Screening of calpain-3 autolytic activity in LGMD muscle: a functional map of CAPN3 gene mutations

Abstract

Background: The diagnosis of calpainopathy is obtained by identifying calpain-3 protein deficiency or CAPN3 gene mutations. However, in many patients with limb girdle muscular dystrophy type 2A (LGMD2A), the calpain-3 protein quantity is normal because loss-of-function mutations cause its enzymatic inactivation. The identification of such patients is difficult unless a functional test suggests pursuing a search for mutations. Materials and methods: A functional in vitro assay, which was able to test calpain-3 autolytic function, was used to screen a large series of muscle biopsy specimens from patients with unclassified LGMD/ hyperCKaemia who have previously shown normal calpain-3 protein quantity. Results: Of 148 muscle biopsy specimens tested,17 samples (11%) had lost normal autolytic function. CAPN3 gene mutations were identified in 15 of 17 patients (88%), who account for about 20% of the total patients with LGMD2A diagnosed in our series. Conclusions:The loss of calpain-3 autolytic activity is highly predictive of primary calpainopathy, and the use of this test as part of calpainopathy diagnosis would improve the rate of disease detection markedly. This study provides the first evidence of the pathogenetic effect of specific CAPN3 gene mutations on the corresponding protein function in LGMD2A muscle and offers new insights into the structural-functional relationship of the gene and protein regions that are crucial for the autolytic activity of calpain-3.

Natural history of LGMD2A for delineating outcome measures in clinical trials

“…Indeed, patients carrying two null mutations that result in the premature truncation of protein synthesis are invariably characterized by a higher disease severity than patients with at least one missense mutation 2, 22, 34. Our study is no exception since, for a large number of parameters, the null mutations tend to lead not only to a more severe presentation, but we also observed that the M2 group corresponds to the less severe group (Table 2C).…”

How to tackle the diagnosis of limb-girdle muscular dystrophy 2A

“…31,32 Furthermore, we feel that because of its high sensitivity (12% of false-negative results) and easy procedure, the functional test for calpain-3 autolytic activity is a valuable tool in the diagnostic algorithm of LGMD2A when protein quantity is normal.…”

“…31 In brief, muscle cryostat sections from controls and patients were quickly dissolved in saline solution and incubated at room temperature for 5 min, before the reaction was blocked by adding loading buffer containing EDTA chelating Ca þ þ ions. All samples were then processed as described for conventional calpain-3 immunoblot.…”

Análise molecular dos genes CAPN3 e FKRP em pacientes com distrofia muscular tipo cinturas

“…No caso 7 foram identificadas as mutações (nonsense) c.328C>T/ p.R110X e c.1468C>T/ p.R490W (missense) no éxon 11 (Richard et al, 1997;Chrobakova et al, 2004;Hermanova et al, 2005;Fanin et al, 2007;Nascimbeni et al, 2010). A mutação c.1468C>T faz com que a enzima seja mais dependente de Ca 2+ e que demore mais para sofrer autólise, promovendo um comprometimento de sua função proteolítica (Ono et al, 1998).…”

“…Entretanto, é relatado que cerca de 20 a 30% dos pacientes com LGMD2A apresentam quantidades normais da CAPN3 (Fanin et al, 2003;Fanin et al, 2007;Groen et al, 2007;Milic et al, 2007), dessa forma, o padrão ouro na confirmação do diagnóstico de calpainopatia é através da demonstração de mutações ao longo do gene CAPN3 (Bushby, 1999).…”