title: Enhanced Detection and Sizing of the HTT CAG Repeat Expansion in Huntington Disease Using an Improved Triplet-Primed PCR Assay.
abstract: Accurate determination of the CAG repeat number is crucial for diagnostic and predictive testing for Huntington disease (HD). Current PCR-based assays can accurately size up to ∼110 HTT CAG repeats.
  
  
  
  To develop an improved assay capable of detecting larger CAG repeat expansions.
  
  
  
  A triplet-primed PCR (TP-PCR) assay was optimized and validated on 14 HD reference DNAs, including a sample carrying a large expansion of ∼180 CAG repeats.
  
  
  
  All 14 HD reference samples showed 100% concordance with the previously verified allele sizes. For alleles under 45 CAGs, identical repeat sizes were obtained, while alleles larger than 46 CAGs were sized to within ±1 CAG. The improved TP-PCR assay successfully detected the ∼180 CAG repeat allele in an affected sample.
  
  
  
  This method extends the detection limit of large expanded alleles to at least ∼175-180 CAG repeats, thus reducing the likelihood of requiring Southern blot analysis for any HD-affected sample.
authors.affiliation: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Mingjue
authors.initials: M
authors.lastname: Zhao
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; D019075:Electrophoresis, Capillary / Q000379:methods; D006801:Humans; D000071058:Huntingtin Protein / Q000235:genetics*; D006816:Huntington Disease / Q000175:diagnosis* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019680:Trinucleotide Repeat Expansion
chemical_list: C086055:HTT protein, human; D000071058:Huntingtin Protein