Strategy for mutation analysis in the autosomal recessive limb-girdle muscular dystrophies

https://doi.org/10.1016/S0960-8966(00)00154-1Get rights and content

Abstract

We describe a strategy for molecular diagnosis in the autosomal recessive limb-girdle muscular dystrophies, a highly heterogeneous group of inherited muscle-wasting diseases. Genetic mutation analysis is directed by immunoanalysis of muscle biopsies using antibodies against a panel of muscular dystrophy-associated proteins. Performing the molecular analysis in this way greatly increases the chance that mutations will be found in the first gene examined. The use of this strategy can significantly decrease the time involved in determining the genetic fault in a patient with a clinical diagnosis of recessive limb-girdle muscular dystrophy, as well as having a feedback effect, which is useful in helping clinicians to identify subtle clinical differences between the subtypes of the disease. The use of this approach has so far helped us to identify mutations in ten sarcoglycanopathy (limb-girdle muscular dystrophy 2C–2F) patients, and seven calpainopathy (limb-girdle muscular dystrophy 2A) patients.

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.

References (47)

  • L.V.B Anderson

    Optimized protein diagnosis in the autosomal recessive limb-girdle muscular dystrophies

    Neuromusc Disord

    (1996)
  • I Naom et al.

    Laminin alpha 2-chain gene mutations in two siblings presenting with limb-girdle muscular dystrophy

    Neuromusc Disord

    (1998)
  • L Merlini et al.

    Decreased expression of laminin beta 1 in chromosome 21-linked Bethlem myopathy

    Neuromusc Disord

    (1999)
  • H Sorimachi et al.

    Structural and physiological functions of ubiquitous and tissue-specific calpain species

    Adv Biophys

    (1996)
  • D.J Duggan et al.

    Autosomal recessive muscular dystrophy and mutations of the sarcoglycan complex

    Neuromusc Disord

    (1996)
  • M.C Speer et al.

    Confirmation of genetic heterogeneity in limb-girdle muscular dystrophy: linkage of an autosomal dominant form to chromosome 5q

    Am J Hum Genet

    (1992)
  • A.J van der Kooi et al.

    Genetic localization of a newly recognized autosomal dominant limb-girdle muscular dystrophy with cardiac involvement (LGMD1B) to chromosome 1q11-21

    Am J of Hum Genet

    (1997)
  • C Minetti et al.

    Mutations in the caveolin-3 gene cause autosomal dominant limb-girdle muscular dystrophy

    Nat Genet

    (1998)
  • A Muchir et al.

    Identification of mutations in the gene encoding lamin A/C in the autosomal dominant form of limb-girdle muscular dystrophy with cardiac involvement (LGMD 1B)

    Neuromusc Disord

    (1999)
  • G Bonne et al.

    Mutations in the gene encoding lamin A/C cause autosomal dominant Emery–Dreifuss muscular dystrophy

    Nat Genet

    (Date?)
  • E.M McNally et al.

    Caveolin-3 in muscular dystrophy

    Hum Mol Genet

    (1998)
  • F Fougerousse et al.

    Mapping of a chromosome 15 region involved in limb girdle muscular dystrophy

    Hum Mol Genet

    (1994)
  • R Bashir et al.

    A gene for autosomal recessive limb-girdle muscular dystrophy maps to chromosome 2p

    Hum Mol Genet

    (1994)
  • Cited by (44)

    • Orthopedic Surgery in Neuromuscular Disorders

      2021, Neuromuscular Disorders: Treatment and Management
    • Sarcolemmal alpha and gamma sarcoglycan protein deficiencies in turkish siblings with a novel missense mutation in the alpha sarcoglycan gene

      2014, Pediatric Neurology
      Citation Excerpt :

      Babameto-Laku et al.4 also reported that the SGCA gene must be first evaluated if there is a concomitant absence of both alpha (α)-sarcoglycan and gamma (γ)-sarcoglycan proteins. It is impossible to narrow the differential diagnosis of LGMD-2D, including other LGMDs and dystrophinopathies, on clinical grounds alone; therefore, immunohistochemical staining of the muscle biopsy and molecular genetic analysis are mandatory for the correct diagnosis.3,5,8,9 In this report, we describe a previously unknown point mutation [c.226 C > T (p.L76 F)] in exon 3, adding to the growing spectrum of mutations in the SGCA gene.

    • Muscular Dystrophies

      2013, Emery and Rimoin's Principles and Practice of Medical Genetics
    • Orthopedic Surgery in Neuromuscular Disorders

      2010, Neuromuscular Disorders: Treatment and Management
    • Reverse protein arrays as novel approach for protein quantification in muscular dystrophies

      2010, Neuromuscular Disorders
      Citation 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.

    View all citing articles on Scopus
    View full text