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Desmin myopathy

L. G. Goldfarb, P. Vicart, H. H. Goebel, M. C. Dalakas
DOI: http://dx.doi.org/10.1093/brain/awh033 723-734 First published online: 14 January 2004

Summary

Desmin myopathy is a recently identified disease associated with mutations in desmin or αB‐crystallin. Typically, the illness presents with lower limb muscle weakness slowly spreading to involve truncal, neck‐flexor, facial, bulbar and respiratory muscles. Skeletal myopathy is often combined with cardiomyopathy manifested by conduction blocks and arrhythmias resulting in premature sudden death. Sections of the affected skeletal and cardiac muscles show abnormal fibre areas containing amorphous eosinophilic deposits seen as granular or granulofilamentous material on electron microscopic examination. Immuno‐staining for desmin is positive in each region containing abnormal structures. The inheritance pattern in familial desmin myopathy is autosomal dominant or autosomal recessive, but many cases have no family history. At least some, and probably most, non‐familial desmin myopathy cases are associated with de novo desmin mutations. Age of disease onset and rate of progression may vary depending on the type of inheritance and location of the causative mutation. Multiple mutations have been identified in the desmin gene: point substitutions, insertion, small in‐frame deletions and a larger exon‐skipping deletion. The majority of these mutations are located in conserved α‐helical segments of desmin. Many of the missense mutations result in changing the original amino acid into proline, which is known as a helix breaker. Studies of transfected cell cultures indicate that mutant desmin is assembly‐incompetent and able to disrupt a pre‐existing filamentous network in dominant‐negative fashion. Disease‐associated desmin mutations in humans or transgenic mice cause accumulation of chimeric intracellular aggregates containing desmin and other cytoskeletal proteins. αB‐crystallin serves in the muscle as a chaperone preventing desmin aggregation under various forms of stress. If mutated, αB‐crystallin may cause a myopathy similar to those resulting from desmin mutations. Routine genetic testing of patients for mutations in desmin and αB‐ crystallin genes is now available and necessary for establishing an accurate diagnosis and providing appropriate genetic counselling. Better understanding of disease pathogenesis would stimulate research focused on developing specific treatments for these conditions.

  • αB‐crystallin; cardiomyopathy; desmin; desmin myopathy; desmin‐related myopathy; myofibrillar myopathy
  • LMNA = lamin A (gene); AD = autosomal dominant pattern of inheritance; AR = autosomal recessive pattern of inheritance; AV = atrioventricular; cDNA = complementary DNA; CK = serum creatine kinase; EchoCG = echocardiogram; EM = electron microscopy; FEV1 = forced expired volume in 1 s; FVC = forced vital capacity

Introduction

Desmin‐related myopathy was originally described as skeletal and cardiac myopathy morphologically characterized by abnormal accumulation of desmin within muscle fibres (Goebel, 1995). This definition focused attention on desmin as a key molecule associated with a diverse group of clinically and pathologically related entities. Molecular studies of these disorders demonstrated that some are truly caused by mutations in desmin (Goldfarb et al., 1998; Muñoz‐Mármol et al., 1998), while another form is associated with mutations in αB‐crystallin that normally acts in the muscle as a chaperone stabilizing desmin molecule by preventing its aggregation (Vicart et al., 1998; Fardeau et al., 2000). Myopathic manifestations of disease caused by either desmin or αB‐crystallin mutations are identical; therefore, a systemic disorder caused by mutations in desmin, αB‐crystallin or perhaps other proteins interacting with desmin and causing myopathy by rendering desmin dysfunctional, are designated as ‘desmin myopathy’, leaving the term ‘desminopathy’ for patients showing mutations in desmin and ‘αB‐crystallinopathy’ for patients with mutations in αB‐crystallin (Goebel and Warlo, 2000). The term ‘myofibrillar myopathy’ was proposed to cover a broader spectrum of pathological changes found in muscle biopsy specimens, namely focal dissolution of the myofibrils and accumulation of degradation products including desmin (Nakano et al., 1996; Engel, 1999). Desmin myopathy is a subgroup of myofibrillar myopathy (Dalakas et al., 2000).

Although just over 60 desmin myopathy patients have so far been identified and fully characterized, there is evidence to suggest that this may be a relatively frequent form of myopathy: (i) desmin myopathy has been identified in patients originating from many countries and populations; (ii) highly conserved regions of the desmin gene appear to be hotspots for human mutations; and (iii) several other proteins are known to closely interact with desmin, and if mutated may cause desmin dysfunction and result in an identical or similar phenotype. Currently, many desmin myopathy cases are misdiagnosed. Diagnostic difficulties arise from the fact that the disease is extremely heterogeneous: in some cases, it manifests as a relentlessly progressive skeletal myopathy with no signs of cardiac involvement (Dalakas et al., 2000, 2003), in others cardiomyopathy is the leading (Goldfarb et al., 1998) or even exclusive (Li et al., 1999) feature; respiratory insufficiency may also be a major manifestation and the cause of death (Dalakas et al., 2002; Dagvadorj et al., 2003a). Most of the known mutations are autosomal dominant (AD), but some are autosomal recessive (AR), and a significant number of mutations are generated de novo. Genetic testing is critical for establishing an accurate diagnosis. The true prevalence of desmin myopathy may be assessed only when most or all patients are tested genetically.

Organization and biological functions of desmin and αB‐crystallin

The cytoskeleton is an integrated network consisting of microfilaments (actins), microtubules (tubulins) and intermediate filaments (IFs). The family of IF proteins includes over 60 members (Fuchs and Cleveland, 1998). The main muscle IF is desmin, a 53‐kDa protein expressed in cardiac, skeletal and smooth muscles. Desmin interacts with other IF proteins to form an intracytoplasmic network that maintains spatial relationship between the contractile apparatus and other structural elements of the cell (Lazarides, 1980). In mature skeletal muscle, desmin filaments encircle and interlink myofibrils at the level of the Z disks and connect them to the plasma membrane and nuclear lamina, thus aligning the myofibrils. In the heart, desmin is increased at intercalated discs and is the major component in the Purkinje fibres (Price, 1984). In accordance with its function, the major part of the desmin molecule is a conserved α‐helical rod of 303 amino acid residues (Fig. 1) that maintains a 7‐residue (heptad) repeat pattern with a typical sequence of hydrophobic and hydrophilic amino acids. This heptad repeat structure guides two polypeptides into formation of a homopolymeric coiled‐coil dimer, the elementary unit of the filament. The 2B segment located at the C‐terminal part of the desmin rod domain contains a discontinuity in the heptad repeat pattern, a ‘stutter’ (Fig. 1), which is equivalent to an insertion of four extra residues at the end of the 2B eighth heptad (Brown et al., 1996). The ‘stutter’ is an obligatory feature of all IF proteins, and its position is absolutely conserved. Experimental ‘straightening out’ of the stutter by inserting three ‘missing’ amino acids to restore a continuous heptad repeat leads to inability of this ‘stutterless’ molecule to anneal into longer filaments (Strelkov and Burkhard, 2002). In compensation for the stutter, the coiled coil slightly unwinds in the stutter vicinity. The local unwinding modifies assembly of the protein and its interaction properties. Another thoroughly examined structure is the YRKLLEGEE motif at the C‐terminal end of the 2B helix. The coiled‐coil structure loosens in this area so that the α‐helices gradually separate, eventually bending away from each other at the EGEE level (Herrmann et al., 2000). In vitro data demonstrate that the YRKLLEGEE motif directs the proper formation of tetramers and controls the number of subunits per filament cross section. The ‘tail’ domain containing ∼30% of β‐sheet, with the remainder of the domain having predominantly random structure and lacking the heptad repeat pattern, is involved in the longitudinal head‐to‐tail tetramer assembly (Herrmann et al., 1996) and control of lateral packing, stabilization and elongation of the higher order filament structures (Heimburg et al., 1996; Strelkov et al., 2002). The tail’s other major function is interacting with cytoskeletal proteins in establishing a cytoplasmic IF network (Rogers et al., 1995).

Fig. 1 Organization of the desmin molecule. A highly conserved α‐helical rod of 303 amino acid residues is flanked by globular N‐ and C‐terminal (‘head’ and ‘tail’) structures (Weber and Geisler, 1985). The helical rod is interrupted in several places resulting in four consecutive α‐helical segments, 1A, 1B, 2A and 2B, connected by short non‐helical linkers. Segments 1A and 2B contain regions highly conserved among intermediate filaments (Herrmann et al., 2000; Strelkov et al., 2001, 2002, 2003). The 2B segment contains a discontinuity in the heptad repeat pattern, a ‘stutter’ (Brown et al., 1996). In desmin, the stutter comprises positions 356–357–358–359. The critical for desmin filament assembly YRKLLEGEE motif is located at the C‐terminal end of the 2B helix.

αB‐crystallin, a member of a highly conserved family of small heat‐shock proteins, is a 22‐kDa cytosolic multimeric protein that has chaperone‐like anti‐aggregation properties. A relatively high level of αB‐crystallin expression is found in the lens, but it is also present in a number of other tissues such as skeletal and cardiac muscle, and to lesser extent skin, brain and kidney (Iwaki et al., 1990; Bova et al., 1999). In skeletal myofibrils and cultured cardiomyocytes, αB‐crystallin is co‐localized with desmin at the Z‐bands (Bennardini et al., 1992). Like most small heat‐shock proteins, αB‐crystallin stabilizes and protects target proteins including desmin by preventing their irreversible aggregation and presents a cellular defence against various forms of stress (Clark and Muchowski, 2000; Wang and Spector, 2000). The C‐terminal α‐helical domain of ∼90 residues is highly conserved within the small heat‐shock protein family, and is responsible for chaperone activity (Muchowski et al., 1997).

Mutations in desmin and αB‐crystallin

Desmin is encoded by a single copy gene (DES) and has been identified and sequenced in several mammalian species. The human desmin gene is located in the chromosome 2q35 band (Viegas‐Péquignot et al., 1989); it encompasses nine exons within an 8.4 kb region, and codes for 476 amino acids (Li et al., 1989). The gene is highly conserved among vertebrate species. Human αB‐crystallin gene (CRYAB) is mapped to chromosome 11q22.3‐q23.1 and is composed of three exons that are highly conserved in a variety of species (Brakenhoff et al., 1990). Desmin myopathy‐like phenotype has also been linked to other loci on chromosome 2q, 10 and 12, but to‐date no disease‐causing genes have been identified in these locations.

Twenty‐one pathogenic mutations have been identified in desmin (Table 1), including 16 missense mutations, three small in‐frame deletions of 1–7 amino acids and an insertion of a single nucleotide resulting in translation termination. In addition, four separate mutations have been identified in splice donor or acceptor sites flanking exon 3. Two mutations have recently been detected in the ‘head’ domain (Ser2Ile and Ser46Phe), but not yet fully characterized (Selcen et al., 2002a). No mutations are known in the desmin 1A helix. A homozygous deletion of 21 nucleotides predicting an in‐frame loss of 7 amino acids from Arg173 through Glu179 in the 1B helix caused a severe clinical syndrome and compromised the ability of desmin to assemble into IFs in cell culture (Muñoz‐Mármol et al., 1998). The A213V desmin variant was detected in two unrelated patients, one having restrictive cardiomyopathy (Bowles et al., 2002) and the other affected with progressive skeletal myopathy with no cardiac involvement (M. de Visser, unpublished). Although the A213V substitution was seen in four control individuals out of 199 tested, and functional studies have produced controversial results, the information generated so far supports the idea that this may be a disease‐causing mutation with low penetrance. A heterozygous single‐nucleotide (adenine) insertion mutation occurring at the third position of codon 241 causes a frameshift leading to serial amino acid replacements: Val242Glu, His243Ser and Glu244Ala, and eventually a premature termination signal at codon 245 (numbering according to the updated sequence, GenBank accession no. AF055081). This mutation is predicted to create a truncated desmin molecule with molecular weight of 27 kDa (Schröder et al., 2003). Transfection studies confirmed that this mutation induces collapse of the preexisting desmin cytoskeleton. It also alters the subcellular distribution of mitochondria and affects biochemical properties of mitochondria in affected skeletal muscles (Schröder et al., 2003). The E245D mutation was found in two affected brothers with severe cardiomyopathy (H. H. Goebel, unpublished). A series of mutations has been identified in the highly conserved donor and acceptor splice sites flanking exon 3 (Table 2), all resulting in a deletion of 32 residues from Asp214 through Glu245 but allowing in‐frame fusion between exons 2 and 4 (Park et al., 2000a; A. Shatunov, unpublished). This deletion disrupts the heptad repeat pattern and therefore interferes with the coiled‐coil structure. The presence of the deletion was confirmed on the mRNA level (Park et al., 2000a). Functional analysis indicated that desmin lacking 32 amino acids was incapable of forming a filamentous network in SW13 (vim–) cells. Recent data indicate that a binding site to nebulin (Bang et al., 2002) and perhaps other interacting proteins are located within this segment.

View this table:
Table 1

Myopathy‐causing mutations in desmin and αB‐crystallin

MutationProtein domainType of inheritanceAge at onset [years (range)]Muscle involvementNumber of familiesReferences
Desmin mutations
Ser2Ile‘Head’??Car + Skel1Selcen et al. (2002a)
Ser46Phe‘Head’???1A. G. Engel, personal communication
Del(Arg173–Glu179)1B helixAR15Skel + Car + Sm1Muñoz‐Mármol et al. (1998)
Ala213Val1B helix??Car only1Bowles et al. (2002)
De novo 42Skel only1M. de Visser (unpublished)
Ins(1bp;X245)1B helix De novo 18Skel + Car1Schröder et al. (2003)
Glu245Asp1B helix??Car only1H. H. Goebel (unpublished)
Del(Asp214–Glu245)1B helixAD30sCar + Skel2Park et al. (2000a)
De novo 38Car + Skel2A. Shatunov (unpublished)
Ala337Pro2B helixAD20–37Skel only1Dalakas et al. (2000)
Asn342Asp2B helixAD23–30Skel only1Dalakas et al. (2000)
Leu345Pro2B helixAD24–46Skel + Car1Sjoberg et al. (1999)
Ala357Pro2B helixAD35–45Skel + Resp1Dagvadorj et al. (2003a)
Del(Glu359–Ser361)2B helixAD31–46Skel only2Kaminska et al. (2003)
Ala360Pro2B helixAR2–9Car + Skel1Goldfarb et al. (1998)
Del(Asn366)2B helixAD36Skel + Car1Kaminska et al. (2003)
Leu370Pro2B helixAD28Skel + Resp2Dagvadorj et al. (2003a)
Leu385Pro2B helix De novo 21Skel + Car1Sugawara et al. (2000)
Gln389Pro2B helix De novo 40Skel only1Goudeau et al. (2001)
Asn393Ile2B helixSecond mutation in the A360P familyGoldfarb et al. (1998)
Arg406Trp2B helix De novo 15–24Car + Skel4Park et al. (2000b)
Lys449Thr‘Tail’??Skel only1Selcen et al. (2002a)
Ile451Met‘Tail’AD15–37Car only1Li et al. (1999)
AD20–30Skel only1Dalakas et al. (2003)
αB‐crystallin mutation
Arg120Glyα‐HelixAD26–45Skel + Car + Cataracts1Fardeau et al. (1978) (2000)

AD = autosomal dominant pattern of inheritance; AR = autosomal recessive; Skel = skeletal myopathy; Car = cardiomyopathy; Resp = respiratory muscle involvement; Sm = smooth muscle myopathy. Codon numbering according to updated sequence in GenBank accession no. AF055081.

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Table 2

Splice site mutations resulting in deletion of 32 amino acids encoded by exon 3

SequenceAcceptor site (Intron 2)Exon threeDonor site (Intron 3)Reference
Wild type…tcccagGAC…GAGgtatac…Li et al. (1989)
IVS2‐1g→a…tcccaaGAC…GAGgtatac…Park et al. (2000a)
IVS2‐2a→t…tccctgGAC…GAGgtatac…A. Shatunov (unpublished)
IVS3+1g→a…tcccagGAC…GAGatgtac…A. Shatunov (unpublished)
IVS3+3a→g…tcccagGAC…GAGgtgtac…Park et al. (2000a)

Nucleotide replacements are highlighted in bold type.

A significant clustering of mutations and polymorphisms is observed in exon 6 corresponding to the C‐terminal part of the highly conserved 2B helix: 11 out of 21 mutations occurred within only 15% of the coding region (Fig. 2). Six missense mutations introduce proline. Proline is not normally present in the desmin rod and is known as a potent helix breaker; its dihedral angle is fixed at –65° and creates a kink in the protein structure (MacArthur and Thornton, 1991). In addition, proline destabilizes α‐helix by its inability to form hydrogen bonds. In mutagenesis experiments, the introduction of proline residues resulted in formation of short, thick and kinked abnormally assembled filaments (Raats et al., 1991). Patients carrying dominant proline‐inserting mutations show classical clinical and histopathological features of desminopathy. Surprisingly, proline‐inserting mutations may also be recessive. The Ala357Pro and Ala360Pro mutations, and the small in‐frame deletions del(Glu359–Ser361) and del(Asn366), are expected to disrupt the coiled‐coil geometry within and around the ‘stutter’ (Dagvadorj et al., 2003a; Kaminska et al., 2003). The Arg406Trp mutation identified in four unrelated Western European patients affects arrangements within the highly conserved YRKLLEGEE motif of the 2B helix (Park et al., 2000b; Dagvadorj et al., 2003b). Two mutations have been identified in the ‘tail’ domain, Lys449Thr and Ile451Met. The disease mechanism in patients with the ‘tail’ domain mutation is distinct from the α‐helical rod mutations, since the tail’s major function is interacting with other cytoskeletal proteins to establish a cytoplasmic intermediate filament network. The inability to interact with these proteins triggers disease development (Dalakas et al., 2003).

Fig. 2 Amino acid sequence alignment of the highly conserved region of desmin exon 6 coding for the C‐terminal part of the 2B α‐helical segment between residues 342 and 415 in multiple evolutionarily diverse species. Sequence includes the ‘stutter’ and the YRKLLEGEE motif that are highly conserved. The heptad repeats are denoted ‘abcdefg’. This small but structurally important area contains 11 of 21 desmin pathogenic mutations. The mutations are shown above the human sequence.

The effects of desmin mutations were tested in various cell lines, and each mutation destroyed the intracellular intermediate filament network (see example in Fig. 3), except for the Ile451Met ‘tail’ domain mutation (Dalakas et al., 2003). The total number of reported families with confirmed desmin mutations is currently 30. Only six of 21 mutations have occurred in more than a single family (Table 1). The pattern of inheritance was AD in 13 families, AR in two families and the mutation was generated de novo in 10 patients. The mode of inheritance in the remaining families has not been determined. The high frequency of de novo mutations suggests that the desmin gene, especially exon 6 coding for the C‐terminal part of the 2B helix, is a hot‐spot for mutations.

Fig. 3 Functional analysis of mutant desmin. Expression vectors containing either full‐length wild type desmin cDNA or mutant desmin cDNA were transfected into SW13(vim‐) and BHK21 cells. Cells transfected with a construct containing wild type desmin cDNA show intense well‐structured filament network; cells transfected with a construct containing mutant cDNA display a pattern characterized by aggregation of desmin‐positive material into disorganized clumps scattered throughout the cytoplasm.

A heterozygous A→G transition at the αB‐crystallin codon 120 resulting in replacement of arginine by glycine (Vicart et al., 1998) was identified in the original multigenerational French family (Fardeau et al., 1978). The Arg120 residue is located in the most conserved region shared by other small heat‐shock proteins. Structural and functional studies indicate that the mutant αB‐crystallin has a much larger molecular weight and decreased β‐sheet content compared with the wild‐type αB‐crystallin (Bova et al., 1999). Altered structure results in defective chaperone function, providing insight into the underlying disease mechanism. A frameshift mutation in αB‐crystallin in a patient with myofibrillar myopathy has recently been reported (Selcen et al., 2002b).

Accumulated data indicate that the majority of patients with clinical and histopathological features of desmin myopathy do not show mutations in either desmin or αB‐crystallin (Dalakas et al., 2000; Selcen et al., 2002b; A. G. Engel, personal communication). Scapuloperoneal weakness and cardiomyopathy with the presence of desmin‐reactive deposits was observed in a large pedigree and has been linked to chromosome 12 (Wilhelmsen et al., 1996). An AD syndrome of predominantly distal or generalized weakness and arrhythmogenic right ventricular cardiomyopathy in a Swedish family was mapped to chromosome 10q22.3 (Melberg et al., 1999) and linkage to locus 2q24‐31 was determined in another family (Nicolao et al., 1999). The causative genes have not yet been identified at these locations.

Desmin myopathy phenotypes

Soon after routine desmin and αB‐crystallin mutation screening became available for diagnostic use, convincing evidence emerged suggesting that different mutations result in somewhat distinct clinical phenotypes (Dalakas et al., 2000). Cardiomyopathy, smooth muscle myopathy, neuropathy, respiratory dysfunction, facial paralysis or cataracts may be present in some cases and absent in others. The age of onset and the rate of disease progression are also variable. A review of genetic mechanisms possibly influencing the phenotype shows that there are several reasons for heterogeneity: (i) dominant, recessive and de novo mutations cause distinct syndromes; (ii) desmin is expressed in skeletal, cardiac and smooth muscles and hence combinations of tissue‐specific alterations result in diverse phenotypes; and (iii) the type and location of the mutation may introduce additional phenotypic modifications. This situation is not unique for desmin myopathy. Mutations in the lamin A gene (LMNA) are known to cause several distinct syndromes: Emery–Dreifuss muscular dystrophy, limb‐girdle 1B muscular dystrophy, dilated cardiomyopathy associated with conduction defects but no skeletal muscle involvement, and Dunnigan partial lipodystrophy (Emery, 2002; Van der Kooi et al., 2002).

Phenotypes associated with desmin mutations (desminopathy)

Analysis of patients from 30 reported families/cases with desmin mutations (Table 1) indicates that the most common presentation is distal muscle weakness in the lower limbs, primarily the anterior compartment. Typically, weakness and atrophy in the legs develop slowly and subsequently appear in hands and arms. Weakness spreads to the truncal, neck‐flexor and sometimes facial muscles. Bulbar signs appear in the later stages of illness involving swallowing and respiratory function impairment. In disease variants marked with early onset cardiomyopathy, patients experienced dizziness and syncopal or fainting episodes.

AR inheritance

The earliest age of onset and most severe disease was observed in cases with AR inheritance. A 15‐year‐old patient who was homozygous for del(Arg173–Glu179) developed generalized weakness and atrophy predominantly in distal muscles of the upper extremities, atrioventricular (AV) block requiring implantation of a permanent pacemaker, and intestinal malabsorption. Echocardiogram (EchoCG) showed dilatation of the right cardiac chambers. Disease progression led to cardiac and respiratory failure and intestinal pseudo‐obstruction. The patient died suddenly at age 28 years. Abundant subsarcolemmal crescent‐shaped strongly eosinophilic masses in skeletal myofibres and centrally located eosinophilic bodies in the cardiomyocytes were immuno‐reactive for desmin and ubiquitin. Ultrastructural studies revealed electron‐dense coarse granular and filamentous aggregates continuous with the Z lines (Ariza et al., 1995; Muñoz‐Mármol et al., 1998),

In another family, three siblings were compound heterozygous for the Ala360Pro and Asn393Ile desmin mutations. They presented with syncopal episodes and complete heart block requiring insertion of a permanent pacemaker at the ages of 2, 9 and 10 years. EchoCG showed moderate to severe biatrial dilatation, but normal ventricular size. Cardiac catheterization revealed left ventricle diastolic dysfunction. Between ages 20 and 24 years, all three developed progressive muscle weakness and wasting in the trunk and extremities, weakness in the neck and facial muscles, and swallowing and breathing difficulties. All three developed congestive heart failure and died at 28, 30 and 32 years of age. Histopathological findings consisted of intracytoplasmic accumulation of amorphous desmin immuno‐reactive material with a characteristic subsarcolemmal distribution. Several older family members carrying either the A360P or the N393I mutation had no signs of muscle or heart disease (Goldfarb et al., 1998; Dalakas et al., 2000). Thus, AR inheritance in two families was characterized by disease onset in childhood or the teens, presentation with cardiomyopathy at an early age followed by skeletal and rarely smooth muscle myopathy and sudden death from cardiac complications.

AD inheritance

Patients showing AD inheritance are characterized by later onset and slower progression of illness (Dalakas et al., 2002). Several distinct clinical syndromes were observed in AD desminopathy families: (i) isolated progressive skeletal myopathy; (ii) skeletal myopathy followed by cardiomyopathy; (iii) skeletal myopathy followed by respiratory insufficiency (in the absence of cardiomyopathy); (iv) cardiomyopathy followed by skeletal myopathy; and (v) isolated cardiomyopathy. Examples of these five variants are shortly described in the following subsections.

Group 1: uncomplicated progressive skeletal myopathy. In a family showing a missense Ala337Pro mutation, disease started with gait disturbance and bilateral weakness in the lower limbs. Weakness developed in proximal and later in distal leg muscles; subsequently, the arms, trunk, neck and face muscles became involved. Swallowing also became affected. The ECG was normal. Two patients were wheelchair‐bound by age 40 years, and one of them died at 47 years. Muscle biopsy demonstrated the presence of amorphous intracytoplasmic material staining intensely for desmin and dystrophin and moderately for vimentin (Goldfarb et al., 1998; Dalakas et al., 2000).

In a family with Ile451Met mutation, a mother and two of her daughters were affected by progressive skeletal myopathy. The disease started with weakness in the lower extremities slowly progressing to involve the upper extremities. In two patients, muscle weakness progressed to wheelchair dependency approximately two decades after disease onset. These two patients also developed difficulty swallowing and impaired respiratory function. There was no evidence of cardiomyopathy and the serum creatine kinase (CK) levels were normal. Accumulation of desmin immuno‐reactive deposits in muscle fibres was present in each patient (Dalakas et al., 2003).

Group 2: skeletal myopathy followed by cardiomyopathy. The Leu345Pro mutation was detected in an Ashkenazi‐Jewish family that included 16 members suffering from symmetric weakness in distal leg muscles progressing to arm, bulbar, respiratory and facial muscles. Six of eight studied patients developed cardiac arrhythmias and conduction blocks about 12 years after the appearance of myopathic symptoms. Histopathologically, some skeletal muscle fibres were atrophic and contained vacuoles and coarse granules; abundant desmin‐positive granulofilamentous deposits in the form of a reticular meshwork were observed between individual myofibrils or adjacent to the sarcolemma (Horowitz and Schmalbruch, 1994; Sjoberg et al., 1999).

Group 3: skeletal myopathy followed by respiratory muscle involvement, but no cardiac disease. In a family carrying the missense Ala357Pro mutation, the father suffering from generalized muscle weakness and wasting developed mild swallowing difficulties, breathlessness on exertion and forced vital capacity (FVC) of only 1 l. He died of a chest infection 7 years after disease onset. His son and daughter had symmetrical weakness in all limbs and FVC reduced to 1.72/1.24 l standing and 1.12/0.92 l lying supine, indicative of diaphragmatic weakness. The patients had modest (four times normal) elevation of serum CK and normal ECG and EchoCG. Muscle biopsy showed variation in fibre size, intracytoplasmic eosinophilic patches immunocytochemically identified as desmin deposits. Electron microscopy (EM) showed deposits of dense granular material between myofibrils and in the subsarcolemmal space (Dagvadorj et al., 2003a).

Group 4: cardiomyopathy followed by skeletal myopathy. The older of two brothers with del(Asp214–Glu245) resulting from IVS2‐1g→a mutation (Table 2) developed dilated cardiomyopathy with an enlarged right ventricle, recurrent left‐sided cardiac failure, complete AV block and pulmonary hypertension. Weakness in the legs appeared 10 years after the onset of cardiac illness and progressed to involve both hands. The patient died of cardiac failure at age 52 years. Skeletal muscle fibres showed accumulation of granulofilamentous material in subsarcolemmal areas, cytoplasmic bodies, and patch‐like lesions immuno‐reactive for desmin, αB‐crystallin and dystrophin. The younger brother also developed an AV block that required a pacemaker at age 41 years, but had no skeletal muscle weakness when last examined at age 50 years. He died from cardiac complications (Goebel et al., 1994; Park et al., 2000a).

Group 5: isolated cardiomyopathy. Six members of an AD family bearing the Ile451Met mutation developed cardiac failure between the ages of 15 and 37 years. Two living patients, father and son, showed cardiomegaly and diminished left ventricular ejection fraction. No signs of skeletal myopathy were observed (Li et al., 1999). As detailed above, the same I451M mutation caused isolated skeletal myopathy with no signs of cardiomyopathy in another AD family. This dramatic difference between the phenotypes associated with the same mutation remains unexplained. No obvious alterations in the coding or regulatory region sequences of the desmin gene were detected in these families (A. Dagvadorj, unpublished). Presumably, clinical variability is determined by the transcription factors that bind to the regulatory sequences located upstream of the desmin promoter and known to confer specific developmental control for desmin expression in cardiac or skeletal muscle (Duprey and Paulin, 1995).

De novo desmin mutations

Desminopathy associated with de novo mutations represents a complex group with even wider margins of variability. (Table 1) Four Western European patients with a de novo Arg406Trp mutation presented at ages between 15 and 24 years with cardiac arrhythmia and conduction block followed in quick succession by muscle weakness and atrophy in the limbs, and in some cases trunk, neck and face. Two patients had dysphagia and respiratory weakness. EchoCG revealed dilated atria and biventricular dysfunction. All four became severely incapacitated in their twenties to early thirties, and one of the patients died from decompensated congestive heart failure at age 28 years. Each of the patients required a permanent pacemaker and two were wheelchair bound. Sections of skeletal muscle showed a significant accumulation of amorphous or granular aggregates in subsarcolemmal and central areas of the muscle fibres that were strongly positive for desmin. EM evaluation showed abnormal granulofilamentous aggregates among the myofibrils and beneath the sarcolemma. The causative Arg406Trp mutation was not found in the patients’ parents, while alternative paternity was unequivocally excluded (Dagvadorj et al., 2003b).

Phenotype observed in patients with αB‐crystallin mutation (αB‐crystallinopathy)

Patients in a large French pedigree with a missense Arg120Gly mutation in αB‐crystallin presented with muscle weakness and shortness of breath. Proximal and distal weakness in the lower and upper limbs, velopharyngeal involvement, hypertrophic cardiomyopathy and discrete lens opacities were subsequently observed. Lens opacities were present in 50% of cases. Serum CK levels were moderately elevated, and the EMG showed a myopathic pattern of abnormalities. Disorganization of filamentous network and characteristic regions in which the intermyofibrillar network completely disappeared (rubbed‐out fibres) were seen on muscle biopsy. Affected areas contained abnormal aggregates immuno‐positive for desmin, αB‐crystallin, dystrophin and ubiquitin. A subsarcolemmal and intermyofibrillar accumulation of dense granulofilamentous material with various degenerative changes was observed on EM (Fardeau et al., 1978; Rappaport et al., 1988; Vicart et al., 1998; Fardeau et al., 2000).

Diagnosis

Recognition of desmin myopathy can be difficult because of the heterogeneity of clinical features and non‐specificity of the histopathology. Although desmin is consistently present and is the most abundant component of the intrasarcoplasmic abnormal aggregates (Goebel, 1995; Dalakas et al., 2000), other proteins including lamin B, αB‐crystallin, gelsolin, nebulin, titin, ubiquitin, α1‐antichymotrypsin, NCAM, dystrophin, γ‐sarcoglycan, vimentin, β‐spectrin, N‐terminal epitopes of amyloid precursor protein, and a fragment of Aβ protein may be present in these deposits (De Bleecker et al., 1996; Goebel, 1997; Engel, 1999). Therefore, muscle protein studies alone are insufficient. Measurement of serum concentration of CK is not helpful, since many patients do not show CK elevation. EMG is important to exclude neurogenic causes of weakness. ECG should be used routinely to identify arrhythmias and cardiac conduction defects; EchoCG helps to diagnose or exclude dilated cardiomyopathy. Reduced respiratory function needs to be confirmed by measurements of vital capacity. Negative family history may be misleading, because in a number of studied cases the patients had de novo mutations. Genetic testing has become essential in establishing an accurate diagnosis and reliable genetic counselling. Diagnostic criteria currently used for determining the need for a molecular genetic study are as follows: progressive muscle weakness in the lower limbs spreading to involve upper extremities; cardiomyopathy expressed with conduction blocks, arrhythmias and restrictive dysfunction; myofibres containing amorphous deposits immuno‐reactive for desmin.

Molecular pathogenesis

Identification of pathogenic mutations in desmin and αB‐crystallin genes, analysis of underlying human disease phenotypes and successful modelling of these conditions in cell cultures and transgenic mice have helped to understand the critical pathogenic events. Current knowledge of disease mechanisms is based on firmly established facts that mutant desmin protein is unable to properly assemble into normal filaments. This results in: (i) loss of desmin function; and (ii) accumulation of mutant misfolded desmin into insoluble toxic aggregates that gradually increase in the cytoplasm and eventually destroy the cell. Whether accumulation of aggregates is more important to disease progression than the loss of desmin function (Hoffman, 2003), remains to be determined. Myopathic changes (Fig. 4A) and widespread abundant desmin‐reactive deposits (Fig. 4B) in the cardiac and skeletal muscles are the morphological hallmarks of desmin myopathy. Depending on the shape and location, the multifocal chimeric aggregates have been described as sarcoplasmic bodies, cytoplasmic bodies or spheroid bodies (reviewed by Goebel et al., 1997). In a number of patients, the patchy electron‐dense granulofilamentous aggregates, are scattered throughout the muscle fibre (Fig. 4C), but most prominently present beneath the sarcolemma (Fardeau et al., 1978, 2000). The granular component of these structures is more consistently present than the filaments (Goebel, 1995, 1997). Studied by immunoelectron microscopy with gold grain technique, the filaments are labelled with desmin antibody, whereas the granular material is non‐reactive (Fig. 4D and E). Importantly, destructive alterations are associated with an anomaly of the Z disk described as Z disk streaming (Nakano et al., 1996; Dalakas et al., 2000). This indicates that Z disk disorganization plays a key role in the disease pathogenesis. These changes in combination with frequent structural disturbances of adjacent sarcomeres, result in widespread myofibrillar pathology (Nakano et al., 1997). A few specimens of cardiac tissue from patients with associated cardiomyopathy have shown similar morphological findings in cardiac myocytes (Bertini et al., 1991; Lobrinus et al., 1998). Myocardial desmin aggregates have largely been seen as granulofilamentous deposits. Occasional involvement of smooth muscle cells has been documented, affecting the intestine and urinary bladder (Ariza et al., 1995; Abraham et al., 1998). Desmin co‐aggregates with other proteins of quite diverse origins, sarcomeric, cytoskeletal, enzymatic, and even those not known to be normally expressed in skeletal muscle such as amyloid of the β‐type and the amyloid precursor protein (De Bleecker et al., 1996; Nakano et al., 1997; Amato et al., 1998). Dystrophin and αB‐crystallin are frequent components of the filamentous structures within abnormal aggregates (Fig. 4F). Evidence that kinases are involved in desminopathies has come from observations of CDC2 (cell division cycle 2) and CDK2 (cyclin‐dependent kinase 2) overexpression in the abnormal intracytoplasmic aggregates (Nakano et al., 1997; Caron and Chapon, 1999), but the specific role of kinases in disease pathogenesis has not yet been determined.

Fig. 4 Sections of affected skeletal muscle of a desminopathy patient. (A) Patches of granulofilamentous material present in several muscle fibres, especially beneath the sarcolemma. Modified Gömöri trichrome stain, ×654. (B) Granulofilamentous patches are rich in desmin. Immunostain, ×245. (C) Large patches of granulofilamentous material among cross‐sectioned myofibrils. Dark dots are glycogen. EM, ×25 980. (D) Labelling desmin with immuno‐gold marks filaments outside of the electron‐dense granular material. EM, ×40 650. (E) Silver‐enhanced gold grains label granulofilamentous material outside of the electron‐dense granular component. Immuno‐EM, ×75 000. (F) Granulofilamentous patches are rich in dystrophin, both in the subsarcolemmal and the internal parts of muscle fibres. Immunostain, ×480.

Depending on the type and location of desmin mutations, mutant desmin may be less capable or completely unable to form filaments. Transfection of various cell lines with mutant desmin results in production of filaments that are shorter than normal, thick and often kinked. They tend to aggregate laterally and form dotted staining patterns (Raats et al., 1991). Misfolded desmin protein escapes proteolytic breakdown and attracts other cytoskeletal proteins into high molecular weight insoluble chimeric aggregates (Li and Dalakas, 2001) that grow and become toxic (Yu et al., 1994). Toxic effect of the aggregates may depend on sequestering of essential cellular proteins. It has been shown conclusively that mutant desmin is capable of disrupting a preexisting filamentous network in dominant‐negative fashion (Raats et al., 1996; Sjoberg et al., 1999).

Solid knowledge of the phenomena associated with the loss of desmin function was obtained in studies of knock‐out mice. Although desmin‐null mice are viable and fertile, and their skeletal, cardiac and smooth muscles develop normally (Capetanaki et al., 1997), cell architecture defects such as misaligned muscle fibres, abnormal sarcomeres, swollen mitochondria and unusual distribution of myosins are seen in the early stages of development (Agbulut et al., 1996). In addition, the neuromuscular junctions are markedly disorganized (Agbulut et al., 2001). After birth, irregularities in the myofibrillar organization are mostly observed in the extensively used skeletal muscles such as the tongue, the diaphragm and the soleus muscle (Li et al., 1996; Milner et al., 1996; Thornell et al., 1997). Cardiac muscle is the most susceptible to the lack of desmin. Mice develop cardiomyopathy early in postnatal life manifested as lysis of individual cardiomyocytes, invasion of macrophages, varying degrees of calcification and finally fibrosis (Thornell et al., 1997). Large arteries are also affected (Lacolley et al., 2001). Older animals show morphology fully characteristic of muscle dystrophy (Li et al., 1996). Disorganized, distended and non‐aligned fibres were observed in the diaphragm. Muscle fibres are gradually lost and replaced by fibrosis. Thus, the lack of desmin in growing and adult knock‐out mice results in multi‐organ disorder involving severe disruption of skeletal and cardiac muscle architecture.

Analysis of transgenic mice expressing the human del(Arg173–Glu179) has provided insights into the mechanisms of intracellular protein aggregation. Examination of the myocardium reveal an accumulation of chimeric intracellular aggregates containing desmin and other cytoskeletal proteins normally interacting with desmin. These aggregates clearly disrupt the continuity and overall organization of the desmin network throughout the cell (Wang et al., 2001a). They appear as early as 1 month after birth and reach a maximum by the eighth to tenth week, which corresponds approximately to early adulthood. Misfolded desmin filaments seem to resist turnover by the normal enzymatic machinery: numerous fragmented filaments were found in the immediate area surrounding the aggregates. In cultured satellite cells taken from a patient carrying the L345P mutation, desmin created a fully normal network in early cell passages; however, after 2 months an increasing number of cells spontaneously produced abnormal aggregates of desmin‐positive material with one of three distribution patterns: perinuclear, spot‐like or subsarcolemmal (Carlsson et al., 2002).

Chaperones assist normal protein folding by restoring proteins to their native conformation after they have been partially denatured by heat, ischaemia, chemotoxicity or other cellular stresses (Hartl et al., 1996). In addition, if necessary, chaperones enhance ubiquitylation and proteasomal degradation of abnormally constructed proteins (Hoffman, 2003). In vitro chaperone assays demonstrated that the mutant Arg120Gly αB‐crystallin was functionally deficient (Bova et al., 1999; Perng et al., 1999). Expression of the mutant αB‐crystallin in SW13 and BHK21 cells leads to formation of abnormal aggregates that contain both desmin and αB‐crystallin reactive material and are surrounded by intermediate filaments (Vicart et al., 1998). Transgenic mice expressing Arg120Gly mutant αB‐crystallin also show the presence of abnormal desmin and αB‐crystallin aggregates in the cardiomyocytes (Wang et al., 2001b, 2002). Additional experiments convincingly confirmed that the accumulation of misfolded proteins occurs due to the loss of chaperone function of the mutant αB‐crystallin; adding of wild‐type αB‐crystallin or HSP27 to the system prevents the formation of aggregates (Chavez Zobel et al., 2003).

Treatment and patient management

There is no specific treatment for desmin myopathy, but some complications can be prevented. Early detection and treatment of cardiac arrhythmias and conduction defects is essential, since implantation of a pacemaker can be lifesaving. Detection of cardiomyopathy and timely treatment of heart failure is another important task. In some cases, cardiac transplantation may be needed. Respiratory insufficiency can be treated by intermittent or permanent positive‐pressure ventilation. Risk of chest infection should be considered in these patients. Although physical therapy is generally advised, we recommend caution because excessive exercise in transgenic mice causes fragility of myofibres. Gene and stem‐cell therapy are active areas of research that promises effective treatments in the future.

Concluding remarks

Desmin myopathy is associated with mutations in desmin, αB‐crystallin and perhaps other genes interacting with desmin. Disease‐causing desmin mutations affect amino‐acid residues at sites that are critical for filament assembly. In humans and transgenic mice, they lead to accumulation of chimeric intracellular aggregates containing desmin and other cytoskeletal proteins. Desminopathy manifests with a variety of phenotypes depending on the type of inheritance or the location of mutations within the relatively large and structurally and functionally complex desmin molecule. αB‐crystallin in the muscle serves as a chaperone for desmin, but if mutated may cause myopathy identical to those resulting from mutations in desmin. Current knowledge of the molecular basis of disorders resulting from mutations in desmin and αB‐crystallin genes allows the use of diagnostic genetic testing. The European Neuromuscular Centre website (http://www.enmc.org) carries information on research laboratories capable of testing desmin myopathy patients.

Note added in proof

This review was in press when Selcen and Engel (2003) reported two αB‐crystallin truncating mutations, Del(2bp;X162) and Q151X, in patients with myofibrillar myopathy. Patient 1 presented with ventilatory insufficiency due to paralysis of the right and weakness of the left diaphragm, followed by the development of skeletal muscle weakness. The patient’s mother and brother also had respiratory disease. Patient 2 showed slowly progressive leg weakness. On muscle biopsy in both cases myofibrillar disintegration began at the Z‐disk and resulted in abnormal local accumulation of desmin, αB‐crystallin, dystrophin and CDC2 kinase.

Acknowledgements

P.V. was supported by a grant from the Association Française contre les Myopathies (AFM). H.H.G. gratefully acknowledges the support from the Deutsche Gesellschaft für Muskelkranke, Freiburg, Germany, and the European Neuromuscular Center (ENMC), Baarn, The Netherlands.

References

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