Changes in Fibre Size

Fibres may be round in shape, and all forms show an abnormal variation in fibre size that is usually obvious ( Fig. 11.1 ). Hypertrophied fibres are common and this may be marked, especially in some adults. The hypertrophied fibres often show splits or appear branched in longitudinal sections. Some of the size variation seen in transverse sections is due to this branching. In contrast to DMD, the hypertrophied fibres are rarely hypercontracted and heavily stained in LGMD. Groups of small fibres, as in BMD, are not usually seen but can occur. Multiple splitting may sometimes give the impression of a group of small fibres ( Fig. 11.2 ). In cases of LGMD1B, we have noted a tendency for the type 1 fibres to be generally smaller in diameter than type 2 ( ), but this is not a specific feature.

A quadriceps biopsy from a patient with limb-girdle dystrophy aged 39 years showing (a) a wide range variation in fibre size (10–100 μm) with necrosis (∗), excess internal nuclei (large arrow) and a little endomysial connective tissue (small arrow); and in (b) a necrotic fibre invaded by macrophages (∗) and a cluster of regenerating basophilic fibres (mean diameter 30 μm) with large nuclei and surrounded by mononuclear cells of various types haematoxylin and eosin (H&E).

An area with several splits giving the impression of a group of small fibres (limb-girdle dystrophy 2I; H&E). Fibre diameter range of non-split fibres 50–80 μm.

Changes in Fibre Typing

As with many muscular dystrophies, type 1 fibres (with slow myosin) are often predominant and fibre typing may be more distinct than in DMD (see Fig. 11.5b ). With adenosine triphosphatase (ATPase), type 2B fibres may be deficient, but it is not clear how this observation relates to myosin content as many fibres co-express more than one form of myosin isoform (see below).

Nuclear Changes

Internal nuclei can be profuse in some cases and multiple within the cross-section of one fibre ( Fig. 11.3a ). As with DMD, nuclei are often associated with the internal splits in a fibre ( Fig. 11.3b ). Nuclear clumps may be seen in chronic cases. The nuclei of regenerating, basophilic fibres may be large ( Fig. 11.1b ) and vesicular, with a prominent nucleolus and pale nucleoplasm.

(a) Fibres with multiple internal nuclei (arrow) from a case with limb-girdle dystrophy 2I aged 13 years (H&E); fibre diameter 15–95 μm. (b) A round hypertrophied fibre with internal nuclei associated with splits (arrow) from a case of limb-girdle dystrophy 2I aged 10 years (H&E).

Degeneration and Regeneration

Necrotic fibres and basophilic regenerating fibres that may be in clusters or isolated are frequently seen (see Fig. 11.1 ). Necrotic fibres are not a universal feature, which may reflect sampling problems, but a high prevalence of fibres with developmental myosin suggests muscle damage. Basophilic fibres may be less frequent than in DMD.

Cellular Response

Phagocytosis is associated with necrosis. Inflammatory cells are a particular feature of LGMD2B (dysferlinopathy) and cases have sometimes been misdiagnosed as a myositis ( ). Abundant inflammatory cells are rare in other forms of LGMD but can occur and can respond to steroid therapy ( ). Eosinophils have been reported as a feature in some cases with defects in the gene encoding calpain-3 (see Fig. 4.31 c; ), but they are not a universal feature and macrophages and lymphocytes may be a pronounced feature ( ). Areas of necrosis may appear cellular because of regeneration and the presence of myoblasts/myotubes. Excess endomysial connective tissue is usually present, but the extent is variable ( Fig. 11.4 ).

(a, b) Two cases of limb-girdle dystrophy showing different degrees of fibrosis. In (a) most fibres are surrounded by endomysial connective tissue (arrow); fibre diameter range 15–110 μm; note also a little adipose tissue (∗). However, in (b) there is very little endomysial connective tissue. Fibre diameter range 20–100 μm.

Architectural Changes

Moth-eaten and whorled fibres are common in LGMD, and ring fibres may be seen. The latter, however, are not specific for LGMD. Lobulated fibres can also occur, especially in LGMD2A, but they can also occur in other disorders ( Fig. 11.5a ) ( ). Various degrees of aggregation of tetrazolium stain may be seen, sometimes with some resemblance to lobulation ( Fig. 11.5b ). Vacuoles can be observed in some cases, and they are the rimmed type surrounded by basophilia ( ). There is clinical and pathological overlap with myofibrillar myopathies in some cases with rimmed vacuoles, in particular in some of the dominant LGMD forms (see Ch. 16 ; ).

(a) Lobulated fibres (20–50 μm in diameter) in a case of limb-girdle dystrophy 2A. (b) Fibres with aggregation of stain in a slightly similar manner to lobulated fibres (arrows) in a case of limb-girdle dystrophy 2C (NADH-TR). Fibre diameter range 10–90 μm.

Dominant Limb-Girdle Muscular Dystrophies

As in many other dominant conditions, the primary defect may not lead to a detectable alteration in protein localization or quantity, as the normal allele produces a normal product. Many of the mutations are missense, and protein is still formed from the mutated allele.

In LGMD1A , caused by mutations in the gene for myotilin, immunohistochemical localization of myotilin may appear normal ( ). Myotilin is associated with the Z line, and nemaline rods occur. There is clinical overlap with some molecularly undefined cases of adult-onset nemaline myopathy, which raises the possibility that myotilin might be responsible, and defects in the gene encoding myotilin are known to be responsible for some cases of ‘spheroid body myopathy’ (see Ch. 15 ; ). Only two large pedigrees have been defined as LGMD1A, and there is clinical and morphological overlap with a myofibrillar myopathy (also known as spheroid myopathy) caused by defects in the same gene and they are now considered to be the same disorder (see Ch. 16 ). Rimmed vacuoles are a feature, as well as accumulation of various proteins, including desmin and myotilin (see Ch. 16 ).

In LGMD1B there is no detectable alteration in the expression of the nuclear protein lamin A/C or in emerin in muscle biopsies (se Ch. 13 ). Mutations in the lamin A/C gene are now known to be associated with a wide spectrum of clinical phenotypes in addition to LGMD1B, including autosomal dominant Emery–Dreifuss muscular dystrophy, familial partial lipodystrophy, an axonal neuropathy (Charcot–Marie–Tooth type 2B1), mandibuloacral disease and premature ageing disorders ( ). There is clearly clinical overlap between LGMD1B and autosomal dominant Emery–Dreifuss muscular dystrophy, including cardiac conduction defects, and the two conditions are considered allelic (see Ch. 13 ). Muscle biopsies from some adolescent or adult patients may show a reduction of sarcolemmal lamininβ1, with normal labelling of blood vessels, but this is not specific and can be seen in other conditions ( ).

Mutations in the gene encoding caveolin-3 are responsible for four skeletal muscle disease phenotypes: LGMD1C, rippling muscle disease, a distal myopathy and cases with persistent high creatine kinase (hyperCKaemia) . In addition, cardiomyopathies are also associated with caveolin-3 mutations ( ). The phenotype in patients described as LGMD1C is characterized by mild to moderate proximal muscle weakness, exercise-induced cramps and a very high CK. Muscle hypertrophy can also be a feature. Cramps following exercise are also a feature of rippling muscle disease, the particular feature of which is percussion-induced muscle contraction in a rippling fashion. Patients with hyperCKaemia have minimal muscle weakness or symptoms. Reported cases of all conditions are heterogeneous with considerable overlap between the phenotypes, suggesting a continuum rather than separate disease entities. In contrast to many dominant conditions, a reduction in the primary protein product, caveolin-3, can be observed with immunohistochemistry and immunoblotting ( ). The reduction is particularly pronounced in cases of LGMD1C, and only two rare cases with a mutation in the CAV3 gene with normal immunohistochemical labelling of caveolin-3 have been reported ( ). In normal muscle, caveolin-3 is localized to the sarcolemma, to caveolae, the cholesterol-rich structures seen with electron microscopy as indentations or small sarcolemmal vesicles (see Fig. 3.17 a). Labelling of caveolin-3 is normal in other forms of muscular dystrophy, except cases with a mutation in the cavin-1 gene and patients with an autoimmune disorder of caveolin-3 (see below and Ch. 6 ; Fig. 11.6 ) ( ). Caveolin-3 expression is not affected by polymorphic changes in the caveolin-3 gene ( ). Caveolin-3 co-precipitates with dystrophin but is not a component of the dystrophin-associated protein complex ( ).

(a) Normal labelling of caveolin-3 on all fibres in a case of LGMD2I (diameter range 20–60 μm). Note the indentation of the sarcolemma and the vacuole-like area, which is probably also an indentation (arrow). The small fibres have slight internal labelling (∗) and may be regenerating fibres. (b) Quadriceps biopsy of a girl aged 12 years with a mutation in the PTRF gene, encoding cavin-1, showing a wide variation in fibre size, excess endomysial connective tissue and excess internal nuclei (H&E). (c) Caveolin-3 in the same case is absent from the sarcolemma of most fibres but shows pronounced internal labelling of one fibre.

Caveolin-3 is the muscle-specific member of the caveolin family, which are principal components of caveolae. Small invaginations of the plasma membrane found in many cell types, caveolae are believed to be involved in membrane trafficking and signal transduction (see Ch. 5 ) and to have a role in the formation and maintenance of T tubules ( ). Caveolin-3 interacts with dysferlin and secondary changes in dysferlin occur in patients with a mutation in the caveolin-3 gene ( ). This interaction suggests that caveolin-3, like dysferlin, may be involved in membrane repair. The plasma membrane shows ultrastructural abnormalities when caveolin-3 is mutated ( ; see Ch. 5 ).

In addition to caveolin-3, other members of this family of proteins are present in caveolae. In particular, mutations have been found in the gene encoding for cavin-1 (also known as polymerase I and transcript release factor – PTRF) that cause a myopathy with lipodystrophy ( ). These patients may have an early onset and show lipodystrophy, raised CK and cardiac dysfunction that can result in sudden death ( ). The early onset suggests this disorder could be a form of congenital muscular dystrophy but, as there are morphological changes in caveolin-3 and caveolae, this disorder is discussed here. Muscle biopsies show variation in fibre size, excess internal nuclei and an increase in connective tissue (see Fig. 11.6b ). Necrosis is not a consistent feature. Immunohistochemistry shows a pronounced secondary reduction in caveolin-3 and sometimes also of dysferlin (see Fig. 11.6c ), and reduced sarcolemmal laminin β1 has also been observed, but this can occur in several disorders. Electron microscopy shows a pronounced reduction/absence of caveolae.

Only a few families have been described with other dominant LGMD phenotypes that were initially identified by linkage studies. Nomenclature of LGMD1D and IE is confusing in the literature and in OMIM, but a locus on chromosome 7q36 has been identified and also one on chromosome 6q23. LGMD1D/1E is often used for both. The defective gene in Finnish families linked to the 7q36 locus has been identified as DNAJB6 , which encodes for a member of the heat shock protein family (HSP40) of co-chaperones ( ). In addition, cases with a dominantly inherited LGMD have been found by exome screening techniques to also have mutations in this gene ( ). Muscle pathology in these cases with defects in the DNAJB6 gene shows features seen in other muscular dystrophies as well as rimmed vacuoles and aggregation of proteins such as TDP43, VCP, p62, SMI-31, ubiquitin, BAG3 and DNAJB6 itself, and electron microscopy shows myofibrillar and Z line disorganization ( ). There is therefore overlap with cases classified as myofibrillar myopathies (see Ch. 16 ). There is also confusion with regard to the 6q23 locus as the only family linked to this locus has been shown to have a mutation in the gene encoding desmin on chromosome 2q35 ( ). The gene defect was identified with the aid of laser capture dissection of aggregated material in muscle fibres, followed by mass spectrometry. The muscle pathology is also consistent with a myofibrillar myopathy and shows the typical features seen in cases with accumulation of desmin, in particular the typical granulomatous filamentous material (see Ch. 16 ). The menadione NBT method showed accumulation of positive material in moderate-sized focal areas and, although described in the report as reducing bodies, no electron microscopy was shown to substantiate this ( ). The report illustrates, as shown in Figure 16.4 , that staining with menadione NBT can be useful for identifying accumulation of myofibrillar material as well as reducing bodies (see Chapter 2, Chapter 4 ).

The protein responsible for LGMD1F is transportin 3 and for LGMD1G heterogeneous nuclear ribonucleoprotein D-like ( ). In cases with a mutation in transportin subsarcolemmal and perinuclear accumulation of transportin can be seen ( ). The gene responsible for LGMD1H has not yet been found, only the locus on chromosome 3p23 ( ). Muscle pathology in both LGMD1F and 1G has been reported to show typical dystrophic changes and rimmed vacuoles. In addition, in LGMD1F large nuclei with pale centres were seen with haematoxylin and eosin (H&E), and 18–20 nm nuclear and cytoplasmic filaments with electron microscopy ( ). Muscle pathology in the few cases of LGMD1H linked to chromosome 3p23-p25 that have been studied showed myopathic changes and ragged-red fibres in one case (but the influence of age was not excluded) and was normal in another ( ). There are no significant immunohistochemical findings reported.

Recessive Limb-Girdle Muscular Dystrophies

The recessive forms of LGMD are more frequent than the dominant forms, in particular LGMD2A and LGMD2I in Caucasian populations, but there are geographical differences. Several forms are rare and have only been described in a few families, and there is overlap with allelic disorders associated with severe forms of, for example, congenital muscular dystrophy (see Table 11.2 ). The gene defects affect a variety of proteins, including an enzyme (calpain-3), nuclear function lamin A/C (LAPB1), sarcomeric proteins (telethonin, titin), components of the dystrophin-associated glycoprotein complex, other sarcolemmal proteins (dysferlin), a putative calcium-activated chloride channel (anoctamin 5), cytoskeletal proteins (plectin, desmin), proteins involved in glycosylation, signalling and trafficking pathways. It remains to be seen if there is a common factor that links them.

LGMD2A is caused by defects in the gene encoding the enzyme calpain-3, which is present in the sarcoplasm of the fibre. The disorder usually has a slow progression and distal muscle involvement is common. Serum CK is often very high (10 × normal). Calpain-3 has a nuclear translocation sequence, suggesting nuclear localization, and it also binds to a C-terminal region of titin ( ). Commercial antibodies to calpain-3 can be used for immunohistochemistry to show an absence of calpain-3 ( ), but immunoblotting is more sensitive and is required to take account of secondary changes such as those associated with primary defects affecting dysferlin, caveolin-3 or titin, and those resulting from degradation ( ). A normal quantity of calpain-3 on an immunoblot does not exclude a defect in the gene ( ).

Recently, a group of patients with only one identified founder mutation in the CAPN3 gene has been identified and considered to be a dominantly inherited form of LGMD related to CAPN3 ( ). It has been named LGMD1I in the proposed new nomenclature (see Table 11.3 ). Calpain-3 on immunoblot of muscle from these patients was reduced, which is unusual for a dominant disorder.

LGMD2B and Miyoshi myopathy are clinically distinct but both are caused by defects in the gene encoding for dysferlin. The differences in phenotype are not understood and can even occur within one family with the same mutation. Both are characterized by a very high CK, 10–150 × the normal level. Distal weakness and inability to walk on tip-toe are features of Miyoshi myopathy, and proximal upper and lower limb weakness develops as the disease progresses. In LGMD2B, however, proximal weakness is present at onset. Onset usually occurs between 11 and 40 years of age ( ), but rare cases with a homozygous mutation resulting in an absence of dysferlin have been reported in two siblings presenting at birth with a normal CK ( ). Some patients lose ambulation but cardiac and respiratory symptoms are not usually features.

In normal muscle dysferlin is localized to the sarcolemma and a reduction in intensity of labelling can be seen in LGMD2B and Miyoshi myopathy. Some internal labelling of fibres may also occur, especially in regenerating fibres. The commercial antibodies, however, give a clearer indication of quantity on immunoblots. As pointed out previously, immunoblots are also important for distinguishing secondary alterations in dysferlin, as these can occur when either the gene for calpain-3 or caveolin-3 is defective ( ) and also in other muscular dystrophies ( ).

A secondary feature associated with defects in dysferlin is the occurrence of inflammatory cells and the sarcolemmal expression of major histocompatibility complex class I antigens (MHC-I; ). The presence of complement C5b-9 has also been reported but not substantiated by others ( ). The published images are confusing and the data on Duchenne and Becker samples are not consistent with other studies The distinction from a myositis is then based on clinical history. Amyloid in muscle has also been demonstrated in several cases ( ).

LGMD2C–2F are often referred to as the sarcoglycanopathies, as the mutations affect members of the sarcoglycan complex, which is associated with dystrophin (α-, β-, γ- and δ-sarcoglycan) ( Fig. 11.7 ). All the sarcoglycans are transmembrane glycoproteins with a small intracellular domain, a single transmembrane domain and a large extracellular domain that is glycosylated. The nomenclature of the sarcoglycans has changed and developed over the years, as additional genes have been identified. The Greek lettering (α, β, γ, δ), however, has now been uniformly adopted. Campbell and co-workers originally introduced a terminology that reflected the molecular mass of these proteins and their membership of the dystrophin-associated glycoprotein (DAG) complex (e.g. 50-DAG, 35-DAG; ), whereas Ozawa and the Japanese group, who had also identified the same proteins, used a numerical terminology with subdivisions, with ‘A’ as prefix (A0–A5; e.g. A3a, A3b; ). Confusion arose as the molecular mass of the various proteins identified was similar. The first member of the complex to be identified was the 50-DAG protein (now known as α-sarcoglycan), and absence of the protein was identified before the gene was cloned. It was originally named adhalin after the Arabic name for muscle ( ). The second member of the complex to be identified was named 35-DAG, reflecting its molecular mass (now known as γ-sarcoglycan). This was followed by identification of a protein with a molecular mass of 43 kDa that was associated with 50- and 35-DAG. Confusion then arose as a different protein with a molecular mass of 43 kDa, and also associated with dystrophin, had already been named 43-DAG (now known as β-dystroglycan). Similarly, another protein with molecular mass of 35 kDa was identified (δ-sarcoglycan; see Table 11.5 ). It should be remembered that early papers on ‘43-DAG’ are in fact referring to the protein now known as β-dystroglycan. It was soon appreciated that the proteins responsible for LGMDs act as a complex and were given the name ‘sarcoglycan’ with a Greek prefix (α, β, γ, δ). The dominant LGMDs were designated LGMD1 and the recessive forms LGMD2. The four sarcoglycanopathies, LGMD2C–2F, were assigned letters in the order in which the genes were identified ( Table 11.5 ).

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