Muscle Biopsy

4.1.1 Traditional Studies (H&E, NADH-TR, and ATPase Staining)

Muscle biopsy findings are summarised in Table 2. Conventional staining showed neurogenic changes (neurogenic atrophy, grouped atrophy, and small angular fibres) in three cases, and myopathic changes (increase of central nuclei and variable fibre diameters) in three cases. No abnormal findings were noted in one case. NADH-TR staining and ATPase staining did not show any abnormalities in Cases 10-12. Compared with the previous reports, the severity of these changes may be milder, but may be attributable to the clinical stage at which muscle biopsy was performed.

4.1.2 Nemaline Rods

We have reported a homozygous ChAc case (Case 15 in Table 1) with abnormal histopathological findings in addition to typical clinical features of ChAc [27]. Briefly, a 37-year-old woman developed mild dysarthria and a gait disturbance in addition to the typical hyperkinetic involuntary movements (choreiform movements of the limbs and tics). Although hypotonia with hyporeflexia was noted, neither muscle weakness nor atrophy was noted. Laboratory and radiological findings were compatible with ChAc. H&E staining revealed mild neurogenic changes (grouped atrophy, small angular fibres, and fibres with some internal nuclei), while modified Gomori trichrome staining demonstrated a moderate number of collections of nemaline rods (Fig. 2). The frequency of nemaline rods was quite high and similar to that of adult-onset nemaline myopathy. There were approximately ten rod-containing myofibres per fascicle. Electron microscopy showed the presence of rods in the subsarcolemmal location within myofibres.

Table 2 Summary of the neurophysiological and pathological findings of ChAc cases

Muscle biopsy

Case NCV

Conventional staining (H&E, ATPase, NADH-TR)

Gomori-trichrome, immunohistochemistry, etc

Sural nerve biopsy

11 12

WNL WNL

WNL WNL

ND ND ND

Mild sensory-motor neuropathy WNL

Slight prolongation of ^CV in PNs

High amplitude MUPs, prolonged duration WNL NE

Mild axonal neuropathy ND

ND ND

Neurogenic atrophy with occasional necrotic fibres, internal nuclei

Neurogenic findings Neurogenic atrophy

ND ND ND

Spontaneous MUPs, denervation potentials

ND WNL

ND ND ND

Mild increase of central nuclei and variable fibre diameter

ND ND

ND ND

Increase of anti-tTGase immune activity, amorphous, dense, egg-shaped elements (EM) within muscle fibres

ND ND ND ND ND

Uneven and discontinuous CI along the sarcolemmas, increase of CI in the sarco-lemma

Slight loss of myelinated fibres, many onion bulbs and fibres in regeneration clusters, segmental demyelination

ND ND ND ND ND

Mild decrease of large myelinated fibres in

13 Mild prolongation of

CV in PNs

14 WNL

15 Low CMAP and SNAP

with normal CVs

17-23 Sensory-motor-neuro-pathy (1/6), axonal neuropathy (3/6), normal (2/6)

Short duration MUPs

High amplitude MUPs, reduced inteference

Grouped atrophy with necrotic fibres and clumps of vesicular nuclei, increase of central nuclei (2/2)

Mild increase of central nuclei and variable fibre diameter

Variation in size of muscle fibres, grouped atrophy, small angular fibres, internal nuclei

Uneven and discontinuous CI along the sarcolemma

Uneven and discontinuous CI

along the sarcolemma Nemaline rods located under sarcolemma

Loss of myelinated fibres (6/6), accumulation of membrano-lamellar profiles in the axo-plasm (5/6)

Decrease of large myelinated fibres, a few myelin-ovoids

NCV nerve conduction velocity, normal limit, MUPs motor unit

EMG electromyography, ND not described, NE not examined, CV conduction velocity, PNs peroneal nerves, WNL within potentials, C/chorein immunoreactivity

Fig. 2 Chorein immunostaining in normal control (c) and comparison with ATPase staining (pH = 4.3: a; pH = 4.6: b) and myosin heavy chain fast (MHC-F) immunostaining (d). Chorein was present at the sarcolemma and sarcoplasma. Chorein positive fibres (arrowhead in c) were predominantly present in type I fibres and positive for ATPase (asterisks in a, b) and MHC-F negative fibres (arrow in d). Bar, 50 |im

Fig. 2 Chorein immunostaining in normal control (c) and comparison with ATPase staining (pH = 4.3: a; pH = 4.6: b) and myosin heavy chain fast (MHC-F) immunostaining (d). Chorein was present at the sarcolemma and sarcoplasma. Chorein positive fibres (arrowhead in c) were predominantly present in type I fibres and positive for ATPase (asterisks in a, b) and MHC-F negative fibres (arrow in d). Bar, 50 |im

4.1.3 tTGases

Using immunohistochemical, high pressure liquid chromatography, and Western blot analysis, Melone et al. found increased amounts of tTGase-derived Ne-(-y-glutamyl)lysine isopeptide cross-links in erythrocytes and skeletal muscles from their two ChAc cases, which also showed internal nuclei and some necrotic fibres, suggesting primary myopathic changes [5, 19]. Electron microscopy demonstrated amorphous elements with osmiophilic microfibrils in degenerating myofibres and at the perinuclear level. Combined with these findings, they suggested that tTGase is involved in the production of these inclusion bodies in ChAc muscles.

4.1.4 Immunochemical Staining

We have reported a pedigree with a heterozygous mutation of VPS13A showing apparent autosomal dominant transmission [23]. Using rabbit polyclonal anti-chorein antibodies reactive to two synthetic peptides (amino acids 109-122 and

Fig. 3 Immunostaining for chorein in chorea-acanthocytosis. Accumulation of chorein (arrows in a and upper inset) was localized along the sarcolemma and at the sarcoplasma in ChAc Case 13. In a longitudinal section from Case 13, it was partially weakened and the surface of the myofi-bres was significantly irregular (arrowheads in b). ChAc: chorea-acanthocytosis; Bar, 50 |im

Fig. 3 Immunostaining for chorein in chorea-acanthocytosis. Accumulation of chorein (arrows in a and upper inset) was localized along the sarcolemma and at the sarcoplasma in ChAc Case 13. In a longitudinal section from Case 13, it was partially weakened and the surface of the myofi-bres was significantly irregular (arrowheads in b). ChAc: chorea-acanthocytosis; Bar, 50 |im

2579-2592), immunohistochemical studies of skeletal muscle were performed. In normal muscle from controls, chorein immunoreactivity was found linearly along the sarcolemma and was intense in some fibres or present at a lesser degree in others (Fig. 2c). Compared with the fibre type distribution of NADH-TR and ATPase (pH = 4.6, 4.3) staining and immunohistochemistry with anti-myosin heavy chain fast antibody, chorein labeling was predominantly present in type I fibres and mainly localized in the sarcolemma (Fig. 2a, b, d). These findings suggest that the chorein function may be associated with mitochondrial activity. Muscle tissues from three heterozygous ChAc cases (Cases 10-12) showed uneven and discontinuous chorein immunoreactivity along the sarcolemma, and in some fibres an increase in chorein immunoreactivity in the sarcoplasma was also noted (Fig. 3). Those chorein accumulations along the sarcolemma were negative for ubiquitin immunostaining. These findings are not seen in typical MLS or genetically-confirmed HD (data not shown).

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