell renewal will be assessed. This work will contribute to the understanding of the role of DUX4 in vivo, thus providing a better understanding of FSHD pathogenesis. 3. Testing a therapeutic approach for FSHD: evaluation of the efficacy of AOs blocking DUX4 in a mouse model of isolated myofibres FSHD is considered the most frequent hereditary muscle disorder in adults, affecting one individual in 20,000. It is associated with contractions of the D4Z4 repeat array in the 4q35 subtelomeric region. In non-affected individuals, this array comprises 11-100 tandem copies of the 3.3-kb D4Z4 element while in patients, only 1-10 D4Z4 copies are left (Wijmenga et al., 1992). Our group has identified the double homeobox 4 (DUX4) gene within each unit of the D4Z4 repeat array (Gabriels et al., 1999) and several studies have now demonstrated the causative role of DUX4 in FSHD. We have demonstrate that the stable full-length DUX4 messenger RNA (mRNA) is produced from the last D4Z4 unit in FSHD, using a polyadenylation signal in the flanking pLAM region, located telomeric to the distal repeat (Dixit et al., 2007) as recently confirmed by a study of genetic polymorphisms in hundreds of patients and thousands of non-affected individuals (Lemmers et al., 2010). This polyadenylation site is necessary to develop FSHD on a contracted allele therefore called “permissive chromosome” (Lemmers et al., 2010). The mRNA from this distal D4Z4 unit contain the entire DUX4 open reading frame (ORF) and 1 or 2 alternatively spliced introns in the 3’UTR (DUX4-fl). In addition, a short DUX4 mRNA terminates at the previously described polyadenylation site in the pLAM region but uses a cryptic splice donor site within the DUX4 ORF (DUX4-s). DUX4-fl was only detected in FSHD muscle cells and biopsies, whereas DUX4-s is detected both in control and some FSHD samples (Snider et al., 2010). A long DUX4 mRNA was detected in induced pluripotent stem cells (iPS cells) and human testis where the gene contains 4 additional exons and a more distal polyadenylation signal. Expression of this DUX4 mRNA was suppressed during differentiation of control iPS cells to embryoid bodies whereas expression of full length DUX4 mRNA persisted in differentiated FSHD iPS cells (Snider et al., 2010). These data, together with the conservation of the DUX4 ORF through evolution (Clapp et al., 2007) suggests a possible role of DUX4 in human development. 4. Investigating mouse models of FSHD There is a great need for a valid mouse model for FSHD. Such an animal model would provide a valuable tool for exploring the effects of newly cloned genes and novel proteins on the pathophysiology of this disease. It would also greatly facilitate research towards the development and testing of new therapeutic approaches to FSHD. We propose to examine two possible mouse models of FSHD, the FRG1 over-expressor, from Drs. Davide Gabellini and Rossella Tupler, and mu-crystallin over-expressor, developed by Drs. Patrick Reed and Robert Bloch. I will breed these mice and test them for their physiological and morphological characteristics, and their susceptibility to injury and ability to recover from injury. I will also initiate xenografting studies to create mice with humanized normal and FSHD ankle dorsiflexor muscles, combining methods that are routine in the Bloch laboratory with unique reagents provided by collaborators in the Wellstone Muscular Dystrophy Cooperative Research Center (MDCRC), “Biomarkers for Therapy of FSHD.” These experiments should reveal the usefulness of available transgenic models for the study of FSHD, and promote the development of humanized mouse muscles for the study of the pathophysiology of FSHD in situ. 5. Epigenetic abnormality in FSHD Our preliminary findings indicate that D4Z4 repeat regions indeed interact with other genome regions, and that these interactions are indeed disrupted in FSHD. With a three-month extension of my fellowship, I plan to perform a high-throughput identification of potential target genes that interact with D4Z4 using the recently developed “Chromatin Interaction Analysis using a Paired-End Tag” (ChIA-PET) technique. This strategy enables the genome-wide detection of chromatin interactions mediated by specific factors that are normally assembled at D4Z4. Identification of additional FSHD pathogenic genes other than FRG1 and DUX4 is important to explore future therapeutic targets to improve or prevent the clinical 6. Analysis of DUX4-fl expression We request support from the FSH Society for our pilot project investigating DUX4 expression in unaffected and FSHD subjects. The DUX4-fl expression model for FSHD has not been independently validated, likely due to the lack of quality clinical resources in the field. At this point in FSHD research, validating and expanding upon the DUX4-cytotxicity model for pathogenesis is vital to the entire field and we are best positioned to do the necessary experiments with the unique set of highly controlled reagents being generated by the NIH Wellstone Muscular Dystrophy CRC for FSHD at BBRI. Each Wellstone cohort consists of an FSHD affected subject and an unaffected first-degree relative. Each subject donated two biopsies, one from the biceps and one from the deltoid. A portion of each biopsy was used to derive myogenic cell cultures. Quite surprisingly, in our initial preliminary results using 4 cohorts we found some inconsistencies with the published DUX4 expression results that have warranted further investigation. Therefore we have begun a much larger effort to analyze DUX4-fl mRNA and protein expression in a larger set of Wellstone cohorts using RT-PCR and immunostaining (ICC). However, this project is not funded at all in my lab or in the original Wellstone budget and my lab receives no financial support from the Wellstone Center. The Wellstone has supported us by providing us with cells, which we culture, and RNA which the Louis Kunkel lab purified from biopsies (we do not actually work with the biopsies) and we have been fortunate to receive these Wellstone samples. At this point, to ensure that our results are statistically meaningful, we need to analyze many more cells and biopsy RNAs and it has become cost prohibitive. Therefore I am requesting financial support for consumables and services (DNA sequencing) to conduct these experiments.
Awards for August 2010 Cycle The Scientific Advisory Board (SAB) met on November 22, 2010, to review grant applications received for the August 2010 round of FSH Society grants funding. Below are: 1.) a listing of the funded projects by grant applicants along with project descriptions as submitted by grant applicants. 1. “Small Molecule Screen to Identify Inhibitors of DUX4-mediated Toxicity, Therapeutic Approach for FSHD" PROJECT SUMMARY We and others have shown that DUX4 is toxic to different cell types, and induces FSHD-associated morphological and transcriptional changes in vitro.As a first step towards developing a targeted therapy for FSHD, we have taken advantage of conditional toxicity of DUX4-inducible myoblasts and we developed a small molecule screening platform for identifying inhibitors of DUX4. In our iC2C12-DUX4 inducible myoblasts, we incorporated full length of the last D4Z4 repeat, so prior its induction, we can not exclude that besides DUX4, some other products are not expressed (RNA, spliced proteins). Assay based on rapid cell death within 24 hours induced by high levels of DUX4 was used for high throughput screen of 200,000 chemicals, part of UT Southwestern HTS compound library. We identified more then 586 compounds with significant rescue ability (60 to >100% cell survival). To identify direct inhibitors, we have conducted serial follow up assays, including secondary screens to eliminate compounds which interfere with the rtTA/TRE inducible gene expression system, to distinguish anti oxidants, to confirm reversion of toxicity in other DUX4-expressing cell types. Several classes of compounds reverted toxicity indirectly, including antioxidants. After these secondary screens, we have narrowed down the list to 82 potentially direct DUX4 inhibitors. The goal of this proposal is to discover a chemical compound/s which efficiently inactivates the DUX4 protein and build on that discovery to develop a drug for a therapeutic approach to FSHD. To achieve this we will have to filter our current list (82 compounds) with additional secondary screens. Among them will be an analysis of MyoD expression and stability as well as cellular localization of the DUX4 protein (Aim 1). We reported that DUX4 is a potent inhibitor of MyoD expression. Therefore, a compound that will rescue MyoD expression in DUX4 induced cells is likely to be a therapeutically effective DUX4 inactivator. We assume that compounds which will be able to inactivate DUX4 in our iC2C12-DUX4 system most likely will be able to rescue FSHD myoblast phenotype. FSHD myoblasts were reported to have impaired differentiation, missregulation of myogenic transcription factors and increased susceptibility to oxidative stress. For that reason, as a functional in vitro study, we will test selected compounds for reversion of FSHD myoblast phenotype (Aim 2). Furthermore, we will test whether selected compounds exhibit their effect on inactivation of DUX4 protein or inhibition of DUX4 transcription or translation (Aim 2). At the end the most potent compound /s will be test for pharmacokinetic and pharmacodynamic properties (Aim 3). The aims of our proposed study target the most crucial topic and urgent needs of FSHD patients: specific and direct pharmacological therapy. Aim 1. To narrow our focus to the most promising direct DUX4 inhibitors. + 2. “Defining the Tissue and Cell Specificity of the Human DUX4 promoter in Mice” PROJECT SUMMARY FSHD was formally classified in 1954, and the primary genetic defect, D4Z4 contraction, was identified in 1992, but the pathogenic mechanisms underlying the disease have only recently started to come into focus. One reason for the difficulties in understanding FSHD biology is the lack of a relevant animal model expressing FSHD-permissive D4Z4 arrays. Since animal models, particularly mice, are crucial tools for studying disease pathogenesis and developing potential therapeutics, the absence of an FSHD mouse model is a fundamental problem in the FSHD field. A major goal of the Harper lab is to generate an FSHD mouse model expressing a single FSHD-permissive human D4Z4 repeat, and to use this model to understand the role of the D4Z4-resident gene, DUX4, in FSHD pathogenesis, and develop RNAi therapeutics targeting DUX4. In preliminary data, supported by previous FSH Society Fellowships to the Harper Lab, we delivered DUX4 to mouse muscle using adenoassociated viral vectors (AAV). DUX4 over-expression in muscle caused myopathy, but DUX4 is generally toxic to many non-muscle cells as well. Thus, we hypothesized that if DUX4 over-expression is an underlying pathogenic event in FSHD, it must be preferentially expressed only in affected muscles. We therefore developed transgenic mice expressing the green fluorescent protein (GFP) gene from the human DUX4 promoter (DUX4p-GFP mice), to determine the tissue and cell specificity of DUX4. In preliminary studies, we observed gross GFP expression in the face, shoulder girdle, and limbs of three independent DUX4p-GFP mouse lines. In this proposal, we will more carefully define the developmental and cellspecific expression patterns of DUX4p-GFP mice, and develop an AAV vector to determine whether a viral-mediated vascular delivery approach can produce the same expression patterns. Ultimately, these studies will be important first steps toward developing an AAV-mediated D4Z4 mouse model. Specific Aim 1: To define the developmental and cell-specific expression patterns of the human DUX4 promoter in mice. Mounting evidence supports the hypothesis that over-expression of the D4Z4-resident DUX4 gene is an underlying pathogenic event in FSHD. DUX4 is generally toxic to many cell types, and since FSHD is characterized by dystrophy of very specific muscle groups, we hypothesized that DUX4 is preferentially expressed only in affected muscles. Our newly generated DUX4p-GFP reporter mice grossly express GFP in areas that are preferentially affected in FSHD. In this Aim, we will perform a detailed characterization of GFP expression in our DUX4p-GFP mice. These results will help define the expected expression patterns of DUX4, and ultimately increase our understanding about the role of DUX4 FSHD pathogenesis. Specific Aim 2: To develop an AAV vector-mediated DUX4p-GFP mouse model. Previous endeavors to generate D4Z4 or DUX4 FSHD mouse models using traditional transgenic approaches have been unsuccessful. Although the previous attempts are not published in peer-reviewed literature, the difficulties encountered in generating these models were discussed in abstracts and talks at various scientific meetings over the last several years, including at the FSH Society’s 2008 International Research Consortium and Research Planning Meeting held in Philadelphia, Pennsylvania (http://www.fshsociety.org/assets/pdf/FSHD_ASHG_IRC2008_Philadelphia_11Nov_ProgramAndAbstract s_proof.pdf). Vascular delivery of AAV vectors carrying FSHD-permissive D4Z4 repeats to adult mice may circumvent the early embryonic death or developmental defects arising from germline transmission of D4Z4 repeats using traditional methods. In this Aim, we will test the feasibility of using AAV vectors to drive D4Z4-specific expression patterns in mouse muscle using an AAV.DUX4p-GFP reporter vector. + 3. “Identification of a Novel FSHD Biomarker [an unknown 50 kDa polypeptide highly expressed in FSHD samples]" PROJECT SUMMARY Screening FSHD patient-derivedmyoblasts, control myoblast, and muscle samples for expression changes at the proteomic level produced an unknown 50 kDa polypeptide highly expressed in FSHD samples compared to controls. Interestingly, this polypeptide is equally expressed in both normal and FSHD-patient derived myoblasts and early myotubes, however, unlike in control cells where its expression decreases, this unknown polypeptide remains highly expressed in differentiated muscle suggesting it is developmentally regulated and this regulation is disrupted in FSHD. This proposal will utilize standard biochemical techniques including column chromatography and mass spectrometry to purify and identify this 50 kDa putative FSHD biomarker. Subsequently, specific antibodies will be gerneated and characterized for further use to screen FSHD-derived cells to establish the universality of this biomarker. In addition, regardless of what its eventually identification turns out to be, identifying this protein will provide insight into FSHD pathophysiology, will be a useful FSHD biomarker, and may be one of the first proteins consistently and specifically upregulated in viable FSHD muscle. Therefore, generating specific and standardized antibodies to this protein will provide a useful resource for clinicians and basic FSHD researchers. + 4. “Toward Therapeutics for FSHD: Understanding mRNA Processing" PROJECT SUMMARY Recently, the group of van der Maarel reported in the journal Science their findings of the high These findings suggest a direct line to a novel therapeutic approach. The toxicity leading to + 5. “A multicenter collaborative study on the clinical features, expression profiling, and quality of life of pediatric facioscapulohumeral muscular dystrophy” PROJECT SUMMARY Fascioscapulohumeral dystrophy (FSHD) is the third most common type of muscular dystrophy, with an estimated prevalence of 1 in 15,000 to 20,000 (Kissel, 1999) (Flanigan et al., 2001). It is an autosomal dominant disorder due to a deletion within the D4Z4 repeat region located on the subtelomeric region of chromosome 4q35. FSHD causes progressive atrophy and frequently asymmetrical weakness involving the face, shoulder girdle, upper arm, abdominal, and lower limb muscles. Most affected individuals develop symptoms during their second or third decade, with 20% eventually become wheelchair dependent (Padberg, Lunt, Koch & Fardeau, 1991) (Zatz et al., 1998). Early childhood onset of FSHD may be associated with more severe weakness as well as extra-neuromuscular manifestations such as mental retardation, retinal vasculopathy, and sensorineural hearing loss (Jardine et al., 1994) (Klinge et al., 2006). Although the majority of cases of FSHD are inherited, about 20 - 30% of sporadic cases may occur as a result of spontaneous mutation or mosaicism (van der Maarel & Frants, 2005). Despite recent advances in the understanding of the molecular genetics of FSHD, the exact mechanism responsible for the disease remains unknown, and presently there is no cure (Tawil & Van Der Maarel, 2006) (van der Maarel, Frants & Padberg, 2007). As well, the prevalence, clinical variability, cross cultural presentation, and the psychosocial impact of FSHD on affected individuals constitute a significant public health concern. Emerging therapeutic trials will benefit from the availability of natural history data and reliable outcome measures (Rose & Tawil, 2004) (Tawil, 2008) for both children and adults with FSHD. Purpose of Study The main objectives of this study are: 1) to establish a standardized muscle testing protocol for use in children and youth with FSHD; 2) to describe the clinical phenotypes of pediatric onset FSHD; 3) to evaluate the impact of FSHD on health-related quality of life and disability across different age groups; and 4) to explore potential genetic modifiers of clinical phenotypes and disease progression in FSHD. TEXT SIZE
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