Validation and comparison of two quantitative real-time PCR assays for direct detection of DMD/BMD carriers

Objectives

To develop a robust and reliable assay for direct identification of female carriers of deletions in the dystrophin gene.

Design and methods

We compared two quantitative real-time PCR approaches for the detection of the deletions of exons 4, 17, 47, and 50 in DMD/BMD carriers. One hundred and ten individuals from 26 unrelated families, including 8 large pedigrees characterized by having at least two DMD affected males, were studied. Carrier status of the subjects was also evaluated by MLPA.

Results

The results showed the gene dosage ratio of 0.99 ± 0.14 and 1.09 ± 0.19 for normal individuals and 0.48 ± 0.06 and 0.50 ± 0.10 for carriers in SYBR green and TaqMan probe assays, respectively. Carrier status was accurately attributed in 100% of cases and confirmed by MLPA.

Conclusion

Quantitative real-time PCR can be used as a direct method for carrier detection in female relatives of DMD patients with known deletions. The results are comparable to the MLPA data.