title: Reciprocal changes in DNA methylation and hydroxymethylation and a broad repressive epigenetic switch characterize FMR1 transcriptional silencing in fragile X syndrome.
abstract: BACKGROUND
  Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from the loss of function of the fragile X mental retardation 1 (FMR1) gene. The molecular pathways associated with FMR1 epigenetic silencing are still elusive, and their characterization may enhance the discovery of novel therapeutic targets as well as the development of novel clinical biomarkers for disease status.
  
  
  RESULTS
  We have deployed customized epigenomic profiling assays to comprehensively map the FMR1 locus chromatin landscape in peripheral mononuclear blood cells (PBMCs) from eight FXS patients and in fibroblast cell lines derived from three FXS patient. Deoxyribonucleic acid (DNA) methylation (5-methylcytosine (5mC)) and hydroxymethylation (5-hydroxymethylcytosine (5hmC)) profiling using methylated DNA immunoprecipitation (MeDIP) combined with a custom FMR1 microarray identifies novel regions of DNA (hydroxy)methylation changes within the FMR1 gene body as well as in proximal flanking regions. At the region surrounding the FMR1 transcriptional start sites, increased levels of 5mC were associated to reciprocal changes in 5hmC, representing a novel molecular feature of FXS disease. Locus-specific validation of FMR1 5mC and 5hmC changes highlighted inter-individual differences that may account for the expected DNA methylation mosaicism observed at the FMR1 locus in FXS patients. Chromatin immunoprecipitation (ChIP) profiling of FMR1 histone modifications, together with 5mC/5hmC and gene expression analyses, support a functional relationship between 5hmC levels and FMR1 transcriptional activation and reveal cell-type specific differences in FMR1 epigenetic regulation. Furthermore, whilst 5mC FMR1 levels positively correlated with FXS disease severity (clinical scores of aberrant behavior), our data reveal for the first time an inverse correlation between 5hmC FMR1 levels and FXS disease severity.
  
  
  CONCLUSIONS
  We identify novel, cell-type specific, regions of FMR1 epigenetic changes in FXS patient cells, providing new insights into the molecular mechanisms of FXS. We propose that the combined measurement of 5mC and 5hmC at selected regions of the FMR1 locus may significantly enhance FXS clinical diagnostics and patient stratification.
authors: Preclinical Safety, Translational Medicine, Novartis Institutes for Biomedical Research, Novartis Pharma AG, CH-4057 Basel, Switzerland.
authors: Sarah
authors: S
authors: Brasa
authors: Preclinical Safety, Translational Medicine, Novartis Institutes for Biomedical Research, Novartis Pharma AG, CH-4057 Basel, Switzerland.
authors: Arne
authors: A
authors: Mueller
authors: Service de Génétique Médicale, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland.
authors: Sébastien
authors: S
authors: Jacquemont
authors: Preclinical Safety, Translational Medicine, Novartis Institutes for Biomedical Research, Novartis Pharma AG, CH-4057 Basel, Switzerland.
authors: Florian
authors: F
authors: Hahne
authors: Neuroscience Translational Medicine, Novartis Institutes for Biomedical Research, Novartis Pharma AG, CH-4056 Basel, Switzerland.
authors: Izabela
authors: I
authors: Rozenberg
authors: BioMarker Development, Novartis Institutes for Biomedical Research, Novartis Pharma AG, Cambridge, MA USA.
authors: Thomas
authors: T
authors: Peters
authors: BioMarker Development, Novartis Institutes for Biomedical Research, Novartis Pharma AG, Cambridge, MA USA.
authors: Yunsheng
authors: Y
authors: He
authors: Clinical Diagnostics, Novartis Institutes for Biomedical Research, Novartis Pharma AG, Cambridge, MA USA.
authors: Christine
authors: C
authors: McCormack
authors: Neuroscience, Novartis Institutes for Biomedical Research, Novartis Pharma AG, CH-4057 Basel, Switzerland.
authors: Fabrizio
authors: F
authors: Gasparini
authors: Preclinical Safety, Translational Medicine, Novartis Institutes for Biomedical Research, Novartis Pharma AG, CH-4057 Basel, Switzerland.
authors: Salah-Dine
authors: SD
authors: Chibout
authors: Preclinical Safety, Translational Medicine, Novartis Institutes for Biomedical Research, Novartis Pharma AG, CH-4057 Basel, Switzerland.
authors: Olivier
authors: O
authors: Grenet
authors: Preclinical Safety, Translational Medicine, Novartis Institutes for Biomedical Research, Novartis Pharma AG, CH-4057 Basel, Switzerland.
authors: Jonathan
authors: J
authors: Moggs
authors: Neuroscience Translational Medicine, Novartis Institutes for Biomedical Research, Novartis Pharma AG, CH-4056 Basel, Switzerland.
authors: Baltazar
authors: B
authors: Gomez-Mancilla
authors: Preclinical Safety, Translational Medicine, Novartis Institutes for Biomedical Research, Novartis Pharma AG, CH-4057 Basel, Switzerland.
authors: Rémi
authors: R
authors: Terranova
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D047369:Chromatin Immunoprecipitation; D019175:DNA Methylation; D044127:Epigenesis, Genetic / Q000235:genetics; D057890:Epigenomics; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics* / Q000502:physiology; D005600:Fragile X Syndrome / Q000235:genetics*; D020868:Gene Silencing; D005819:Genetic Markers; D006801:Humans; D008297:Male; D008875:Middle Aged; D034622:RNA Interference; D055815:Young Adult
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; D005819:Genetic Markers; C497683:FMR1 protein, human
keywords: 5-hydroxymethylation (5hmC); Chromatin profiling; Clinical biomarker; Epigenetic silencing, FMR1; Fragile X syndrome (FXS)