The mutations we detected and clinical data are listed in Tables 1a and 1b. Some of these patients were reported in our previous paper [8]. The creatine kinase levels were highly elevated in all cases, with the exception of creatine kinase levels in handicapped patients, where the destroyed muscle leads to creatine kinase level normalization. The main group of patients started walking later than normal children (after 12 months). In two cases: deletion patient #32 and duplication patient #2, a serious delay in starting to walk was registered at around or after second year of age. The duplication patient #2 had very severe symptoms with an early involvement of limbs, limb-girdle and neck muscles and fast disease progression.

The early symptoms in all patients have been very similar: walking on toes, difficulties in climbing stairs, running, getting up. Family history (the presence of other affected family members) was positive in 13 cases.

The deletion patient #23 had been clinically diagnosed as Emery-Dreifuss muscular dystrophy (OMIM#181350; OMIM#310300). The precise diagnosis of BMD was established during the present study.

Of interest is the deletion of the very last exon of the DMD gene (exon 79), detected in deletion patient #6. It is not clear where the deletion ends, but the clinical picture was compatible with DMD phenotype (early onset and fast progression). Deletion patient #29 also had a deletion covering exon 79. He was also severely affected with a fast disease progression. A similar mutation has been described earlier in a BMD patient as a part of a contiguous gene deletion syndrome [9].

Two duplications of exons 2-33 and 13-40 (duplication patients #2 and #4) cover more than one-third of the gene sequence, and result in DMD and BMD phenotypes, respectively. Most probably, the duplication situated close to the N-terminus of the dystrophin is associated with a severe phenotype, while the one in the rod-domain leads to the BMD phenotype [8].

Some patients were pre-screened for deletions in the DMD gene by a standard multiplex set of primers. In deletion patient #14 the deletion of exons 49-50 was detected in this way, but the family requested confirmation with an alternative method. In several cases the deletions were detected by mPCR, but deletion borders were not precisely determined, or carrier status determination in these families required MLPA. In deletion patients #37 and #39, the deletion of exon 44 was not detected by multiplex PCR (most probably because of PCR unspecificity or contamination) and they were not considered as deletions. A number of index patients were classified as unknown mutations after mPCR.

The MLPA analysis/direct sequencing characterized mutation in all cases, which comprised 42 deletions (82%), six duplications (12%) and three point mutations (6%) (see Table 1a). In addition, this analysis in the four families with no living index patient revealed deletions in all families of dead patients diagnosed with DMD, using the DNA samples of their mothers and sisters (see Table 1b).

All deletion/duplication borders were precisely determined by this method as required for adequate gene therapy in the future. The deletion and duplication electrophoretic profiles are presented on Figure 1a and 1b. All deletions were confirmed by PCR amplification.

In our hands, the combination of the MLPA test with direct sequencing of the entire DMD gene, proved to be a powerful diagnostic approach in DMD/BMD families. In point mutation patient #2 only exon 23 was missing on the MLPA electropherogram. The single amplification of exon 23 showed its presence in the patient, but the point mutation c.2991C>G, p.Tyr997X was detected by sequencing of the exon [8]. Point mutation patients #1 and #3 tested negative by MLPA, but sequencing of the entire coding sequence of the DMD gene revealed the nonsense mutations c.8776C>T, p.Gln2926X and c.583C>T, p.Arg195X, respectively [8].

The classical mPCR of 18 exons in the deletion hot-spot region of the DMD gene [2,3] could detect 36 deletions in the present sample (86% of deletions), 15 of which (42%) were only partial, with no clear deletion borders. The mPCR could only detect deletions, and it is applicable only when the index patient is available for study. Moreover, the presented MLPA analysis was successful not only in all the patients tested, but also in all four families with no index patient. We identified four deletions in these families (see Table 1b); all four mothers were deletion carriers, one sister was genetically proved to be a carrier and two were not carriers. The MLPA data is easily interpreted also for direct carrier status determination.

The application of the most recent genetic tests in our routine diagnostic studies, gave us the possibility to genetically clarify a number of Bulgarian DMD/BMD patients. A national web site for clinically and genetically well described neuromuscular cases is available [10], according to the requirements of the Treat-NMD Network.