title: Gene expression during normal and FSHD myogenesis.
abstract: BACKGROUND
  Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35. Within each repeat unit is a gene, DUX4, that can encode a protein containing two homeodomains. A DUX4 transcript derived from the last repeat unit in a contracted array is associated with pathogenesis but it is unclear how.
  
  
  METHODS
  Using exon-based microarrays, the expression profiles of myogenic precursor cells were determined. Both undifferentiated myoblasts and myoblasts differentiated to myotubes derived from FSHD patients and controls were studied after immunocytochemical verification of the quality of the cultures. To further our understanding of FSHD and normal myogenesis, the expression profiles obtained were compared to those of 19 non-muscle cell types analyzed by identical methods.
  
  
  RESULTS
  Many of the ~17,000 examined genes were differentially expressed (>2-fold, p<0.01) in control myoblasts or myotubes vs. non-muscle cells (2185 and 3006, respectively) or in FSHD vs. control myoblasts or myotubes (295 and 797, respectively). Surprisingly, despite the morphologically normal differentiation of FSHD myoblasts to myotubes, most of the disease-related dysregulation was seen as dampening of normal myogenesis-specific expression changes, including in genes for muscle structure, mitochondrial function, stress responses, and signal transduction. Other classes of genes, including those encoding extracellular matrix or pro-inflammatory proteins, were upregulated in FSHD myogenic cells independent of an inverse myogenesis association. Importantly, the disease-linked DUX4 RNA isoform was detected by RT-PCR in FSHD myoblast and myotube preparations only at extremely low levels. Unique insights into myogenesis-specific gene expression were also obtained. For example, all four Argonaute genes involved in RNA-silencing were significantly upregulated during normal (but not FSHD) myogenesis relative to non-muscle cell types.
  
  
  CONCLUSIONS
  DUX4's pathogenic effect in FSHD may occur transiently at or before the stage of myoblast formation to establish a cascade of gene dysregulation. This contrasts with the current emphasis on toxic effects of experimentally upregulated DUX4 expression at the myoblast or myotube stages. Our model could explain why DUX4's inappropriate expression was barely detectable in myoblasts and myotubes but nonetheless linked to FSHD.
authors.affiliation: Human Genetics Program, Tulane Medical School, New Orleans, LA, USA.
authors.forename: Koji
authors.initials: K
authors.lastname: Tsumagari
authors.forename: Shao-Chi
authors.initials: SC
authors.lastname: Chang
authors.forename: Michelle
authors.initials: M
authors.lastname: Lacey
authors.forename: Carl
authors.initials: C
authors.lastname: Baribault
authors.forename: Sridar V
authors.initials: SV
authors.lastname: Chittur
authors.forename: Janet
authors.initials: J
authors.lastname: Sowden
authors.forename: Rabi
authors.initials: R
authors.lastname: Tawil
authors.forename: Gregory E
authors.initials: GE
authors.lastname: Crawford
authors.forename: Melanie
authors.initials: M
authors.lastname: Ehrlich
mesh_terms: D017398:Alternative Splicing; D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002894:Chromosomes, Human, Pair 4; D015536:Down-Regulation; D005091:Exons; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D024510:Muscle Development / Q000235:genetics*; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D020391:Muscular Dystrophy, Facioscapulohumeral / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D032446:Myoblasts / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D059368:RNA Isoforms / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation
chemical_list: D060565:Argonaute Proteins; C121257:DUX4L1 protein, human; D018398:Homeodomain Proteins; D059368:RNA Isoforms