Myofibrillar Myopathies and Other Myopathies with Rimmed Vacuoles





Introduction


A vacuole is a membrane-bound area of a muscle fibre that may contain various cellular components or enzymes. The type and function of a vacuole vary, and the properties of a vacuole can be a useful pathological indicator. The membrane may be revealed with antibodies to sarcolemmal proteins, or may only be visible with electron microscopy. The nature of the contents of a vacuole may also only be seen with electron microscopy, although some stains (e.g. PAS and acid phosphatase) and some antibodies (e.g. to markers of autophagy or proteins related to ubiquitinylation) can give an indication of the material within a vacuole. It is important to distinguish vacuoles from unstained areas in histologically stained sections (e.g. with haematoxylin and eosin (H&E) and Gomori trichrome), which relate to lipid droplets or ice crystal damage. In some muscle biopsies vacuoles may only be a minor feature, whereas in others they are numerous. In the past, the type of vacuole surrounded by basophilic material that stains red with the Gomori trichrome stain (commonly known as rimmed vacuoles), in particular, was thought to be restricted to disorders such as inclusion body myositis, but it is now apparent that this type of vacuole can occur in a wide variety of disorders, including myopathies, muscular dystrophies and neuropathies. They probably relate to common pathological pathways such as misfolding of proteins, the proteasome and autophagic pathways and various chaperone and co-chaperone proteins ( ). In this chapter we discuss disorders in which vacuoles, together with additional pathological features and the clinical phenotype, can be useful diagnostic clues. In particular, we cover the pathology of myofibrillar myopathies and other disorders with an accumulation of proteins, and some distal myopathies, including those described as hereditary inclusion body myopathies. The pathology and associated phenotype of other disorders where vacuoles of various types are a feature are described in other chapters: glycogenoses (see Ch. 17 ), ion channel disorders (see Ch. 20 ), inclusion body myositis (see Ch. 22 ), myosin 2A myopathy (see Ch. 15 ), oculopharyngeal muscular dystrophy (see Ch. 14 ), LGMD2G (TCAP; see Ch. 11 ) and toxic myopathies (see Ch. 23 ).


Myofibrillar Myopathies


Over the years a number of disorders with various types of inclusions have been described, such as spheroid bodies ( ), sarcoplasmic bodies ( ), cytoplasmic bodies ( ) and granulofilamentous material ( ). More recently, it has been appreciated that these disorders share similar histopathological features, in particular accumulation of several proteins, especially desmin. This led to the use of terms such as ‘desminopathies’, ‘desmin-related myopathies’ and ‘protein aggregate myopathies’ ( ). Several other proteins in addition to desmin also accumulate, several of which are also seen in inclusion body myositis (see Ch. 22 ) and neurodegenerative disorders. The terms ‘hereditary inclusion body myopathy’ or ‘protein surplus myopathies’ have also been suggested ( ), as well as the more general term of ‘myofibrillar myopathies’ ( ). The molecular basis of several of these conditions has now been determined. Although it is now known that defects in several proteins associated with the sarcomere can cause a neuromuscular disorder (see Fig. 8.2 ), the term ‘myofibrillar myopathy’ is usually restricted to conditions in which proteins of the Z line, or associated with it, are implicated. These share some similar pathological features, which include deposition of amorphous or granular aggregates consisting of ectopic proteins, such as desmin, αB-crystallin, myotilin, dystrophin and others, presence of rimmed or non-rimmed vacuoles and focal loss of oxidative enzyme activity ( ). The genes and proteins considered in this section are listed in Table 16.1 .



TABLE. 16.1

The Defective Genes and Proteins Known to Cause a Myofibrillar Myopathy




































































































Gene Symbol Protein Inheritance Cardiac Involvement Clinical Features
DES Desmin AD Yes Adult onset; distal > proximal muscle weakness, axial weakness; respiratory failure
DES Desmin AR Yes Infantile or childhood onset; proximal and distal muscle weakness
CRYAB αB-crystallin AD Yes Distal > proximal muscle weakness; cataracts
CRYAB αB-crystallin AR No Infantile onset; axial and abdominal muscle stiffness and weakness; respiratory failure
MYOT Myotilin AD Yes Adult or late onset; distal and proximal muscle weakness
MYOT Myotilin AR Yes Adult onset; proximal weakness of lower extremities
FLNC Filamin C AD Yes Adult onset; distal < proximal muscle weakness
LDB3 LDB3 ( ZASP ) AD Yes Adult or late onset, occasional childhood onset; distal > proximal muscle weakness; sometimes neuropathy
BAG3 BAG3 AD Yes Early onset; severe progression; neuropathy may occur
FHL1 FHL1 XD Yes Broad phenotype; childhood-onset cases with severe progression
TTN Titin (FN3 119 domain) AD No Adult onset; proximal and distal muscle weakness; early respiratory failure (HMERF)
PLEC Plectin AR Yes Infantile/childhood/adult onset of muscle weakness with proximal and distal muscle weakness; skin blistering
ACTA1 Skeletal muscle
α-actin
AD No Onset in infancy; upper > lower limb; contractures
HSPB8 HSPB8 AD No Adult onset; distal > proximal muscle weakness; peripheral motor neuropathy
DNAJB6 DNAJB6 AD No Childhood/adult onset; distal and proximal muscle weakness

DDB3, LIM domain-binding protein 3; ZASP, Z line alternatively spliced PDZ motif-containing protein; BAG3, Bcl-2-associated athanogene 3; FHL1, four and00 a half LIM domains 1 protein; HSPB8, heat-shock 22-kd protein 8; DNAJB6, DNAJ/HSP40 homolog subfamily B member 6; AD, autosomal dominant; AR, autosomal recessive; XD, X-linked dominant; HMERF, hereditary myopathy with early respiratory failure.


Defects in the proteins that are included in this group of disorders were historically desmin, αB-crystallin, myotilin, Z-line alternatively spliced PDZ motif-containing protein (ZASP), filamin C and bcl-2-associated athanogene 3 (BAG3). More recently, the definition has been broadened to include more protein defects ( ). Four-and-a-half-LIM domain 1 protein (FHL1) is often included, although this is not a protein of the Z line but localizes to the myofibrils and sarcolemma ( ) and also titin, skeletal muscle α-actin, heat-shock 22-kd protein 8 (HSPB8) and DNAJ/HSP40 homolog subfamily B member 6 (DNAJB6) and plectin. Plectin is closely associated with desmin and the Z line, and desmin accumulation occurs when there is a mutation in the gene in common with myofibrillar myopathies. Although vacuoles are not a frequent pathological feature of plectinopathy, it may be grouped among the myofibrillar myopathies because of several other important pathological features ( ). Telethonin is also a protein of the Z line and biopsies can have vacuoles but this is discussed with limb-girdle muscular dystrophies (LGMD2G, ; see Ch. 11 ).


Many cases are sporadic, but when inheritance can be determined, the majority of cases show autosomal dominant inheritance, although rare recessive mutations in the genes encoding desmin, αB-crystallin and myotilin have been identified ( ), and disorders associated with FHL1 are X-linked with mainly dominant inheritance. Myopathies caused by mutations in PLEC are inherited in an autosomal recessive manner.


Clinical Features


With identification of the various molecular defects, the spectrum of clinical features associated with myofibrillar myopathies is expanding. Age of onset is variable and can be in childhood, adolescence or adulthood. Although there is a spectrum of presentation, and many identified cases are of adult onset, often late (beyond the fourth decade), particularly in cases with ZASP, myotilin or filamin C defects, cases with defects associated with the desmin gene tend to present earlier than those with ZASP, myotilin or filamin C defects. Other gene defects, such as BAG3 , CRYAB and ACTA1, frequently present in childhood.


Muscle weakness is often slowly progressive but may be rapid in early-onset cases such as those with FHL1 or BAG3 defects. Childhood cases with these mutations can be particularly severe, with rapid progression. Weakness may be proximal or, more frequently, distal, or both, or scapuloperoneal. Weakness may be asymmetrical in some cases with FHL1 defects. Muscle weakness may be accompanied by muscle wasting, stiffness or aching, cramps and sensory symptoms. Facial weakness is uncommon, but dysarthria and swallowing difficulties may occur in some older patients. Wasting of hand muscles occurs in cases with mutations in the actin-binding domain of filamin C but the cases reported do not show the typical pathology of myofibrillar myopathies: in particular, no vacuoles ( ). Evidence of peripheral neuropathy is present in a significant proportion of patients, particularly in cases with BAG3 and HSPB8 mutations, and some show myotonic discharges. Cardiac involvement is common, particularly in those with a mutation in the gene encoding desmin, when it may precede or coincide with skeletal muscle weakness. Cardiac involvement may be in the form of arrhythmia, or dilated or hypertrophic cardiomyopathy. Cardiomyopathy is a feature of BAG3 cases with childhood onset and occurs in cases with mutations affecting filamin C and FHL1 but is more rare in those with defects in myotilin. Respiratory failure is present in several cases of myofibrillar myopathies, especially those that present early, and is a common feature of hereditary myopathy with early respiratory failure (HMERF) associated with mutations in the gene encoding titin. Cataracts are a feature associated with mutations in the αB-crystallin gene and can be a useful clinical distinction from other forms of myofibrillar myopathy, although they may not be detected in cases presenting early ( ). Other features that can occur are rigid spine, scoliosis and contractures, and scapular winging is common ( ). When defects in the gene encoding myotilin were first identified the disorder was described as limb-girdle muscular dystrophy 1A but it is now appreciated that this is the same disorder as that now known as a myofibrillar myopathy ( ).


Serum creatine kinase (CK) activity is either normal or mildly elevated, but some cases, such as BAG3- or myotilin-related cases, may have a pronounced increase. Muscle magnetic resonance imaging (MRI) patterns, in conjunction with clinicopathological studies, can be helpful in distinguishing the subtype of myofibrillar myopathy ( ), but there is also overlap between them, and the stage of disease has to be taken into account.


Histopathology


The spectrum of pathology in myofibrillar myopathies is broad, ranging from minimal to pronounced. Fibre-size variability with atrophy and hypertrophy, however, is present in many biopsies ( Fig. 16.1 ). Some fibres may look basophilic and granular (see Fig. 16.1a ). Other myopathic features include fibre splitting, excess internal nuclei (which may be multiple within a fibre) and proliferation of endomysial connective tissue and adipose tissue. Multiple internal nuclei can be a prominent feature in some cases with a mutation in the gene encoding desmin. A few inflammatory cells may be present but inflammation is not a pronounced feature, and necrosis and regeneration may occur but are not usually extensive.




Fig. 16.1


Quadriceps biopsies from (a) a man aged 38 years with a mutation in the gene encoding desmin and (b) a 69-year-old man with a mutation in the gene encoding myotilin. Note in both the fibre-size variability, the increase in internal nuclei and eosinophilic areas, which are particularly pronounced in (b) (large arrows); (a) also shows small slightly basophilic fibres (small arrow) and a granular fibre (∗) and in (b) there are atrophic fibres with rimmed vacuoles (green arrow) (H&E). Fibre diameter range: (a) 35–95 μm; (b) 10–105 μm.


The Gomori trichrome stain is particularly useful for assessing myofibrillar myopathies and reveals areas within fibres that are more darkly stained than the surrounding myofibrils ( Fig. 16.2 ). These may have a more bluish or darker green colour with the trichrome stain, and with H&E they are often eosinophilic (see Figs. 16.1 and 16.2 ). Some inclusions may be stained red with the Gomori trichrome and are cytoplasmic bodies or spheroid bodies of irregular shape ( Fig. 16.3 ), or reducing bodies. Reducing bodies can be distinguished from cytoplasmic bodies using the menadione nitroblue tetrazolium (NBT) method without substrate, which stains reducing bodies and accompanying accumulated protein darkly (see Fig. 15.30 ). Reducing bodies are only seen in cases with an FHL1 mutation in the LIM domain 2 region, most of which are the severe childhood-onset cases ( ), but some myofibrillar material may also appear darkly stained with menadione NBT ( Fig. 16.4 ). The female carriers with FHL1 mutations may also show reducing bodies, but are often clinically milder, possibly with X-inactivation having a role. Reducing bodies are not a feature of milder cases with mutations in other domains, but performing the menadione NBT stain is worthwhile (see Fig. 16.4 ). The dark areas lack mitochondria and therefore do not stain for oxidative enzymes. Some are also congophilic but, in contrast to amyloid deposits in inclusion body myositis, they are not metachromatic with the crystal violet stain ( ). The Congo red stain is best viewed under fluorescence, using an excitation filter in the red range, as for rhodamine or Texas red ( Fig. 16.5 ).




Fig. 16.2


Darkly stained areas in the quadriceps biopsy from the 69-year-old man shown in Fig. 16.1b (Gomori trichrome). Fibre diameter range 10–105 μm.



Fig. 16.3


A red-stained cytoplasmic body and irregular-shaped spheroid-like body with a pale center (arrows) in a quadriceps biopsy from a 28-year-old man with a mutation in the gene encoding myotilin (Gomori trichrome). Fibre diameter range 25–50 μm .



Fig. 16.4


(a) Quadriceps biopsy from the 69-year-old male with a mutation in the gene encoding myotilin shown in Fig. 16.1b stained with menadione NBT without substrate showing dark stained areas of myofibrillar material (arrows). (b) Quadriceps biopsy from a 3-year-old boy with a mutation in the FHL1 gene stained with menadione NBT without substrate showing reducing bodies (red arrow) and a few very intensely labelled fibres (arrow).



Fig. 16.5


Congo red staining viewed with fluorescence using a 545–580 nm excitation filter in the biopsy from the case shown in Fig. 16.1b . Note the bright fluorescent areas (arrows).


Vacuoles, rimmed or un-rimmed, are a particular feature of myofibrillar myopathies (see Figs. 16.1b and 16.2 ). Rimmed vacuoles have a rim of granular basophilia with H&E that is red with Gomori trichrome ( Fig. 16.6 ), reflecting the membranous whorls of the vacuoles, and they may show increased acid phosphatase activity.




Fig. 16.6


Quadriceps biopsy from the 69-year-old man with a mutation in the gene encoding myotilin shown in Fig. 16.1b showing prominent vacuoles with basophilic granules at the periphery which are myelin-like whorls (arrows) (H&E).


Fibre type grouping and groups of atrophic fibres of both types may be present, consistent with a peripheral neuropathy ( Fig. 16.7 ), and nerves may show loss of myelin and increased fibrosis. Neurogenic features can be particularly apparent in BAG3 -related cases. A predominance of type 1 fibres may occur in some cases.




Fig. 16.7


Quadriceps biopsy from an 11-year-old boy with a mutation in the BAG3 gene showing a small cluster of atrophic fibres (small arrow) and occasional angulated fibres (large arrow) consistent with denervation (H&E).


Loss of oxidative enzyme activity in large areas of fibres gives a ‘rubbed-out’ appearance (also referred to as ‘wiped-out’; see Figs. 16.8, 16.10, 16.12 and 16.13 ). These have been reported to be more frequent in cases with a mutation in the genes encoding desmin and αB-crystallin, whereas myotilin- and ZASP-related cases have a higher frequency of vacuoles (see Fig. 16.11 ) ( ). Some fibres may show uneven oxidative enzyme staining, core-like areas ( Fig. 16.8 ) and mitochondrial dysfunction may be apparent, including the presence of cytochrome c oxidase (COX)-negative fibres, although in older cases this may be age-related ( ).




FIG. 16.8


Quadriceps biopsy from a male with a mutation in the gene encoding ZASP stained for cytochrome c oxidase showing ‘wiped-out’ areas (large arrow), large core-like areas (∗) and fibres with focal loss of enzyme activity (small arrows).



Fig. 16.9


Immunolabelling of (a) ubiquitin, (b) αB-crystallin, (c) desmin, (d) myotilin and (e) phosphorylated tau in the biopsy of a case with a mutation in the gene encoding myotilin, showing accumulation of all these proteins; (f) labelling of dystrophin inside a vacuolated fibre in a case with a mutation in the gene encoding ZASP.



Fig. 16.10


Quadriceps biopsy from a woman with a mutation in the gene encoding desmin. (a) There are several split fibres with internal nuclei and eosinophilic protein deposits (arrows) and fibre with rimmed vacuoles (arrowhead). (b) Protein deposits are either dark green or purple in Gomori trichrome staining (arrows) and a fibre with rimmed vacuoles (arrowhead). (c) Large ‘wiped out’ regions in NADH-TR staining. (d–g) Abnormal and partly ectopic protein deposition in muscle fibres (d, desmin; e, dystrophin; f, γ-sarcoglycan; g, N-CAM). (h) Electron microscopy illustrating characteristic intermyofibrillar deposition of granulofilamentous material.



Fig. 16.11


Quadriceps biopsy from a 70-year-old woman with ZASPopathy. (a) Protein aggregates are eosinophilic and stain red in H&E (arrow), and there are numerous fibres with rimmed vacuoles (arrowheads). (b) Protein aggregates appear as dense purple inclusions (arrows) in Gomori trichrome and rimmed vacuoles (arrowheads) are frequent. (c) Many vacuoles contain myelin-like degradation material. (d) With electron microscopy some protein aggregates appear with a dense core of fibrillar material (arrow) surrounded by more loosely arranged filaments. (e) Small rod-like inclusions (arrows) are present as well as (f) clusters of abnormal mitochondria (arrows) with paracrystalline inclusions.



Fig. 16.12


Quadriceps biopsy from a 5-year-old girl with BAGopathy including rigid spine, progressive muscle weakness and cardiomyopathy (a) H&E staining demonstrates eosinophilic protein aggregates (arrow). (b) The protein deposits (arrows) are dark green in Gomori trichrome and result in (c) large regions with loss of oxidative enzyme activity (arrow) revealed by staining of NADH-TR. (d and e) Electron microscopy demonstrating accumulation of Z line and granulofilamentous material between the myofibrils.



Fig. 16.13


Quadriceps biopsy from a 33-year-old man with hereditary myopathy with early respiratory failure (HMERF) showing (a) focal areas with marked structural changes with split fibres, internal nuclei and eosinophilic inclusions (H&E), (b) red-dark purple-stained inclusions (Gomori trichrome), (c) ‘wiped-out’ regions of myofibrillar lesions (NADH-TR) and (d) characteristic distribution of cytoplasmic bodies in a row beneath the sarcolemma forming an incomplete ring (arrows) and a fibre with rimmed vacuoles (arrowhead; Gomori trichrome).


Immunohistochemistry


The abnormal fibres contain an accumulation of several proteins, and immunohistochemistry is useful for assessing them. The proteins detected in the abnormal fibres include desmin, αB-crystallin, syncoilin, ubiquitin, myotilin, filamin C, caveolin-3, dystrophin, β-amyloid precursor protein, Xin, filamentous actin, gelsolin, heat-shock proteins, neural cell adhesion molecule (N-CAM), phosphorylated tau, TDP43, p62 and prion protein ( Figs. 16.9 and 16.10 ) ( ). From a diagnostic point of view it is rarely necessary to demonstrate a wide number of proteins. The antibodies we find most useful are to dystrophin, desmin, myotilin, αB-crystallin and p62, but it is important to distinguish both artefactual accumulation of label and accumulation of proteins that occurs in cores from the abnormal accumulation in myofibrillar myopathies. The desmin aggregation can be variable and may be punctuate, be only present at the periphery of the fibre, or be seen as large areas in various locations ( ). Some of the same proteins identified are also seen in inclusion body myositis, hereditary inclusion body myopathies caused by defects in the GNE gene (UDP- N -acetylglucosamine 2-epimerase/ N -acetylmannosamine kinase) and the valosin-containing protein (VCP) and other myopathies with distal involvement (see below). This may cause diagnostic confusion, but in inclusion body myopathy caused by GNE mutations, dystrophin and desmin accumulation are not usually associated with the vacuolated fibres. Up-regulation of MHC-I on the sarcolemma is not usually a pronounced feature of myofibrillar myopathies, in contrast to inclusion body myositis, but traces may occur. Cases with mutations in FHL1 can show accumulation of FHL1 protein, particularly if reducing bodies are not present ( ), and a reduction of FHL1 has been demonstrated on immunoblots ( ).


Electron Microscopy


Electron microscopy is a useful tool for the study of myofibrillar myopathies and reveals various degrees of myofibrillar disruption. This includes Z line streaming, accumulation of Z line material (see Fig. 16.14b ), accumulation of characteristic granulofilamentous material (see Figs. 16.10h, 16.12d, e) ), except in FHL1-related cases (see Fig. 5.42 ), 15–18 nm tubulofilamentous inclusions (see Fig. 22.21 ) and various other types of inclusions ( Fig. 16.11 ) ( ). The inclusions include cytoplasmic bodies with a halo of filaments radiating from them, or dark dense spheroid bodies, myelin-like whorls and autophagic debris (see Fig. 16.11c ), and reducing bodies, which are the specific feature of severe FHL1 -related cases. Nemaline rod-like structures may also be seen (see Figs. 16.11e and 16.14a ), and nuclear rods have been observed in some cases with defects in genes encoding ZASP and myotilin ( ). In one patient with a αB-crystallin mutation a zebra body was observed ( ). Clusters of mitochondria, frequently with various inclusions, are common (see Fig. 16.11f ), and apoptotic nuclei may be seen, particularly in BAG3 -related cases.




Fig. 16.14


Electron micrographs from a patient with HMERF showing (a) globular deposits of dense material surrounded by fibrillar material resembling cytoplasmic bodies; note also thickened Z lines and rod-like structures and (b) extensive Z line streaming.



Fig. 16.15


Quadriceps biopsy from a 7-year-old girl with mutations in the kyphoscoliosis peptidase gene, KY . There is slightly increased variability in muscle fibre size, increase of interstitial connective tissue, occasional internalized nuclei and scattered very atrophic, dark fibres with staining for NADH-TR. (a) H&E. (b) NADH-TR.


Molecular Defects


The defective genes that have been identified are shown in Table 16.1 . The number of cases with an identified mutation is increasing and several large families have been described.


Desmin ( DES ) mutations were the first identified ( ); most are dominantly inherited, although a few recessive cases have also been reported ( ). The cases reported by were heterozygous for two null mutations that interfered with desmin aggregation, and no desmin was detectable on sections or immunoblots. Desmin is a highly conserved intermediate filament of skeletal, cardiac and smooth muscle. It is localized to the periphery of the Z line and the subsarcolemmal cytoskeleton and the filaments are 10 nm in diameter, originally designated as intermediate between actin and myosin. Desmin is linked to the Z line and associates with plectin to link the myofibrils to each other, to the sarcolemma, to mitochondria and to nuclei. The various mutations identified, mostly missense variants, impair assembly of the desmin filaments ( ). Cardiomyopathy is frequent in desminopathies and important for the prognosis in individual cases, and respiratory involvement is also common in desminopathy. Muscle pathology is typical for myofibrillar myopathies with dystrophic features, myofibrillar lesions with protein aggregates including ectopic proteins and rubbed-out fibres on oxidative enzyme staining, fibres with rimmed vacuoles and intermyofibrillar accumulation of granulofilamentous material (see Fig. 16.10 ).


α B-crystallin is a cytoplasmic small heat-shock protein that has a chaperone role in protecting the intermediate filament network from stress-induced damage ( ). Mutations in the αB-crystallin gene, CRYAB , which were first described by , interfere with this chaperone function. Two α-crystallin forms (A and B), encoded by different genes, have been identified and both are abundant in the lens, where they prevent the formation of cataracts. The presence of cataracts is a distinguishing feature of cases with a mutation in the αB-crystallin gene that is not found in the other myofibrillar myopathies, but they are not a consistent feature in all cases. αB-crystallinopathy is a rare cause of myofibrillar myopathy with usually dominant inheritance and adult onset, but a recessive variant was described in Canadian native infants and another child with muscle stiffness as a predominant symptom ( ).


Myotilin is a Z line protein that interacts with several other Z line proteins and is essential for the myofibrillar assembly. Mutations in the myotilin gene, MYOT , are rare causes of myofibrillar myopathy (see Figs. 16.1–16.6 and 16.9 a–e ) but also have been identified in limb-girdle muscular dystrophy 1A (LGMD1A; see Ch. 11 ). These are now considered to be part of a spectrum of the same disorder. The majority of mutations occur in the serine-rich N-terminal domain of exon 2, but a rare case with a mutation in exon 9 has been reported ( ). Cardiomyopathy is an inconsistent finding that was reported in some cases, but was not present in a case with recessive myotilin mutations who showed sinus arrhythmia as the only sign of a cardiac manifestation ( ).


Filamin C is part of a family of proteins, and its expression is restricted to skeletal and cardiac muscle. It cross-links and stabilizes actin and binds to several Z line proteins and to γ- and δ-sarcoglycan at the sarcolemma. Patients with mutations in the actin-binding domain have been reported to have a distal phenotype but do not show the typical muscle pathology of myofibrillar myopathies ( ). Many of the patients with a mutation in the rod domain, however, do show the typical pathological features and have a proximal myopathy ( ). Respiratory involvement is frequent, and cardiomyopathy may occur in approximately half of the patients ( ).


Z line alternatively spliced PDZ motif-containing protein (ZASP) or LIM domain-binding protein 3 (LDB3) is also predominantly expressed in skeletal and cardiac muscle and exists in different isoforms. It binds to α-actinin-2 in the Z line. There are two founder mutations, and mutations in LDB3 have been shown to be responsible for a type of myofibrillar myopathy ( ) and the late-onset distal myopathy described by ( ). Although ZASPopathy is generally a late-onset disorder with distal muscle weakness starting in the anterior compartment of the lower extremities, a childhood-onset case of distal myopathy has been described ( ). Cardiomyopathy is infrequent and usually mild, and neuropathy may be present. Muscle pathology shows the hallmarks of myofibrillar myopathies with frequent rimmed vacuoles (see Figs. 16.9 f and 16.11 ). Late onset distal muscle weakness and rimmed vacuoles are clinicopathological features that may be similar to sporadic inclusion body myositis ( ).


Bcl-2-associated athanogene-3 (BAG3) is a co-chaperone protein and is also part of a family of proteins. It is highly expressed in skeletal and cardiac muscle but is also present at low levels in other tissues. It participates in the degradation of misfolded or aggregated proteins by complexing with heat-shock proteins. It has a role not only in muscle but also in neural tissues and tumour cells and various cellular activities ( ). It also has a role in apoptosis, which is consistent with the finding of apoptotic nuclei in cases with a mutation. A BAG3 mutation as a rare cause of myofibrillar myopathy was first described by ; subsequently, additional cases have been reported, nearly all with either a P209Q or P209S mutation ( ), but recently another position was identified in BAG3 myofibrillar myopathy, P470S ( ). Other variants have been associated with pure cardiomyopathy. The clinical features in BAG3-associated myofibrillar myopathy are characterized by childhood onset of progressive muscle weakness and respiratory failure, often combined with rigid spine and contractures and in most cases also cardiomyopathy. Peripheral neuropathy may be present. Muscle pathology shows typical alterations of myofibrillar myopathies with intermyofibrillar deposition of granulofilamentous material ( Fig. 16.12 ) and small angular fibres suggestive of denervation (see Fig. 16.7 ).


Four and half LIM domain 1 protein (FHL1) is another protein that is part of a family of proteins. Its function is not fully known but it is believed to participate in cell growth, differentiation, and sarcomeric assembly and to be a regulator of muscle mass ( ). It has three isoforms, and mutations affecting all isoforms result in a more severe phenotype. The severity of the phenotype and presence of reducing bodies in a biopsy correlate with the domain that the mutation affects, with mutations in the LIM domain 2 region being associated with the severe phenotype ( ). Reducing body myopathy was originally classified as a congenital myopathy because of the presence of a structural defect. It is now apparent, however, that this disorder is significantly different from other congenital myopathies (see Ch. 15 ) as onset is often in early childhood, not at birth, and the disease is much more progressive than most congenital myopathies. The pattern of contractures, muscle weakness and cardiac involvement in some patients with mutations in the FHL1 gene has been assigned to a form of Emery–Dreifuss muscular dystrophy ( ), but the clinical and pathological features in these cases are similar to cases of myofibrillar myopathy with scapuloperoneal weakness. These patients could therefore be considered as part of the spectrum associated with mutations in FHLI ( ), rather than as a form of Emery–Dreifuss muscular dystrophy, which have defects in nuclear membrane proteins (see Ch. 13 ). More recently, a patient presenting with muscle hypertrophy and a deletion of the complete FHL1 gene added to the wide spectrum of FHL1-associated myopathies ( ). This case showed non-specific muscle pathology and not changes typical for myofibrillar myopathy.


Autosomal dominant myopathies with extensive myofibrillar lesions and eosinophilic deposits associated with adult onset of disease and early respiratory failure, hereditary myopathy with early respiratory failure (HMERF) , have been described in sporadic and familial cases linked to various chromosomal regions ( ). In two studies from Sweden and UK, patients with HMERF have been found to carry the same pathogenic variant in the region of TTN that encodes the titin 119th fibronectin-3 domain (FN3 119) , which is located in the A band of the sarcomeres ( ). Additional pathogenic variants were described in the same titin domain, indicating mutational vulnerability of TTN exon 343 and that HMERF is caused by pathogenic variants in this particular titin domain ( ).


The clinical features described by in seven Swedish families with HMERF included proximal muscle weakness in the upper and lower extremities and an early involvement of respiratory and neck flexor muscles, and also in some cases of ankle dorsiflexors. and described three families from the UK with HMERF. Age of onset and the degree of muscle weakness were highly variable. All had onset in adulthood but presented with either weakness of ankle dorsiflexors, proximal muscle weakness or respiratory symptoms. Weakness was generally more severe in the lower extremities, particularly ankle dorsiflexion and hip flexion. The disease was progressive over years, and ambulation was affected in many patients needing either a walking aid or wheelchair. Characteristic MRI findings in 21 patients included involvement of semitendinous and peroneus longus muscles in the majority of patients, also in presymptomatic cases. described three Swedish families with HMERF associated with the same variant TTN in the 119th fibronectin-3 domain. The presentation was from early adulthood to 40 years of age, with either weakness in shoulder or pelvic girdle muscles, weakness of ankle dorsiflexors, myalgia or respiratory symptoms. At examination the characteristic muscle weakness distribution included weakness of ankle dorsiflexion, hip flexion, neck flexors and trunk muscles. All had calf hypertrophy. Seven of eight investigated patients had respiratory symptoms and four of these needed non-invasive night ventilation. MRI of the legs in two cases showed a more severe involvement of the proximal compared to the distal parts of the thighs. In the lower legs the anterior and lateral compartments were severely involved. Cardiac involvement with cardiac conduction abnormalities and cardiomyopathy may occur in some cases of HMERF ( ).


The characteristic histopathological features in HMERF include variability in muscle fibre size with atrophic and hypertrophic fibres, many fibres with numerous internal nuclei and focal areas with frequent split fibres ( Fig. 16.13a ). Eosinophilic inclusions or deposits are typical. The deposits are red or dark green in trichrome-stained sections and some of them have the appearance of cytoplasmic bodies ( Fig. 16.13b ). Staining for NADH-TR shows frequent unstained, ‘rubbed-out’ regions ( Fig. 16.13c ). A common feature (but not universal) is arrangement of the cytoplasmic bodies in rows beneath the sarcolemma ( Fig. 16.13d ) ( ), and muscle fibres with rimmed vacuoles are usually present (see Fig. 16.13d ).


The characteristic pathological changes are focal, frequently with groups of several fibres showing marked alterations, whereas other regions are less affected, or even normal. There may be considerable variation in the degree of pathology between different muscle biopsy samples, and some biopsy specimens may reveal marked fatty infiltration. Immunohistochemical analysis shows numerous N-CAM-positive fibres, and in fibres with structural changes there is often accumulation of various proteins such as desmin and dystrophin. In the cytoplasmic bodies there is accumulation of actin, myotilin and αB-crystallin ( ). The cytoplasmic body-like inclusions label positively for filamentous actin and bind phalloidin.


Electron microscopy shows frequent Z line alterations with streaming and regions with extensive flag-like semi-dense extensions of the Z lines and rod-like structures ( Fig. 16.14 ). Flag-like Z line alterations have also been reported in other myofibrillar myopathies ( ). There are also large regions with myofibrillar disruption and irregular electron-dense deposits. Some cytoplasmic bodies surrounded by a halo of radially arranged thin filaments may be seen. Accumulation of granulofilamentous material or dappled dense bodies, as seen in some myofibrillar myopathies discussed previously, are not typical features ( ). Structural alterations corresponding to rimmed vacuoles show degradation products and lamellar myeloid structures. Collections of 15–20 nm tubulofilamentous inclusions, as seen in inclusion body myositis, may be observed (see Fig. 22.21 ). Most mitochondria are structurally normal, but a few may show paracrystalline inclusions.


It should be noted that the characteristic changes with eosinophilic cytoplasmic inclusions may be lacking in a muscle biopsy, and the cause of disease may then be indicated by other features such as clinical or MRI findings.


Plectin is a large protein expressed in many tissues but mainly in stratified squamous epithelia, muscle and brain. It has a central role in the functional organization of the cytoskeleton by being fundamental for the organization of intermediate filaments and their association with other cellular components ( ). Mutations in the PLEC gene are associated with a wide range of diseases, including muscular dystrophy type 2Q and epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) ( ). In a study on the histopathological changes in three cases of EBS-MD, found high variability of muscle fibre size, a large proportion of muscle fibres with internal nuclei, ‘rubbed-out’ aspects with oxidative enzyme staining, disrupted desmin network and desmin aggregates. In one case, multiple fibres showed rimmed vacuoles. They concluded that this type of plectinopathy was caused by plectin deficiency, with secondary disruption of the cytoskeleton associated with mitochondrial network abnormalities and dystrophic features. The morphological changes justified the disease to be grouped among the myofibrillar myopathies.


Mutations in ACTA1 encoding skeletal muscle α -actin are associated with a variety of muscle diseases, mainly congenital myopathies (see Ch. 15 ). One patient described by showed pathological features consistent with a diagnosis of myofibrillar myopathy. The patient had neonatal onset of muscle hypotonia and weakness, predominantly affecting the upper extremities. He had contractures in his fingers, high arched palate and facial muscle weakness and was dependent on a ventilator. He died at 3 years of age. The biopsy at 26 months old demonstrated dystrophic features with some vacuoles and amorphous regions in the fibres showing ectopic expression of proteins such as desmin, myotilin, dystrophin and αB-crystallin. No electron microscopy was performed to look for the presence of rods, nor staining with phalloidin to identify F-actin. Aggregation of mutant actin, however, was demonstrated in vitro .


Heat-shock 22-kd protein 8 (HSPB8) is a small heat-shock protein associated with Charcot–Marie–Tooth type 2L and also distal hereditary motor neuropathy type IIa. described two families with a distal myopathy presenting in adulthood and with myofibrillar pathological changes on muscle biopsy associated with dominant mutations in HSPB8 . The muscle pathological changes were a mixture of neurogenic alterations with fibre type grouping and group atrophy and myopathic alterations including rimmed and non-rimmed vacuoles, cytoplasmic bodies and protein aggregates. They also had a peripheral axonal neuropathy. No heart disease was reported.


DNAJ/HSP40 homolog subfamily B member 6 (DNAJB6) is associated with dominantly inherited limb muscular dystrophy (LGMD1D) ( ). Morphological examination of muscle has revealed that there are several features indicating that this myopathy can be included among the myofibrillar myopathies. Thus, there is myofibrillar disintegration with protein aggregates containing various ectopic proteins similar to other myofibrillar myopathies in addition to rimmed vacuoles ( ). DNAJB6 is a co-chaperone functioning with HSP70 proteins and is probably involved in chaperone-assisted selective autophagy (CASA). The clinical characteristics include adult-onset limb muscle weakness that may be predominantly distal in a few cases ( ). No associated cardiomyopathy was reported ( ).


Pathological Differential Diagnosis


Differential diagnosis in the disorders discussed previously is complicated by overlapping clinical and pathological features, and it can be difficult if the typical pathological features are not present in a biopsy. In the myofibrillar myopathies ‘rubbed-out’/‘wiped-out’ fibres are reported to be a consistent feature of cases with mutations in the genes encoding desmin and αB-crystallin, and cases related to ZASP and myotilin are reported to show more vacuoles ( ), but the number of cases studied is not extensive. The large accumulations of proteins and the dark-staining material seen with Gomori trichrome are features of myofibrillar myopathies and are not seen in inclusion body myositis, and MHC-I up-regulation is less in myofibrillar myopathies compared with inclusion body myositis. Inclusion body myositis and myofibrillar myopathies show congophilic amyloid deposits; however, these are not a consistent feature of other myopathies with vacuoles but have been observed in cases with mutations in VCP ( ) (see below). However, not all cases in the literature have been studied with the same panel of techniques. Some electron microscopy studies have revealed subtle differences in relation to the genotypes of myofibrillar myopathies ( ). The granulofilamentous material is a feature of myofibrillar myopathies, but the tubulofilamentous inclusions can occur in several myopathies with vacuoles. As with all neuromuscular disorders, clinical correlations are essential and muscle MRI may have an increasing role ( ). The most useful panel of techniques for studying these disorders is H&E, Gomori trichrome, oxidative enzymes, Congo red, desmin, myotilin, αB-crystallin, p62, VCP, supplemented by menadione NBT, if reducing bodies are thought to be present, dystrophin and electron microscopy. In HMERF, identification of actin with phalloidin in the cytoplasmic-like bodies can be useful.


Other Myopathies with Autophagic Vacuoles and/or Protein Aggregates


Kyphoscoliosis peptidase (KY) encodes a transglutaminase-like peptidase that interacts with muscle cytoskeletal proteins and is part of a Z line protein complex. A neuromuscular disease in mice caused by a spontaneous, homozygous mutation in the kyphoscoliosis peptidase gene ( Ky ) was described as a model for kyphoscoliosis and was the source of the gene name ( ). Although the protein is mainly expressed in muscle, a mixed myopathic and neurogenic pathogenesis was discussed. and described an early-onset neuromuscular disease in three individuals associated with truncating variants in KY. Although mainly myopathic features were identified, electrophysiological studies and some aspects of the muscle pathology, including fibre type grouping and targetoid fibres, indicated a neurogenic component ( ). In one child the muscle MRI showed marked fatty change of the calf muscles but a quadriceps muscle biopsy showed mainly non-specific changes including fibre-size variation, atrophic fibers and mild increase in interstitial connective tissue ( Fig. 16.15 ). The clinical manifestations included congenital equinovarus foot deformity in two cases and early-onset Achilles tendon contractures in all three. Muscle weakness was accompanied by joint contractures and kyphosis that developed in young adulthood in two individuals. Because of some structural abnormalities in muscle biopsies, including Z line disorganization and other sarcomeric lesions, the disease has sometimes, e.g. in the Online Mendelian Inheritance in Man (OMIM) database, been grouped together with myofibrillar myopathies in spite of lack of protein aggregates and vacuoles. A combined neurogenic and myopathic pathogenesis was later further supported by the identification of a Bedouin kindred with hereditary spastic paraplegia associated with a homozygous KY variant ( ).


Mutations in the gene coding titin ( TTN ) have been found in a variety of disorders described in the literature. These include autosomal recessive forms of limb-girdle muscular dystrophy (LGMD2J; see Ch. 11 ), dominant tibial muscular dystrophy and HMERF (see above),and others. Tibial muscular dystrophy is particularly prevalent in Finland, because of a founder mutation, but it also occurs in other populations ( ). CK is usually normal or only slightly elevated, and muscle MRI of the lower limbs is useful for differential diagnosis ( ).


Muscle pathology in titin distal myopathy shows features of a muscular dystrophy with variation in fibre size, fibrosis and adipose tissue, and internal nuclei, but necrotic fibres are rare ( ). Rimmed vacuoles are seen in the tibial muscles, which may show accumulation of ubiquitin, but the other proteins and Congo red seen in myofibrillar myopathies are not present. Immunohistochemistry using exon-specific antibodies to the C-terminal M line M8/M9 region shows an absence of titin but it is detected with antibodies to other regions ( ). Titin has a binding site for calpain-3 and immunoblots show a secondary reduction of calpain-3.


Rimmed vacuoles are a feature of several other disorders, some of which have a myopathy that predominantly affects the distal muscles ( ) and others with proximal weakness. In this section we discuss myopathies in which vacuoles, in particular the rimmed type, are a feature of the pathology, although as pointed out previously, rimmed vacuoles can be observed in many disorders. The pathology of other disorders characterized by prominently distal weakness in which rimmed vacuoles are not a pronounced feature is discussed in other chapters (see Chapter 11, Chapter 14, Chapter 15 ).


Mutations in the gene encoding UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) are responsible for a distal myopathy with or without rimmed vacuoles. The disorder is referred to as Nonaka myopathy, quadriceps-sparing inclusion body myopathy, autosomal recessive hereditary inclusion body myopathy (HIBM) or GNE myopathy. Inheritance is autosomal recessive, and a founder homozygous mutation, originally identified in the Persian Jewish population, is also present in other countries in the Middle East ( ). Affected individuals in other populations are either compound heterozygous or homozygous for other mutations ( ). Onset of the disorder is usually in the third decade, although onset before 20 years of age or after 45 years can occur. Weakness is bilateral and distal, gradually spreading proximally. The characteristic feature is sparing of the quadriceps, which remain strong even with progressive weakness of other muscles, and when patients are wheelchair bound. A few rare cases with defects in the GNE gene may show weakness of the quadriceps and involvement of the upper limbs may occur in advanced stages of the disorder. Serum CK is usually only mildly elevated.


Muscle biopsies from cases with GNE defects show variability in fibre size, some angular fibres, increased internal nuclei, and some fibrosis but necrosis is rare. Rimmed vacuoles and p62-positive inclusions are a feature, but the number, even in affected muscles, varies ( Fig. 16.16 ). Inflammation, as in other disorders with vacuoles discussed in this chapter, is minimal or absent. This has been used to distinguish these disorders from inclusion body myositis, but inflammation in any disorder may be focal and sampling may influence its presence or absence, and some cases with GNE mutations have been reported to show inflammation ( ). Although a number of the same proteins seen in myofibrillar myopathies and inclusion body myositis also accumulate in GNE myopathy, congophilia is reported to be absent ( ). Electron microscopy also shows the similar cytoplasmic 15–21 nm tubulofilamentous inclusions seen in inclusion body myositis and myofibrillar myopathies.


Feb 23, 2021 | Posted by in MUSCULOSKELETAL MEDICINE | Comments Off on Myofibrillar Myopathies and Other Myopathies with Rimmed Vacuoles
Premium Wordpress Themes by UFO Themes