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

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34 Citations (Scopus)

Abstract

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 (ΔΔCt). 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.

Original languageEnglish
Pages (from-to)145-150
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume339
Issue number1
DOIs
Publication statusPublished - Jan 6 2006

Fingerprint

Dystrophin
Multiplex Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
Exons
Genes
Polymerase Chain Reaction
X-Linked Genes
Gene Dosage
Duchenne Muscular Dystrophy
Assays
Screening
Genetic Testing
Testing
Mutation

Keywords

  • Carrier diagnosis
  • Deletions
  • Duchenne muscular dystrophy
  • Duplications
  • Real-time PCR

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

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abstract = "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 (ΔΔCt). 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.",
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AU - Traverso, Monica

AU - Malnati, Mauro

AU - Minetti, Carlo

AU - Regis, Stefano

AU - Tedeschi, Silvana

AU - Pedemonte, Marina

AU - Bruno, Claudio

AU - Biassoni, Roberto

AU - Zara, Federico

PY - 2006/1/6

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N2 - 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 (ΔΔCt). 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.

AB - 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 (ΔΔCt). 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.

KW - Carrier diagnosis

KW - Deletions

KW - Duchenne muscular dystrophy

KW - Duplications

KW - Real-time PCR

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