Identification of deletions and duplications in the Duchenne muscular dystrophy gene and female carrier status in western India using combined methods of multiplex polymerase chain reaction and multiplex ligation-dependent probe amplification

BACKGROUND: The technique of multiplex ligation-dependent probe amplification (MLPA) assay is an advanced technique to identify deletions and duplications of all the 79 exons of DMD gene in patients with Duchenne/Becker muscular dystrophy (DMD/BMD) and female carriers. AIM: To use MLPA assay to detect deletions which remained unidentified on multiplex polymerase chain reaction (mPCR) analysis, scanning 32 exons of the "hot spot" region. Besides knowing the deletions and/or duplications, MLPA was also used to determine the carrier status of the females at risk. MATERIALS AND METHODS: Twenty male patients showing no deletions on mPCR and 10 suspected carrier females were studied by MLPA assay using P-034 and P-035, probe sets (MRC Holland) covering all the 79 exons followed by capillary electrophoresis on sequencing system. RESULTS: On MLPA analysis, nine patients showed deletions of exons other than 32 exons screened by mPCR represented by absence of peak. Value of peak areas were double or more in four patients indicating duplications of exons. Carrier status was confirmed in 50% of females at risk. CONCLUSION: Combining the two techniques, mPCR followed by MLPA assay, has enabled more accurate detection and extent of deletions and duplications which otherwise would have remained unidentified, thereby increasing the mutation pick up rate. These findings have also allowed prediction of expected phenotype. Determining carrier status has a considerable significance in estimating the risk in future pregnancies and prenatal testing options to limit the birth of affected individuals.