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HAMILTON, Ontario, CANADA L8N 4A6
PHONE: (905) 521-6141
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http://www.fhs.mcmaster.ca/hrlmp/
Issue No. 67
QUARTERLY NEWSLETTER
February 2003
Muscular Dystrophies, Recent Advances and
the
Use of Immunohistochemistry in Diagnosis
Background
The muscular dystrophies represent a large diverse group of inherited muscle diseases characterized
generally by slowly progressive muscle weakness and atrophy. In these muscle disorders, the underlying
defect is felt to be intrinsic to the skeletal muscle fibres. Duchenne’s muscular dystrophy was the first to
be characterized. Most cases have an early childhood onset and are phenotypically severe. The
responsible gene was isolated, cloned and localized to chromosome XP21, and the gene protein
subsequently characterized (dystrophin). This protein was shown to be a sarcolemmal protein in the
muscle fibres and by immunohistochemistry, was shown to be absent in the more severe cases
(Duchenne’s form), and reduced in the less severe phenotypic form (Becker’s muscular dystrophy). From
the clinical point of review, there remained a large group of milder, later onset muscle dystrophy diseases
in which the underlying gene and protein abnormalities were unknown. However, during the last 10 years,
as a consequence of the genetic revolution, a number of underlying responsible genes and muscle
proteins have been identified in which defects underlie specific subtypes of this large group of muscular
dystrophies clinically referred to as limb girdle muscular dystrophies (see table 1). As a result, recently,
the application of routine immunohistochemistry to skeletal muscle biopsy specimens is allowing the
specific subtyping of some of these forms of limb girdle muscular dystrophy. The Hamilton Regional
Laboratory Medicine Program, through the Pathology/Neuropathology Department, receives a large
number of muscle biopsies per annum (greater than 150), making it one of the larger neuromuscular
referral pathology centers in North America. There is a strong clinical need then to characterize as
accurately as possible from the pathologic point of view, the dystrophy sub-type of these cases.
Recent Advances in Sub-classification of Limb Girdle Muscular Dystrophy
Clinically defined limb girdle muscular dystrophy has been shown to be genetically quite heterogeneous,
currently more than 10 different underlying genetic defects have been described, resulting in both
autosomal dominant and recessive forms of muscle disease. A number of these genes have had their
proteins isolated and characterized (see table 1). Many of these proteins are muscle fibre membrane
proteins and their absence or significant reduction can be defined by routine immunohistochemistry from
frozen skeletal muscle biopsy tissue (i.e. laminin alpha 2, dysferlin, sarcoglycan complexes) (see Fig. 1).
Those proteins which are located in the muscle fibre sarcolemma would lead to an absent membrane
staining pattern as a diagnostic abnormality. This is analogous to the earlier genetic and
immunohistochemical work showing deficient membrane staining for dystrophin in Duchenne’s muscular
dystrophy. Technically, this requires careful immunohistochemical characterization of the normal muscle
fibre membrane staining pattern for these proteins. In addition, when test immunohistochemistry is being
performed, staining for spectrin is performed, as an integral membrane protein. This serves as a useful
control so that negative staining for the particular test protein, i.e. dysferlin, can be considered to be a
specific protein deficiency if spectrin remains positive, so that it does not simply reflect generalized
damage or loss of membrane proteins (see figure 2a and 2b). This immunohistochemical analysis
potentially allows a rapid and routine diagnostic approach to be utilized on the biopsy tissue already
available for the general pathology work-up.
Current Applications
In addition to dystrophin immunohistochemistry, used to diagnose Duchenne’s and Becker’s forms of
muscular dystrophy, the Pathology/Immunohistochemistry laboratory can currently diagnose protein
deficiencies of dysferlin, the sarcoglycan complex (gamma, alpha, beta and delta) laminin alpha 2
(Merosin), while caveolin-3 and calpain-3 are currently in a state of work in progress. This allows the
potential specific diagnosis of six additional forms of limb girdle muscular dystrophy/distal muscular
dystrophy, which would otherwise be considered part of the larger rubric of non-specific limb girdle
muscular dystrophies. These are largely autosomal recessive muscle diseases with varying clinical
courses and phenotypes (limb girdle muscular dystrophy type 2B, 2C, 2D, 2E and 2F, as well as the distal
muscle dystrophy disease Miyoshi myopathy and congenital muscular dystrophy). Although presently
specific therapies do not exist for these muscle diseases, delineating the specific form present, is
clinically useful in predicting the subsequent behaviour and phenotype as well as for familial and genetic
counseling. Furthermore, as the normal protein function of these molecules becomes better understood,
specific therapeutic interventions may become apparent in the future.
Figure 2a and 2b
Figure 1 Legend
Schematic characterization of the molecular organization of the
integral membrane and associated proteins involved in skeletal
muscle fibres in muscular dystrophies.
Muscle biopsy from a limb girdle muscle
dystrophy patient showing maintained
sarcolemmal staining for spectrin with
absent sarcolemmal membrane staining for
dysferlin, diagnostic of limb girdle muscular
dystrophe type 2B.
Table 1
Protein
Immunohistochemistry
Dystrophy Diagnosis
Pattern
Dystrophin
Membrane, absent
Duchenne’s
Dystrophin
Membrane, reduced
Becker’s
Emerin
Nuclear, absent
Emery-Dreifuss
Merosin (alpha-2
Membrane, absent
Classic form, congenital muscular dystrophy
Membrane, absent
Limb girdle muscular dystrophy type 2B (Miyoshi
laminin)
Dysferlin
myopathy)
Gamma
sarcoglycan
Membrane, absent
Limb girdle muscular dystrophy type 2C
Alpha
Membrane, absent
Limb girdle muscular dystrophy type 2D
Beta sarcoglycan
Membrane, absent
Limb girdle muscular dystrophy type 2E
Delta
Membrane, absent
Limb girdle muscular dystrophy type 2F
Caveolin-3
Membrane, absent
Limb girdle muscular dystrophy type 1C
Calpain-3
Cytoplasmic, absent
Limb girdle muscular dystrophy type 2A
sarcoglycan
sarcoglycan
Table 1 Legend: A partial listing of the proteins and correlated immunohistochemical staining pattern, used in the diagnosis of some of the
major forms of muscular dystrophies.
References
1. Cohn RD et al. Molecular basis of muscular dystrophies. Muscle & Nerve 2000;23:1456-1471.
2. Anderson LV et al. Characterization of monoclonal antibodies to calpain 3 and protein expression in muscle from patients with
limb-girdle muscular dystrophy type 2A. Am J Pathol 1998;153:1169-1179.
3. Lim LE et al. The sarcoglycan complex in limb-girdle muscular dystrophy. Curr Opin Neurol 1998;11:443-452.
4. Matsuda C et al. Dysferlin is a surface membrane-associated protein that is absent in Miyoshi myopathy. Neurology
1999;53:1119-1122.
5. McNally EM et al. Caveolin-3 in muscular dystrophy. Hum Mol Genet 1998;7:871-877.
Dr. J. Provias
Discipline of Anatomic Pathology
Hamilton Regional Laboratory Medicine Program
Hamilton General Hospital Site