title: High-resolution methylation polymerase chain reaction for fragile X analysis: evidence for novel FMR1 methylation patterns undetected in Southern blot analyses.
abstract: OBJECTIVE
  Fragile X syndrome is associated with the expansion of CGG trinucleotide repeats and subsequent methylation of the FMR1 gene. Molecular diagnosis of fragile X currently requires Southern blot analysis to assess methylation. This study describes the evaluation of a polymerase chain reaction-only workflow for the determination of methylation status across a broad range of FMR1 genotypes in male and female specimens.
  
  
  METHODS
  We evaluated a novel method that combines allele-specific methylation polymerase chain reaction and capillary electrophoresis with eight cell line and 80 clinical samples, including 39 full mutations. Methylation status was determined using a three-step workflow: (1) differential treatment of genomic DNA using a methylation-sensitive restriction enzyme; (2) polymerase chain reaction with two sets of dye-tagged primers; and (3) amplicon sizing by capillary electrophoresis. All samples were analyzed by both methylation polymerase chain reaction and Southern blot analysis.
  
  
  RESULTS
  FMR1 methylation status and CGG repeat sizing were accurately and reproducibly determined in a set of methylation controls and genomic DNA samples representing a spectrum of CGG repeat lengths and methylation states. Moreover, methylation polymerase chain reaction revealed allele-specific methylation patterns in premutation alleles that were unobtainable using Southern blot analysis.
  
  
  CONCLUSIONS
  Methylation polymerase chain reaction enabled high throughput, high resolution, and semiquantitative methylation assessments of FMR1 alleles, as well as determinations of CGG repeat length. Results for all samples were concordant with corresponding Southern blot analyses. As a result, this study presents a polymerase chain reaction-based method for comprehensive FMR1 analysis. In addition, the identification of novel methylation mosaic patterns revealed after polymerase chain reaction and capillary electrophoresis may be relevant to several FMR1 disorders.
authors: Diagnostic Research and Technology Development, Asuragen, Inc., Austin, Texas 78744, USA.
authors: Liangjing
authors: L
authors: Chen
authors: Diagnostic Research and Technology Development, Asuragen, Inc., Austin, Texas 78744, USA.
authors: Andrew
authors: A
authors: Hadd
authors: Diagnostic Research and Technology Development, Asuragen, Inc., Austin, Texas 78744, USA.
authors: Sachin
authors: S
authors: Sah
authors: Diagnostic Research and Technology Development, Asuragen, Inc., Austin, Texas 78744, USA.
authors: Jeffrey F
authors: JF
authors: Houghton
authors: Diagnostic Research and Technology Development, Asuragen, Inc., Austin, Texas 78744, USA.
authors: Stela
authors: S
authors: Filipovic-Sadic
authors: Department of Biochemistry and Molecular Medicine, University of California, School of Medicine, Davis, California 95616, USA.|M.I.N.D. Institute, University of California Davis Medical Center, Sacramento, California 95817, USA.
authors: Wenting
authors: W
authors: Zhang
authors: Department of Biochemistry and Molecular Medicine, University of California, School of Medicine, Davis, California 95616, USA.|M.I.N.D. Institute, University of California Davis Medical Center, Sacramento, California 95817, USA.
authors: Paul J
authors: PJ
authors: Hagerman
authors: Department of Biochemistry and Molecular Medicine, University of California, School of Medicine, Davis, California 95616, USA.|M.I.N.D. Institute, University of California Davis Medical Center, Sacramento, California 95817, USA.
authors: Flora
authors: F
authors: Tassone
authors: Diagnostic Research and Technology Development, Asuragen, Inc., Austin, Texas 78744, USA.
authors: Gary J
authors: GJ
authors: Latham
mesh_terms: D015139:Blotting, Southern; D002460:Cell Line; D019175:DNA Methylation; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005600:Fragile X Syndrome / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D008297:Male; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; C497683:FMR1 protein, human