title: Methylation of novel markers of fragile X alleles is inversely correlated with FMRP expression and FMR1 activation ratio.
abstract: The fragile X syndrome (FXS) is caused by silencing of the fragile X mental retardation gene (FMR1) and the absence of its product, fragile X mental retardation protein (FMRP), resulting from CpG island methylation associated with large CGG repeat expansions (more than 200) termed full mutation (FM). We have identified a number of novel epigenetic markers for FXS using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), naming the most informative fragile X-related epigenetic element 1 (FREE1) and 2 (FREE2). Methylation of both regions was correlated with that of the FMR1 CpG island detected using Southern blot (FREE1 R = 0.97; P < 0.00001, n = 23 and FREE2 R = 0.93; P < 0.00001, n = 23) and negatively correlated with lymphocyte expression of FMRP (FREE1 R = -0.62; P = 0.01, n = 15 and FREE2 R = -0.55; P = 0.03, n = 15) in blood of partially methylated 'high functioning' FM males. In blood of FM carrier females, methylation of both markers was inversely correlated with the FMR1 activation ratio (FREE1 R = -0.93; P < 0.0001, n = 12 and FREE2 R = -0.95; P < 0.0001, n = 9). In a sample set of 49 controls, 18 grey zone (GZ 40-54 repeats), 22 premutation (PM 55-170 repeats) and 22 (affected) FXS subjects, the FREE1 methylation pattern was consistent between blood and chorionic villi as a marker of methylated FM alleles and could be used to differentiate FXS males and females from controls, as well as from carriers of GZ/PM alleles, but not between GZ and PM alleles and controls. Considering its high-throughput and specificity for pathogenic FM alleles, low cost and minimal DNA requirements, FREE MALDI-TOF MS offers a unique tool in FXS diagnostics and newborn population screening.
authors.affiliation: Chromosome and Chromatin Research Laboratory, The Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria 3052, Australia. david.godler@mcri.edu.au
authors.forename: David Eugeny
authors.initials: DE
authors.lastname: Godler
authors.forename: Flora
authors.initials: F
authors.lastname: Tassone
authors.forename: Danuta Zuzanna
authors.initials: DZ
authors.lastname: Loesch
authors.forename: Annette Kimball
authors.initials: AK
authors.lastname: Taylor
authors.forename: Freya
authors.initials: F
authors.lastname: Gehling
authors.forename: Randi Jenssen
authors.initials: RJ
authors.lastname: Hagerman
authors.forename: Trent
authors.initials: T
authors.lastname: Burgess
authors.forename: Devika
authors.initials: D
authors.lastname: Ganesamoorthy
authors.forename: Debbie
authors.initials: D
authors.lastname: Hennerich
authors.forename: Lavinia
authors.initials: L
authors.lastname: Gordon
authors.forename: Andrew
authors.initials: A
authors.lastname: Evans
authors.forename: K H
authors.initials: KH
authors.lastname: Choo
authors.forename: Howard Robert
authors.initials: HR
authors.lastname: Slater
mesh_terms: D000368:Aged; D000483:Alleles; D001483:Base Sequence; D002460:Cell Line; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics* / Q000378:metabolism; D005600:Fragile X Syndrome / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D015533:Transcriptional Activation
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; C497683:FMR1 protein, human