title: Validation of a robust PCR-based assay for quantifying fragile X CGG repeats.
abstract: BACKGROUND
  Sizing of FMR1 trinucleotide repeats in the clinical laboratory requires the use of capillary sequencer by PCR, or by a labor intensive measurement using Southern blot method. Our aim was to validate an accurate and robust PCR assay for quantification of CGG repeats.
  
  
  METHODS
  We performed an analytical and clinical validation of a new PCR-based method that utilizes a low-cost capillary electrophoresis instrument and the FragilEase™ reagent kit. First, analytical performance was demonstrated on 12 Coriell reference samples comprising normal through full mutations. Subsequently, a cohort of 112 archived clinical DNA samples, enriched for premutation and full mutations, was analyzed.
  
  
  RESULTS
  All samples were amplified successfully. Quantification of repeat numbers was interpreted by the use of standards with known repeats. Twenty-five full-mutation samples were successfully amplified with the largest allele size measured at 1380 repeats. The repeat numbers from the new assay were concordant with those obtained with the reference method. The intra-assay (CV<2.5%) and inter-assay imprecision was within 1 CGG repeat.
  
  
  CONCLUSIONS
  This new PCR-based method is reproducible and capable of identifying all Fragile X alleles. It is an accurate and robust method that facilitates Fragile X testing in a broader spectrum of clinical laboratories.
authors.affiliation: Department of Obstetrics and Gynaecology, Chinese University of Hong Kong, Hong Kong Special Administrative Region. Electronic address: kky254@ha.org.hk.
authors.forename: Yvonne K
authors.initials: YK
authors.lastname: Kwok
authors.affiliation: Department of Obstetrics and Gynaecology, Chinese University of Hong Kong, Hong Kong Special Administrative Region. Electronic address: cherrywong@cuhk.edu.hk.
authors.forename: Kit Man
authors.initials: KM
authors.lastname: Wong
authors.affiliation: Clinical Genetic Service, Department of Health, Hong Kong Special Administrative Region. Electronic address: dr.ivanlo@gmail.com.
authors.forename: Fai Man
authors.initials: FM
authors.lastname: Lo
authors.affiliation: Department of Obstetrics and Gynaecology, Chinese University of Hong Kong, Hong Kong Special Administrative Region. Electronic address: gracekong@cuhk.edu.hk.
authors.forename: Grace Wing Shan
authors.initials: GWS
authors.lastname: Kong
authors.affiliation: PerkinElmer Health Sciences, Inc., 940 Winter Street, Waltham, MA 02451, USA. Electronic address: Kent.Moore@PerkinElmer.com.
authors.forename: J Kent
authors.initials: JK
authors.lastname: Moore
authors.affiliation: PerkinElmer Health Sciences, Inc., 940 Winter Street, Waltham, MA 02451, USA. Electronic address: Shaoping.WU@PERKINELMER.COM.
authors.forename: Shaoping
authors.initials: S
authors.lastname: Wu
authors.affiliation: Clinical Genetic Service, Department of Health, Hong Kong Special Administrative Region. Electronic address: taksumlam@gmail.com.
authors.forename: Stephen T S
authors.initials: STS
authors.lastname: Lam
authors.affiliation: PerkinElmer Health Sciences, Inc., 940 Winter Street, Waltham, MA 02451, USA. Electronic address: Mack.Schermer@perkinelmer.com.
authors.forename: Mack
authors.initials: M
authors.lastname: Schermer
authors.affiliation: Department of Obstetrics and Gynaecology, Chinese University of Hong Kong, Hong Kong Special Administrative Region. Electronic address: tyleung@cuhk.edu.hk.
authors.forename: Tak Yeung
authors.initials: TY
authors.lastname: Leung
authors.affiliation: Department of Obstetrics and Gynaecology, Chinese University of Hong Kong, Hong Kong Special Administrative Region; CUHK-Utrecht University Joint Center of Language, Mind and Brain Genetics Core, Hong Kong Special Administrative Region; Angsana Molecular & Diagnostics Laboratory (HK) Ltd., Hong Kong Special Administrative Region. Electronic address: richardchoy@cuhk.edu.hk.
authors.forename: Kwong Wai
authors.initials: KW
authors.lastname: Choy
mesh_terms: D004586:Electrophoresis; D005260:Female; D005600:Fragile X Syndrome / Q000235:genetics*; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D018911:Trinucleotide Repeats / Q000235:genetics*
keywords: Capillary electrophoresis; FMR1; Fragile X; Full mutation; PCR; Trinucleotide repeats