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.

Well-devised quantification analysis for duplication mutation of Duchenne muscular dystrophy aimed at preimplantation genetic diagnosis

“…Although many duplication mutations differing in their localization and extent have been described so far in DMD patients (the Leiden muscular dystrophy pages), a versatile and effective detecting method for PGD has not been developed and mutation diagnosis at PGD is not available for about 10% of DMD patients with duplication mutation. In recent years, techniques for the detection of duplications have multiplied (reviewed in Armour et al [28]), and very few protocols have been published proposing quantitative real-time PCR for measuring copy number changes in patients with duplications spanning one or more exons [29]. Conventional PCR strategies only analyze final PCR products without visualizing the kinetics of the reaction and are not adequate for quantification due to their inability to indicate the exponential phase.…”

“…Although fluorescencelabeled TaqMan-probes or LUX-primers are a little complicated and expensive to synthesize, SYBR-green dye is cost-effective and reliable. In addition, Joncourt et al [29] have described that fluorescence signals of the hybridization probes decreased over time even when probes were stored as stock solutions at a constant temperature of −20 • C and the stability of the probes was difficult to control. We also experienced instability of quantification analysis using fluorescence-labeled TaqMan-probes and LUX-primers.…”

Quantitative Assay of Deletion or Duplication Genotype by Capillary Electrophoresis System: Application in Prader–Willi Syndrome and Duchenne Muscular Dystrophy

“…We used a quantitative real-time PCR assay to verify the status of the deleted carriers and nondeleted carriers identified by the multiplex quantitative PCR with HDA capillary electrophoresis system analysis (48 ). Results of allele dose analysis for exons 6 and 51 according to their deletion rates in the DMD patients are shown in Fig.…”

Quantitative 1-Step DNA Methylation Analysis with Native Genomic DNA as Template

“…1B). Values for ⌬⌬C q (where C q is the quantification cycle) of the locus of interest and the reference locus, as well as of the sample to be investigated and the calibrator sample, are used to quantify copy number, as has previously been described (22,23 ).…”