Tackling the diagnosis of limb-girdle muscular dystrophy 2A

Autosomal recessive limb-girdle muscular dystrophies (LGMD) cause the progressive wasting of proximal limb-girdle muscles. These are a group of disorders that include at least 14 different genetic forms, the most common of which are LGMD2I found many in Northern Europe and LGMD2A also widespread in many European countries, Japan, Russia and Australia (Fanin et al. 2009). The LGMD2A type of the disorder is caused by mutations of the CAPN3 gene which encodes for a muscle-specific proteolytic enzyme called calpain-3. This enzyme plays a roll in the process of sarcomere (basic unit of muscle filaments) remodelling.
Due to its high frequency there is an increasing demand for the molecular diagnosis of LGMD2A. The current way this is done is via mutational analysis of the gene. But this is a cumbersome task as CAPN3 is a large gene. To improve this process Marina Fanin, Anna Nascimbeni, Elisabetta Tasca and Corrado Angelini designed a new approach. Their diagnosis of LGMD2A was biased on a preliminary quantitative protein analysis of calpain-3 from a muscle biopsy to identify patients with abnormal levels due to the mutations in the CAPN3 gene. This was combined with this was an analysis of calpain-3 autolytic activity when levels were found to be normal. A mutation screening of the CAPN3 gene by single strand conformational polymorphism was then used as a final tool to identify patients who showed normal levels and autolytic activity of the calpain-3 enzyme but still my have LGMD2A due to mutations of the CAPN3 gene and to confirm the findings of the first 2 tests.
Fanin et al. study found protein defect in 87 of 519 patients. A further 108 patients who showed normal calpain-3 levels and LGMD phenotypes had the analysis of calpain-3 autolytic activity levels which identified a further 17 cases with loss of autolytic function. The mutations screening was conducted on 282 patients which included the 87 with protein deficiencies, the 17 patients with loss of autolytic function and another 178 patients with normal protein levels but with LGMD phenotypes. They identified 66 different mutations of the CAPN3 gene including 6 new mutations that had not been previously reported. Mutations were found in 80% of cases with calpain-3 defects and in 88% of cases displaying the loss of the enzymes autolytic function. A further 10 patients, with normal calpain-3 and autolytic function levels but with LGMD phenotypes, were identified via the mutation screening.
This research shows that the more cost affective methods of calpain-3 analysis combined with autolytic activity analysis may be used to initially identify patients likely to have LGMD2A. Also the uses of autolytic function analysis enable the ability to identify patients with mutations that render the calpain-3 protein inactive but still produce it in normal level. This would allow larger scale testing of patients prior to the cumbersome process of mutation screening.

Reference:
Fanin, M., Nascimbeni, A.C., Tasca, E. and Angelini, C. (2009) How to tackle the diagnosis of limb-girdle musclar dystrophy 2A. European journal of Human Genetics. 17, p598-603.

Full article at: http://www.nature.com.ezproxy.library.uq.edu.au/ejhg/journal/v17/n5/pdf/ejhg2008193a.pdf