title: Individual epigenetic status of the pathogenic D4Z4 macrosatellite correlates with disease in facioscapulohumeral muscular dystrophy.
abstract: BACKGROUND
  Both forms of facioscapulohumeral muscular dystrophy (FSHD) are associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite. Chromatin changes due to large deletions of heterochromatin (FSHD1) or mutations in chromatin regulatory proteins (FSHD2) lead to relaxation of epigenetic repression and increased expression of the deleterious double homeobox 4 (DUX4) gene encoded within the distal D4Z4 repeat. However, many individuals with the genetic requirements for FSHD remain asymptomatic throughout their lives. Here we investigated family cohorts of FSHD1 individuals who were either affected (manifesting) or without any discernible weakness (nonmanifesting/asymptomatic) and their unaffected family members to determine if individual epigenetic status and stability of repression at the contracted 4q35 D4Z4 array in myocytes correlates with FSHD disease.
  
  
  RESULTS
  Family cohorts were analyzed for DNA methylation on the distal pathogenic 4q35 D4Z4 repeat on permissive A-type subtelomeres. We found DNA hypomethylation in FSHD1-affected subjects, hypermethylation in healthy controls, and distinctly intermediate levels of methylation in nonmanifesting subjects. We next tested if these differences in DNA methylation had functional relevance by assaying DUX4-fl expression and the stability of epigenetic repression of DUX4-fl in myogenic cells. Treatment with drugs that alter epigenetic status revealed that healthy cells were refractory to treatment, maintaining stable repression of DUX4, while FSHD1-affected cells were highly responsive to treatment and thus epigenetically poised to express DUX4. Myocytes from nonmanifesting subjects had significantly higher levels of DNA methylation and were more resistant to DUX4 activation in response to epigenetic drug treatment than cells from FSHD1-affected first-degree relatives containing the same contraction, indicating that the epigenetic status of the contracted D4Z4 array is reflective of disease.
  
  
  CONCLUSIONS
  The epigenetic status of the distal 4qA D4Z4 repeat correlates with FSHD disease; FSHD-affected subjects have hypomethylation, healthy unaffected subjects have hypermethylation, and nonmanifesting subjects have characteristically intermediate methylation. Thus, analysis of DNA methylation at the distal D4Z4 repeat could be used as a diagnostic indicator of developing clinical FSHD. In addition, the stability of epigenetic repression upstream of DUX4 expression is a key regulator of disease and a viable therapeutic target.
authors.affiliation: Department of Neurology and Department of Cell and Developmental Biology, The Wellstone Program, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655 USA.
authors.forename: Takako I
authors.initials: TI
authors.lastname: Jones
authors.affiliation: Department of Neurology and Department of Cell and Developmental Biology, The Wellstone Program, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655 USA ; The Eunice Kennedy Shriver National Institute of Child Health and Human Development, Sen. Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, 31 Center Drive, Bethesda, MD USA.
authors.forename: Oliver D
authors.initials: OD
authors.lastname: King
authors.affiliation: Department of Neurology and Department of Cell and Developmental Biology, The Wellstone Program, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655 USA ; The Eunice Kennedy Shriver National Institute of Child Health and Human Development, Sen. Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, 31 Center Drive, Bethesda, MD USA.
authors.forename: Charis L
authors.initials: CL
authors.lastname: Himeda
authors.affiliation: Neuromuscular Biology & Disease Group, Departments of Neurology and Physiology & Biophysics, Boston University School of Medicine, 72 E Concord St, Boston, MA 02118 USA.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Homma
authors.affiliation: Department of Neurology and Department of Cell and Developmental Biology, The Wellstone Program, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655 USA ; The Eunice Kennedy Shriver National Institute of Child Health and Human Development, Sen. Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, 31 Center Drive, Bethesda, MD USA.
authors.forename: Jennifer C J
authors.initials: JC
authors.lastname: Chen
authors.affiliation: Neuromuscular Biology & Disease Group, Departments of Neurology and Physiology & Biophysics, Boston University School of Medicine, 72 E Concord St, Boston, MA 02118 USA.
authors.forename: Mary Lou
authors.initials: ML
authors.lastname: Beermann
authors.affiliation: Department of Neurology and Department of Cell and Developmental Biology, The Wellstone Program, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655 USA ; Key Lab of Swine Genetics and Breeding, Ministry of Agriculture, College of Animal Science and Technology, Huazhong Agricultural University, No.1, Shizishan Street, Wuhan, 430070 People's Republic of China.
authors.forename: Chi
authors.initials: C
authors.lastname: Yan
authors.affiliation: Department of Neurology and Department of Cell and Developmental Biology, The Wellstone Program, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655 USA ; The Eunice Kennedy Shriver National Institute of Child Health and Human Development, Sen. Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, 31 Center Drive, Bethesda, MD USA.
authors.forename: Charles P
authors.initials: CP
authors.lastname: Emerson
authors.affiliation: Neuromuscular Biology & Disease Group, Departments of Neurology and Physiology & Biophysics, Boston University School of Medicine, 72 E Concord St, Boston, MA 02118 USA.
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Miller
authors.affiliation: The Eunice Kennedy Shriver National Institute of Child Health and Human Development, Sen. Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, 31 Center Drive, Bethesda, MD USA ; The Hugo W. Moser Research Institute, Kennedy Krieger Institute, and the Departments of Neurology and Neuroscience, The Johns Hopkins School of Medicine, 733 N Broadway, Baltimore, MD 21205 USA.
authors.forename: Kathryn R
authors.initials: KR
authors.lastname: Wagner
authors.affiliation: Department of Neurology and Department of Cell and Developmental Biology, The Wellstone Program, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655 USA ; The Eunice Kennedy Shriver National Institute of Child Health and Human Development, Sen. Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, 31 Center Drive, Bethesda, MD USA.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Jones
keywords: D4Z4; DNA methylation; DUX4; Decitabine; Disease modifier; Epiallele; Epigenetic modifier; FSHD; Muscular dystrophy