title: Improved fluorescent PCR-based assay for sizing CGG repeats at the FRAXA locus.
abstract: Fragile X syndrome is the most frequent heritable genetic disease involving mental retardation and is usually caused by an expanded CGG repeat in the first exon of the FMR1 gene. Therefore, searching for CGG expansion at the FRAXA locus among the mentally retarded has become a routine investigation in neuro-paediatric practice. Consequently, we have developed a fluorescent PCR-based assay for sizing repeats as an alternative to laborious and time-consuming Southern blot. The procedure utilises a reverse fluorescent labelled primer, and the Expand Long Template PCR system (Roche) with addition of dimethylsulfoxide and 7-deaza-dGTP It allows precise determination of the CGG repeat number in males and females for alleles from normal to premutation size range and detection of full mutations in males. We believe that this PCR protocol, allowing a high sample throughput, is useful for first-line screening among mentally retarded males, possibly complemented by Southern blot analysis to assess the methylation status of large mutated alleles.
authors.affiliation: Laboratoire de Biochimie et Biologie Moléculaire, Hôpital Necker Enfants-Malades, Paris, France. biochimie.trousseau@trs.ap-hop-paris.fr
authors.forename: C
authors.initials: C
authors.lastname: Houdayer
authors.forename: A
authors.initials: A
authors.lastname: Lemonnier
authors.forename: M
authors.initials: M
authors.lastname: Gerard
authors.forename: C
authors.initials: C
authors.lastname: Chauve
authors.forename: M
authors.initials: M
authors.lastname: Tredano
authors.forename: T B
authors.initials: TB
authors.lastname: de Villemeur
authors.forename: P
authors.initials: P
authors.lastname: Aymard
authors.forename: J P
authors.initials: JP
authors.lastname: Bonnefont
authors.forename: D
authors.initials: D
authors.lastname: Feldmann
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D005600:Fragile X Syndrome / Q000175:diagnosis* / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D008607:Intellectual Disability / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D018911:Trinucleotide Repeats