Journal of the NeurologicalSciences, 109 (1992) 1-10 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00 JNS 03739

Peripheral neuropathy with giant axons and cardiomyopathy associated with desmin type intermediate filaments in skeletal muscle Mario Sabatelli a, Enrico Bertini b Enzo Ricci a, Giovanni Salviati c, Stefania Magi a Manuela Papac¢i a and Pietro Tonali" a Neurological Institute, Catholic Universityand UILDM Sez. Laziale, Rome, Italy, b Department of Neuropediatries, Hospital Bambino Gesd, Rome, Italy, and c NRC Unitfor Muscle Biology and Physiopathology, c / o lustimte of General Pathology, Universityof Padova, Pndova, Italy (Received 19 July, 1991) (Revised, received 12 November, 1991) (Accepted 13 November, 1991)

Key words: Giant axonal neuropathy; Cardiomyopatby; Desmin; Neurofilament Summary A sporadic case (female, aged 14 years) is reported who was affected by myopathy, restrictive cardiomyopathy and sensory motor polyneuropatby. A muscle biopsy showed accumulation of osmiophilic granular and filamentous material on electron microscopy, which stained positively in immunofluorescence for desmin. Increased desmin phosphorylated isoforms have been demonstrated by one- and two-dimensional electrophoresis. Sural nerve biopsy showed a peripheral neuropatby with giant axons, f l i e d with closely packed neurofilaments. Clinical and morphological aspects of this new disease entity are discussed with regards to the classical form of giant axonal neuropathy and to other conditions of peripheral neuropathy with giant axons.

Introduction

Case relmrt

Since the reports of Berg et al. (1972) and Asbury et ai. (1972), on a case of giant axonal neuropathy, there has been growing evidence that disorders of intermediate filaments (IF) may be responsible for a heterogeneous group of diseases. Two main distinct clinicalpathological entities have been described: (1) peripheral neuropathy (with or without central nervous sistem involvement) associated with neurofilament accumulations (Tandan et al. 1987; Buissoniere et al. 1989; Ouvrier et al. 1989), and (2) myopathy, cardiomyopathy, or both, associated with desmin storage (Pellissier et ai., 1989). We report here a previously undescribed condition, characterized by peripheral neuropathy with giant axons in the sural nerve biopsy and hypertrophic and restrictive cardiomyopathy with evidence of desmin storage in muscle.

F.F. was admitted at age 14 years to our Neurological Institute. She was the only child of non-consanguineous parents. Pregnancy and delivery were reported as normal. Motor and intellectual milestones were normal: she could walk unassisted at 10½ months of age. Around the age of 10 years, the parents noticed that she walked on tip toes. An orthopaedic examination at age 12 years disclosed that the right leg was l cm shorter than the left. There was also bilateral pes equinus, cavus and slight dorsal scoliosis. At admission, neurological examination showed an alert girl of normal intelligence. Cranial nerves were not involved. She walked with a steppage gait, on the tips of her toes. Passive excursion of the tibio-tarsal joints was limited bilaterally with retraction of Achilles tendons. Weakness was limited m head flexors and peroneal muscles. The calls were enlarged and felt "rubbery". Tendon jerks were absent. Romberg sign was negative. Sensory examination showed mild loss of vibratory sensation distally in both legs. She had diffuse livedo reticularis. Chest x-ray showed mild generalized cardiac enlargement and electrocardiography showed signs of severe right and left atrial hypertrophy. Echocardiography disclosed a hyperthrophlc and restrictive eardiomyopathy with secondary enlargement of both atria. Routine haematological exams were normal. Serum CK was 805 IU/I (~rmal < 230) and LDH was 610 IU/I (normal 230-460). Nerve conduction studies showed delayed conduction velocities and decreased motor and sensory amplitudes (Table 1). Electromyography (EMG) of the right deltoid and biceps braehii showed clear "myopathic" abnormalities whflc EMG of the right tibialis anterior and medial gastrocnemius showed fibrillation potentials and few high

Corresl~ondence to: Dr. Mario Sabatelli, MD, Neurological Institute, Catholic University, Policlinico A. Gemelli, Largo Gemelli 8, 1-00168 Rome, Italy. Tel.: (06) 30154279; Fax: (06) 305 1343.

TABLE 1 NEUROGRAPHIC STUDIES All studies are performed on the right side.

Material and methods

Nerve

MCV (m/s)

CMAP ampl.

SCV SAP

Median Ulnar Deep peroneal Tibial Median Ulnar Radial

(mV) 8 7 0.5 0.5

(~V)

34 42 30 30.8

Sural

33.6 NE NE NE

SAP

1.15 NE NE NE

MCV: motor conduction velocity; SCV: sensory conduction velocity; CMAP: compound motor action potential; SAP: sensory action potential; NE: not evocable; m/s: meters per second; mV: millivolt; /zV: microvolt.

frequency giant motor unit potentials together with increased brief, low amplitude, polyphasicpotentials. A right sural nerve biopsy and a right quadriceps femoris muscle biopsy were performed and are described below.

Nerve A whole sural nerve biopsy specimen was f'uced in 2.5% glutaraldehyde in phosphate buffer and post-fixed in 1% phosphate-buffered osmium tetroxide. T h e tissue was dehydrated, infiltrated and embedded in S p u r t resin. Sections 1 / ~ m thick were stained with toluidine blue. Thin sections were stained with uranyl acetate and lead citrate prior to examination in a Philips E M 400 electron microscope. Muscle The muscle biopsy was divided into two parts. O n e sample was quickly frozen in liquid nitrogen-cooled isopentane and stained with standard techniques (Dubowitz 1985) and with menadione-nitro-blue tetrazolium reaction; another portion was frozen and preserved at - 8 0 ° C for biochemical studies. Four # m - t h i c k cryostat sections of the muscle biopsy were double labelled first with commercially available monoclonal antibodies against desmin-fluorescein (Dako) and then with phalloidin-tetramethylrhodamin (Molecular Probes), which are highly specific for F-actin

Fig. 1. 1-pm thick transverse section of sural nerve biopsy showing a reduced density of myelinated fibers and one giant axon surrounded by a thin myelin sheath. Toluidine blue stain. × 1074.

(Faulstich et al. 1988). For double labelling, sections from a control case and from the patient's muscle were incubated for 1 h with the monoclonai anti-desmin antibody (1 : 100), washed 3 times with PBS containing 1% BSA, incubated for half an hour with anti-mouse IgG-fluorescein (1 : 50), washed 3 times in PBS containing 1% BSA and finally incubated half an hour with phalloidin-tetramethylrhodamin. As a control, one section was treated only with the anti-mouse IgG-fluorescein (1:50). All sections were examined and photographed with a Leitz Ortoplan microscope equipped

with epi-illumination. Other specimens were fixed in phosphate-buffered 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, embedded in Epon, counterstained with uranyl acetate and lead citrate and observed with a Philips EM 400 electron microscope.

Muscle polyacrylamide gel electrophoresis One-dimensional polyacrylamide gel electrophoresis and two-dimensional electrophoresis were performed as previously described (Bertini et ai. 1991).

Fig. 2. (a) Electronmicrograph of the sural nerve showing a giant axon containing tightly packed whorls of neurofilaments, x 21000.

4 Results

Nerve morphology Light microscopic examination of transverse sections showed a reduction in the number of myelinated fibres (Fig. 1). Morphometric analysis showed 4000 myelJnated fibres/mm 2 with a loss of the normal second peak at 10-12 /tin. Acute axonal degeneration was absent. Sporadic onion bulbs formations and regenerating clusters were seen. Some fascicles revealed occasional enlarged myelinated fibres with thin myelin sheath. By electron microscopy, the axonal swellings were composed of densely packed 10 nm filamentous s!r,actures (Fig. 2a, b). Accumulations of 10 nm filaments were not present within the Schwann cell cytoplasm, unmyelinated fibres and fibroblasts. Unmyeli-

nated fibers were reduced. Many endoneurial capillaries presented marked thickening of the basement membrane. One hundred teased fibers were analyzed. Many of them revealed segmental demyelination and remyelination (62%) and myelit3 irregularities (34%). So,he fibres (12%) showed fusifor:~, focal swellings (~ig. 3).

Muscle morphology The number of muscle fibres showing pathological changes varied in different fascicles and were generally grouped in clusters. Many fibres showed nuclear centralization and increased fibre diameter variability. Some fibers showed accumulation of dark purple-green amorphous dense material in .modified Gomori trichrome (Fig. 4a). This material showed no reducing

Fig. 2. (b) Higher magnification of the filamentous material. × 96 000.

activity with the menadione-nitroblue tetrazolium reaction. Rare fibres showing the dark purple-green amorphous material contained typical cytoplasmic bodies (CBs) appearing as oval or round, strongly fucsinophilic, bright red structures with modified Gomori trichrome stain. With oxidative enzyme histochemistry, multiple negative areas of various sizes were observed resembling mult~ or minicore formations. The ATPase reaction did not show type grouping as seen in neurogenic amyotrophy. Immunofluorescence showed multiple round areas strongly positive for F-actin and negative for desmin in some fibres where desmin accumulation was prominent (Fig. 4b, c). The signal for desmin was increased in the sarcoplasm and in subsarcolemmal spaces of those fibres that showed dark-purple

amorphous material in serial sections stained with modified Gomori trichrome. Strongly positive round areas for F-actin corresponded to the central core of CBs as they appeared in modified Gomori stained serial sections. Ultrastructurally most muscle fibres contained highly electron dense material both in subsarcolemmal and intermyofibrillar spaces. At high magnification, this material had a mixed, filamentous and granular appearance and appeared to have similar electron density to the Z-bands (Fig. 5).

Muscle gel electrophoresis SDS-gel electrophoresis showed that the amount of an approximately 53-kDa protein band was greatly

Fig. 3. Teased nerve fiber of the sural nerve showing focal axonal swellings and a demyelinated segment, x 618.

enhanced in the patient's muscle. This protein band was selectively immunostained by anti-desmin monoclonal antibody.

Two-dimensional electrophoresis (Fig. 6) showed several isoforms of desmin with different p H present both in normal and in pathological muscle. However,

Fig. 4. (a-c) Right quadriceps femoris muscle. (b) and (c) show the same section double labelled for F-actin and desmin respectively,a: a serial section stained with modified trichrome; many fibers disclose accumulation of multiple polymorphous dark purple-green sarcoplasmicmaterial and some bright red spots corresponding to cytoplasmicbodies (double arrows). These cytoplasmicbodies are strongly positive for F-actin (b) (double arrows) and are surrounded by a ring of desmin enhancement (c) (double arrows).

Fig. 4. (c) Desminis increasedin manybut not all fibersand particularlyin those that showthe presenceof cytoplasmicbodies(doublearrows). x450. the most acidic isoforms of desmin were more abbundant in the patient's muscle when compared to the control. This indicated that desmin isoforms were more phosphorylated in the patient than in normal muscle. Although immunofluorescence showed many areas strongly positive for F-actin, two-dimensional electrophoresis showed a similar amount of actin in the patient and the control.

Discussion

In 1972 Berg et al. and Asbury et al. first described a chronic progressive peripheral neuropathy in a 6year-old girl with peculiar "kinky" hair. The sural nerve biopsy disclosed masses of neurofdaments distending many of the axons to giant proportions. Other cases reported since then (for review see Tandan et al. 1987; Buissoniere et al. 1989) had similar clinical and pathological characteristics suggesting that giant axonal neuropathy (GAN) represents a distinct clinical-pathological entity. GAN is an autosomal recessive disease characterized by a chronic sensory motor neuropathy with onset of symptoms within 4 years of age. The hair of these patients is usually curly and involvement of the central nervous system is common. Giant axons have also been observed in sural nerve biopsies from patients with other conditions (Table 2). Because clinical and histopathological features in these

cases are quite different from GAN we prefer tO classify them as "Peripheral neuropathies with giant axons" (PNGA). Vogel et al. (1985) described a kinship with dominantly inherited sensory motor neuropathy type II. Sural nerve biopsies showed occasional giant axons distended by large aggregates of neurofilaments and 3 out of 6 patients showed signs of hypertrophic cardiomyopathy. Muscle biopsy was not performed in these patients. These cases differed from GAN because they had dominant inheritance, milder clinical course, absence of CNS involvement and no hair abnormalities. The patients described by Ben Hamida et al. (1990) also seem to represent a separate entity distinct both from GAN and from the condition reported by Vogel et al. (1985). These cases had peripheral neuropathy and multisystem degeneration, progression was very slow, and inheritance was autosomal recessive. In 1974, Liu and Gumbinas reported a post-mortem study of a 13-year-old boy with cardiomyopathy, generalized muscle weakness and clinical and electromyographic signs of mild peripheral neuropathy. Pathological examination revealed axonal spheroids due to neurofilament accumulations in the dorsal gray horn, dorsal columns of the spinal cord, tegmentum of the medulla and dorsal spinal roots. Myocardium and, to a lesser extent, skeletal muscle contained electron-dense material which, however, was not further characterized.

Giant axons have also been described in many toxic and experimental neuropathies such as acrylamide (Prineas 1969; Spencer and Schaumburg 1977), nhexane (Korobldn et al. 1975; Towfigi et al. 1976; Schaumburg and Spencer 1976), n-butyl ketone (Mendel et al. 1974; Spencer et al. 1975), /3,/3-iminodiproprionitrate (IDPN)(Chou and Hartman 1965), ~

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carbon disulphide (Linnoila et al. 1975; Juntunen et al. 1977). Our case seems to represent a new clinical-pathological entity, because it differs both from GAN and from all reported cases of PNGA. Neuromuscular symptoms started much later and the severity of clinical manifestations was milder than reported in GAN. ~i i i i ~ i ~

i~!

¸ ~

!~i

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Fig. 5. Electronmicrograph of muscle showing accumulation of osmiophilic granular and filamentous material between myofibrils, x35 700.

TABLE 2 Classification of peripheral neuropathies with giant axons (PNGA) (1) Peripheral neurnpathy and cardiomyopathy (Vogel et al. 1985) (AD) (2) Peripheral neumpathy with multisystem degeneration (Ben Hamida et al., 1990) (AR) (3) Peripheral neuropathy and cardiomyopathy with desmin storage (present report and Bertini et al. 1991) (S) (4) Toxic and experimental neuropathies (n-hexane; methylbuthyl ketone; acrylamide, IDPN, CS2) (for references see text) AD: autosomal dominant transmission; AR: autosomal recessive transmission; S: sporadic.

Furthermore, our patient had normal hair and no clinical or EEG signs of CNS involvement and cardiomyopathy was a prominent feature. Pathological findings of the sural nerve biopsy also differ from those of GAN patients. Light microscopy revealed only occasional swollen axons, and some fascicles were devoid of giant axons. The diameter of the giant axons never exceeded 2~p,m. The myelin sheath was very thin but no swollen axon was completely devoid of myelin. In (}AN numerous giant axons are present in each fascicle and many of them are larger than 2 5 / t m in diameter. The enlarged axons show a thin myelin sheath and many of them are devoid of myelin (Tandan et al. 1987). The histological changes in sural nerve biopsies reported by Vogel et al. (1985) and Ben Hamida et al. (1990) mostly resemble those seen in our case more than the abnormalities generally observed in GAN. The most interesting finding in our case is the association of a cardiomyopathy and a myopathy with desmin storage in the muscle biopsy. Myopathy associated with storage of desmin intermediate filaments is a rare familial disorder characterized by intrasarcoplasmatic accumulation of material that reacts specifically with anti-desmin antibodies.

Clinical manifestations appear to be quite heterogeneous in age of onset, distribution of muscle involvement, pattern of inheritance (Fardeau et al. 1978; Edstrom et ~1. 1980; Porte et al. 1980; Pellissier et al. 1989). Desmin accumulation can be found either in cases with isolated myopathy, isolated cardiomyopathy or with both. Bertini et al. (1991) have reported a sporadic female patient with the combination of a myopathy, a restrictive cardiomyopathy and clinical and electrophysiological signs of a peripheral sensory-motor neuropathy; sural nerve biopsy was not performed. Muscle and heart showed desmin accumulation with increased phosphorylated isoforms as in our case. In all other reported cases of muscle a n d / o r myocardial desmin storage disorders there is no clear evidence of a polyneuropathy. Our findings demonstrate a new disorder of intermediate filaments involving both peripheral nerves and striated muscles suggesting an abnormality of a common metabolic pathway affecting neurofilamets and desmin. Finally, we want to remark that we have not found desmin accumulation by immunofluorescence and electron mycroscopy in the muscle biopsy of a patient affected by a typical GAN.

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IEF

CONTROL"

1 O"

O

o

actin

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PAT I E NT

actin--

WESTERN

BLOT

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COOM ASSI E

BLUE

Fig. 6. Two-dimentional electropboresis stained with Coomassie brilliant blue (on the fight), transferred to nitrocellulose sheets and immunostained with mouse anti-dcsmin monoclonal antibody (on the left). Increased amount of desmin phosphorylated isoforms in the patient's muscle (dots on the right of the Western blot). The amount of actin is similar in the patient and the control. SDS: sodium dodecyl sulfate; ]EF: isoelectfic focusing.

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