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| Status |
Public on Sep 29, 2011 |
| Title |
Expression profiling FSHD vs. control myoblasts and myotubes |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
The gene expression pathways leading to muscle pathology in facioscapulohumeral dystrophy (FSHD) remain to be elucidated. This muscular dystrophy is caused by a contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35.2. We compared expression of control and FSHD myoblasts and myotubes (three preparations each) on exon microarrays (Affymetrix Human Exon 1.0 ST) and validated FSHD-specific differences for representative genes by qRT-PCR on additional myoblast cell strains. The FSHD and control myoblasts used for these experiments were shown to grow and differentiate into myotubes equally efficiently as control myoblasts. There were no significant FSHD-control differences in RNA levels for MYOD1 and MYOG at the myoblast and myotube stages and for MYF5 and MYF6 at the myoblast stage. In contrast, 295 other genes were dysregulated at least 2-fold in FSHD vs. control myoblasts (p <0.01, adjusted for multiple comparisons).
Remarkably, only 10% of the FSHD-associated gene dysregulation at the myoblast stage was downregulation. At the myotube stage, about ten times as many genes exhibited FSHD-associated downregulated as at the myoblast stage and twice as many genes displayed FSHD-associated upregulation. The FSHD-related changes in RNA levels appear to be due to posttranscriptional as well as transcriptional alterations. Among the prominently dysregulated pathways were signaling and oxidative stress pathways. By comparing expression profiles of control myoblasts and myotubes to each other and to 19 non-muscle cell types profiled identically, our study also revealed many new myogenesis associations for genes not previously annotated as muscle-specific.
Keywords: Disease state analysis and time course for differentiation
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| Overall design |
For the microarray, two normal-control myoblast-myotube sample pairs and one disease-control pair (sporadic inclusion body myositis; IBM), which are previously described samples GSM443914_CM33j_ Undiff_06042009_, GSM443913_CM33j_Diff_06042009_, GSM443916_CM01154_002_Undiff, GSM443915_CM01154_002_Diff_06012009, GSM443918_CM01201_001_Undiff_06012009, GSM443917_CM01201_001_Diff_06012009, were compared to three myoblast-myotube sample pairs generated from moderately affected skeletal muscle from patients with molecularly confirmed FSHD. Myoblasts were harvested at about 70% confluency, and myotubes were harvested at maximal myotube formation, 5 or 7 days of differentiation. The control myoblast and myotube biological replicates 1-3 were from a normal-control 23 year-old male (quadriceps biopsy), a normal-control 27 year-old female (quadriceps biopsy), and a 74 year-old female (deltoid biopsy), respectively. The FSHD myoblast biological replicates 1-3 were from a 45 year-old female (quadriceps biopsy), 22 year-old female (orthopedic scapular fixation rhomboid tissue), and 13 year-old male (quadriceps biopsy) and contained six, five, and three copies of the D4Z4 repeat unit in their disease-linked arrays, respectively. The FSHD myotube biological replicates 1-3 were from the same batch of cells as for the FSHD myoblasts with the exception of biological replicate 3, which was from a 14 year-old female (deltoid biopsy, 2 copies of the disease-linked D4Z4 repeat unit). For qRT-PCR validation, 4 - 8 FSHD myoblast or myotube samples from different patients with a molecularly confirmed diagnosis were compared to 4 - 8 analogous control samples from different unaffected individuals. Most of these were from quadriceps biopsies, and all but one had not been used on the microarray. Methods for generation of myoblast cell strains, propagation of myoblasts, and induction of differentiation by serum limitation were previously described (http://genome.ucsc.edu/ENCODE/protocols/cell/human/HSMM_HSMMtube_Crawford_protocol.pdf). Each batch of myoblasts and myotubes was checked by immunocytochemical staining for the quality of the cell preparation. The myoblast preparations for the microarray or qRT-PCR validation had >85% desmin-positive cells as determined with an antibody preparation confirmed to have selectivity for myogenic cells (Labvision/Thermo Scientific) using DAPI counterstaining. The myotube preparations had >70% of the nuclei in desmin-positive cells containing more than 2 nuclei per cell. Five of the six preparations for the microarray analysis (all but FM1795) were also immunostained with a monoclonal antibody to myosin heavy chain (MF20, a gift of Stephen D. Hauschka) and had >70% of the nuclei in multinucleated myosin heavy chain-positive cells.
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| Contributor(s) |
Ehrlich M, Tsumagari K |
| Citation(s) |
21951698 |
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| Submission date |
Dec 17, 2010 |
| Last update date |
Feb 18, 2019 |
| Contact name |
Sridar V Chittur |
| E-mail(s) |
schittur@albany.edu
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| Phone |
518-591-7215
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| Organization name |
SUNY-University at Albany
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| Department |
Center for Functional Genomics
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| Lab |
Microarray Core
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| Street address |
One Discovery Drive, CRC 342G
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| City |
Rensselaer |
| State/province |
NY |
| ZIP/Postal code |
12144 |
| Country |
USA |
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| Platforms (1) |
| GPL5175 |
[HuEx-1_0-st] Affymetrix Human Exon 1.0 ST Array [transcript (gene) version] |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA135293 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE26145_RAW.tar |
272.9 Mb |
(http)(custom) |
TAR (of CEL, CHP) |
| Processed data included within Sample table |
| Processed data provided as supplementary file |
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