Strategy for mutation analysis in the autosomal recessive limb-girdle muscular dystrophies
Introduction
The limb-girdle muscular dystrophies (LGMD) are a highly heterogeneous group of progressive muscle wasting diseases, which may be inherited in either an autosomal dominant (LGMD type 1) or autosomal recessive (LGMD type 2) fashion [1]. Of the three dominant forms so far identified [2], [3], [4], two of the genes have been identified; the lamin A/C gene responsible for LGMD 1B (also responsible for autosomal dominant Emery–Dreifuss muscular dystrophy) [5], [6], and the caveolin-3 gene responsible for LGMD 1C [7].
The recessive forms are even more heterogeneous, with nine genetically distinct forms identified (LGMD 2A–2I) [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. At the molecular level, they can be divided into the sarcoglycanopathies [11], [18], [19], [20] and the non-sarcoglycanopathies [21], [22], [23], based on whether they are caused by mutations in a gene encoding a member of the sarcoglycan (SG) component of the dystrophin-associated protein complex or not. This complex is located at the muscle cell membrane, and is thought to provide a mechanical link between the cell cytoskeleton and the extracellular matrix [24]. The sarcoglycans (α-, β-, γ- and δ-SG) form a subcomplex whose components are interdependent to varying degrees. Evidence suggests that β- and δ-SG are most tightly associated together, with γ-SG less tightly bound, and α-SG is the most loosely associated member of the complex [25]. In most cases, mutations in one of the sarcoglycan genes causes a secondary reduction of the other three proteins that can vary from partial deficiency to total absence [26].
The non-sarcoglycanopathies, as their name implies involve proteins which are not members of the sarcoglycan complex. Of the five loci currently known (LGMD 2A, 2B, 2G–2I), two genes have so far been isolated, those of the muscle-specific calpain (CAPN3) [21] responsible for LGMD 2A, and the dysferlin gene [22], [23] responsible for LGMD 2B and its allelic variant Miyoshi myopathy. The functions of both these genes and their proteins are currently being investigated. In general, the non-sarcoglycanopathies show a later onset and milder disease progression than the sarcoglycanopathies, though many exceptions have been reported [27].
The level of genetic heterogeneity seen in this group of diseases, together with the possibility of clinical overlap with other neuromuscular disorders demands that the approach to mutation analysis be as efficient as possible in terms of both time and money, if LGMD analysis is to be introduced into the diagnostic setting. Detection of the mutation, and thereby confirmation of the primary molecular pathological event in any particular patient is necessary for absolute diagnosis, but specifically for genetic counselling or prenatal diagnosis. Similarly, any gene-based therapy will depend upon knowledge of the mutation. All of the recessive LGMD genes cloned so far are multi-exonic, with few, if any recurrent mutations. Here we present a strategy for immunologically guided mutation analysis that greatly increases the chance that mutations will be found in the first gene examined in any particular case. We illustrate our approach by describing some results of our own analyses in the sarcoglycans and CAPN3.
Section snippets
Patients
The clinical diagnostic criteria for the recessive limb-girdle muscular dystrophies have been comprehensively reviewed by [27].
Thirteen patients were selected for mutation analysis in the CAPN3 gene, based on the criteria that they had abnormal labelling of the calpain 3 protein on western blots, and normal dysferlin labelling. Fourteen patients had abnormal labelling for the sarcoglycans and were selected for mutation analysis in the sarcoglycan genes.
Patients with abnormal dysferlin labelling
The sarcoglycanopathies
Fourteen patients presented with a protein profile indicative of primary mutations in one of the sarcoglycan genes (Table 2). Of these, five individuals (γ-1–γ-5) appeared to have γ-sarcoglycan more severely reduced than the other three proteins (Fig. 2B,D). These five patients were therefore examined for mutations in the γ-sarcoglycan gene. At least one pathogenic sequence alteration was found in each of the five patients (Table 2). One of the patients (γ-4, Fig. 2D) showed labelling for
Discussion
The highly heterogeneous nature of the autosomal recessive limb-girdle muscular dystrophies demands that an efficient strategy be developed to direct the process of searching for genetic mutations within the six known genes. We suggest that complete examination of the protein profile (in combination with a comprehensive clinical examination) is an excellent starting point for the determination of genetic diagnosis in LGMD.
The strategy presented here is intended to provide the best advice on
Acknowledgements
This work is funded by the Muscular Dystrophy Campaign.
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2010, Neuromuscular DisordersCitation Excerpt :In DMD/BMD patients, the absence or reduction of dystrophin is often coupled with secondary deficiencies in proteins of the dystrophin–glycoprotein complex DGC [14]. Reductions of one of the sarcoglycans in LGMD patients can be coupled with secondary reductions in dystrophin and/or in the other sarcoglycans of such variable extent that the application of one sarcoglycan antibody is not sufficient to discriminate sarcoglycanopathies from other muscular dystrophies [15]. In some cases, primary dysferlinopathies (LGMD2B) have been reported to be associated with secondary calpain-3 deficiencies [16], a muscle-specific protease that is mutated in LGMD2A.