title: A novel FMR1 PCR method for the routine detection of low abundance expanded alleles and full mutations in fragile X syndrome.
abstract: BACKGROUND
  Fragile X syndrome (FXS) is a trinucleotide-repeat disease caused by the expansion of CGG sequences in the 5' untranslated region of the FMR1 (fragile X mental retardation 1) gene. Molecular diagnoses of FXS and other emerging FMR1 disorders typically rely on 2 tests, PCR and Southern blotting; however, performance or throughput limitations of these methods currently constrain routine testing.
  
  
  METHODS
  We evaluated a novel FMR1 gene-specific PCR technology with DNA templates from 20 cell lines and 146 blinded clinical samples. The CGG repeat number was determined by fragment sizing of PCR amplicons with capillary electrophoresis, and results were compared with those for FMR1 Southern blotting analyses with the same samples.
  
  
  RESULTS
  The FMR1 PCR accurately detected full-mutation alleles up to at least 1300 CGG repeats and consisting of >99% GC character. All categories of alleles detected by Southern blotting, including 66 samples with full mutations, were also identified by the FMR1 PCR for each of the 146 clinical samples. Because all full mutation alleles in samples from heterozygous females were detected by the PCR, allele zygosity was reconciled in every case. The PCR reagents also detected a 1% mass fraction of a 940-CGG allele in a background of 99% 23-CGG allele-a roughly 5- fold greater sensitivity than obtained with Southern blotting.
  
  
  CONCLUSIONS
  The novel PCR technology can accurately categorize the spectrum of FMR1 alleles, including alleles previously considered too large to amplify; reproducibly detect low abundance full mutation alleles; and correctly infer homozygosity in female samples, thus greatly reducing the need for sample reflexing to Southern blotting.
authors.affiliation: Diagnostic Research & Technology Development, Asuragen, Inc., Austin, TX 78744, USA.
authors.forename: Stela
authors.initials: S
authors.lastname: Filipovic-Sadic
authors.forename: Sachin
authors.initials: S
authors.lastname: Sah
authors.forename: Liangjing
authors.initials: L
authors.lastname: Chen
authors.forename: Julie
authors.initials: J
authors.lastname: Krosting
authors.forename: Edward
authors.initials: E
authors.lastname: Sekinger
authors.forename: Wenting
authors.initials: W
authors.lastname: Zhang
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hagerman
authors.forename: Timothy T
authors.initials: TT
authors.lastname: Stenzel
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Hadd
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Latham
authors.forename: Flora
authors.initials: F
authors.lastname: Tassone
mesh_terms: D000483:Alleles; D005260:Female; D005600:Fragile X Syndrome / Q000175:diagnosis / Q000235:genetics*; D006720:Homozygote; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity