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550delA mutation in the calpain 3 (CAPN3) gene: DMD/BMD, SMA, or LGMD2A—clinically misdiagnosed cases

Limb-girdle muscular dystrophy type 2A (LGMD2A, calpainopathy, OMIM#253600) is an autosomal recessive muscular disorder caused by mutations in the calpain 3 (CAPN3, OMIM#114240) gene localized on chromosome 15q15.1-15.3. [Beckmann et al., 1991; Allamand et al., 1995]. A great variety of mutations scattered along the whole gene sequence has been described so far [Richard et al., 1997, 1999]. Clinically, LGMD2A is characterized by symmetrical and selective atrophy of the pelvic, scapular and trunk muscles, elevated serum creatin kinase and a necrotic regeneration pattern on muscular biopsies. There are no mental, cardiac, or facial manifestations. Patients have frequent falls, difficulties in running and climbing stairs. Scapular winging is usually present from the early stages. Several of these symptoms are common among neuromuscular disorders including Duchenne/Becker muscular dystrophy (DMD/ BMD), and spinal muscular atrophy (SMA) [Bushby, 1997, 1999]. It is possible that LGMD2A accounts for a significant proportion of the autosomal recessive LGMD cases in Bulgaria, as has already been established in other countries [Dincer et al., 1997; Richard et al., 1997; Urtasun et al., 1998]. Until now calpainopathy has been poorly recognized by clinicians in Bulgaria. We suspected that some of our patients with genetically unconfirmed DMD/BMD or SMA could have been misdiagnosed. We hypothesized that the 550delA mutation detected in exon 4 of theCAPN3 gene [Richard et al., 1995] would be frequently found among the Bulgarian LGMD2A patients, as such a high frequency has already been published for several Slavonic and neighboring populations [Dincer et al., 1997; Pogoda et al., 2000; Canki-Klan et al., 2004]. To test this hypothesis, we screened by PCR/SSCP analysis exon 4 of the CAPN3 gene. Direct sequence analysis (ABI PRISM 310 Genetic Analyzer, Applied Biosystems) was used when an abnormal migration pattern for exon 4 was observed. The screening involved three groups of patients from our genetic register: 39 patients (of 37 families) with unknown type autosomal recessive LGMD or undefined type progressive muscular disorder, 59 genetically unconfirmed sporadic DMD/ BMD cases, and 49 genetically unconfirmed SMA cases. In ten DMD/BMD cases and in two SMA cases, the DNA sample of the proband was not available, and we screened their parents for the mutation. The 550delA mutation was found in homozygous or heterozygous state in seven LGMD patients (six families), in four cases with preliminary diagnosis DMD/BMD, and in two cases previously registered as SMA. In one LGMD patient, we found a new allelic variant in exon 4 in heterozygous state: 505C>G (Arg169Gly) (Table I). Direct sequence analysis of the whole coding gene sequence (24 exons including the splice-junction sites) was performed on five of the patients for finding their second mutation. In two cases we found the mutation 967G>T (Glu323X) in exon 7. In three patients the second mutation was not found (Table I). A probable explanation could be incidental heterozygous status or a large deletion on the second chromosome. The clinical diagnosis in these three cases need to be precisely reevaluated and additional tests on RNA level need to be performed in order to provide adequate genetic counseling and management. Large deletions covering the whole exon 4 of the gene were suspected in two patients homozygous for the 550delA mutation (the analysis in their parents showed that only mothers were mutation carriers and non-paternity was excluded in both cases). Our preliminary data on RNA level confirmed the presence of a large deletion in one of these cases [Todorova, unpublished data]. To determine the frequency of the 550delA mutation in the Bulgarian population, we screened 388 control DNA samples from unrelated individuals of Bulgarian origin, but no carriers were found. This result was unexpected and remains unexplainable considering that 550delA mutation occurred as a relatively frequent cause for LGMD2A in our patients. A new polymorphism in exon 4 of the CAPN3 gene 606T>C(Ser202Ser) with frequency 1.8% was identified in the control sample. The substitution Arg169Gly was not detected among the control sample, which favors the hypothesis that this allelic variant is a private mutation. Our data showed that LGMD2A due to 550delA mutation in homozygous and heterozygous state accounts for about 30% of all autosomal recessive LGMD cases in Bulgaria. We found that 4% of the SMA patients and 7% of the DMD/BMD patients from our genetic register were misdiagnosed. It is reasonable to consider LGMD2A as a possible diagnosis in genetically unconfirmed DMD/BMD and SMA cases with unconvincing clinical data. Bilyana Georgieva and Albena Todorova contributed equally to this work.