title: Methylation analysis of the fragile X syndrome by PCR.
abstract: The fragile X syndrome is predominantly caused by a large expansion of a CGG trinucleotide repeat in the promoter region of the FMR1 gene, which is associated with methylation and downregulation of transcription. The molecular diagnosis of this disorder is based on repeat size and methylation analysis of the FMR1 gene usually by Southern blot analysis. We describe a PCR-based method for the analysis of methylation of the FMR1 gene, which involves bisulfite treatment of DNA prior to amplification. Fifty-two normal and 48 affected, premutation, or mosaic males were analyzed in a blinded study by this method. A prospective study of 30 males suspected of fragile X was also performed. Amplification specific for the methylated FMR1 sequence was readily observed in all individuals with a full mutation, whereas all normal and premutation individuals showed only amplification-specific for the unmethylated sequence, thus, allowing affected and unaffected males to be distinguished. A full mutation in the presence of mosaicism was also detectable by this method. Methylation-specific PCR appears to be a rapid and reliable tool for the diagnosis of fragile X males.
authors.affiliation: Department of Human Genetics, University of Chicago, IL 60637, USA.
authors.forename: S
authors.initials: S
authors.lastname: Das
authors.forename: T
authors.initials: T
authors.lastname: Kubota
authors.forename: M
authors.initials: M
authors.lastname: Song
authors.forename: R
authors.initials: R
authors.lastname: Daniel
authors.forename: E M
authors.initials: EM
authors.lastname: Berry-Kravis
authors.forename: T W
authors.initials: TW
authors.lastname: Prior
authors.forename: B
authors.initials: B
authors.lastname: Popovich
authors.forename: L
authors.initials: L
authors.lastname: Rosser
authors.forename: T
authors.initials: T
authors.lastname: Arinami
authors.forename: D H
authors.initials: DH
authors.lastname: Ledbetter
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D019175:DNA Methylation; D017931:DNA Primers / Q000235:genetics; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1; D005600:Fragile X Syndrome / Q000175:diagnosis* / Q000235:genetics*; D006801:Humans; D008297:Male; D009030:Mosaicism; D009419:Nerve Tissue Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D016601:RNA-Binding Proteins; D018911:Trinucleotide Repeats
chemical_list: D004247:DNA; D017931:DNA Primers; D051860:Fragile X Messenger Ribonucleoprotein 1; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; C497683:FMR1 protein, human