validation and comparison of two novel quantitative methods for carrier detection of dmd/bmd

mutations in the dystrophin gene cause duchenne muscular dystrophy (dmd), the most commonly inherited neuromuscular disorder, and becker muscular dystrophy (bmd), the milder allelic form of the disease. the mutation spectrum within this gene is unusual in that deletion of one or more exons are found in ~65% of cases. since no effective treatment is so far available for these diseases, the identification of carrier females is critical to prevent the birth of affected males. pcr approach is not applicable in detecting female carriers in whom deletions are masked by the amplification of the normal x chromosome. thus, identification of female carriers must be carried out by different strategies. in this study we used two quantitative assays, real-time pcr and multiplex ligation probe amplification (mlpa), to detect carriers. first, mlpa analysis was performed using salsa mlpa kit p034 / p035 and the raw data were analyzed using coffalyser software. this method allows simultaneous analysis of all specific sequences using multiplex pcr reactions. in order to confirm the mlpa results by another approach, we optimized and evaluated two quantitative real-time pcr methods using sybr green i and taqman probe chemistry (applied biosystems). the quantitative real-time pcr assay was performed using comparative threshold cycle method (δδct). carrier status was precisely attributed in 100% of cases. interestingly, the sybr green assay was shown to be as accurate as probe based method. moreover the mlpa results are comparable to the corresponding results obtained from real-time pcr assays. in conclusion, these quantitative assays have the potential to be used as an alternative direct method for carrier detection in female relatives of dmd patients especially in families with no living affected males. also may easily be adapted for other genetic conditions involving gene deletions and duplications.