title: Rapid identification of female carriers of DMD/BMD by quantitative real-time PCR.
abstract: Recently developed PCR systems offer online-monitoring of amplification and allow simple and reliable DNA quantification. We have used the LightCycler system to develop a simple and rapid method for direct identification of female carriers of deletions and duplications in the dystrophin gene. The challenge resides in the ability to identify the presence of a deleted or duplicated allele over the background contributed by the normal allele. Quantification is based on the determination of the ratio between potentially deleted/duplicated dystrophin exons and non-deleted/-duplicated reference exons using the unspecific dsDNA-dye SYBRgreen I. In a retrospective study, we evaluated our method in female relatives of DMD/BMD patients with known carrier status by comparative analysis of deleted or duplicated versus non-deleted/-duplicated exons. Carrier status was accurately attributed in 100% of cases, the mean ratios being 0.52+/-0.12 for deletion carriers (expected value: 0.5) and 1.56+/-0.18 for duplication carriers (expected value: 1.5) vs. 1.022+/-0.17 for non-carriers (expected value: 1.0). The method proved to be simple, rapid, reliable, and cost-effective. It may be used for direct determination of deletions/duplications in potential DMD/BMD carriers and may easily be adapted for other genetic conditions involving deletions and duplications.
authors.affiliation: Division of Human Genetics, Children's University Hospital, Inselspital, Bern, Switzerland. franziska.joncourt@dkf2.unibe.ch
authors.forename: Franziska
authors.initials: F
authors.lastname: Joncourt
authors.forename: Barbara
authors.initials: B
authors.lastname: Neuhaus
authors.forename: Kristin
authors.initials: K
authors.lastname: Jostarndt-Foegen
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kleinle
authors.forename: Bernhard
authors.initials: B
authors.lastname: Steiner
authors.forename: Sabina
authors.initials: S
authors.lastname: Gallati
mesh_terms: D052160:Benzothiazoles; D003959:Diamines; D016189:Dystrophin / Q000235:genetics*; D005260:Female; D005456:Fluorescent Dyes; D020440:Gene Duplication; D006580:Genetic Carrier Screening / Q000379:methods*; D006801:Humans; D020388:Muscular Dystrophy, Duchenne / Q000175:diagnosis*; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D015203:Reproducibility of Results; D017384:Sequence Deletion; D013997:Time Factors
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D016189:Dystrophin; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I