title: Simplified molecular diagnosis of fragile X syndrome by fluorescent methylation-specific PCR and GeneScan analysis.
abstract: BACKGROUND
  Fragile X syndrome (FXS), the most common cause of inherited mental impairment, is most commonly related to hyperexpansion and hypermethylation of a polymorphic CGG trinucleotide repeat in the 5' untranslated region of the FMR1 gene. Southern blot analysis is the most commonly used method for molecular diagnosis of FXS. We describe a simplified strategy based on fluorescent methylation-specific PCR (ms-PCR) and GeneScan analysis for molecular diagnosis of fragile X syndrome.
  
  
  METHODS
  We used sodium bisulfite treatment to selectively modify genomic DNA from fragile X and normal lymphoblastoid cell lines and from patients. We then performed ms-PCR amplification using fluorescently-labeled primers complementary to modified methylated or unmethylated DNA. Amplification products were resolved by capillary electrophoresis. FMR1 mutational status was determined by a combination of fluorescent peak sizes and patterns on the GeneScan electropherogram.
  
  
  RESULTS
  DNA samples from male and female persons with known NL, PM, and FM FMR1 CGG repeats were analyzed. Each FMR1 genotype produced a unique GeneScan electropherogram pattern, thus providing a way to identify the various disease states. The number of CGG repeats in all NL and PM alleles were determined accurately. Analysis by both the new assay and Southern blot of a family segregating with FXS showed complete concordance between both methods.
  
  
  CONCLUSIONS
  This simplified molecular diagnostic test, based on fluorescent methylation-specific PCR, may be a suitable alternative or complement to Southern blot analysis for the diagnosis of FXS.
authors.affiliation: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, and Children's Medical Institute, National University Hospital, Singapore. zhouyouyou@fudan.edu.cn
authors.forename: Youyou
authors.initials: Y
authors.lastname: Zhou
authors.forename: Josephine M S
authors.initials: JM
authors.lastname: Lum
authors.forename: Gare-Hoon
authors.initials: GH
authors.lastname: Yeo
authors.forename: Jennifer
authors.initials: J
authors.lastname: Kiing
authors.forename: Stacey K H
authors.initials: SK
authors.lastname: Tay
authors.forename: Samuel S
authors.initials: SS
authors.lastname: Chong
mesh_terms: D019175:DNA Methylation; D019075:Electrophoresis, Capillary; D005260:Female; D005456:Fluorescent Dyes; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005600:Fragile X Syndrome / Q000235:genetics*; D006801:Humans; D007202:Indicators and Reagents; D008297:Male; D025202:Molecular Diagnostic Techniques; D009154:Mutation; D016133:Polymerase Chain Reaction; D013447:Sulfites
chemical_list: D005456:Fluorescent Dyes; D051860:Fragile X Messenger Ribonucleoprotein 1; D007202:Indicators and Reagents; D013447:Sulfites; C497683:FMR1 protein, human; C009279:sodium bisulfite