title: Single-tube methylation-specific duplex-PCR assay for rapid and accurate diagnosis of Fragile X Mental Retardation 1-related disorders.
abstract: OBJECTIVE
  Molecular diagnosis of fragile X syndrome demands assessment of fragile X mental retardation 1 (FMR1) CGG repeat size and methylation status, while predicting disease transmission risk requires determination of AGG interruption pattern. There is currently no single assay that provides all three categories of information. We describe a single-tube methylation-specific triplet-primed PCR assay for concurrently assessing methylation state, repeat size and structure of CGG repeat(s).
  
  
  METHODS
  Differentially labeled primers specific for methylated and unmethylated FMR1 alleles were used to amplify bisulfite-modified DNA, followed by capillary electrophoresis. Twenty-four reference DNAs and 107 patient samples were analyzed to evaluate assay performance.
  
  
  RESULTS
  Repeat size, AGG interruption pattern and methylation state were correctly identified in all tested samples. The assay also detected skewed X-inactivation when present in females, and somatic mosaicism in fragile X males.
  
  
  CONCLUSIONS
  When used in a molecular diagnostic setting, this novel assay could significantly minimize the need to reflex patient samples for Southern analysis.
authors.affiliation: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119074, Singapore.
authors.forename: Indhu-Shree
authors.initials: IS
authors.lastname: Rajan-Babu
authors.forename: Clara R L
authors.initials: CR
authors.lastname: Teo
authors.forename: Mulias
authors.initials: M
authors.lastname: Lian
authors.forename: Caroline G
authors.initials: CG
authors.lastname: Lee
authors.forename: Hai-Yang
authors.initials: HY
authors.lastname: Law
authors.forename: Samuel S
authors.initials: SS
authors.lastname: Chong
mesh_terms: D000483:Alleles; D002460:Cell Line; D019175:DNA Methylation; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005600:Fragile X Syndrome / Q000175:diagnosis* / Q000235:genetics*; D005838:Genotype; D006801:Humans; D008297:Male; D009030:Mosaicism; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D018911:Trinucleotide Repeats; D049951:X Chromosome Inactivation
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; C497683:FMR1 protein, human
keywords: AGG interruption; CGG repeat; FMR1; methylation; triplet-primed PCR