December 2009
The FSH Society's Scientific Advisory Board (SAB)
recently approved the funding of six (6) new post-doctoral research awards.
1.
Daphne Cabianca, M.S.
Mentor: Davide Gabellini, Ph.D.
Division of Regenerative Medicine, Fondazione Centro San Raffaele del Monte Tabor
Milano, Italy
"A ncRNA regulating the epigenetic switch at the basis of FSHD"
Awarded the FSH Society New York Music and Song Fellowship Grant
$45,000 USD, 18 months
[Abstract Provided by Applicant]: Facioscapulohumeral muscular dystrophy (FSHD), the 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. The mechanism through which contraction of D4Z4 repeats causes muscular dystrophy is currently not clear, but there is a general agreement that reduction of D4Z4 activates an epigenetic cascade leading to 4q35 chromatin re-organization and altered gene expression.
My preliminary results suggest that a non-protein coding RNA (ncRNA) transcribed proximally to D4Z4 regulates 4q35 gene expression in FSHD. Furthermore, I found that the trithorax protein Ash1 is recruited to the region selectively in FSHD patients and is involved in 4q35 gene de-repression. It is tempting to speculate that production of the ncRNA activates an epigenetic switch culminating with 4q35 gene de-repression in FSHD. An attractive hypothesis would be that transcription of the region proximal to D4Z4 plays a role in de-condensation of the 4q35 genomic region, setting the stage for activation of 4q35 genes and, most importantly, preventing re-repression of the region. Here, I propose to rigorously investigate the role played by the ncRNA in regulating the epigenetic state of D4Z4 and in 4q35 gene de-repression.
My specific aim is:
1. To elucidate the mechanism underlying control of gene expression at 4q35. Understanding the mechanism through which the ncRNA is inducing 4q35 gene de-repression in FSHD will generate novel insights into the biological role of ncRNAs in chromatin structure regulation in higher eukaryotes. Moreover, it will help to elucidate the molecular pathways that become altered in FSHD, provide useful molecular markers of FSHD and favor the identification of potential therapeutic targets.
2.
Julie Dumonceaux, Ph.D.
Mentor: Gillian Butler Browne
Association Institut de Myologie
Paris, France
"Molecular mechanisms involved in FSHD"
Awarded the FSH Society Delta
Railroad Construction Company Fellowship Grant
$37,800 USD, 1 year
[Abstract Provided by Applicant]: The global deregulation of muscle genes in facioscapulohumeral dystrophy (FSHD) is still poorly understood : despite the identification of a contraction in the D4Z4 repeats in the chromosome 4 shared by the patients, the molecular mechanisms responsible for the disease have not yet been resolved. Our aim is to increase our understanding of these mechanisms. We will focus our studies on the abnormal expression of miRNA and on DUX4 expression in FSHD myoblasts and myotubes in order to determine the transcriptional alterations mediated by the D4Z4 contraction observed in the FSHD patients.
The effects of DUX4 over-expression will be analyzed in normal and immuno-deficient mice after transduction of the whole muscle by a DUX4 coding AAV vector. The DUX4 gene has been cloned under the control of a tetracycline dependent promoter (collaboration with Alexandra Belayew). This system allows us to control the expression level of DUX4 mRNA and to stop the DUX4 over-expression at any time. Our preliminary results shows that after a massive over-expression of DUX4, the majority of the muscle fibers have a centrally located nuclei, suggesting a toxicity of the transgene. We will now confirm this result on more mice and determine if this DUX4 over-expression induces a miss-regulation of other genes (FRG1, p21, PITX1, etc.) or of miRNAs. We will also modulate the expression of DUX4 to a lower and less toxic level.
Using immortalized FSHD myoblasts we have observed that some miRNAs are miss-regulated in these FSHD compared to DMD or control clones. We would like to confirm these results using a unique and rare material: immortalized clones of myoblasts generated from mosaic human muscle biopsies which will allow us to eliminate the extremely high inter individual variations classically observed among FSHD patients. Many clones have been isolated and have been sent to our collaborators Silvere Van der Maarel who provided the muscle biopsies and Stephen Tapscott. We will also receive from Nicolas Levy a skin biopsis from 2 highly interesting cases: identical twins of 32 years old, of whom one is totally asymptomatic whereas the other one is in a wheelchair. Both of them carry the same deletion: a southern blot of the peripheral lymphocytes has revealed that they both carry 2 D4Z4 units. We would like to understand how 2 identical twins have a totally different phenotype. We will immortalized the skin fibroblasts, transduced them using a lentivirus encoding MyoD under the control of an inducible promoter and analyse the expression of DUX4, FRG1, p21 etc in these cells. Moreover, all the immortalized clones we will generate (from the mosaic patients, as well as from the twins) will be injected into regenerating tibialis anterior muscles of immuno-deficient mice to analyse their fusion potential and the mRNA and miRNA mis-regulation in an in vivo context.
This work will contribute to a better understanding of the molecular mechanisms leading to FSHD. Some therapeutic targets as well as some bio-markers of the disease may be identified which would be essential for a cure.
3.
Scott Harper, Ph.D.
Center for Gene Therapy, The Research
Institute at Nationwide Children's Hospital, Ohio State University
"Investigating DUX4 Structure, Function, Expression Using Rational Mutagenesis and the Human DUX4 Promoter"
Awarded an FSH Society [ Cape Cod Walk 'n' Roll Fund, Conners/Jacobs Families Research Fund, and Kelly Family Research Fund ] Research Fellowship Grant
$40,000 USD, 1 year
[Abstract Provided by Applicant]: The pathogenic mechanisms underlying facioscapulohumeral muscular dystrophy (FSHD) are unclear. We hypothesize that DUX4 over-expression in muscle contributes to FSHD development. In our pilot study which was partially funded by FSH Society in 2008 (FSHD-LCT-002; $10,000), we showed the first in vivo evidence that DUX4 caused apoptosis and phenotypes associated with muscular dystrophy in zebrafish and mice. In a follow-up study, also funded by the FSH Society in 2009 (FSHS-JJFR-001; $40,000), we began to define the biochemical function of the DUX4 protein as it related to apoptosis and muscle toxicity, using rational mutagenesis. We have so far demonstrated that DUX4-mediated cell death and dystrophy were dependent upon its ability to bind DNA, and presumably transctivate downstream pro-apoptotic cascades. These results suggested that DUX4 toxicity is related to increased activity of natural DUX4 function and not simply to non-specific effects caused by over-loading cells with excessive protein.
Specific Aim 1: To define DUX4 domains necessary for stimulating apoptosis and muscle toxicity in vitro. In our preliminary work, we over-expressed DUX4 in mouse muscle using adeno-associated viral vectors (AAV). In parallel experiments, we also generated DUX4-expressing zebrafish embryos. Our data support that DUX4 induced apoptosis in vitro and caused dystrophic phenotypes in two different animal models (mice and zebrafish) in vivo. To gain a better understanding of DUX4 structure and function, we rationally mutagenized 8 predicted DUX4 functional domains or residues to investigate the functional effects of these changes on DUX4-induced apoptosis. In our most definitive data to date, we showed that DNA binding by the DUX4 homeodomain 1 (HOX1) is required to induce cell death in vitro and dystrophy-associated phenotypes in vivo. In this Aim, we will continue to investigate DUX4 structure function relationships pertaining to DUX4 pro-apoptotic activity in vitro using DUX4 mutants we have already generated or new constructs that we will generate. These studies will be an important step toward understanding DUX4 structure and function relationships as they pertain to stimulation of apoptosis and muscle toxicity.
Specific Aim 2: To define DUX4 domains necessary for stimulating apoptosis and muscle toxicity in vivo using the human DUX4 promoter. DUX4 has been detected in human FSHD patient muscle biopsies, but its normal expression pattern in humans is unknown. Our preliminary data support that DUX4 induces apoptosis and phenotypes associated with muscular dystrophy in two different animal models, suggesting it contributes to FSHD development. For our preliminary studies, we expressed DUX4 using vectors containing the ubiquitously active CMV promoter or the engineered, muscle-specific MHCK7 promoter. Both of these promoters have well-characterized expression patterns, but it is unknown whether their cell-type and developmental specificity overlaps with that of natural DUX4. In our previous fellowship, we proposed to express DUX4 or mutant DUX4 constructs in zebrafish using the engineered muscle-specific MHCK7 promoter. Here, we have modified this expression strategy to more faithfully model DUX4 expression. We hypothesize that in vivo expression of DUX4 from its own promoter will produce phenotypes associated with muscular dystrophy in vivo. In this Aim, we will first investigate the developmental and cell-type expression patterns of the human DUX4 promoter by generating eGFPreporter zebrafish. We will then investigate DUX4 structure-function relationships pertaining to DUX4- induced muscular dystrophy in vivo by expressing wild type and mutant human DUX4 constructs from the DUX4 promoter in zebrafish. This work will help us understand temporal and cell-type specificity of DUX4 expression, and ultimately better define a potential role for DUX4 in FSHD pathogenesis.
4.
Michael Kyba, Ph.D.
Minnesota Medical Foundation, University of Minnesota
"FSHD iPS cells: bioinformatics support"
Awarded the
FSH Society New York Music and Song Fellowship Grant
$45,000 USD, 1 year
Scientific relevance
Dr. Kyba’s research in Minnesota uses the recent scientific discovery of reprogramming somatic cells from adults into an embryonic stem (ES)-like state called induced pluripotent stem cells [iPS cells] to create an opportunity to model human diseases in a new way. Reverse engineering adult stem cells into embryonic stem cells creates an opportunity whose potential is greatest for diseases such as FSHD whose early regenerative and developmental processes are hard to study using tissue samples of mature differentiated adult muscle and whose mechanisms are currently unknown.
Scientific Relevance to FSHD – Achieving a Link between the FSHD the Genetic Mutation and Molecular Mechanism that causes FSHD muscles to be weak
FSH Society funds are facilitating the use and study of the first iPS cell lines developed carrying the FSHD mutation. iPS cell lines aid studies to watch and observe the epigenetics of the affected FSHD chromosome in muscle development and myogenic differentiation. Researchers are probing the epigenetics and the associated function of the FSHD associated D4Z4 repeat through each developmental point in the differentiation hierarchy (pluripotent cells, mesoderm, myogenic stem cells, myogenic progenitors, and myotubes) looking for changes that distinguish normal unaffected muscle from that of muscle affected by FSHD.
Clinical and Therapeutic Relevance to the Treatment and Cure of FSHD
Using induced pluripotent cells [iPS cells] derived from FSHD patient cells, researchers aim to:
1.) link the genetic mutation and molecular mechanism to provide a roadmap to a rational pharmacological intervention, and
2.) test the feasibility of genetic cure of FSHD in cells for the purpose of developing an autologous cell therapy.
FSH Society is funding these studies to address what we believe are the three key roadblocks:
1.) understanding the chromatin mechanics of the 4q35.2 locus,
2.) understanding the myogenic defect in FSHD, and,
3.) testing strategies to genetically repair the FSHD causing genetic defect on chromosome 4.
[Abstract Provided by Applicant]: Facioscapulohumeral muscular dystrophy (FSHO) is a genetically dominant progressive myopathy affecting approximately 25,000 individuals in the United States. It is the third mostcommon muscular dystrophy by incidence with a prevalence near or surpassing Duchenne's. The DNA lesion associated with this disease is a contraction within a series of 3.3 kb repeats - (D4Z4 repeats) near the telomere of 4q. It is not understood how this contraction results in disease, however it appears to modify the chromatin configuration of 4q35.2 and this has been proposed to lead to de-repression of nearby genes. There is currently no animal model bearing the actual FSHD mutation (D4Z4 contraction), and the lack of a suitable model system to study the effects of this mutation has severely hampered progress in understanding FSHD.
In an effort to shed light on the disease mechanism and to speed a potential cell therapy, we have recently derived induced pluripotent cells [iPS cells] from myoblast cultures taken from FSHD patients and controls. The overall goal of this research program is to take advantage of the unique tool represented by pluripotent FSHD-affected cells to accelerate our path towards a molecular understanding of this disease. To address this goal, we will combine in vitro differentiation of iPS cells with assays for chromatin status and gene expression at 4q35.2. We will use a combination of high throughput, in some cases whole genome assays, which will generate a large quantity of bioinformatics data. Funding is requested to support a bioinformatics specialist to generate chromatin maps based on this data, which will allow us to pinpoint where in the genome, and at what stage in development, and in what developmental lineages, chromatin changes take place in FSHD. This data will provide a critical and currently missing link between the genetic damage which ultimately causes FSHD (the D4Z4 repeat array contraction) and the eventual myopathic phenotype.
5.
Alberto Rosa, M.D., Ph.D.
Cellular and Molecular Biology, Fundacion Allende
Cordoba, Argentina
"DUX4-mediated control of Pitx1 gene expression"
Awarded the FSH Society Tango Fellowship Grant
$3,649.85 USD, 1 year
[Abstract Provided by Applicant]: A small research project request to identify specific amino acids from H1/H2 homeodomains controlling the expression of the Pitx1 gene. Hypothesis: Specific amino acids from the DUX4 H1/H2 homeodomains bind the promoter region of the gene Pitx1 to control its expression. Rationale: DUX4 wild type binds the sequence CGGATGCTGTCTTCTAATTAGT-TTGGACCC, located at the promoter region of the gene Pitx1. Homeobox motifs bind a core motif TAAT present in the promoter region of their specific target genes. In this project we will identify amino acids from H1 and/or H2 motifs participating in the control of Pitx1 gene expression mediated by DUX4.
6.
Yi Xing, Ph.D.
The University of Iowa
"Genome-wide analysis of FRG1-mediated splicing defects in FSHD and IFSHD"
Awarded the FSH Society Aubrie Lee Family Research Grant for Infantile FSHD & Fire Island Fellowship
$39,998 USD, 1 year
Researchers at the University of Iowa (UI) will lead an investigation on infantile facioscapulohumeral muscular dystrophy, or IFSHD, thanks to a one-year, $39,998 grant from the FSH Society, Inc.
Led by Yi Xing, Ph.D., UI assistant professor of internal medicine, biomedical engineering, and biostatistics, the team will use cutting-edge genomic technologies, including exon array chips and ultra-deep mRNA-sequencing, to identify RNA splicing differences among healthy people and people with FSHD or the infantile form of FSHD. RNA splicing differences affect how the genetic code is assembled and translated, and these differences can end up creating defective messenger RNAs or proteins.
This research opportunity comes, in part, as a result of the IFSHD clinic that Katherine Mathews, M.D., ran in conjunction with the FSH Society patient meeting at the University of Iowa in July 2008 and the data collected at that time from IFSHD patients. Xing is col¬laborating with Dr. Mathews, UI professor of pediatrics and neurology, who has collected skin fibroblast cells from FSHD patients, iFSHD patients and healthy controls made available through the efforts of the University of Iowa NIH-Funded Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center cell core run by Steven Moore, M.D., Ph.D., and the efforts of the FSH Society in recruiting patient donors.
[Abstract Provided by Applicant]: The goal of this project is to systematically examine FRG1-mediated splicing defects in FSHD and infantile FSHD (iFSHD). FRG1 is a component of the spliceosome and several lines of evidence suggest its involvement in RNA processing. It is proposed that due to the deletion of a transcriptional silencer within D4Z4, FRG1 is overexpressed in the skeletal muscle of FSHD patients. Transgenic mice selectively overexpressing FRG1 in skeletal muscle develop phenotypes that resemble FSHD in human patients. Moreover, in skeletal muscle of FRG1 transgenic mice and FSHD patients, aberrant splicing of two muscle-expressed genes (Tnnt3, Mtmr1) was observed. However, the global impact of FRG1 overexpression on splicing, as well as its role in FSHD pathogenesis, remains poorly understood, and other models of pathogenesis are also attractive. We plan to test the hypothesis that overexpression of FRG1 in FSHD patients will result in global disruption of pre-mRNA splicing. Further, if this hypothesis is correct, we would predict that the severe clinical phenotype of iFSHD is correlated with more profound and widespread transcriptome dysregulation at the level of splicing than is seen in milder, adult onset FSHD.
To test these hypotheses, we will utilize a high-density Affymetrix exon array (HJAY array), with 4.6 million probes for 315,137 exons and 260,488 exon junctions in the human genome to identify aberrant splicing events in FSHD and iFSHD. Our collaborator Katherine Mathews has collected skin fibroblast cells from FSHD patients, iFSHD patients and healthy controls and these are available through the Iowa Wellstone Center Core B, run by Steven Moore. After MyoD-induced myodifferentiation of fibroblasts, we will extract RNA and use exon arrays to identify splicing differences between healthy controls, FSHD patients and iFSHD patients. We intend to examine at least four individuals from each group. Following exon array analysis, candidate disease specific splicing events will be validated by RT-PCR/qPCR. We will also test the hypothesis that disease specific aberrant splicing events preferentially impact genes important for muscle or other organs known to be affected in FSHD/iFSHD. Together, results from this project will lead to improved understanding of FSHD/iFSHD pathophysiology, and reveal novel disease markers and therapeutic targets.
