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Abstract Desmin (DES) mutations have been recognizedas a cause of desmin-related myopathy (OMIM 601419),or desminopathy, a disease characterized by progressivelimb muscle weakness and accumulation of desmin-reac-tive granular aggregates in the myofibers. We have stud-ied three families with skeletal or cardioskeletal myopa-thy caused by small in-frame deletions in the desmin gene.The newly identified in-frame deletions E359_S361deland N366del alter the heptad periodicity within a critical2B coiled-coil segment. Structural analysis reveals that

the E359_S361 deletion introduces a second stutter imme-diately downstream of the naturally occurring stutter, thusdoubling the extent of the local coiled-coil unwinding.The N366del mutation converts the wild-type stutter intoa different type of discontinuity, a stammer. A stammer, asopposed to a stutter, is expected to cause an extra over-winding of the coiled-coil. These mutations alter the coiled-coil geometry in specific ways leading to fatal damage todesmin filament assembly. Expression studies in two celllines confirm the inability of desmin molecules with thischanged architecture to polymerize into a functional fila-mentous network. This study provides insights into mo-lecular pathogenetic mechanisms of desmin mutation-as-sociated skeletal and cardioskeletal myopathy.

Introduction

Desminopathy is a recently identified neuromuscular dis-order typically characterized by progressive skeletal mus-cle weakness and cardiomyopathy; the disease is associ-ated with the presence of mutations in the desmin gene(Dalakas et al. 2000). Sections of the affected skeletal andcardiac muscles show atrophic fibers and areas containingaggregates of granular or granulofilamentous material;immunostaining for desmin is positive in each region con-taining abnormal inclusions (Goebel et al. 1994; Horowitzand Schmalbruch 1994; Nakano et al. 1996; Dalakas et al.2000). Desmin is a 53-kDa intermediate filament proteinof the heart, skeletal, and smooth muscle cells. Desminfilaments encircle the Z discs connecting the myofibrils toeach other and to the plasma membrane; this integratingrole of desmin is critical for sarcomere stability in the con-tracting muscle (Lazarides 1980). In accordance with itsfunction, the desmin molecule contains an extended α-he-lical coiled-coil “rod” domain. The rod domain features a seven-residue (heptad) repeat pattern abcdefg, where aand d positions are mostly occupied by apolar residues.This pattern allows two helices to twist around each otheryielding a homopolymeric coiled-coil dimer, the elemen-tary unit of the filament. The heptad repeat pattern has in-

Anna Kaminska · Sergei V. Strelkov · Bertrand Goudeau · Montse Olivé · Ayush Dagvadorj · Anna Fidzianska ·Monique Simon-Casteras · Alexey Shatunov · Marinos C. Dalakas · Isidro Ferrer · Hubert Kwiecinski ·Patrick Vicart · Lev G. Goldfarb

Small deletions disturb desmin architecture leading to breakdown of muscle cells and development of skeletal or cardioskeletal myopathy

Hum Genet (2004) 114 : 306–313DOI 10.1007/s00439-003-1057-7

Received: 2 June 2003 / Accepted: 21 October 2003 / Published online: 27 November 2003

ORIGINAL INVESTIGATION

Electronic database information: nucleotide and amino acid sequence data are available in the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank) under accession nos. AY114212 forE359_S361del and AF21879 for N366del mutations

A. Kaminska · A. FidzianskaNeuromuscular Unit, Medical Research Center, Polish Academy of Sciences, Warsaw, Poland

A. Kaminska · H. KwiecinskiDepartment of Neurology, Medical University of Warsaw, Warsaw, Poland

S. V. StrelkovM. E. Müller Institute for Structural Biology, Biozentrum Basel,CH-4056 Basel, Switzerland

S. V. StrelkovSchemyakin-Ovchinnikov Institute of Bioorganic Chemistry,Moscow, Russia

B. Goudeau · M. Simon-Casteras · P. VicartLaboratoire Cytosquelette et Développement, UMR CNRS 7000,Faculté de Médecine Pitié-Salpétrière, 75013 Paris, France

M. Olivé · I. FerrerInstitut de Neuropatologia, Ciutat Sanitària i Universitària de Bellvitge, Hospitalet de Llobregat, Barcelona, Spain

A. Dagvadorj · A. Shatunov · M. C. Dalakas · L. G. Goldfarb (✉)Building 10, Room 4B37, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 10 Center Drive, MSC 1361, Bethesda, MD 20892-1361, USATel.: +1-301-4021480, Fax: +1-301-4966341,e-mail: goldfarbl@ninds.nih.gov

© Springer-Verlag 2003

terruptions at three sites dividing the rod into four seg-ments, viz., 1A, 1B, 2A, and 2B (Fuchs and Weber 1994).In addition, the coiled-coil segment 2B of all intermediatefilament proteins contains a conserved discontinuity ofthe heptad repeat called a “stutter”, which is equivalent toan insert of four residues. Another known type of discon-tinuity in coiled-coils is a “stammer”, which correspondsto a three-residue insert. Although they cause local distor-tion of coiled-coil geometry, both stutters and stammerscan be tolerated within coiled-coil structures (Brown et al.1996). The atomic structure of the elementary dimer ofvimentin, an intermediate filament protein closely relatedto desmin, has recently been characterized by X-ray crys-tallography (Strelkov et al. 2001, 2002, 2003) and servesas a basis for modeling desmin. The assembly of desminfilaments is driven by lateral interactions of the elemen-tary dimers in an antiparallel fashion. This association ofdimers results in the so-called unit-length filaments thatthen anneal longitudinally, ultimately yielding mature fil-aments (Herrmann and Aebi 1999; Strelkov et al. 2003).Dimer-dimer interactions are extremely specific, and theassembly of the filament is highly dependent on the prop-erties of the elementary dimer (Fuchs and Weber 1994).

Convincing evidence has accumulated suggesting thatmultiple clinical subtypes of desmin myopathy are associ-ated with the pattern of inheritance and the type and loca-tion of the disease-associated mutations in the desminmolecule. Most of the known disease-causing desmin mu-tations are located in the coiled-coil 2B segment at the C-terminal end of the rod domain (Goldfarb et al. 1998;Dalakas et al. 2002). Mutant desmin has been shown tobecome assembly-incompetent and capable of disruptinga preexisting filamentous network in a dominant-negativefashion (Raats et al. 1996; Sjoberg et al. 1999). Disease-associated desmin mutations in humans or transgenic micecause an accumulation of chimeric intracellular aggregatescontaining desmin and other cytoskeletal proteins (Dala-kas et al. 2000; Wang et al. 2001). Misfolded desmin re-sists turnover by the enzymatic machinery. Another mus-cle protein, αB-crystallin encoded by the CRYAB gene,serves as a chaperone for desmin and, if mutated, causes asimilar form of desmin myopathy (Vicart et al. 1998).

Muñoz-Mármol et al. (1998) have described a homo-zygous deletion resulting in an in-frame loss of sevenamino acids in helix 1B, from R173 through E179. Thisdeletion causes generalized muscular weakness and atro-phy, predominantly in distal muscles of the upper extremi-ties, atrioventricular block requiring implantation of a per-manent pacemaker, and intestinal malabsorption. TheR173_E179del mutation compromises the ability of desminto assemble into intermediate filaments when expressed incell culture.

We have conducted a molecular study of two Polishand a Spanish family, in which 16 individuals are affectedwith skeletal or cardioskeletal myopathy. Studies includepedigree analysis, mutation detection, molecular model-ing, and expression of candidate desmin mutations in cellcultures.

Materials and methods

Clinical and histopathologic studies

We have identified two families from Poland segregating progres-sive skeletal myopathy (Fig. 1). These Polish families are notknown to be related. The third studied family was a family fromSpain manifesting a combination of skeletal and cardiac myopa-thy. After obtaining informed consent, seven myopathy patientsand five unaffected family members were neurologically exam-ined. Skeletal muscle biopsy was performed on each of the sevenpatients. Studies were carried out on 5-µm-thick transverse sec-tions of fresh-frozen and paraffin-embedded muscle biopsy tissue.Serial sections were stained with hematoxylin and eosin, modifiedGomori trichrome stain, and antibodies against desmin (clone D33,DAKO, Glostrup, Denmark) diluted 1:100. Electron-microscopicexamination was performed according to a previously describedprotocol (Dalakas et al. 2000). Blood samples were collected fromfive patients and five unaffected family members. Genetic studieswere approved by the Institutional Review Board of the NationalInstitute of Neurological Disorders and Stroke, NIH.

Mutation detection and mutation screening

Patients and their unaffected family members were screened forthe presence of mutations in the desmin (DES) and αB-crystallin(CRYAB) genes. Genomic DNA was used as the template for poly-merase chain reaction (PCR) amplification with primers con-structed for desmin gene exons 1–9 and αB-crystallin gene exons1–3. Amplification was carried out by using optimal proceduresdesigned for each separate segment. PCR-generated fragments ofeach desmin and αB-crystallin exon were purified by QIAquickPCR Purification Kit (Qiagen) and directly sequenced with aDyeTerminator sequencing protocol on an automated ABI Prism3100 Genetic Analyzer (Applied Biosystems). For precise identifi-cation of small deletions, exon 6 was subsequently amplified fromgenomic DNA of patient II-1 of Polish family KP-1, patient II-3from Polish family RP-2, and the patient from the Spanish family.The resulting 399-bp fragments were subcloned into pCR2.1-TOPO vector (Invitrogen) and transfected into TOP 10 One-ShotCompetent Cells (Invitrogen). Eight clones from each patient weresequenced in both directions. Additionally, desmin exon 6 of eachpatient and of their unaffected family members and 96 control in-dividuals of predominantly European origin was PCR-amplifiedby using a forward primer fluorescently labeled with 6-FAM (Re-search Genetics). Fragment size was determined by electrophore-sis on the ABI Prism 3100 Genetic Analyzer and using the GSAnalysis software v. 3.7 (Applied Biosystems).

Molecular modeling

The crystal structure of human vimentin segment 2B has been de-termined to 2.3-Å resolution (Protein Data Bank, http://www.rcsb.org/pdb/cgi/explore, entry 1gk4 – crystal structure of human vi-mentin, 2B coil; Strelkov et al. 2002). Since human desmin and vi-mentin share about 75% sequence identity, the vimentin crystalstructure served as a basis for three-dimensional modeling of seg-ment 2B of the wild-type desmin dimer by using the previously de-scribed methodology (Strelkov et al. 2002). Models of mutant desmindimers were then derived from the wild-type model by applyinggeometrical distortions to compensate for the emerging heptad re-peat discontinuities (Brown et al. 1996; Strelkov and Burkhard2002). Correspondingly, E359_S361del was modeled by a 45° lo-cal unwinding of the coiled-coil in the vicinity of residue 360,whereas N366del was modeled by increasing the left-handed wind-ing of the coiled-coil by a total of 90° within the 347–366 region. Theconstructed models were further analyzed by using the TWISTERprogram (Strelkov and Burkhard 2002).

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Expression of full-length cDNA in cell cultures

Mutations identified in the studied families were introduced intofull-length human desmin cDNA by site-directed mutagenesis (Hoet al. 1989). The resulting fragments containing either mutant or

wild-type desmin cDNA were subcloned into pcDNA3 vector (In-vitrogen). The mutant constructs were verified by sequencing andtransfected into SW13(vim–), a human epithelial cell line that doesnot express intermediate filaments, and BHK21, a cell line thatwas derived from baby hamster kidney epithelial cells and that ex-presses desmin, vimentin, and keratins. After being washed withphosphate-buffered saline, the transfected cells were analyzed byimmunofluorescence microscopy as previously described (Goudeauet al. 2001).

Tracing ancestral origins of the Polish mutation

Genotyping of patients and unaffected members of the Polish fam-ilies was carried out with the following microsatellite markersflanking the desmin gene on chromosome 2q35: centromere –

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Fig. 1 Pedigrees of two Polish families, KP-1 and RP-2, affectedby adult-onset, slowly progressive myopathy. Filled circles orsquares Individuals diagnosed with desminopathy according toclinical and histopathologic criteria, empty symbols unaffectedfamily members, filled bars below symbols disease-associated hap-lotype obtained by using the results of genotyping with micro-satellite markers flanking the desmin gene and intragenic single-nucleotide polymorphisms. The disease-associated haplotypes areidentical in families KP-1 and RP-2, indicating a common heritage

D2S2382 – 1.22 cM – D2S2248 – 1.07 cM – D2S1338 – 2.67 cM – D2S163 – desmin gene – D2S126 – 3.20 cM – D2S133 – 3.21 cM– D2S2354 – telomere spanning 14 cM. PCR amplification of themicrosatellite markers was performed by using standard procedures.The PCR-amplified fragments were tested on the ABI Prism 3100Genetic Analyzer, and size (in nucleotides) was determined by GSAnalysis software v. 3.7 (Applied Biosystems). In addition, intra-genic single nucleotide polymorphisms (SNPs) at nucleotide posi-tions 828, 1014, and 1104 of the desmin gene were genotyped inall four patients and five unaffected family members. The haplotypewas determined based on pedigree analysis and sequencing results.

Results

Clinical features

The disease onset in the affected members of the KP-1family (mother, her daughter, niece, and nephew; Fig. 1)was between 39 and 45 years of age, with gait disturbanceand bilateral weakness in the lower limbs. The mother anddaughter first developed weakness in the proximal andlater in the distal leg muscles, whereas the niece had thissequence reversed. Weakness and wasting slowly spreadto other muscle groups of the lower extremities and even-tually involved the upper limbs. The mother became wheel-chair-dependent 18 years after disease onset. On examina-tion, each patient had moderate weakness and wasting inthe lower and upper extremities, except for the daughterwho showed only mild symmetrical weakness in the prox-imal leg muscles. The trunk, neck, and facial muscles wereintact; swallowing and respiration were not impaired. Ten-don reflexes were preserved, no sensory deficits were de-tected. Electrocardiographic (ECG) and echocardiographic(EchoCG) studies were unremarkable. Electromyography

(EMG) showed a myopathic pattern in each patient butwas normal in the daughter. Motor and sensory conductionvelocities were normal. Serum creatine kinase (CK) levelswere 2–7 times normal. Sections of skeletal muscle biopsytissue stained with Gomori trichrome method showed cyto-plasmic inclusions immunoreactive for desmin. Electron-microscopic evaluation showed abnormal granulofila-mentous aggregates among the myofibrils (Fig. 2B).

Two siblings (brother and sister), members of a secondPolish family (RP-2, Fig. 1), presented with difficultyclimbing stairs and raising their arms because of symmet-ric weakness in the proximal muscles of their upper andlower extremities. Neurological examination revealed prox-imal upper and lower limb weakness; reflexes were nor-mal, and sensation was preserved. The disease onset inthis family was somewhat earlier than in the KP-1 family,viz., between 31 and 33 years of age, and had a slower paceof progression. Again, no truncal, facial, bulbar, or respi-ratory muscles were involved. Cardiovascular examina-tion including ECG and EchoCG showed no abnormalities.Serum CK was not significantly elevated. Histopatho-logically, some of the skeletal muscle fibers were atrophicand contained desmin-reactive granules. Many fibers showedimmunoreactivity for αB-crystallin. Ultrastructural stud-ies revealed multiple electron-dense coarse granular andfilamentous aggregates located under the sarcolemma orwithin the sarcoplasm at the level of the Z line.

Patient Bar-1 from Spain presented with symmetricaldistal lower limb weakness at the age of 36 years. Weak-ness progressed to involve the proximal muscles of the legsand arms. By the age of 45 years, weakness and diffusemuscle atrophy had become pronounced in all extremi-ties. Facial or bulbar muscles were not involved. Reflexes

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Fig. 2 Left Cross-section ofaffected skeletal muscle from apatient of Family Bar-1. Im-munostaining for desmin inparaffin sections with lighthematoxylin counterstainingshows abnormal desmin-reac-tive focal deposits within thefibers (dark material) and be-low the sarcolemma. One re-generating fiber is uniformlydesmin-positive. Right Elec-tron micrograph of affectedskeletal muscle from a patientof Family KP-1 showing gran-ular electron-dense material lo-cated below the sarcolemmaand to a lesser degree betweenthe myofibrils. Z-disc stream-ing, which is mild in this mi-crograph, was prominent inother areas

were present in the upper but absent in the lower limbs.The patient became wheelchair-dependent at the age of50. ECG and 24-h Holter monitoring showed a left ante-rior hemiblock and minimal prolongation of the PR inter-val; EchoCG was normal. Respiratory function test showedmoderate restrictive lung dysfunction (FVC 58.8%; FEV155.8%). Serum CK was not significantly elevated. Onelectrophysiologic examination, motor and sensory nerveconduction velocities were normal. Needle EMG showedpositive sharp waves, fibrillation potentials, and occasionalmyotonic discharges in the proximal and distal muscles ofeach limb, and a myopathic recruitment pattern. The pa-tient died suddenly at the age of 56 years, most probablyfrom cardiac complications. On muscle biopsy, many myo-fibers contained cytoplasmic inclusions that were stronglyimmunoreactive for desmin (Fig. 2A). An electron-micro-scopic study demonstrated abnormal granulofilamentousaggregates between the myofibrils. The patient’s motherand maternal grandmother suffered from a similar diseaseand also died suddenly at the age of 60 and 63 years, re-spectively. The patient’s brother and sister were unaffected.

Analysis of desmin and αB-crystallin gene sequences

The analysis was complicated by the presence of a repeat-ing six-nucleotide sequence GCCAGT in exon 6. The up-stream repeat encodes residues A357–S358, whereas thedownstream GCCAGT repeat codes for A360–S361 (Ta-ble 1). To determine precisely which of the two repeatswas missing in the Polish patients, the entire exon 6 wascloned and sequenced. Patients of both Polish familiesshowed an identical deletion of the GAGGCCAGT se-quence, which included the downstream GCCAGT repeat.This alteration predicts an in-frame deletion of the E359–A360–S361 segment (Table 1). The affected area of thedesmin gene is known to be conserved in evolution, andno irregularities in this region were found in 96 matched

control individuals (192 chromosomes) studied by frag-ment-size analysis. This suggests that the E359_S361delmutation is associated with the disease.

Similar analysis of the patient from the Spanish familyled to the identification of a three-nucleotide sequence dele-tion in desmin exon 6. The deletion comprises the secondand third nucleotides of codon 366 (AC) and the first nu-cleotide of codon 367 (A), which would leave the ATT se-quence coding for Ile at codon 367 intact, but result in anin-frame deletion of the Asn residue at position 366. Se-quencing and size-fragment analysis of exon 6 in two sib-lings of the Spanish patient and 96 unrelated controls failedto uncover deletions in exon 6, suggesting that N366del isthe candidate mutation associated with desmin myopathyin the Spanish patient. Both mutations are located in anevolutionary conserved area of the desmin gene (Fig. 3).Sequencing of the αB-crystallin gene did not show muta-tions in any of the studied patients.

Functional analysis of mutant desmin

To characterize pathogenic potentials of the mutant desminprotein carrying either the E359_S361del or the N366delmutation, constructs containing the wild-type and mutantdesmin cDNA were expressed in SW13(vim–) and BHK21cells and were studied by immunocytochemistry withanti-desmin antibodies. Cell lines transiently transfectedwith plasmid containing wild-type desmin cDNA formedan extensive network of cytoplasmic filaments that re-acted positively with desmin-specific antibody as expectedof a normal intermediate filament network (Fig. 4a, d). Cellstransfected with a construct containing mutant cDNAwith either the E359_S361del or the N366del mutationdemonstrated disrupted coarse desmin-reactive aggregatesand clumps scattered throughout the cytoplasm, suggest-ing that mutant desmin was unable to form functional fil-aments (Fig. 4b, e, and Fig. 4c, f, respectively).

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Fig. 3 Amino acid sequencealignment of a coiled-coil seg-ment of desmin in various spe-cies across evolution. The hep-tad repeats are marked abcdefg;the normally occurring stutteris highlighted. The three-residue E359_S361del and thesingle-residue N366del muta-tions are shown above the hu-man sequence

Table 1 Nucleotide and aminoacid sequences of the desmingene fragment containing re-peating blocks (bold). The lo-cation of the E359_S361delmutation is given in italics

Deleted sequence E359_S361del

Codon no. 356 357 358 359 360 361 362Nucleotide triplets TTT GCC AGT GAG GCC AGT GGCAmino acids Phe Ala Ser Glu Ala Ser Gly

Origin of the mutant chromosome in the affected Polish families

Genotyping of patients and unaffected members of bothPolish families was carried out with the use of microsatel-lite markers flanking the desmin gene and intragenic SNPsat nucleotide positions 828, 1014, and 1104. The resultswere used for haplotype analysis (Fig. 1). A combined mi-crosatellite and SNP haplotype was unambiguously deter-mined. Myopathy patients of both Polish families share a unique disease-associated haplotype 1–1–2–3–c–g–g–E359_S361del–1–5–4, suggesting that the Polish familiesinherited the mutant chromosome from a common ances-tor. The unaffected family members do not show the samehaplotype or the presence of the E359_S361del mutation.

Structural models of mutant desmin

In order to evaluate the effects of the E359_S361del andN366del mutations at the structural level, we have gener-ated three-dimensional molecular models of the wild-typedesmin and both mutants. The desmin coiled-coil segment2B contains a naturally occurring stutter insert that hasbeen highly conserved during evolution (four residuesfrom F356–E359; Fig. 3). As a compensation for the nat-ural stutter, the coiled-coil unwinds in the vicinity of thestutter (Fig. 5). The E359_S361del mutation creates a sec-ond stutter adjacent to the normally existing stutter. Thisshould cause additional local unwinding of the coiled-coil(Fig. 5). The N366del mutation, which also occurs in thevicinity of the wild-type stutter, converts the latter into a

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Fig. 4a–f Functional analysis of mutant desmin. Expressionvectors containing either full-length wild-type desmin cDNA or mutant desmin cDNA withE359_S361del or N366del mu-tations were transfected intoSW13(vim-) and BHK21 cells.Both types of cells transfectedwith a construct containing thewild-type desmin cDNA (a, d)show an intense well-structuredfilament network; cells transfectedwith a construct containing eitherthe E359_S361del (b, e) or theN366del (c, f) mutation display acompletely different pattern, char-acterized by aggregation of des-min-positive material into coarsegranules and disorganized clumpsscattered throughout the cytoplasm

Fig. 5 Molecular models ofdesmin coiled-coil segment2B. The wild-type (WT) struc-ture is based on analogy withcrystallographic data for hu-man vimentin. Arrows Pre-dicted positions of the twostutters for the E359_S361delmutant and the stammer for theN366del mutant, star mutationsite. Note the change of the an-gular position of the tail do-mains with respect to the dimeraxis

heptad repeat stammer, which should cause an overwind-ing of the coiled-coil (Fig. 5; Brown et al. 1996; Strelkovand Burkhard 2002). Both E359_S361del and N366delare thus expected to result in altered coiled-coil geometrywithin segment 2B relative to the wild-type. As a conse-quence, the angular position of the tail domain with re-spect to the dimer axis will be changed.

Discussion

Small in-frame deletions in the desmin gene have beenidentified in three families with autosomal dominantskeletal or cardioskeletal myopathy. The disease onset inmembers of two families from Poland was in adulthoodwith muscle weakness in the lower limbs that progressedslowly to involve upper extremities. The patient from theSpanish family had similar symptoms of skeletal myopa-thy, but later during the course of illness she developedcardiomyopathy expressed as cardiac conduction blockand moderate respiratory insufficiency. Pathological find-ings in all cases consisted of abnormal desmin deposits inthe cytoplasm of muscle fibers and ultrastructural patternscharacterized by destructive alterations of the myofibrilsat the level of the Z discs, as previously described (Goebel1995; Nakano et al. 1996).

The small deletions identified in our patients are lo-cated within the coiled-coil segment 2B of desmin rod do-main. The last 32 residues of segment 2B are highly con-served across the intermediate filament proteins and ap-pear to be critically important for desmin filament assem-bly (Fuchs and Weber 1994; Strelkov et al. 2002). In ad-dition, desmin coiled-coil segment 2B contains a naturallyoccurring stutter (residues F356–E359, see Fig. 3). As acompensation for the stutter, the coiled-coil unwinds inthe stutter vicinity (Strelkov et al. 2002). The local un-winding caused by the stutter is crucial for the correctfunctional conformation of the dimer. Indeed, the removalof the stutter in human vimentin by the insertion of threeamino acids (SAT) aimed at restoring a continuous heptadrepeat leads to the inability of this “stutterless” moleculeto anneal into longer filaments (Herrmann and Aebi1999).

Unlike the previously reported seven-amino-acid dele-tion (Muñoz-Mármol et al. 1998), which apparently doesnot disturb the heptad periodicity, the three-residue andsingle-residue deletions reported here introduce disconti-nuities into the wild-type heptad pattern. Deletion of thethree residues adds a second stutter immediately down-stream of the naturally occurring stutter. As a conse-quence, the extent of the local coiled-coil unwinding inthe E359_S361del mutant becomes about twice as large asthat in the wild-type structure. In contrast, the N366delmutation occurring in the heptad adjacent to the wild-typestutter converts the stutter into a different type of disconti-nuity, a stammer. A stammer, as opposed to a stutter, is ex-pected to cause an extra overwinding of the coiled-coil.Therefore, both E359_S361del and N366del are expectedto result in altered coiled-coil geometry of the 2B segment

relative to the wild-type. These structural changes arelikely to have a pronounced overall effect on the three-di-mensional structure of the rod domain. Indeed, the localunwinding or overwinding results in a different angularposition, with respect to the dimer axis, at the ends of theN-terminal and C-terminal domains. As a consequence ofthe changes in the structure of the elementary dimers, theirpairwise interactions governing the filament assemblyprocess will be altered (Strelkov et al. 2002). The ultimateoutcome is a drastic divergence from the normal assemblypathway evidenced by pathological changes observed inpatients’ muscle biopsy and cell culture experiments.

As predicted, both deletion mutations identified in ourpatients severely affect the ability of desmin to form a fil-amentous network in SW13(vim–) cells. BHK21 cells areknown to have an intrinsic intermediate filament network;expression of mutant desmin in these cells results in thedismantling of the pre-existing network and accumulationof disorganized desmin-containing intracytoplasmic ag-gregates. Since the mutation is heterozygous, the mostlikely pathogenic mechanism is a dominant negative ef-fect, as demonstrated for other desmin mutations in invitro peptide assembly assays (Raats et al. 1996) and ob-served in patients with a missense desmin mutation(Sjoberg et al. 1999). The newly identified desmin muta-tions interfere with the ability of the cell to form the inter-mediate filament network that is critical for maintainingcellular and tissue integrity.

Short repeating sequences are frequent in the genomeand sometimes make the region unstable by allowingdeletions or insertions of the repeating sequences. Dele-tion or expansion of short repeats have been associatedwith a number of neurological disorders such as Hunting-ton’s disease, spinocerebellar ataxias (La Spada et al.1994), and prion disease (Gambetti et al. 1999). The two-nucleotide (ag) interruption between the GCCAGTG re-peats in the desmin gene might make the region more sta-ble compared with other DNA regions containing repeat-ing sequences.

This study provides evidence that small deletions iden-tified in three families disturb the coiled-coil structure ofdesmin in a highly specific way and are responsible for fa-milial skeletal and cardioskeletal myopathy characterizedby desmin deposits in myofibers. Molecular analysis ofthese three desminopathy families improves our under-standing of this disorder and permits a more precise mo-lecular diagnosis. Moreover, knowledge of the structuraldamage inflicted by these desmin mutations should makeit possible to design therapeutic compounds capable ofrestoring the normal desmin filament assembly in dis-eased muscle.

Acknowledgements The authors are grateful to the members ofthe affected families for their enthusiastic participation in thisstudy. Dr. Strelkov acknowledges support from the Swiss NationalScience Foundation and the M.E. Müller Foundation of Switzer-land. The work of Drs. Goudeau, Simon-Casteras, and Vicart wassupported by a grant from the Association Française contre lesMyopathies (AFM). Drs. Olivé and Ferrer were recipients of FIS02-0005 and SAF2001-4681-E grants and were generously sup-ported by the Centro Vasco Txoco Lagun-Artea.

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