Rapid identification of female carriers of DMD/BMD by quantitative real-time PCR

✍ Scribed by Franziska Joncourt; Barbara Neuhaus; Kristin Jostarndt-Foegen; Stephanie Kleinle; Bernhard Steiner; Sabina Gallati

• Publisher: John Wiley and Sons
• Year: 2004
• Tongue: English
• Weight: 236 KB
• Volume: 23
• Category: Article
• ISSN: 1059-7794
• DOI: 10.1002/humu.20007

✦ Synopsis

Communicated by Johan den Dunnen

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 0.5270.12 for deletion carriers (expected value: 0.5) and 1.5670.18 for duplication carriers (expected value: 1.5) vs. 1.02270.17 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. Hum Mutat 23:385-391, 2004 r 2004 Wiley-Liss, Inc.