Fixation of Sections

Many antibodies when applied to muscle sections do not require fixation, and this is our method of choice. Acetone or methanol, however, is frequently used for studies of muscle sections, but in our experience this is often unnecessary for the study of human muscle. Formaldehyde or paraformaldehyde, at various concentrations, is also sometimes used. Acetone or methanol, or permeabilization with Triton X-100 (0.05–0.2%) or saponin is often necessary for localization of intercellular antigens in cultured muscle cells and can also be used on sections. A fixative or permeabilization solution may affect the epitope of an antibody and if fixation is required, the most suitable, and its concentration, has to be determined experimentally.

Immunolabelling and Washing

For manual immunolabelling, dry sections are placed in a moist atmosphere to prevent evaporation of the small volumes of reagents, as described in Chapter 1 . Use of a hydrophobic pen to draw round each section is a convenient way to reduce the volume of reagents used and to prevent reagents spreading. Coverslips or slides can be placed in racks and washed collectively in a dish, or they can be rinsed individually. Three rinses of buffer are required, but the timing of each is variable. It can be from 3 to 5 minutes, or in the case of individual coverslips, 10-second dip washes are adequate. The staining tray shown in Figure 1.10 has a magnetic strip which holds the slides in place and is a convenient way to wash a batch of slides by gently expelling buffer from a ‘squeezy’ bottle over the sections for about 30 seconds. Sections must not be allowed to dry out at any stage or false results and artefact will arise. It is also important to ensure that there are no bubbles in the antibody solution when applied, as no labelling will occur under the bubble.

Blocking Agents

Non-specific background, which reduces the signal-to-noise ratio, may be a problem. Additives to the buffer, such as bovine serum albumin (BSA; 2%) or detergents (0.2% Triton X), or normal serum from the same species as the secondary antibody, can be used to reduce non-specific binding of secondary antibodies but, as with fixatives, they may affect the epitope of the antibody. Some tissue components may contain endogenous enzymes, such as peroxidase or alkaline phosphatase. For example, macrophages contain endogenous peroxidase and blocking agents are needed if cluster of differentiation (CD) markers are localized with peroxidase. Endogenous peroxidase can be blocked with hydrogen peroxide (1%) and endogenous alkaline phosphatase can be blocked with levamisole (1 mM), although the EnVision kit contains a peroxidase blocker. It can sometimes be useful to observe endogenous peroxidase activity and to know macrophages are present without having to use specific cell markers (see Fig. 6.8 ). Endogenous biotin can be blocked by applying unconjugated avidin followed by biotin, available as a commercial kit. We find that there is rarely an advantage in using blocking agents for the study of human muscle; good washing and optimal dilution of the antibodies are more critical factors. Phosphate-buffered saline (0.1 M or 0.2 M) at pH7.2–7.4 is usually used for washing, and commercially available tablets are now a convenient way to make this up.

Primary Antibodies

A wide variety of primary antibodies to muscle proteins are now commercially available, in particular those that detect primary defects in recessive muscular dystrophies. Several companies sell relevant primary antibodies, and most companies now have a website. There are also various directories and hybridoma banks (see Appendix 2 ). Several companies market the same antibody clone, so purchasing an antibody from a different company does not necessarily mean it is a different antibody. Attention should be paid to the characterization of a commercial antibody as this is not always complete on data sheets, and also to the recommended storage of an antibody. If freezing is recommended, an antibody should be divided into aliquots of convenient volume to avoid repeated cycles of freeze–thawing. Company websites (e.g. Abcam) offer useful advice on storage and the length of time that an antibody can be used, which is increasingly important for laboratory accreditation. When possible, characterization of an antibody should be checked in the original publication describing the use of the antibody, or the information obtained from the company. Dilution of a primary antibody should always be assessed by titration in each laboratory, and is dependent on such factors as supplier, poly- or monoclonal antibody, secondary antibody, time and temperature of incubation, and the detection system used. As for washing, phosphate-buffered saline at pH7.2–7.4 is used for diluting antibodies. Incubation times can vary from 30 to 60 minutes to overnight at 4°C. The latter sometimes reduces background and lower dilutions of antibody may be possible. Both monoclonal and polyclonal antibodies are available. The former, released from hybridoma cells, recognize a single epitope and are highly specific. Mouse monoclonal antibodies have been used for many years but rat and rabbit monoclonal antibodies are also now available. Polyclonal antibodies are usually purified from serum following injection of the antigen into an animal species (e.g. rabbit, goat or sheep) and recognize several epitopes on the antigen. A control not using the primary antibody is particularly important when using polyclonal antibodies (see below). Double labelling using primary antibodies raised in different species is relatively straightforward, but if the primary antibodies are both monoclonals from the same species kits are available (e.g. Zenon immunolabelling kits). For double/multiplex labelling it is often possible to use a cocktail of antibodies applied at the same time, provided they do not interfere with each other, and appropriate secondary antibodies then applied for each. If multiple antibodies are applied sequentially, the saturation of the first primary must be considered and any possible binding of the unsaturated first primary antibody with subsequent secondary antibodies used.

Detection Systems

It is common to use an indirect detection method to visualize the primary antibody, rather than directly labelling each primary antibody with a marker, and several commercial kits are now available. Indirect labelling gives more flexibility as it involves applying a secondary antibody against the appropriate immunoglobulin of the species in which the primary antibody was raised, and the same one can then be used to visualize several different primary antibodies. Most antibodies are immunoglobulin G (IgG) immunoglobulins but some are IgM. It is essential to use a secondary antibody to the appropriate immunoglobulin or to use one that recognizes all classes. Secondary antibodies to different IgG classes can be useful for multiplex labelling ( ). The secondary antibody may be directly conjugated to biotin, an enzyme or a fluorochrome. Biotin conjugates are then followed by streptavidin labelled with a marker of choice. This amplification technique has the advantage of enhancing the signal because of the total number of bound avidin molecules. Streptavidin directly labelled with a fluorochrome or a preformed avidin–biotin complex (ABC) may be used. Good enhancement is also achieved with commercial kits (e.g. EnVision or Menarini) and the tryamide system (Zenon immunolabelling technology) in which peroxidase is used to catalyze the deposition of biotin or fluorochrome-labelled tryamide close to the antigen–antibody binding site. The deposited biotin is then visualized with streptavidin conjugated to peroxidase or a fluorochrome.

The choice of label to visualize the antibody is often a matter of personal preference but it is also governed by the type of microscope available, the need for a permanent preparation, the amount and localization of the antigen and the affinity of the antibody used. Enzyme labels, such as peroxidase or alkaline phosphatase, provide permanent results and it is easier to see the overall structure of the tissue, particularly if sections are counterstained after immunolabelling. With fluorescent labels, however, it may be easier to distinguish small areas of localized antibody which appear bright against a dark background, and specificity can easily be checked by changing the excitation filter. Methods for quantifying fluorescent signals are also being developed (see below). Aqueous mountants have now improved which reduce fading, and fluorescence can be retained for several weeks, months or sometimes even years. Good fluorescent nuclear counterstains are also now available (see below). Fluorescent methods are usually quicker to apply and avoid the use of hazardous substances such as diaminobenzidine (DAB). Double and multiplex labelling is easier using fluorochromes and the relationship of different antigens closely localized at the same site is more precisely determined; however, brightfield double labelling is also being used ( ). Illustrative examples of the use of both peroxidase and fluorochromes are given in this book.

Fluorescent Labels

Several fluorochromes are now available, such as fluorescein, rhodamine, Texas Red, AMCA, Cy3 and Cy5 and Alexa probes. The choice is often one of personal preference, but factors such as cost, intensity, filters fitted to the microscope and fading also need to be considered. Fluorescein isothiocyanate (FITC) fades rapidly on excitation and FITC–Ig conjugates bind non-specifically to muscle components. Its use is diminishing in favour of Cy and Alexa probes (marketed by Molecular Probes) which give a particularly high signal-to-background ratio and are resistant to fading on storage and with excitation. Bleaching of sections can occur when high-power objectives are used. Storage of the labelled sections in the dark and at 4°C helps to reduce fading. Autofluoresence in muscle, such as that associated with blood vessels and lipofuscin, should be identified by exchanging excitation filters. The development of new labels encourages users to change their methods. While this has obvious advantages, pathological comparisons of intensity are important and new baselines may have to be established if changes are made. Our method of choice was previously a biotin–streptavidin–Texas Red system but we now routinely use streptavidin–Alexa 594 or 488 when labelling by hand with fluorescent markers, particularly for localizing small areas or when double labelling is required. For the majority of antibodies we now routinely use a peroxidase-based method on a Ventana (Roche) Discovery Ultra or a Dako Autostainer machine. Methods have had to be adapted and reagents varied in order to obtain good reproducible results with some antibodies.

Enzyme Labels

Secondary antibodies can be conjugated to an enzyme that can then be histochemically detected at the site of the antigen by deposition of a coloured, insoluble reaction product. Peroxidase detected with DAB has traditionally been the most used enzyme label. It produces a brown end product which is stable for long periods at room temperature and can be enhanced with silver or nickel to produce a black end product. Other substrates that can be used include aminoethyl carbazole (red end product) or 4-chloro-1-naphthol (blue end product). A common alternative to peroxidase is alkaline phosphatase, which, with the appropriate substrate, can be visualized as a red, blue or black end product. Glucose oxidase and β-galactosidase are also useful, particularly for double labelling of two different antibodies. Amplification, as mentioned previously, can be obtained with a biotin–avidin system, either with a preformed complex, directly conjugated to streptavidin alone, or various commercial kits. Peroxidase antiperoxidase also provides enhancement but is now less commonly used since the wider availability of biotinylated systems. As with fluorochromes it is necessary to establish baselines and become accustomed to levels of intensity of peroxidase labelling with each antibody used. It is important to know how a particular antibody works in your laboratory with your method.

Counterstaining and Mounting

Counterstains provide an overall impression of the morphology of the tissue and can be nuclear or cytoplasmic—more commonly the former. They are not essential, and their use is one of individual choice. In sections where labelling is expected to be absent, such as with dystrophin in DMD or mature muscle with no immature myosin isoforms, a nuclear counterstain can be helpful. In Emery–Dreifuss muscular dystrophy the absence of emerin from the nuclei using a peroxidase detection system is easier to assess without a counterstain. With fluorescence, however, a nuclear counterstain at a different wavelength to the one used to detect emerin can be helpful to show where the nuclei are. The choice of counterstain is dependent on the colour of the end product or fluorochrome. Haematoxylin and methyl green are common nuclear counterstains used after peroxidase. If too much haematoxylin is present, it may give a pale delineation of the sarcolemma and care is then needed in assessing any very low levels of sarcolemmal proteins. For example, when assessing dystrophin in peroxidase-labelled sections from cases of DMD the low levels of minor transcripts may be obscured or low levels of sarcolemmal major histocompatibility complex (MHC) class I may be difficult to identify if the haematoxylin counterstain is too strong. Fluorescence is then an advantage, as the low levels are visible against the dark background. For fluorescence, the nuclear counterstains most commonly used are 4′6-diamidino-2-phenylindole.2HCl (DAPI) (1 μg/mL), Hoechst dye (10 μg/mL), which are both blue under ultraviolet light, or ethidium bromide (1 μg/mL) or TOTO, which are red (all available from Molecular Probes). Mountants containing a counterstain are also commercially available (e.g. with DAPI from Vector Laboratories).

The choice of mountant is also determined by the visualization marker. Sections labelled with enzymes such as peroxidase can be dehydrated in graded alcohols, cleared and mounted in synthetic resins. Fluorescent markers require aqueous mountants and several commercial ones contain antifading agents. We have found Hydromount (National Diagnostics) to be useful as this dries to hold the coverslip in place and fluorescence lasts for several weeks or months. Mountants from Vector Labs are also useful. If a glycerol-based mountant is used, which does not dry, the coverslip should be sealed and held in place by nail varnish.

Membrane-Associated Proteins

Several defective proteins associated with the sarcolemma are responsible for various forms of muscular dystrophy. The gene encoding dystrophin was cloned in 1987 and was the first gene defect to be identified in a neuromuscular disorder (see Ch. 10 ; ). Mutations in the dystrophin gene on chromosome Xp21 cause Duchenne or Becker muscular dystrophy (see ). Dystrophin is a cytoskeletal protein, and immunolabelling of normal muscle shows it is localized to the sarcolemma of every fibre ( Fig. 6.5a ). Abnormal labelling is seen in Xp21 conditions. A secondary reduction, however, can occur in some limb-girdle dystrophies and congenital muscular dystrophies (see below). The majority of cases of DMD show an absence of dystrophin from most fibres or a very marked reduction ( Fig. 6.5b ; see Ch. 10 ). This is thought to be due to disruption of the reading frame by the mutation. Isolated fibres, or small clusters, may show a normal intensity and are known as revertant fibres ( Fig. 6.5c ). These are fibres in which the reading frame has been maintained by skipping the mutation. In Becker muscular dystrophy the reading frame is also maintained and labelling is usually of reduced intensity on all fibres, or uneven on many fibres ( Fig. 6.5d ; see Ch. 10 ). In some Becker muscular dystrophy cases, however, dystrophin immunolabelling may be indistinguishable from normal and, then, assessment of secondary changes is useful. Abnormalities related to a defect in the dystrophin gene can also be seen in manifesting female carriers of DMD ( Fig. 6.6 ) and occasionally in Becker muscular dystrophy carriers ( ), because of random X-inactivation patterns (see Ch. 10 ).

Dystrophin is a large protein, and it is important to use more than one antibody for assessment to avoid false results. If the epitope of an antibody lies within a region of the protein encoded by a deleted region of the gene, dystrophin will not be detected, giving the impression of DMD. If the reading frame is maintained, however, other domains including the C-terminal region will be present, consistent with Becker muscular dystrophy. It is therefore important when assessing a case of muscular dystrophy to use antibodies that recognize N- and C-terminal regions of dystrophin, and it is usual to also include one to the rod domain of the protein. Although the majority of cases of Duchenne and Becker dystrophy conform to the frame shift hypothesis, there are exceptions, and clinical severity should not be judged from dystrophin immunolabelling (see Ch. 10 ; ). Several commercial monoclonal and polyclonal antibodies are available, but their affinity is variable, and caution in comparing results is then needed.

Dystrophin is associated with complexes of proteins that collectively have been called the dystrophin-associated proteins (DAPs) ( ). All of these localize to the sarcolemma in immunohistochemical analysis. Some of these proteins are glycosylated, some are transmembrane or extracellular; others are intracellular. The DAPs are thought to provide a structural link between the subsarcolemmal actin cytoskeleton and the extracellular matrix, stabilizing the membrane during repeated contraction and relaxation. The complex may also stabilize various receptors and ion channels. The DAPs are grouped into subcomplexes ( Fig. 6.7 ): α- and β-dystroglycan, which are post-translational products of the same gene, the sarcoglycans (α, β, γ, δ) and sarcospan, the syntrophins, dystrobrevin and nNOS ( ). Two additional genes with homology to sarcoglycans have also been identified (ε and ζ), and their proteins also localize to the sarcolemma ( ), but they are part of different complexes with roles in other locations, including the neuromuscular junctions, retina and brain ( ). ε-Sarcoglycan is homologous to α-sarcoglycan and is thought to replace it in smooth muscle, where it is closely associated with β- and δ-sarcoglycan ( ). Mutations in ε-sarcoglycan cause myoclonus-dystonia syndrome ( ), but no pathogenic mutations in ζ-sarcoglycan in humans have been found. Defects in the genes for α-, β-, γ- and δ-sarcoglycan have been identified in forms of limb-girdle muscular dystrophies (see Ch. 11 ). Rare mutations in the gene encoding dystroglycan ( DAG1 ) have been identified and are responsible for a form of limb-girdle muscular dystrophy ( ), and rare cases with a congenital muscular dystrophy phenotype have been reported (see Chapter 11, Chapter 12 ; ). The major defect associated with dystroglycan is the secondary post-translational modification of α-dystroglycan by glycosylation, and it is recognized as an important pathogenic mechanism in several forms of congenital muscular dystrophy (see below and Ch. 12 ). The syntrophins are believed to be important in anchoring ion channels ( ), but no mutations in the genes encoding syntrophins, dystrobrevin or nNOS have yet been found, only secondary alterations in their expression (see below).

The sarcoglycans (α, β, γ, δ) function as a group, so that a defect in one results in reduced expression of all four. Complete absence of one sarcoglycan, or the most severe reduction, is usually an indicator of the defective gene and helps to direct molecular analysis ( Fig. 6.8 ). The other three sarcoglycans then show variable degrees of abnormal immunolabelling, ranging from pronounced to minimal. An absence of the whole sarcoglycan complex may occur and is usually associated with a defect in the β-sarcoglycan gene ( ). Immunoanalysis, however, does not always predict the genotype ( ). A secondary reduction of sarcoglycans occurs in DMD (see below).

Other forms of limb-girdle muscular dystrophy are caused by mutations in other genes coding for proteins localized to the sarcolemma, and a reduction can be detected with immunohistochemistry. An absence or marked reduction of the plasma membrane protein dysferlin can be seen in some cases of limb-girdle 2B dystrophy and Miyoshi myopathy. Dysferlin is believed to be involved in membrane fusion and repair ( ). Currently available commercial antibodies do not always give a clear result with immunohistochemistry, and secondary changes in dysferlin expression can also occur in other muscular dystrophies (see below). Abnormalities in dysferlin expression are often clearer on immunoblots, and several proteins should be examined simultaneously to ensure the reduction is not secondary to a primary defect in another protein such as calpain-3 or caveolin-3 ( ).

Caveolin-3 is the muscle-specific form of caveolin, and defects in its gene occur in a dominant form of limb-girdle dystrophy (LGMD1C), in rippling muscle disease, in association with exercise intolerance and rhabdomyolysis and some rare cases with only elevated serum creatine kinase (CK) (see Ch.11 ; ). Caveolin-3 is a plasma membrane protein, and in normal muscle it is seen localized to the sarcolemma. An absence or reduction of the protein can be detected by immunohistochemistry and is a rare example of a detectable reduction of protein resulting from a dominant mutation. Patients with an autoimmune, non-genetic form, of rippling muscle disease have also been identified and muscle biopsies show a mosaic pattern of caveolin expression on muscle fibres ( ).

Extracellular matrix proteins have been shown to have a major role in neuromuscular disorders. Defects in the LAMA2 gene encoding the laminin α 2 chain of merosin (laminin 211) occur in the severe MDC1A form of congenital muscular dystrophy (‘merosin-deficient’ congenital muscular dystrophy; ; see Ch. 12 ). In normal muscle the laminin α2 chain is localized to the basal lamina of every muscle fibre. It is not present in the basal lamina round blood vessels of skeletal muscle, nor of smooth muscle, although a thin layer of labelling of the inner layer adjacent to the lumen of large vessels may be seen and may relate to the tunica intima or inner endothelial layer. This is in contrast to the laminin α5, β1, β2 and γ1 chains ( Fig. 6.9a–e ; and see Fig. 6.32 ) and to blood vessels of the brain ( ). Laminins are heterotrimers, the three chains of which are arranged in a cruciform shape and encoded by separate genes. To date, five α, four β and three γ chains have been identified, which are assembled into at least 15 different forms with a variable tissue distribution ( ). Early nomenclature referred to the chains as A, B1, B2 but this was revised to accommodate the growing number of different chains, and a further revision is now often used ( ). The main variants localized to the sarcolemma are laminin-2 (often referred to as merosin; α2-β1-γ1 chains, also referred to as 211) and laminin-4 (S-merosin; α2-β2-γ1 chains, laminin 221). The laminin β2 chain has an organizational role at the neuromuscular junction, where it is concentrated ( ). Contrary to common belief, however, appreciable amounts of laminin β2 are detected on the extrajunctional sarcolemma, particularly in mature muscle (see Fig. 6.9e ; ). The difference probably relates to the use of different antibodies in the various studies. The laminin α5 chain is detected on blood vessels and is highly expressed on immature fibres. It is down-regulated during development and only a little is seen on the sarcolemma of mature human muscle fibres (see Fig. 6.9b ). The clinical severity of cases with a defect in the LAMA2 gene that encodes the laminin α2 chain of laminin 211 (merosin) varies from severe to mild and this is usually reflected in the amount of protein that can be detected. Severe cases usually show an absence, or only very slight traces, of laminin α2 on the sarcolemma ( Fig. 6.10a–c ; ). Milder cases show only a reduction of the protein and it is then necessary to assess both N- and C-terminal epitopes of this large protein ( ; see Ch. 12 ). Secondary changes in laminin α2, α5 and β1 chains are discussed below. No defects in laminin γ1 have been identified and it is therefore useful for assessing the integrity of the basal lamina.

Defects in the genes that encode three chains of collagen VI are responsible for Bethlem myopathy and the Ullrich form of congenital muscular dystrophy (see Ch. 12 ; ). Distinct labelling of collagen VI associated with the sarcolemma is seen in normal muscle ( Fig. 6.11 ), and it is also seen in the perimysium and endomysium when there is excess fibrosis. In the milder cases of Bethlem myopathy, many of which are dominantly inherited, an alteration in collagen VI immunolabelling is rarely seen ( Fig. 6.12 ; but see Fig. 12.9 ). In recessive cases of Ullrich congenital muscular dystrophy and cases intermediate between Ullrich congenital muscular dystrophy and Bethlem myopathy, however, collagen VI may be absent or severely reduced (see Fig. 6.12b, c ). In some cases, the reduction may only be a subtle change that is only apparent at the sarcolemma (see Fig. 6.12c, d ). It is important that the good preservation of the basal lamina/basement membrane is controlled with parallel double-labelling studies of another basal lamina/basement membrane protein, such as collagen IV, collagen V or perlecan. Collagen V has a similar localization to collagen VI, and perlecan interacts with collagen VI (see below).

Missense mutations and in-frame insertions in the gene for perlecan have been reported in Schwartz–Jampel syndrome, but the protein is still present ( ). Complete absence of the protein is lethal and occurs in dyssegmental dysplasia with severe lack of bone growth. Perlecan has a major role in clustering of acetylcholine esterase to the neuromuscular junction and connects the basal lamina to the plasma membrane via integrins.

Mutations in others collagen genes, including collagen I, III and V. are associated with Ehlers–Danlos syndrome in which neuromuscular symptoms can be present ( ). Heterozygous mutations in the COL42A gene that encodes the 2A chain of collagen IV have been reported in a few patients with a myopathic phenotype resembling muscle-eye-brain disease/Walker–Warburg syndrome, as well as being associated with other phenotypes ( ). Mutations in collagen IX are associated with epiphysial dysplasia and a myopathy ( ), but immunohistochemical studies of the defective proteins in muscle are limited. Mutations in collagen XII cause a disorder similar to Ullrich CMD and abnormalities in expression of the protein can be detected (see ch.12). An abnormality in the expression of tenascin–X has also been shown in some patients with Ehlers–Danlos syndrome, a disorder that is associated with a variety of genetic defects ( ).

Another primary defect in a membrane-associated protein is integrin α 7 , which interacts with laminin α2 ( ). Defects in the gene cause a mild disorder classified as a congenital muscular dystrophy but it is generally accepted that the features are ‘myopathic’ rather than ‘dystrophic’. Absence of integrin α7 is seen with a concomitant decrease in its partner, integrin β1D. This disorder is very rare, and the original studies were not performed with a commercial antibody (see Ch. 12 ). Integrin α7β1D forms a transmembrane complex that interacts with several proteins, including laminin α2, and secondary alterations in integrins occur (see below).

The lysosomal-associated membrane protein 2 (LAMP-2) is a transmembrane lysosomal protein that is defective in Danon disease (see Ch. 16 ). This X-linked disorder is associated with cardiomyopathy, a vacuolar myopathy and variable degrees of mental retardation ( ). Immunohistochemistry and immunoblots show a virtual absence of LAMP-2 protein ( ), as well as several secondary abnormalities (see Ch. 16 ).

A protein of the endoplasmic reticulum membrane required for the assembly of the vacuolar ATPase complex, encoded by the VMA21 gene, is responsible for the X-linked myopathy with excessive autophagy (XMEA; ). The muscle pathology shares some similarities with Danon disease (see Ch. 16 ), but there are no immunohistochemical studies of the primary gene product.

Primary Defects in Nuclear Proteins

The nuclear envelope is composed of an outer and inner membrane, nuclear pore complexes and a lamina. The outer membrane is continuous with the endoplasmic reticulum, and a number of proteins are integral components of the inner membrane. Several proteins of the nuclear cytoskeleton and nuclear envelope interact and form a complex ( ). Some of these proteins are known to have pathogenic consequences in neuromuscular disorders (emerin, lamin A/C, nesprin 1 and 2, TMEM43), while others (e.g. SUN1 and SUN2) are modifiers (see Ch. 13 ). Emerin is anchored to the inner nuclear membrane ( Fig. 6.13 ), and mutations in the gene are responsible for the X-linked form of Emery–Dreifuss muscular dystrophy (see Ch. 13 ). The majority of mutations in the EMD gene encoding emerin result in truncation of the protein, which lacks the C-terminal domain, and result in a total absence of the protein from all nuclear membranes. This absence is easily detected by immunohistochemistry of muscle, skin or buccal cells ( ). Very rare exceptions to this have been reported, and emerin was seen to be reduced ( ) or mislocalized in the muscle cytoplasm ( ).

Lamins are a principal component of the nuclear lamina, and mutations in the gene for lamin A/C ( LMNA ) are associated with several phenotypes, including the autosomal dominant form of Emery–Dreifuss muscular dystrophy (see Ch. 13 ), dilated cardiomyopathy, limb-girdle muscular dystrophy 1B, familial partial lipodystrophy, an axonal neuropathy (Charcot–Marie–Tooth type 2B1), mandibuloacral disease and, premature ageing disorders ( ). Immunolabelling of lamin A/C in dominant Emery–Dreifuss muscular dystrophy of muscle shows no detectable difference from controls. Secondary changes in laminin β1, however, may be seen (see below).

The nuclear membrane proteins nesprin-1 , nesprin-2 and TMEM43 (encoded by SYNE-1 , SYNE-2 and TMEM43 , respectively) have also been shown to be associated with myopathic disorders (see ). Nesprin-1 has been shown to be associated with a severe recessive disorder with arthrogryposis ( ), and in patients with cardiac conduction defects and an Emery–Dreifuss muscular dystrophy phenotype ( ). An absence of nesprin-1 can be demonstrated in some patients (see Figure 13.10 ). In control muscle labelling of nuclear membrane proteins is similar in peripheral and internal nuclei ( Fig. 6.13 ) except in regenerating fibres labelled with antibodies to nesprin-1 (see Ch.13 ). Heterozygous mutations in TMEM43 have also been identified in two cases with an Emery–Dreifuss muscular dystrophy phenotype ( ). Immunohistochemical studies in these cases have been limited, but fibroblasts and cells expressing the mutant protein have shown reduced or abnormal immunolabelling of nuclear membrane proteins. A muscle biopsy from one of the cases with a TMEM43 mutation was reported to show reduced nuclear labelling of TMEM43 ( ).

Mutations in the gene encoding the nuclear matrix protein matrin-3 cause an autosomal dominant distal myopathy with vocal cord and pharyngeal weakness ( ). Matrin-3 immunohistochemical labelling showed reduced labelling of some nuclei in the only biopsy available for study, but the sample was of end-stage muscle.

Other disorders are also caused by defects in nuclear proteins, but immunohistochemistry currently has a limited role in studies of the primary protein defect. These include the survival motor neurone (SMN) protein in spinal muscular atrophy, the expansion of the genes responsible for both forms of myotonic dystrophy and the expansion of the poly(A)-binding protein nuclear 1 gene ( PABPN1 ) in oculopharyngeal muscular dystrophy (OPMD). Antibodies to PABPN1 have been shown to localize to the nuclear inclusions in patients with OPMD (see Ch. 14 ; ). Antibodies to PABPN1, however, can also label nuclei in controls, and high potassium is needed to show specificity for OPMD (see Ch. 14 ).

Primary Defects in Myofibrillar and Cytoskeletal Proteins

An increasing number of defective genes have been identified in a number of proteins related to myofibrillar proteins. They result in myofibrillar disorganization and, collectively, the term ‘myofibrillar myopathy’ has been used for one of these groups of disorders (see Ch. 16 ). The myofibrillar myopathies highlight the importance of proteins that interact with Z line proteins and maintain sarcomeric structure ( ). Myofibrillar proteins affected by mutations in the corresponding gene include myosin heavy chain isoforms, skeletal actin, telethonin, myotilin, ZASP, Bag3, αB-crystallin, filamin C, FHL1, troponin isoforms, tropomyosin, titin, nebulin and the intermediate filaments desmin and plectin. The gene for the intermediate filament protein syncoilin has also been screened as a possible candidate for a myofibrillar myopathy, but no mutations have been found ( ). These defective proteins can all be detected by immunohistochemistry but as some relate to a dominant condition, and/or the mutations are often missense, a total absence of the protein is rarely seen. Sometimes, however, an accumulation of protein occurs—for example, desmin, myotilin or slow myosin (see below)—and additional secondary changes can be detected (see Ch. 16 ).

Myosin heavy chains are encoded by a multigene family and exist in several isoforms that are regulated in a tissue and developmental specific manner ( ). The physiological fast or slow characteristics of the different fibre types relate to the respective isoforms. The major myosin heavy chain isoform in slow, type 1 fibres is encoded by the MYH7 gene on chromosome 14. This is also the major isoform in cardiac ventricles (β-cardiac myosin heavy chain) and mutations are a cause of familial cardiomyopathy ( ). Mutations in the rod domain of MYH7 have been identified in patients with no overt cardiomyopathy but a skeletal muscle myopathy, sometimes with myosin storage ( ). Immunohistochemistry shows these inclusions are in type 1 fibres and contain slow myosin (see Ch. 15 ). This disorder has been classified as a protein surplus myosin storage myopathy ( ). And mutations in the 3′ domain of MYH7 have been shown to cause a distal myopathy ( ). Thus, mutations in different domains of this gene lead to dominant disorders with varying phenotype. A few patients with recessively inherited mutations in the MYH7 gene have also been reported ( ).

Both dominant and recessive inheritance of mutations in the fast myosin heavy chain IIa gene ( MYH2 ) on chromosome 17 have been found (see Ch. 15 ; ). In addition to the presence of rimmed vacuoles, immunolabelling of fast myosin shows the fast 2A fibres are selectively affected, being atrophic, or absent ( ). Mutations in genes encoding developmental myosins ( MYH8 and MYH3 ) are associated with syndromes that present with distal arthrogryposis (see Ch. 15 ; ), but there is no information on their immunohistochemical localization in muscle samples. Similarly, immunohistochemical studies of myosin-binding protein C in cases with mutations in the corresponding gene are limited ( ).

Mutations in the ACTA1 gene encoding sarcomeric skeletal muscle actin are now known to be associated with a broad spectrum of clinical phenotypes in forms of nemaline myopathy, ranging from severe to mild (see Ch. 15 ; ). Mutations can give rise to actin accumulation, with or without nemaline rods, which may be nuclear and/or cytoplasmic. Mutations in the ACTA1 gene also result in congenital fibre type disproportion without any other structural defect ( ) and in a dominant congenital myopathy characterized by cores devoid of mitochondria and disrupted myofibrils and type 1 fibre predominance, as in central core disease, but no nemaline rods ( ). A mutation in the ACTA1 gene has also been found in the rare case described as ‘zebra body myopathy’, and actin accumulation as well as nemaline rods and zebra bodies were observed ( ).

Immunohistochemical studies of tropomyosin isoforms in human muscle as a primary defect are limited, but mutations in the genes for α-tropomyosin and β-tropomyosin are responsible for rare dominant forms of nemaline myopathy and cap-like structures (see ). Mutations in the gene for β-tropomyosin can also cause distal arthrogryposis without the presence of rods ( ). The primary role of tropomyosin in relation to cardiomyopathies has also been established ( ).

Isoforms of α -actinin have been characterized and mainly studied in relation to fibre typing and the rods in nemaline myopathy, rather than as a primary cause of a muscle disorder. Antibodies to α-actinin-2 label all muscle fibre types, while antibodies to α-actinin-3 only label a subset of fast fibres. A common, non-pathogenic polymorphism has been found in the gene for α-actinin-3 ( ACTN3 ) which results in an absence of α-actinin-3 from all fibres and may have some athletic advantage ( ).

Titin and nebulin are two of the largest proteins known, both of which exist as multiple isoforms resulting from differential splicing ( ). They have a role in maintaining myofibrillar alignment and length and contribute to the elasticity of the myofibrils. The genes for both proteins are located on chromosome 2q, and mutations in the nebulin gene are responsible for one of the more common forms of nemaline myopathy ( ; ). Commercial antibodies to nebulin in patients with mutations in the NEB gene show retention of the protein, but research studies with an antibody corresponding to the SH3 region of nebulin have shown an absence in a few severe cases of nemaline myopathy, which can help direct molecular analysis ( ). Studies have also shown a reduction of nebulin on immunoblots in some severe cases with NEB mutations ( ). Recently, monoclonal antibodies that specifically recognize exon 143 or 144 of nebulin have been raised ( ). These two exons are differentially spliced in human muscle and studies suggest that they are developmentally regulated and that exon 143 is more highly expressed in fibres with fast myosin ( Fig. 6.14 ; ). Titin, encoded by the TTN gene, is involved in neuromuscular disorders with varying phenotype, including limb-girdle dystrophy 2J, dominant hereditary myopathy with early respiratory failure (HMERF), some early-onset cases with a fatal cardiomyopathy and a congenital myopathy as well as cardiomyopathies (see Chapter 11, Chapter 15, Chapter 16 ; ). As with nebulin, absence of the whole titin protein does not occur, but a non-commercial antibody raised against the transcript from the defective last exon of this giant gene has shown an absence in LGMD2J ( ). Secondary abnormalities in nebulin expression occur in DMD and, prior to the identification of the dystrophin gene, nebulin was thought to be the primary defective gene product ( ), but this was subsequently found to be a secondary effect.

Mutations affecting other myofibrillar proteins in which immunohistochemistry has been used to study the primary protein include telethonin , responsible for limb-girdle muscular dystrophy 2G, myotilin , responsible for limb-girdle muscular dystrophy 1A and a myofibrillar myopathy, and slow troponin T , responsible for a rare form of nemaline myopathy ( ). The prevalence of these defects is not known, as only a few affected patients have been reported. Limb-girdle muscular dystrophy 2G was thought to be confined to the Brazilian population, but rare cases elsewhere in the world have been identified which show an absence of telethonin protein with immunolabelling ( ) or, occasionally, retention of telethonin ( ). Studies of cases with mutations in the gene for myotilin suggest they may be a common cause of myofibrillar myopathy in some populations (see Ch. 16 ; ). Mutations in the gene for a fast isoform of troponin I on chromosome 11 cause a form of distal arthrogryposis ( ), but there are currently no immunohistochemical data on the expression of the protein although abnormalities have been detected in immunoblots ( ).

The cytoskeletal protein desmin is involved both primarily and secondarily in myofibrillar myopathies often collectively referred to as ‘desminopathies’, ‘desmin-related myopathies’ or ‘myofibrillar myopathies’ (see Ch. 16 ; ). Both missense mutations and deletions in the gene for desmin have been identified ( ). Inheritance is often dominant but rare recessive cases are also known. Age of onset is variable, and cardiomyopathy is a frequent complication. Antibodies to desmin show accumulation in fibres that do not express fetal myosin, distinguishing them from regenerating fibres with high desmin. No desmin was detected in the muscle by immunohistochemistry and immunoblotting from two siblings heterozygous for null mutations in the DES gene ( ). A characteristic accumulation of electron-dense granulofilamentous material is seen at the ultrastructural level (see Ch. 5 ), and additional features include secondary accumulation of a variety of proteins and cytoplasmic and spheroid bodies (see Ch. 16 ). Similar pathological features are also seen when other genes in this group of disorders causing a myofibrillar myopathy are affected ( ). The gene encoding plectin is mutated in the rare skin blistering recessive disorder, epidermolysis bullosa simplex with muscular dystrophy, and is associated with a range of neuromuscular phenotypes ( ). The blistering may not always be marked ( ), and the gene can also be associated with a myasthenic syndrome (see Ch. 21 ; ) and a form of limb-girdle muscular dystrophy (see Ch. 11 ; ). Plectin has a role in mitochondrial function, in organizing the intermediate filament cytoarchitecture, including anchoring desmin to the nuclear membrane, to the periphery of Z lines and at the sarcolemmal costameres ( ). Immunolabelling of skin and muscle with antibodies to plectin shows an absence or reduction and that a secondary accumulation of desmin can occur in epidermolysis bullosa simplex ( ). Plectin also interacts with β-dystroglycan, dystrophin and utrophin ( ) and secondary abnormalities occur in other disorders (see below). Alternative splicing of plectin transcripts gives rise to eight protein isoforms, of which four are highly expressed in muscle (1, 1b, 1d and 1f). Mutations affecting the 1f isoform have been found in a recessively inherited form of limb-girdle muscular dystrophy, and a reduction in sarcolemmal plectin immunolabelling was reported, and also of the corresponding RNA ( ). Internal labelling of plectin was retained in muscle fibres, and studies of mouse muscle, using isoform-specific antibodies, have shown that this internal labelling is higher in 2A fibres ( ). This publication also showed that the plectin 1d isoform is localized to the Z line. We are not aware of studies with isoform-specific antibodies in diseased human muscle, and those published have been with antibodies that recognize all isoforms, but alterations in plectin localization can be observed with them.

Primary Defects in Enzymes

Several primary enzyme defects are known to cause a neuromuscular disorder, but it is often more important to examine the activity of the enzyme biochemically rather than its localization by immunohistochemistry. Immunoblots, however, are useful for studying enzymes such as calpain-3 , which is defective in limb-girdle muscular dystrophy 2A ( ). This disorder is unusual among the limb-girdle muscular dystrophies in not being caused by a membrane-associated protein. Calpain-3 is an enzyme located in the cytosol of the fibre and in the nucleus, and it binds to the C-terminus of titin. Absence of calpain-3 can be detected on sections but a reduction is more difficult to assess and requires immunoblots when secondary changes affecting dysferlin and caveolin-3 can also be assessed ( ).

The valosin-containing protein (VCP, also known as p97) and UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) are two enzymes associated with myopathies often characterized by vacuoles (see Ch. 16 ). Mutations in the VCP gene cause inclusion body myopathy with Paget bone disease and frontotemporal dementia (IBMPFD) and the phenotype now also includes amyotrophic lateral sclerosis (ALS), Parkinson disease, cardiomyopathy and Charcot–Marie–Tooth disease ( ). VCP is a ubiquitously expressed triple A ATPase that has an important role in the ubiquitin proteosome protein degradation system and aggresome formation, as well as other cell functions that include the cell cycle, nuclear envelope reconstruction, postmitotic Golgi reassembly, DNA damage response and suppression of apoptosis ( ). Immunohistochemistry of muscle biopsies shows accumulation of nuclear and sarcoplasmic VCP and TDP-43. MHC I is sometimes up-regulated and sometimes inflammatory cells are present ( ), but these are not consistent features. Lobulated fibres, ring fibres and some polyglucosan-like bodies have also been reported in a patient with a mutation in the VCP gene who had asymmetrical scapuloperoneal weakness and head drop without Paget disease or dementia ( ).

The myopathy caused by mutations in the gene encoding GNE (also known as Nonaka myopathy, distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy) is a distal myopathy that spares the quadriceps, a feature that led to the disease also being called ‘quadriceps-sparing myopathy’ (see Ch. 16 ; ). GNE is an enzyme involved in the biosynthesis pathway of sialic acids and the reduced enzyme activity of GNE is thought to involve hyposialylation of glycoproteins, although this does not explain all of the pathogenic mechanism ( ). A variety of research studies have examined sialylation of various proteins in GNE myopathy that include β1-integrin, NCAM, α-dystroglycan, although the latter is not considered to have a major role in GNE myopathy ( ).

Immunohistochemical studies of primary defects in mitochondrial enzymes are increasing. Abnormalities in mitochondrial oxidative phosphorylation are associated with several multisystem disorders (see Ch. 18 ). The traditional method to study this in muscle biopsies has been histochemical analysis of succinic dehydrogenase and cytochrome c oxidase (see Chapter 18, Chapter 3, Chapter 4 ) which localize the activity of these mitochondrial specific enzymes ( ). Antibodies to complex I–IV, however, are proving useful in some conditions but do not detect defects in all mitochondrial-related disorders ( ). Complex I deficiency can be identified by antibodies to this component (see Ch. 18 ), and a quadruple multiplex immunofluorescent method has been developed to examine complex I, complex IV and mitochondrial mass within fibres labelled for laminin ( ). Antibodies to TOMM22 and porin are useful for labelling all mitochondria ( ). Antibodies to mitochondria show a fibre-typing pattern comparable to staining to cytochrome c oxidase but are particularly useful in identifying a deficiency of complex I (see Ch. 18 ).

Primary Defects of Ion Channels

The main ion channels that have been studied by immunohistochemistry are the ryanodine receptor 1 (RYR1), the dihydropyridine receptor (DHPR) and the sarcoendoplasmic reticulum calcium ATPase (SERCA) (see Ch. 20 ). Mutations in the RYR1 gene on chromosome 19q occur in core myopathies and malignant hyperthermia (see Ch. 15 ; ). Malignant hyperthermia susceptibility is also linked to several other loci and genes (see Ch. 20 ; ). Not all cases of RYR1 -related core disease appear to be susceptible to malignant hyperthermia but patients are always advised of the potential risk. The ryanodine receptor is a large ligand-gated calcium-release channel with an essential role in excitation–contraction coupling ( ). It has several C-terminal transmembrane loops in the membrane of the sarcoplasmic reticulum and cytoplasmic regions that interact with the voltage-gated dihydropyridine receptor of the T tubule. Genotype–phenotype correlations suggest that mutations in the transmembrane C-terminal region of RYR1 are more commonly, but not exclusively, associated with central core disease, whereas malignant hyperthermia is more often associated with N-terminal hot spots ( ). Many mutations in the RYR1 gene are missense and dominantly inherited, so total absence of the protein is not seen, nor in the patients with recessively inherited mutations. Limited immunohistochemical studies of the RyR1 protein have shown a reduction in some cases but they are not currently used for diagnosis. A secondary accumulation of several proteins, however, can be useful for visualizing the cores (see below).

Brody disease, characterized by stiffness and exercise-induced pain ( ), is caused by mutations in the ATP2A1 gene encoding the sarcoplasmic reticulum calcium ATPase, SERCA1 ( ). SERCA1 is confined to fast fibres and SERCA2 to slow fibres. Thus, antibodies to these show a checkerboard pattern in normal muscle. In Brody disease an absence of SERCA1 from the fast fibres can be detected. Proteins, such as sarcolipin, that are associated with SERCA are candidates for Brody syndrome, which clinically resembles Brody disease, but no mutations have yet been found ( ).