Nina Canki-Klain

Extensive scanning of the calpain-3 gene broadens the spectrum of LGMD2A phenotypes

Background: The limb girdle muscular dystrophies (LGMD) are a heterogeneous group of Mendelian disorders highlighted by weakness of the pelvic and shoulder girdle muscles. Seventeen autosomal loci have been so far identified and genetic tests are mandatory to distinguish among the forms. Mutations at the calpain 3 locus (CAPN3) cause LGMD type 2A. Objective: To obtain unbiased information on the consequences of CAPN3 mutations. Patients: 530 subjects with different grades of symptoms and 300 controls. Methods: High throughput denaturing HPLC analysis of DNA pools. Results: 141 LGMD2A cases were identified, carrying 82 different CAPN3 mutations (45 novel), along with 18 novel polymorphisms/variants. Females had a more favourable course than males. In 94% of the more severely affected patient group, the defect was also discovered in the second allele. This proves the sensitivity of the approach. CAPN3 mutations were found in 35.1% of classical LGMD phenotypes. Mutations were also found in 18.4% of atypical patients and in 12.6% of subjects with high serum creatine kinase levels. Conclusions: A non-invasive and cost–effective strategy, based on the high throughput denaturing HPLC analysis of DNA pools, was used to obtain unbiased information on the consequences of CAPN3 mutations in the largest genetic study ever undertaken. This broadens the spectrum of LGMD2A phenotypes and sets the carrier frequency at 1:103.

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.