title: Performance evaluation of two methods using commercially available reagents for PCR-based detection of FMR1 mutation.
abstract: The current workflow for clinical Fragile X testing is time consuming and labor intensive. Recently developed PCR-based methods simplify workflow, amplify full mutation alleles, and improve sensitivity for detecting low-level mosaicism. We evaluated the performance characteristics and workflow of two methods using commercially available reagents for determining FMR1 mutation status. We also tested each method's ability to detect mosaicism (range, 100% to 1% for males; 50% to 1% for females). One method used reagents from Asuragen (AmplideX FMR1 PCR, research use only). The second method used analyte specific reagents from Abbott Molecular, including FMR1 Primer 1 (for repeat sizing) and FMR1 Primer 2 (for screening of expanded alleles). Each reaction was evaluated for accuracy, precision, correlation with previous results, and workflow. Both methods performed equally well in accuracy and precision studies using NIST standards and previously characterized Coriell samples. Both methods showed 100% concordance with results from a previous consensus study and for previously analyzed patient samples. The Asuragen reagents were able to detect full mutation mosaicism down to 5% and premutation mosaicism to 1%. The Abbott Molecular Primer 2 reagents were able to detect both full mutation and premutation mosaicism down to 25%. Both PCR-based methods for the determination of FMR1 mutation status performed well, with expected results in their final diagnoses, and differed significantly only in their workflow.
authors: Division of Molecular Diagnostics, Department of Pathology, Molecular Diagnostics Laboratory, Virginia Commonwealth University, Richmond, Virginia 23298-0248, USA.
authors: Jane S
authors: JS
authors: Juusola
authors: Paula
authors: P
authors: Anderson
authors: Fernanda
authors: F
authors: Sabato
authors: David S
authors: DS
authors: Wilkinson
authors: Arti
authors: A
authors: Pandya
authors: Andrea
authors: A
authors: Ferreira-Gonzalez
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005838:Genotype; D006801:Humans; D008297:Male; D009030:Mosaicism; D009154:Mutation; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; D011933:Reagent Kits, Diagnostic; C497683:FMR1 protein, human