title: Simple fluorescent PCR assay for discriminating FRAXA fully mutated females from normal homozygotes.
abstract: Fragile X syndrome linked to the FRAXA locus is the most common inherited genetic disease accounting for mental retardation and is usually caused by the expansion of an unstable CGG repeat in the first exon of the FMR1 gene on the X chromosome. Despite its robustness, Southern blot is not suitable for large-scale routine screening as part of neuropediatric practice. PCR appears as an interesting alternative, and various protocols have been successfully applied to molecular screening in mentally retarded boys and girls. Unfortunately, as of this date these protocols are unable to detect the expanded allele in FRAXA females reliably, thereby failing to discriminate between fully mutated females from normal homozygotes. Therefore, we opted for an alternative approach in designing a semiquantitative PCR assay, based on the amplification of the sole wild-type allele. This method allowed us to detect the presence of one or two normal alleles with the same sizes, thereby discriminating between a FRAXA fully mutated female or a normal homozygote, respectively. A trial on 95 DNA samples from normal and mutated females demonstrated the reliability of the procedure. We believe this simple PCR assay is a powerful approach that would reduce the recourse to Southern blotting in females with mental retardation of unknown etiology.
authors.affiliation: Biochimie et Biologie Moléculaire, Hôpital d'Enfants Armand-Trousseau, AP-HP, Paris, France.
authors.forename: Claude
authors.initials: C
authors.lastname: Houdayer
authors.forename: Julie
authors.initials: J
authors.lastname: Lourdaux
authors.forename: Thierry
authors.initials: T
authors.lastname: Billette de Villemeur
authors.forename: Ghislaine
authors.initials: G
authors.lastname: Royer-Legrain
authors.forename: Michel
authors.initials: M
authors.lastname: Bahuau
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Bonnefont
authors.forename: Delphine
authors.initials: D
authors.lastname: Feldmann
authors.forename: Rémy
authors.initials: R
authors.lastname: Couderc
mesh_terms: D001483:Base Sequence; D002648:Child; D041321:Chromosomes, Human, X; D017931:DNA Primers; D005091:Exons; D005260:Female; D005600:Fragile X Syndrome / Q000235:genetics*; D006580:Genetic Carrier Screening; D006720:Homozygote; D006801:Humans; D008607:Intellectual Disability / Q000235:genetics; D008297:Male; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012016:Reference Values; D015203:Reproducibility of Results; D018911:Trinucleotide Repeats
chemical_list: D017931:DNA Primers