Multiplex real-time PCR for detection of deletions and duplications in dystrophin gene

• Published in: Biochemical and biophysical research communications Vol. 339; no. 1; pp. 145 - 150
• Main Authors: Traverso, Monica, Malnati, Mauro, Minetti, Carlo, Regis, Stefano, Tedeschi, Silvana, Pedemonte, Marina, Bruno, Claudio, Biassoni, Roberto, Zara, Federico
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06.01.2006
• Subjects: Carrier diagnosis; Deletions; DNA Primers; Duchenne muscular dystrophy; Duplications; Dystrophin - genetics; Exons; Female; Gene Deletion; Gene Duplication; Genetic Carrier Screening; Humans; Male; Muscular Dystrophy, Duchenne - genetics; Polymerase Chain Reaction; Real-time PCR; Duplications; Real-time PCR; Carrier diagnosis; Duchenne muscular dystrophy; Deletions
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2005.11.006

Genetic testing of Duchenne and Becker muscular dystrophies (DMD/BMD) is a difficult task due to the occurrence of deletions or duplications within dystrophin ( DMD) gene that requires dose sensitive tests. We developed three multiplex quantitative real-time PCR assays for dystrophin exon 5, 45, and 51 within two major hotspots of deletion/duplication. Each exon was co-amplified with a reference X-linked gene and the copy number of the target fragment was calculated by comparative threshold cycle method (ΔΔ C t). We compared the performance of this method with previously described end-point PCR fluorescent analysis (EPFA) by studying 24 subjects carrying DMD deletions or duplications. We showed that Q-PCR is an accurate and sensitive technique for the identification of deletions and duplications in DMD/BMD. Q-PCR is a valuable tool for independent confirmation of EPFA screening, particularly when deletions/duplications of single exons occur or for rapid identification of known mutations in at risk carriers.