Desmin-related myopathy: clinical, electrophysiological, radiological, neuropathological and genetic studies

Journal of the Neurological Sciences 219 (2004) 125 – 137 www.elsevier.com/locate/jns

Desmin-related myopathy: clinical, electrophysiological, radiological, neuropathological and genetic studies Montse Olive´ a,*, Lev Goldfarb b, Dolores Moreno a, Encarna Laforet a, Ayush Dagvadorj b, Nyamkhishig Sambuughin b, Juan Antonio Martı´nez-Matos c, Francesca Martı´nez d, Josefina Alio´ e, Eva Farrero f, Patrick Vicart g, Isidro Ferrer a b

a Institut de Neuropatologia, Hospital de Bellvitge, C/Feixa Llarga s/nj, 08907 Hospitalet de Llobregat, Barcelona, Spain Clinical Neurogenetics Unit, National Institute of Neurological Disorders and Stroke, National Institute of Health, Bethesda, MD, USA c Neuromuscular Unit, Department of Neurology, Hospital Univeritari de Bellvitge, Barcelona, Spain d Department of Radiology, Hospital Univeritari de Bellvitge, Barcelona, Spain e Department of Cardiology, Hospital Univeritari de Bellvitge, Barcelona, Spain f Department of Respiratory Medicine, Hospital Univeritari de Bellvitge, Barcelona, Spain g Laboratoire Cytosquelette et De´veloppement, Faculte´ de Me´decine Pitie´-Salpe´trie`re, Paris, France

Received 28 March 2003; received in revised form 16 December 2003; accepted 5 January 2004

Abstract Ten Spanish patients from six unrelated families diagnosed with desmin-related myopathy (DRM) were studied. The pattern of DRM inheritance was autosomal dominant in three families, autosomal recessive in one, and there was no family history in two cases. The disease onset was in early adulthood. Cardiac myopathy was the initial presentation in two patients, respiratory insufficiency in one, and lower limb weakness in all others. Cardiac involvement was observed in four patients. Lens opacities were found in four. CK level was normal or slightly elevated, and electrophysiological examination was consistent with myopathy. Muscle biopsies identified intracytoplasmic desminimmunoreactive inclusions. In addition to desmin, synemin, actin, gelsolin, ubiquitin, aB-crystallin and amyloid hA4 were also present in the deposits. Ultrastructural examination revealed areas of myofibrillary disruption, abnormal electron-dense structures and accumulations of granulofilamentous material. A missense R406W mutation and a novel single amino acid deletion in the desmin gene were identified in two patients; the other patients did not show mutations in desmin, synemin, syncoilin or aB-crystallin genes. Analysis of 10 Spanish DRM cases illustrates a wide clinical, myopathological and genetic spectrum of DRM, reinforcing the need for further exploration of genetic causes for this group of disorders. D 2004 Elsevier B.V. All rights reserved. Keywords: Desmin-related myopathy; Desmin; Mutations; Synemin; Syncoilin; aB-crystallin

1. Introduction Desmin-related myopathies (DRM) are a clinically and genetically heterogeneous group of inherited or sporadic muscle diseases characterised by progressive distal and proximal weakness often associated with cardiomyopathy [1,2]. DRM constitute a newly designated group of myopathies marked by intracellular accumulation of desmin, the intermediate filament specific to muscle fibres that in some

* Corresponding author. Fax: +34-93-2607-503. E-mail address: [email protected] (M. Olive´). 0022-510X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2004.01.007

pathologic conditions may accumulate in cytoplasmic bodies [3], sarcoplasmic bodies [4], spheroid bodies [5] and granulofilamentous material [6]. Muscle biopsies from patients affected by DRM are characterized by: (A) the formation of inclusion bodies, i.e. cytoplasmic, sarcoplasmic and spheroid bodies and patches of ‘‘hyaline structures’’; and (B) granulofilamentous material by EM or ‘‘non-hyaline structures’’ by immunohistochemistry [7]. The term myofibrillar myopathy has been proposed to cover a wider spectrum of pathological changes found in muscle biopsy specimens, namely focal dissolution of the myofibrils and accumulation of the products of myofibrillar degradation [7].

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Immunohistochemical studies have demonstrated intracytoplasmic accumulation of several proteins, with desmin being the most commonly found [7– 9]. According to a latest suggestion, DRM was classified as ‘‘protein surplus myopathies’’ [10,11]. Desmin is the principal intermediate filament of mature skeletal and cardiac muscle. Located at the Z-lines, it plays a pivotal role in maintaining the structural integrity of the myofibrils by connecting adjacent myofibrils laterally at the Z-line level and linking peripheral myofibrils to the sarcolemma and nuclei [12]. Synemin and syncoilin are novel members of the intermediate protein superfamily [13,14]. Both proteins have recently been found accumulated in muscle fibres of DRM patients [15 –17]. Mutations in two genes, desmin and aB-crystallin, were shown to cause DRM [18 – 20]. In addition, linkage to other genetic loci on chromosomes 10q22.3, 12q and 2q24 – 31 has been found in isolated families [21 – 23]. However, in the majority of patients suffering from DRM, the causative genes remain to be discovered. It is not known at present whether mutations in synemin or syncoilin may be causing DRM in some patients. The present study provides a detailed description of the clinical, electrophysiological, radiological, neuropathological and molecular studies in DRM patients. Genetic studies include analysis of genes previously shown to be involved in DRM, desmin and aB-crystallin, as well as novel candidate genes encoding synemin and syncoilin. Studies were conducted in a series of 10 patients from 6 unrelated families.

2. Patients and methods 2.1. Patients Ten patients from six unrelated families were studied. The diagnosis was established after examination of the muscle biopsies from at least one affected patient per family. The inclusion criteria were the presence of nonhyaline and hyaline desmin-reactive cytoplasmic inclusions, and the presence of electron-dense granular aggregates at the ultrastructural level. The following investigations were carried out on each patient: pedigree analysis, clinical neuromuscular exam performed with the use of the Medical Research Council (MRC) grading scale [24], routine laboratory tests that included evaluation of serum creatine kinase and electrophysiological studies (sensory and motor nerve conduction tests and concentric needle electromyography), cardiological examination consisting of ECG, 24-h Holter monitoring and echocardiography, respiratory function tests (FVC), slit lamp examination for lens opacity, and muscle CT scans at mid-thigh and mid-lower-leg levels performed to determine the severity of muscle atrophy (mild, moderate, severe) [25].

2.1.1. Muscle biopsy Open muscle biopsy was performed on seven patients after informed consent was obtained. Part of the muscle sample was immediately frozen in liquid nitrogen-cooled isopentane and processed for routine histochemical reactions and immunohistochemistry for desmin, synemin, dystrophin, utrophin, spectrin and a-sarcoglycan. Another part of the muscle sample was fixed in 4% paraformaldehyde for 24 h at 4 jC and embedded in paraffin. Dewaxed transverse 4-Am-thick sections were processed for desmin, synemin, gelsolin, actin, aB-crystallin, ubiquitin and amyloid hA4 immunohistochemistry. Acetone-fixed cryostat sections, 7 Am thick, and dewaxed paraffin sections, 4 Am thick, were processed using the avidin – biotin – peroxidase method (ABC kit, Vectastain, Vector Labs, Burlingame, CA, USA). After blocking endogenous peroxidase with 1% H2O2 and 10% methanol for 15 min, the sections were incubated overnight at 4 jC with the primary antibody. The sections were subsequently incubated with anti-mouse or anti-rabbit IgG secondary antibodies diluted to 1:100 for 1 h at room temperature, and then with the ABC at a dilution of 1:100 for 1 h at room temperature. The peroxidase reaction was visualised with 0.05% diaminobenzidine and 0.01% hydrogen peroxide. Paraffin sections were slightly counterstained with haematoxylin. In order to rule out nonspecific immunoreactions, consecutive sections in every case were routinely incubated with secondary anti-mouse or anti-rabbit IgG, without the primary antibodies. In addition, some sections were processed with rabbit or mouse IgG at increasing concentrations, and then incubated with the corresponding secondary antibody, to rule out nonspecific staining due to non-immune IgGs. The use of several IgG concentrations was considered because of the lack of information about the exact concentrations of immune IgG in most of the commercial antibodies used in the present study. A list of antibodies used in the study and the dilution at which they were applied is presented in Table 1. The anti-synemin antibody was kindly provided by Prof. D. Paulin. Finally, a small piece of biopsy tissue was processed for ultrastructural examination according to standard methods.

Table 1 Antibodies used in the present study Antigen

Antibody

Species

Source

Dilution

Desmin Sinemin Dystrophin a-Sarcoglycan Spectrin Utrophin Actin Gelsolin Ah4 Ubiquitin aB-crystallin

monoclonal polyclonal monoclonal monoclonal monoclonal monoclonal monoclonal monoclonal monoclonal polyclonal polyclonal

mouse rabbit mouse mouse mouse mouse mouse mouse mouse rabbit rabbit

Dako D. Paulin Novocastra Novocastra Novocastra Novocastra Dako Sigma Novocastra Dako Novocastra

1:15 1:500 1:20 1:200 1:100 1:10 1:100 1:500 1:50 1:100 1:500

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2.1.2. Genetic studies To detect mutations in desmin, aB-crystallin, synemin and syncoilin genes, genomic DNA was extracted from the 10 patients’ blood samples by using the Wizardk Genomic DNA Purification Kit (Promega). Genomic DNA served as template for amplification of each exon with intronic primers. Amplification was carried out using an optimal procedure designed for each separate segment. Desmin and aB-crystallin gene analysis was conducted by direct sequencing of PCR-produced fragments using the DyeTerminatork Sequencing Protocol (Applied Biosystems) on an automated ABI PrismR 3100 Genetic Analyzer (Applied Biosystems). For synemin and syncoilin genes, preliminary testing was performed by DHPLC analysis of each PCR fragment on a Transgenomic WAVE DNA Fragment Analysis System using DNASep column (Transgenomic, Omaha). The melting profile of the PCR product was determined using Wavemaker Software (Transgenomic) at several different temperatures. Samples showing heteroduplex pattern were then sequenced using the DyeTerminatork Sequencing Protocol (Applied Biosystems) on an automated ABI PrismR 310 Genetic Analyzer according to the manufacturer’s protocol (Applied Biosystems).

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3. Results 3.1. Inheritance Two cases (1 and 3) were sporadic. These patients were born to healthy, non-consanguineous parents and had no family history of muscle or cardiac disease. In one patient (case 2), the inheritance was autosomal recessive, whereas in the rest of the patients (cases 4 through 10) the disease was inherited as an autosomal dominant trait. Patients 5, 6, and 7, and patients 8, 9 and 10 are members of two multiplex families (Fig. 1). 3.2. Clinical features and outcomes Three of the patients were females and seven males. The age of disease onset ranged from 12 to 50 years (mean 35). Motor milestones were normal in each patient. In two patients (cases 1 and 2) cardiomyopathy was the first manifestation of the disease, whereas in patient 3 a severe respiratory insufficiency was the presenting symptom. In other patients, the initial complaint was limb muscle weakness. In two patients (cases 2 and 3), the distribution of muscle weakness was mainly proximal. In these cases, as

Fig. 1. Pedigree of families 1 and 2. Top: patients 5, 6 and 7 correspond to individuals II: 3, II: 4 and II: 1, respectively. Bottom: patients 8, 9 and 10 correspond to individuals III: 1, III: 2 and II: 6, respectively.

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well as in patient 1, axial muscles were also affected. In six patients (case 4, and cases 6 through 10), distal muscles of the lower limbs were most markedly affected. However, as the disease progressed, all muscles of the lower limbs, and distal and proximal muscles of upper extremities, also became weak. Patients 4 and 10 had been wheelchair-bound from the age of 42 and 58, respectively. Two patients had a nasal voice and complained of dysphagia due to bulbar involvement. Patients 1 and 4 developed bilateral ankle contractures, whereas patient 2 had a very mild rigidity of the spine. Facial and extraocular muscles were not affected. Deep tendon reflexes were absent at the ankle in seven patients (cases 1, 2 and 5– 9), at the knee and ankle in two cases (3 and 4), and in four limbs in one patient (case 10); sensation was intact (Table 2).

3.4. Respiratory involvement Patient 3 suffered from a severe respiratory insufficiency necessitating nocturnal non-invasive ventilatory support. In three patients (cases 1, 2 and 3), respiratory function test showed a mild degree of restrictive lung function, whereas in other patients respiratory function tests were normal. 3.5. Ophthalmologic examination Slit lamp examination showed bilateral punctiform lens opacities in two patients (cases 1 and 2). A bilateral cortical cataract in patient 4 and a unilateral nuclear cataract in patient 5 were detected, apparently unrelated to myopathy. 3.6. Laboratory test

3.3. Cardiac involvement Cardiac involvement was detected in four patients. Patient 1 had suffered from repetitive syncopal episodes due to a complete atrio-ventricular conduction block at the age of 15, requiring a permanent pacemaker implantation. The patient developed dilated restrictive cardiomyopathy and died suddenly at the age of 28 years. Patient 2 complained of dyspnea on exertion from the age of 12 and was diagnosed with non-obstructive hypertrophic cardiomyopathy at 18. Patient 3 showed signs of impaired right ventricular function, which clearly improved after the introduction of non-invasive mechanical ventilation. Finally, in patient 4 ECG recordings showed left anterior hemiblock with a minimal prolongation of the PR interval. Although there was no obvious cardiac dysfunction, the patient died suddenly at the age of 56. In the rest of the patients, no cardiac abnormality was detected (Table 2).

CK levels were normal or slightly elevated; the highest CK level was seen in patient 2, at five times above normal values (Table 3). 3.7. Electrophysiological studies Electroneurography and concentric needle electromyography (EMG) were performed in each case except for patients 6 and 10. Sensory (sural and median nerve) and motor (common peroneal nerve) nerve conduction studies were normal. On EMG examination, spontaneous activity at rest with positive sharp waves, fibrillation potentials and complex repetitive discharges were recorded in all cases. During voluntary contraction, a myopathic recruitment pattern was observed in four patients (cases 1, 2, 4 and 5), whereas a mixture of small and large polyphasic motor unit potentials was recorded in cases 3, 7, 8 and 9 (Table 3).

Table 2 Summary of clinical features in patients included in this study Case

Sex/age at onset

Current age

Inheritance

First symptom

Weakness distribution

Cardiac involvement

Respiratory insufficiency

Slit lamp examination

1

F/15

28*

Spo

Syncope

Proximal = distal bulbar, axial

Mild

Bilateral pulvurulent lens opacities

2

M/12

47

AR

Dyspnea on exertion

Proximal, axial

Complete a-v block, pacemaker, restrictive cardiomyopathy, sudden death Hypertrophic cardiomyopathy

No

3

M/42

52

Spo

Resp. insuf.

Right-side heart failure, RBBB

Severe

4

F/36

56*

AD

Foot drop

Axial, bulbar, hip adductors Distal>proximal

Bilateral pulvurulent lens opacities Normal

Mild

Minimal bilateral cortical cataract

5

M/30

55

AD

Difficulties on stairs

Proximal = distal

Left anterior hemiblock, minimal prolongation PR, sudden death No

No

6 7 8

M/35 F/30 M/52

53 63 54

AD AD AD

Distal>proximal Distal>proximal Distal

No No No

No No No

9 10

M/50 M/48

58 82*

AD AD

Difficulties on stairs Difficulties on stairs Inability to walk on tiptoe and heels Foot drop Foot drop

Unilateral nuclear cataract Normal Normal Normal

Distal>proximal Distal>proximal

No No

No No

Normal ND

F: female, M: male, Spo: sporadic, AR: autosomal recessive, AD: autosomal dominant, a-v: atrio-ventricular, RBBB: right bundle branch block, ND: not done. * Age at death.

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Table 3 Summary of the laboratory, electrophysiological and radiological data CT scan Case

CK levels

Electrophysiological studies

Thigh

ENG

Anterior

Posterior

Hip adductors

Anterior

Posterior

+ + +++ + ND ND

++ ++ + ++ + ND ND

ND

+ ND

++ ++ +++ +++ + ND ND + ++ ND

++ + + +++ + ND ND + ++ ND

+ ++ + +++ + ND ND ++ +++ ND

EMG SA

1 2 3 4 5 6 7 8 9 10

2 5 2 1.5 1 1 1 2 2 2

N N N N N N N N N ND

F, PSW, F, PSW, F, PSW, F, PSW, F, PSW ND F, PSW, F, PSW, F, PSW, ND

Leg

RP CRD CRD CRD CRD

CRD CRD CRD

Myogenic Myogenic Mixed Myogenic Myogenic ND Mixed Mixed Mixed ND

CK levels: number above normal values, N: normal, ND: not done, F: fibrillation potentials, PSW: positive sharp waves, CRD: complex repetitive discharges; muscle CT scan hypodensity: + = mild, + + = moderate, ++ + = severe.

3.8. CT scan At the mid-thigh level, the adductor magnus and muscles of the posterior compartment were the most frequently affected. Quadriceps was relatively well preserved in all, except for patient 4. In the calf, both the anterior and posterior compartment muscles were affected to a variable degree in each studied patient (Table 3 and Fig. 2). 3.9. Muscle biopsy Muscle biopsy was performed in each patient except for cases 7 and 10. The most striking finding was the presence of typical non-hyaline and hyaline cytoplasmic inclusions. They were single or multiple and present in a variable number of fibres. The inclusions stained pink on H and E and blue – red with the modified Gomori stain and devoid of ATPase and oxidative enzyme activity. In addition to these

specific changes, myopathic abnormalities of variable severity were seen. Variation of the fibre size was mild in cases 1 and 3 but pronounced in the rest of the patients. In three instances (cases 5, 6 and 8), large numbers of internal nuclei and pyknotic nuclear clumps were observed. Only in one case (case 8), there were foci of necrotic fibres undergoing phagocytosis. Rimmed vacuoles were frequently seen (cases 3, 4, 5, 6 and 8) (Fig. 3). In six patients (cases 1 to 6), variable numbers of annular fibres were seen on paraffin sections stained with anti-desmin or anti-synemin antibodies. Moderate endomysial fibrosis was observed in patients 4, 5, 6 and 8. Immunocytochemical studies revealed intracytoplasmic desmin-immunoreactive aggregates in all cases. In patients 1 to 4, desmin immunoreactivity was seen predominantly in the subsarcolemmal regions, as well as in the form of isolated rounded inclusions at the centre of the fibres. By contrast, in cases 5, 6 and 8, desmin inclusions were more frequently observed as focal, often

Fig. 2. CT scan at mid-lower-leg level of patient 1. There is marked hypodensity seen in the muscles of the anterior compartment, whereas less severe change is present in the posterior compartment muscles.

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Fig. 3. Cryostat sections stained with haematoxylin and eosin (A, B, C and D), modified Gomori (E and F), NADH (G and H) and ATPase pre-incubated at pH 4.35 (I). Single (A) or multiple (B) cytoplasmic inclusions are seen in several fibres. A muscle fibre containing a hyalin lesion and a rimmed vacuole (C). Foci of degenerating and regenerating fibres with phagocytosis (C). A large single inclusion in a fibre and a rimmed vacuole (D). On modified trichrome stains, several areas contain irregularly shaped blue – red inclusions (E and F) as well as some nemaline-like rods (E). The inclusions are devoid of oxidative enzyme activity (G), whereas in other fibre areas strong oxydative activity is seen (H). The inclusions are devoid of ATPase activity (I). Bar in F = 25 Am, bar in I = 50 Am.

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Fig. 4. Patterns of desmin immunoreactivity. Desmin aggregates in the subsarcolemmal areas (A, B), presented as single inclusions (B and C) and multiple aggregates or diffuse patches, especially in atrophic fibres (D, E and F). Desmin aggregates (G) co-localised with synemin aggregates (H). A ring fibre immunostained with anti-desmin antibodies is seen in I. Bar in F = 50 Am, bar in I = 25 Am.

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Fig. 5. Ectopic immunoreactivity to dystrophin (A) and a-sarcoglycan (B). Several areas containing actin (C), gelsolin (D), nebulin (E), ubiquitin (F), aBcrystallin (G and H) and Ah-4 (I). A and B cryostat sections; C – I paraffin sections. Bar = 50 Am.

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multiple inclusions and as diffuse aggregates occupying the totality of the fibre. Desmin co-localized with synemin as seen on serial consecutive sections immunostained with anti-desmin or anti-synemin antibodies (Fig. 4). In addition to desmin and synemin, some areas of the cytoplasm were immunoreactive for dystrophin, a-sarcoglycan, actin, gelsolin, ubiquitin, aB-crystallin and amyloid hA4 in each case (Fig. 5). Foci of myofibril disruption associated with dense structures of Z-line origin were seen on EM (Fig. 6). A matrix of granulofilamentous material lying at the level of Z band was visible under the sarcolemma or between the myofibrils. In some cases, particularly in cases 5, 6 and 8, a large number of small nemaline rods was observed. Focal aggregates of mitochondria and residual bodies containing degenerating membranous organelles were other common findings (Fig. 7). Muscle biopsy abnormalities are summarised in Table 4. 3.10. Identification of sequence alterations in desmin, aBcrystallin, synemin and syncoilin genes Analysis of exon-by-exon nucleotide sequences of the desmin coding region resulted in identification of desmin

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mutations in two patients. Patient 1 showed a C-to-T transition in codon 406, changing the codon sequence from CGG to TGG and resulting in predicted amino acid substitution of tryptophan for arginine. This mutation has previously been identified in a patient with a very similar phenotype and proved to be pathogenic in the SW13 (vim-) cells [26]. The R406W mutation was independently confirmed by restriction analysis with BsaWI endonuclease, which recognizes this alteration. Direct sequencing of exon 6 amplified from genomic DNA-, and subsequent restriction analysis with BsaWI-, failed to detect this mutation in the patient’s father and mother. To investigate whether the R406W desmin mutation is a common DNA variation, 123 healthy unrelated control individuals (246 chromosomes) of European origin were screened with the BsaWI endonuclease and found to be negative (data not shown), making it highly unlikely that this mutation represents a normal human polymorphism. Arginine at the 406th position of desmin protein is conserved across species from zebrafish to humans. Sequence analysis of the desmin gene in patient 4 demonstrated a deletion of 3 nucleotides (AAC) in sequence CAGGACAACATTGCA, which corresponds to an inframe single amino acid (asparagine) deletion at position

Fig. 6. Granulofilamentous material at the level of Z lines is seen between the myofibrils (A). Electron-dense structures and Z bodies in abnormal fibre regions in B, C and D. Bar = 1 Am.

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Fig. 7. Small nemaline bodies (A and B) and aggregates of mitochondria (C). A muscle fibre showing some areas completely replaced by foci of degraded myofibrillar elements (D). Bar = 1 Am.

366: GlnAspAsnIleAla. Again, this alteration in desmin gene was not seen in 123 healthy control individuals. Asparagine at this position is highly conserved in evolution. Cell line transfected with the mutant desmin construct lacking asparagine at desmin position 366 has demonstrated the inability of mutant desmin to form a filament network. Detailed expression analysis will be reported elsewhere [27]. Nucleotide sequencing of coding regions in aB-crystallin, synemin and syncoilin genes did not reveal variations that could be associated with the observed phenotypes.

4. Discussion We are reporting herein clinical, electrophysiological, radiological neuropathological and genetic findings in 10 patients affected by DRM. Mutations in desmin gene were identified in two of our patients, whereas the remaining eight patients had no mutations in desmin, aB-crystallin, synemin or syncoilin genes. These findings are in accordance with previous studies indicating that mutations in desmin and aB-crystallin genes account for only a minority of patients with DRM.

Table 4 Summary of muscle biopsy findings Case

1 2 3 4 5 6 8 9

Site of biopsy

Deltoid Biceps braquii Quadriceps Deltoid Biceps braquii Biceps braquii Soleus Biceps braquii

Variation of fibre size

Internal nuclei

+ ++ + +++ ++ ++ +++ +

+/ ++ +/ ++ +++ +++ +++

+ = mild, + + = moderate, ++ + = severe.

Necrosis

+

Rimmed vacuoles

+ ++ ++ + ++

Ring fibres

Characteristics of desmin aggregates

+ + + ++ + ++

+ + + ++ ++ ++ ++

Diffuse/large

Multiple

Subsarcolemmal

+ ++ ++ +++ +

++ ++ ++ ++ + ++ + +

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Mutations in the desmin gene were first identified in 1998 [18,19]. To date, 12 different missense mutations, 1 insertion mutation, 2 splicing defects and 3 deletions have been reported in 18 affected families, including the present series [2,18,19,26,28 – 34]. These results support the view that desminopathy is a separate subgroup among the myofibrillar myopathies. Of the patients in whom a mutation in the desmin gene was identified, including the two from the present series, eight are sporadic, eight families have an autosomal dominant and two families an autosomal recessive mode of inheritance [2,18,19,26,28 –34]. These findings indicate a high rate of de novo mutations in desminopathy patients (Table 5). No differences in the age of onset could be established between patients with mutations in desmin and patients with no such mutations. Cardiac involvement with conduction defects and/or cardiomyopathy were present in four patients of the present series. In two of them, cardiomyopathy was the presenting symptom. One patient suffered from severe respiratory insufficiency. In the remaining cases, muscle weakness in the extremities was the major manifestation. In contrast to previous observations, none of our patients had facial weakness [19,20,26,28,30]. The two patients with desmin mutations died suddenly. It is worth stressing that almost all patients in whom a desmin mutation has been

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identified, suffered from cardiomyopathy thus indicating that although cardiomyopathy is not uniquely characteristic of desminopathy, it seems to be more frequent in this subgroup of patients [2,18,19,26,28 –34]. In addition to desmin, a mutation in the aB-crystallin gene has been reported in two families [20,35]. Members of the family described by Fardeau et al. suffered from an autosomal dominant cardiac and skeletal myopathy [20]. aB-Crystallin, a member of the small heat-shock proteins family, is abundant in lens where it has a structural role in maintaining transparency, as well as in skeletal and cardiac muscles where it helps to maintain the structural integrity of the myofibrils during mechanical stress [36]. Interestingly, members of this family were found to suffer from discrete lens opacities and therefore the presence of cataracts was initially thought to be a distinguishing feature in patients with aB-crystallinopathy. Our observations demonstrate that lens opacities is not a specific feature of aB-crystallinopathy. In one study, neuropathic features have been reported in up to 49% of DRM cases based on clinical and EMG examinations [35]. However, the study does not provide data on sensory and motor nerve conduction. Yet the documentation of involvement of the peripheral nervous system on pathological grounds has not been reliable, with

Table 5 Mutations reported in the desmin gene Reference

Inheritance

Type of mutation

Main clinical findings Onset

Distribution of myopathy

Cardiomyopathy

[18] [18]

AD AR

Early adulthood Childhood

Generalised Generalised

[19]

AR

ALA337PRO ALA360PRO ASN393ILE 21-BP DEL

Adolescence

Generalised, including smooth muscle

[32] [28]

AD AD

ILE451MET LEU345PRO

Unknown Early/middle adulthood

None Generalised

[2]

AD

ILE451MET

Early adulthood

Generalised

[2] [2] [2] [29] [26] [26] [31]

AD Spo Spo Spo Spo Spo Spo

ASN342ASP ARG406TRP Splicing defect LEU385PRO ARG406TRP Splicing defect GLN389PRO

Early adulthood Early adulthood Adulthood Early adulthood Early adulthood Early adulthood Adulthood

Generalised 4 limbs distal>proximala 4 limbs distal>proximala Generalised Generalised Generalised Generalised

[33] [34]

Spo AD

c.5141_5143insA A357P

Adulthood Adulthood

[34]

AD

L370P

Early adulthood

Present series

Spo

ARG406TRP

Adolescence

4 limbs distal>proximal 4 limbs distal>proximal, respiratory weakness 4 limbs distal>proximal, respiratory weakness Generalised

RBBB A-V block, pacemaker, restrictive cardiomyopathy A-V block, pacemaker, restrictive cardiomyopathy, persistent ductus arteriosus Dilated cardiomyopathy A-V block, pacemaker, RBBB, congestive heart failure Mitral valve prolapse, mitral and tricuspide regurgitation No Conduction defects, pacemaker Conduction defects, pacemaker A-V block, pacemaker A-V block, pacemaker A-V block, pacemaker RBBB, mitral, tricuspid and pulmonary regurgitation Ventricular arrhythmia, defibrillator No

Present series

AD

Single aa deletion

Adulthood

4 limbs distal >proximal

RBBB: right bundle-branch block. Generalised: limbs, bulbar, facial, respiratory. a Facial, bulbar and respiratory involvement unknown.

No A-V block, pacemaker, restrictive cardiomyopathy, persistent ductus arteriosus Left anterior hemiblock, PR long

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the exception of just a few cases [37 – 39]. In all our EMG examined cases, distal latencies, amplitudes of the sensory and motor evoked potentials, as well as nerve conduction velocities, were normal. However, EMG examination showed spontaneous activity at rest in all studied cases. Although nerve biopsies were not performed in this series of patients, the normality of nerve conduction studies together with the lack of sensory symptoms make the possibility of an underlying peripheral neuropathy very unlikely. Although muscle CT scan did not show a selective pattern of involvement, the quadriceps muscle was relatively well preserved in most of the cases, whereas muscles of the posterior thigh, especially adductor magnus and the distal muscles of the lower leg, were always affected. An identical pattern of involvement was observed in one previously reported DRM patient [29]. We identified typical cytoplasmic desmin-reactive inclusions in each patient. In most of the cases, some ring fibers were observed, a finding not previously reported in DRM. Desmin aggregates were mainly observed in the subsarcolemmal regions, sometimes as single independent intracytoplasmic inclusions, but most often as multiple aggregates or diffuse patches. In addition to desmin, ectopic expression of dystrophin and a-sarcoglycan, as well as areas containing actin, gelsolin and aB-crystallin, were observed, analogous to previously reported results [8,9]. Taken together, present observations illustrate the wide clinical, pathological and genetic spectrum of DRM. Further molecular studies in patients affected by DRM are necessary to fully understand the genetic bases of these diseases.

Acknowledgements This work was supported by FIS grant 02-0005 and SAF2001-4681-E, and by the Centro Vasco Txoco LagunArtea. We wish to thank the patients and their families for their collaboration and Tom Yohannan for editorial advice.

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