Anna Chiara Nascimbeni

Loss of Calpain-3 Autocatalytic Activity in LGMD2A Patients with Normal Protein Expression

The diagnosis of limb girdle muscular dystrophy (LGMD) type 2A (due to mutations in the gene encoding for calpain-3) is currently based on protein analysis, but mutant patients with normal protein expression have also been identified. In this study we investigated 150 LGMD patients with normal calpain-3 protein expression, identified gene mutations by an allele-specific polymerase chain reaction test, and analyzed the mutant calpain-3 catalytic activity. Four different mutations were found in eight patients (5.5%): a frame-shifting deletion (550 A del) and three missense (R490Q, R489Q, R490W). Patients with normal calpain-3 protein expression on Western blot are a considerable proportion (20%) of our total LGMD2A population. While in control muscle the calpain-3 Ca(++)-dependent autocatalytic activity was evident within 5 minutes and was prevented by ethylene diaminetetraacetic acid, in all mutant patient samples the protein was not degraded, indicating that the normal autocatalytic function had been lost. By this new functional test, we show that conventional protein diagnosis fails to detect some mutant proteins, and prove the pathogenetic role of R490Q, R489Q, R490W missense mutations. We suggest that these mutations impair protein activity by affecting interdomain protein interaction, or reduce autocatalytic activity by lowering the Ca(++) sensitivity.

Frequency of LGMD gene mutations in Italian patients with distinct clinical phenotypes.

Background: The frequency of various limb-girdle muscular dystrophy (LGMD) molecular diagnoses has previously been investigated only in cohorts of patients presenting LGMD phenotype. Methods: A total of 550 muscle biopsies underwent multiple protein screening (including calpain-3 functional assay) and extensive gene mutation analysis to examine the frequency of LGMD subtypes in patients with distinct clinical phenotypes (severe childhood-onset LGMD, adult-onset LGMD, distoproximal myopathy, and asymptomatic hyperCKemia). Results: The percentage of molecularly ascertained cases directly relates with the degree of clinical involvement: 60% of total LGMD (77% of childhood-onset, 46% of adult-onset, 66% of distoproximal myopathy) and 14% of hyperCKemia. The higher number of molecular diagnoses in severe phenotypes might suggest that genes selected for our screening are those more frequently associated with severe LGMD, and that the hyperCKemia group includes heterogeneous diagnoses. The probability of obtaining a molecular diagnosis increases when a protein defect is found in a muscle biopsy: in such cases, we diagnosed 87% of LGMD and 76% of hyperCKemia. Conclusions: Diagnosing 77% of childhood-onset limb-girdle muscular dystrophy (LGMD) and 60% of total LGMD is an important result. The missing identification of gene mutations in about 40% of patients with typical LGMD phenotype suggests that unknown genetic or nongenetic etiologies are still to be recognized. Dysferlin, caveolin-3, and emerin protein defects invariably corresponded to primary disorders (100%), whereas a lower correlation was found for sarcoglycans (77%) and calpain-3 (84%). The different efficiency of genetic diagnosis after the identification of a protein defect in the various disorders is possibly due to different pathogenetic effects of mutations.

The frequency of limb girdle muscular dystrophy 2A in northeastern Italy

Limb-girdle muscular dystrophy 2A (LGMD2A) is considered to be the most frequent LGMD. Our study surveyed an area in northeastern Italy where an almost complete ascertainment was possible. To identify LGMD2A patients we used a new diagnostic approach, including several molecular and biochemical methods. In 84 screened patients from northeastern Italy, we identified 39 LGMD2A patients, the prevalence of LGMD2A being 9.47 per million. In the Venezia district it appears higher than in other districts of the Veneto region, and in the Friuli region it is three times higher than in Veneto, due to the recurrence of single mutation. Haplotype analysis suggested a founder effect. The population from Venezia and Friuli has a higher risk of being heterozygote for these two mutant alleles than people from the rest of northeastern Italy. Our results indicate that LGMD2A is one of the most frequent autosomal recessive disorders, thus finding its molecular characterization becoming increasingly important.

Molecular pathology and enzyme processing in various phenotypes of acid maltase deficiency

Objectives: To examine at molecular, biochemical, and muscle pathology level the striking clinical heterogeneity resulting from acid α-glucosidase deficiency. Methods: We investigated 23 patients with infantile-onset or late-onset glycogen storage disease type II by enzyme activity, protein expression by immunoblotting, GAA gene mutations, and muscle pathology including immunolabeling for Golgi and sarcolemmal proteins. Results: The enzyme activity was absent or minimal in infantile-onset cases and variably reduced in late-onset patients. Genotype-phenotype correlation (seven novel mutations were found) showed that most late-onset patients had the heterozygous IVS1 leaky splicing mutation (one patient was homozygous), but the course of the disease was often difficult to predict on the basis of the mutations alone. All patients showed an abnormal pattern of enzyme protein processing, with increased amounts of the inactive forms and very low or absent amounts of the mature forms. The molecular weight of the mature and the intermediate forms appeared higher in patients’ samples than in the control muscle. We observed a Golgi proliferation in muscle fibers possibly caused by the retention of inactive forms of enzyme protein that cannot be correctly targeted from Golgi to lysosomes. The vacuolar membranes expressed sarcolemmal proteins in late-onset but not in infantile-onset patients, suggesting an extensive autophagy and vacuolar membrane remodeling in late-onset patients. Conclusions: The different protein molecular weight between patients and controls could be due to an excessive sialylation of mutant enzyme: this might be possibly caused by a delayed transport and longer transit of the inactive proteins in the Golgi apparatus.

Impaired autophagy contributes to muscle atrophy in glycogen storage disease type II patients

The autophagy-lysosome system is essential for muscle cell homeostasis and its dysfunction has been linked to muscle disorders that are typically distinguished by massive autophagic buildup. Among them, glycogen storage disease type II (GSDII) is characterized by the presence of large glycogen-filled lysosomes in the skeletal muscle, due to a defect in the lysosomal enzyme acid α-glucosidase (GAA). The accumulation of autophagosomes is believed to be detrimental for myofiber function. However, the role of autophagy in the pathogenesis of GSDII is still unclear. To address this issue we monitored autophagy in muscle biopsies and myotubes of early and late-onset GSDII patients at different time points of disease progression. Moreover we also analyzed muscles from patients treated with enzyme replacement therapy (ERT). Our data suggest that autophagy is a protective mechanism that is required for myofiber survival in late-onset forms of GSDII. Importantly, our findings suggest that a normal autophagy flux is important for a correct maturation of GAA and for the uptake of recombinant human GAA. In conclusion, autophagy failure plays an important role in GSDII disease progression, and the development of new drugs to restore the autophagic flux should be considered to improve ERT efficacy.

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

Limb-girdle muscular dystrophy (LGMD) 2A (calpainopathy) is the most frequent form of LGMD in many European countries. The increasing demand for a molecular diagnosis makes the identification of strategies to improve gene mutation detection crucial. We conducted both a quantitative analysis of calpain-3 protein in 519 muscles from patients with unclassified LGMD, unclassified myopathy and hyperCKemia, and a functional assay of calpain-3 autolytic activity in 108 cases with LGMD and normal protein quantity. Subsequently, screening of CAPN3 gene mutations was performed using allele-specific tests and simplified SSCP analysis. We diagnosed a total of 94 LGMD2A patients, carrying 66 different mutations (six are newly identified). The probability of diagnosing calpainopathy was very high in patients showing either a quantitative (80%) or a functional calpain-3 protein defect (88%). Our data show a high predictive value for reduced-absent calpain-3 or lost autolytic activity. These biochemical assays are powerful tools for otherwise laborious genetic screening of cases with a high probability of being primary calpainopathy. Our multistep diagnostic approach is rational and highly effective. This strategy has improved the detection rate of the disease and our extension of screening to presymptomatic phenotypes (hyperCKemia) has allowed us to obtain early diagnoses, which has important consequences for patient care and genetic counseling.

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

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.

The clinical course of calpainopathy (LGMD2A) and dysferlinopathy (LGMD2B).

Abstract Objective: Autosomal recessive limb girdle muscular dystrophies (LGMD type 2) are a clinically and genetically heterogeneous group of disorders, characterized by progressive involvement and wasting of limb girdle muscles. In order to describe the peculiar clinical features of LGMD2A (calpainopathy) and LGMD2B (dysferlinopathy), the most frequent forms of LGMD in European countries, we analysed and compared the phenotype and the clinical course in two relatively large groups of these patients. Methods: We selected 22 patients with a molecular diagnosis of LGMD2A and 21 patients with LGMD2B and reported their clinical data collected from both clinical history and during periodical neuromuscular examinations: age and distribution of muscle involvement at onset, clinical functional score by the use of ten-point modified scale of Gardner–Medwin and Walton at onset and at last clinical examination, and the rate of disease progression. Results: LGMD2A group included patients with different ages at onset (early-onset or late-onset), different phenotypes (upper girdle in Erb LGMD or lower girdle in Leyden–Moebius LGMD) and different disease progressions (rapid or slow course). LGMD2B patients differed for pattern of muscle involvement at onset (distal in Miyoshi dystrophy or proximal in Leyden–Moebius LGMD) but they had a rather homogeneous age at onset (in the second/third decade) and rate of disease progression. Discussion: Our data show that besides the clinical differences within each group of patients, the two forms of LGMD present distinctive clinical features. The various phenotypes and courses can be attributed to specific pathogenetic mechanisms and might suggest differential therapeutic strategies.

Muscle protein analysis in the detection of heterozygotes for recessive limb girdle muscular dystrophy type 2B and 2E.

The diagnosis of isolated heterozygotes for recessive LGMD is quite difficult because no specific biochemical or protein assays are available, and the molecular analysis is not feasible due to the wide genetic heterogeneity. We investigated a series of definite heterozygotes for different forms of LGMD to determine whether the carrier status will result in a detectable protein defect in muscle biopsy. Definite heterozygotes from 4 families (3 LGMD2B and 1 LGMD2E) underwent quantitative immunoblot analysis of mutant protein in their muscle. While the quantity of beta-sarcoglycan was nearly normal in the LGMD2E carrier, the levels of dysferlin protein were reduced to 50% of controls in the carriers of LGMD2B. We have demonstrated the value of protein analysis in the identification of both familial and isolated LGMD2B heterozygotes, and suggested the use of dysferlin protein testing to select muscle biopsies from suspected carriers for a subsequent mutation analysis. Muscle protein analysis would be used to screen asymptomatic patients who underwent muscle biopsy because of unexplained hyperCKemia.

Correlations between clinical severity, genotype and muscle pathology in limb girdle muscular dystrophy type 2A

Background: Limb girdle muscular dystrophy type 2A (LGMD2A) is characterised by wide variability in clinical features and rate of progression. Patients with two null mutations usually have a rapid course, but in the remaining cases (two missense mutations or compound heterozygote mutations) prognosis is uncertain. Methods: We conducted what is to our knowledge the first systematic histopathological, biochemical and molecular investigation of 24 LGMD2A patients, subdivided according to rapid or slow disease progression, to determine if some parameters could correlate with disease progression. Results: We found that muscle histopathology score and the extent of regenerating and degenerating fibres could be correlated with the rate of disease course when the biochemical and molecular data do not offer sufficient information. Comparison of clinical and muscle histopathological data between LGMD2A and four other types of LGMD (LGMD2B–E) also gave another important and novel result. We found that LGMD2A has significantly lower levels of dystrophic features (ie degenerating and regenerating fibres) and higher levels of chronic changes (ie lobulated fibres) compared with other LGMDs, particularly LGMD2B. These results might explain the observation that atrophic muscle involvement seems to be a clinical feature peculiar to LGMD2A patients. Conclusions: Distinguishing patterns of muscle histopathological changes in LGMD2A might reflect the effects of a disease-specific pathogenetic mechanism and provide clues complementary to genetic data.