Astrid Milić

Interactions with M-band Titin and Calpain 3 Link Myospryn (CMYA5) to Tibial and Limb-girdle Muscular Dystrophies

Mutations in the C terminus of titin, situated at the M-band of the striated muscle sarcomere, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy (LGMD) type 2J. Mutations in the protease calpain 3 (CAPN3), in turn, lead to LGMD2A, and secondary CAPN3 deficiency in LGMD2J suggests that the pathomechanisms of the diseases are linked. Yeast two-hybrid screens carried out to elucidate the molecular pathways of TMD/LGMD2J and LGMD2A resulted in the identification of myospryn (CMYA5, cardiomyopathy-associated 5) as a binding partner for both M-band titin and CAPN3. Additional yeast two-hybrid and coimmunoprecipitation studies confirmed both interactions. The interaction of myospryn and M-band titin was supported by localization of endogenous and transfected myospryn at the M-band level. Coexpression studies showed that myospryn is a proteolytic substrate for CAPN3 and suggested that myospryn may protect CAPN3 from autolysis. Myospryn is a muscle-specific protein of the tripartite motif superfamily, reported to function in vesicular trafficking and protein kinase A signaling and implicated in the pathogenesis of Duchenne muscular dystrophy. The novel interactions indicate a role for myospryn in the sarcomeric M-band and may be relevant for the molecular pathomechanisms of TMD/LGMD2J and LGMD2A.

A third of LGMD2A biopsies have normal calpain 3 proteolytic activity as determined by an in vitro assay

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive muscular disorder caused by mutations in the gene coding for calpain 3, a calcium-dependent protease. We developed an in vitro assay that can detect the proteolytic activity of calpain 3 in a muscle sample. This assay is based on the use of an inactive calpain 3 as a substrate for active calpain 3 molecules. A total of 79 human biopsies have been analysed using an unbiased single blind method. Results were confronted with the molecular diagnosis for confirmation. Proteolytic activity was either reduced or absent in 68% of LGMD2A biopsies. In the remaining 32%, normal proteolytic activity was found despite the presence of calpain 3 mutation(s), suggesting that other calpain 3 properties might be impaired to give rise to the LGMD2A phenotype. Our assay is easily adaptable to routine and appears to be more sensitive than common analysis by immunodetection.

Prevalence of the 550delA mutation in calpainopathy (LGMD 2A) in Croatia

Mutations in the calpain 3 (CAPN3) gene are responsible for limb‐girdle muscular dystrophy (LGMD) type 2A. We report five causal mutations: 550delA, ΔFWSAL, R541W, Y357X and R49H found on 45/50 of alleles studied in 25 unrelated families from Croatia. The 550delA mutation was present on 76% of CAPN3 chromosomes that led us to screen general population for this mutation; 532 random blood samples from three different regions were analyzed using allele‐specific PCR. Four healthy 550delA heterozygous were found suggesting a frequency of 1 in 133. All four carriers detected originated from an island and mountain region close to the Adriatic Sea. These findings combined with haplotype analysis confirm that our general population is rather “closed” with a probable founder effect in some parts of the country. In addition, the high frequency of 550delA mutation found in some neighboring European countries together with the easy detection of the 550delA mutation should streamline genetic analysis, especially bearing in mind the geographic and ethnic origin of the patients. Our results, combined with published haplotype studies suggest that 550delA originated in the Eastern Mediterranean from which it has probably spread widely across Europe. Extending this study to other areas would help to address this epidemiological question. Our data are relevant to accurate genetic counseling and patient testing since we lack sensitive and specific biopsy screening methods for detecting patients with calpainopathy. Thus, detection of patients relies on the direct detection of gene mutation and our findings may be helpful in establishing diagnostic screening strategy.

Non-invasive protein analysis in the first dysferlinopathy Croatian families

Mutations in human dysferlin (DYSF) gene cause both limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), also named dysferlinopathy. They are autosomal recessive muscular dystrophies characterized by degeneration and weakness of proximal and/or distal limb girdle muscles caused by partial or complete absence of a sarcolemmal protein dysferlin. The size and large mutational spectrum of DYSF impose a multistep diagnosis strategy before gene analysis. Here we report the first three patients from two unrelated Croatian families in which diagnosis of dysferlinopathy was suggested on the basis of clinical picture, family history and linkage analysis. In order to confirm the presumed diagnosis, we performed a blood-based assay in which dysferlin expression is screened in blood monocytes. All three tested patients showed complete absence of dysferlin expression, giving strong evidence of dysferlinopathy that was recently confirmed by mutation analysis. In conclusion, we would suggest the presented diagnostic strategy as a reliable and non-invasive method to be used as an alternative to muscle tissue protein analysis in routine diagnostics of dysferlinopathies, prior to the more complex and demanding search for causative DYSF mutations. This non-aggressive approach seems especially useful in situation in which multiplex Western blot (WB) analysis of different muscular dystrophy proteins on muscle sample is not available.