title: Reliable and sensitive detection of fragile X (expanded) alleles in clinical prenatal DNA samples with a fast turnaround time.
abstract: This study evaluated a large set of blinded, previously analyzed prenatal DNA samples with a novel, CGG triplet-repeat primed (TP)-PCR assay (Amplidex FMR1 PCR Kit; Asuragen, Austin, TX). This cohort of 67 fetal DNAs contained 18 full mutations (270 to 1100 repeats, including 1 mosaic), 12 premutations (59 to 150 repeats), 9 intermediate mutations (54 to 58 repeats), and 28 normal samples (17 to 50 repeats, including 3 homozygous female samples). TP-PCR accurately identified FMR1 genotypes, ranging from normal to full- mutation alleles, with a 100% specificity (95% CI, 85.0% to 100%) and a 97.4% sensitivity (95% CI, 84.9% to 99.9%) in comparison with Southern blot analysis results. Exact sizing was possible for a spectrum of normal, intermediate, and premutation (up to 150 repeats) alleles, but CGG repeat numbers >200 are only identified as full mutations. All homozygous alleles were correctly resolved. The assay is also able to reproducibly detect a 2.5% premutation and a 3% full-mutation mosaicism in a normal male background, but a large premutation in a full male mutation background was masked when the amount of the latter was >5%. Implementation of this TP-PCR will significantly reduce reflex testing using Southern blot analyses. Additional testing with methylation-informative techniques might still be needed for a few cases with (large) premutations or full mutations.
authors.affiliation: Center for Medical Genetics, UZ Brussel, Brussels, Belgium. sara.seneca@uzbrussel.be
authors.forename: Sara
authors.initials: S
authors.lastname: Seneca
authors.forename: Willy
authors.initials: W
authors.lastname: Lissens
authors.forename: Kristof
authors.initials: K
authors.lastname: Endels
authors.forename: Ben
authors.initials: B
authors.lastname: Caljon
authors.forename: Maryse
authors.initials: M
authors.lastname: Bonduelle
authors.forename: Kathleen
authors.initials: K
authors.lastname: Keymolen
authors.forename: Marjan
authors.initials: M
authors.lastname: De Rademaeker
authors.forename: Urielle
authors.initials: U
authors.lastname: Ullmann
authors.forename: Patrick
authors.initials: P
authors.lastname: Haentjens
authors.forename: Kim
authors.initials: K
authors.lastname: Van Berkel
authors.forename: Sonia
authors.initials: S
authors.lastname: Van Dooren
mesh_terms: D000483:Alleles; D004247:DNA / Q000235:genetics; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005820:Genetic Testing / Q000191:economics / Q000379:methods*; D005838:Genotype; D006801:Humans; D008297:Male; D009030:Mosaicism; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000191:economics / Q000379:methods*; D015203:Reproducibility of Results; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D013997:Time Factors; D018911:Trinucleotide Repeats
chemical_list: D004247:DNA; D051860:Fragile X Messenger Ribonucleoprotein 1; C497683:FMR1 protein, human