title: A simple, high-throughput assay for Fragile X expanded alleles using triple repeat primed PCR and capillary electrophoresis.
abstract: Population screening has been proposed for Fragile X syndrome to identify premutation carrier females and affected newborns. We developed a PCR-based assay capable of quickly detecting the presence or absence of an expanded FMR1 allele with high sensitivity and specificity. This assay combines a triplet repeat primed PCR with high-throughput automated capillary electrophoresis. We evaluated assay performance using archived samples sent for Fragile X diagnostic testing representing a range of Fragile X CGG-repeat expansions. Two hundred five previously genotyped samples were tested with the new assay. Data were analyzed for the presence of a trinucleotide "ladder" extending beyond 55 repeats, which was set as a cut-off to identify expanded FMR1 alleles. We identified expanded FMR1 alleles in 132 samples (59 premutation, 71 full mutation, 2 mosaics) and normal FMR1 alleles in 73 samples. We found 100% concordance with previous results from PCR and Southern blot analyses. In addition, we show feasibility of using this assay with DNA extracted from dried-blood spots. Using a single PCR combined with high-throughput fragment analysis on the automated capillary electrophoresis instrument, we developed a rapid and reproducible PCR-based laboratory assay that meets many of the requirements for a first-tier test for population screening.
authors.affiliation: Pathology Department, University of Utah, Salt Lake City, Utah, USA. lyone@aruplab.com
authors.forename: Elaine
authors.initials: E
authors.lastname: Lyon
authors.forename: Thomas
authors.initials: T
authors.lastname: Laver
authors.forename: Ping
authors.initials: P
authors.lastname: Yu
authors.forename: Mohamed
authors.initials: M
authors.lastname: Jama
authors.forename: Keith
authors.initials: K
authors.lastname: Young
authors.forename: Michael
authors.initials: M
authors.lastname: Zoccoli
authors.forename: Natalia
authors.initials: N
authors.lastname: Marlowe
mesh_terms: D000483:Alleles; D001800:Blood Specimen Collection; D004587:Electrophoresis, Agar Gel; D019075:Electrophoresis, Capillary / Q000379:methods*; D005240:Feasibility Studies; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005600:Fragile X Syndrome / Q000235:genetics*; D005838:Genotype; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019680:Trinucleotide Repeat Expansion / Q000235:genetics*
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1