From proteins to genes: immunoanalysis in the diagnosis of muscular dystrophies

Abstract

Muscular dystrophies are a large heterogeneous group of inherited diseases that cause progressive muscle weakness and permanent muscle damage. Very few muscular dystrophies show sufficient specific clinical features to allow a definite diagnosis. Because of the currently limited capacity to screen for numerous genes simultaneously, muscle biopsy is a time and cost-effective test for many of these disorders. Protein analysis interpreted in correlation with the clinical phenotype is a useful way of directing genetic testing in many types of muscular dystrophies. Immunohistochemistry and western blot are complementary techniques used to gather quantitative and qualitative information on the expression of proteins involved in this group of diseases. Immunoanalysis has a major diagnostic application mostly in recessive conditions where the absence of labelling for a particular protein is likely to indicate a defect in that gene. However, abnormalities in protein expression can vary from absence to very subtle reduction. It is good practice to test muscle biopsies with antibodies for several proteins simultaneously and to interpret the results in context. Indeed, there is a degree of direct or functional association between many of these proteins that is reflected by the presence of specific secondary abnormalities that are of value, especially when the diagnosis is not straightforward.

Figure 1

Control tests for necrosis and regeneration. Expression of various markers of sarcolemmal integrity (β-spectrin) and regeneration (neonatal myosin heavy chain (Neo-MHC), utrophin and laminin α5) in serial sections of control, Duchenne muscular dystrophy (DMD) and limb girdle muscular dystrophy (LGMD)2B muscle. Arrows indicate abnormal immature fibres positive for Neo-MHC; stars highlight regenerating fibres at different stages. The pattern of expression of the regeneration markers is less defined in DMD muscle due to the overall upregulation of utrophin and laminin α5 and secondary loss of neuronal nitric oxide synthase (nNOS).

Figure 2

The dystrophin glycoprotein complex (DGC). Schematic representation of the core DGC and associated proteins.

Figure 3

Primary and secondary protein abnormalities in dystrophin glycoprotein complex (DGC)-related disorders. A full description is given in the text. BMD = Becker muscular dystrophy; α-DG = α-dystroglycan; β-DG = β-dystroglycan; Dys-C = dystrophin C-terminal; Dys-N = dystrophin N-terminal; lam-α2 = laminin α2; LGMD2D = sarcoglycanopathy with primary defect in the SGCA gene; LGMD2I = dystroglycanopathy with primary defect in the FKRP gene; α-SG = α-sarcoglycan.

Figure 4

Comparative expression of dysferlin, caveolin 3 and calpain 3. Expression of these proteins is primarily or secondarily affected in patients with defects in the caveolin 3 (CAV3) gene (limb girdle muscular dystrophy (LGMD)1C), the dysferlin (DYSF) gene (LGMD2B) and the calpain 3 (CAPN3) gene (LGMD2A). In a patient with mosaic expression of caveolin 3, fibres with loss of the protein also show reduced sarcolemmal labelling for dysferlin (a). (b) Immunoblot is a more reliable technique for diagnosis of LGMD2A and 2B. β-DG = β-dystroglycan; Dys-C = dystrophin C-terminal; α-SG = α-sarcoglycan.