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A Mutation in the Dimerization Domain of Filamin C Causes a Novel Type of Autosomal Dominant Myofibrillar Myopathy

2005; Elsevier BV; Volume: 77; Issue: 2 Linguagem: Inglês

10.1086/431959

1537-6605

Matthias Vorgerd, Peter F. M. van der Ven, Vera Bruchertseifer, Thomas Löwe, Rudolf A. Kley, Rolf Schröder, Hanns Lochmüller, Mirko Himmel, Katrin Koehler, Dieter O. Fürst, Angela Huebner,

Skin and Cellular Biology Research

Myofibrillar myopathy (MFM) is a human disease that is characterized by focal myofibrillar destruction and pathological cytoplasmic protein aggregations. In an extended German pedigree with a novel form of MFM characterized by clinical features of a limb-girdle myopathy and morphological features of MFM, we identified a cosegregating, heterozygous nonsense mutation (8130G→A; W2710X) in the filamin c gene (FLNC) on chromosome 7q32.1. The mutation is the first found in FLNC and is localized in the dimerization domain of filamin c. Functional studies showed that, in the truncated mutant protein, this domain has a disturbed secondary structure that leads to the inability to dimerize properly. As a consequence of this malfunction, the muscle fibers of our patients display massive cytoplasmic aggregates containing filamin c and several Z-disk–associated and sarcolemmal proteins. Myofibrillar myopathy (MFM) is a human disease that is characterized by focal myofibrillar destruction and pathological cytoplasmic protein aggregations. In an extended German pedigree with a novel form of MFM characterized by clinical features of a limb-girdle myopathy and morphological features of MFM, we identified a cosegregating, heterozygous nonsense mutation (8130G→A; W2710X) in the filamin c gene (FLNC) on chromosome 7q32.1. The mutation is the first found in FLNC and is localized in the dimerization domain of filamin c. Functional studies showed that, in the truncated mutant protein, this domain has a disturbed secondary structure that leads to the inability to dimerize properly. As a consequence of this malfunction, the muscle fibers of our patients display massive cytoplasmic aggregates containing filamin c and several Z-disk–associated and sarcolemmal proteins. Myofibrillar myopathy (MFM) is a neuromuscular disorder, usually with adult onset and an autosomal dominant inheritance pattern, that results in slowly progressive weakening of limb muscles. MFM may lead to multisystem involvement (eye, peripheral nerve, and heart), and progressive cardiorespiratory complications are potentially lethal (Goldfarb et al. Goldfarb et al., 1998Goldfarb LG Park KY Cervenakova L Gorokhova S Lee HS Vasconcelos O Nagle JW Semino-Mora C Sivakumar K Dalakas MC Missense mutations in desmin associated with familial cardiac and skeletal myopathy.Nat Genet. 1998; 19: 402-403Crossref PubMed Scopus (428) Google Scholar, Goldfarb et al., 2004Goldfarb LG Vicart P Goebel HH Dalakas MC Desmin myopathy.Brain. 2004; 127: 723-734Crossref PubMed Scopus (191) Google Scholar; Selcen and Engel Selcen and Engel, 2003Selcen D Engel AG Myofibrillar myopathy caused by novel dominant negative α-B-crystallin mutations.Ann Neurol. 2003; 54: 804-810Crossref PubMed Scopus (222) Google Scholar; Bär et al. 2005). Typically, skeletal-muscle fibers of patients with MFM show Z-disk pathologies in association with cytoplasmic accumulation of several Z-disk and cytoplasmic proteins (Selcen and Engel Selcen and Engel, 2004bSelcen D Engel AG Myofibrillar myopathy.in: Engel AG Franzini-Armstrong C Myology. McGraw-Hill, New York2004: 1187-1202Google Scholar). MFM is genetically heterogeneous. Thus far, four genes have been associated with MFM: the genes encoding desmin (DES [MIM 125660]) (Goldfarb et al. Goldfarb et al., 1998Goldfarb LG Park KY Cervenakova L Gorokhova S Lee HS Vasconcelos O Nagle JW Semino-Mora C Sivakumar K Dalakas MC Missense mutations in desmin associated with familial cardiac and skeletal myopathy.Nat Genet. 1998; 19: 402-403Crossref PubMed Scopus (428) Google Scholar), myotilin (TTID [MIM 604103]) (Hauser et al. Hauser et al., 2000Hauser MA Horrigan SK Salmikangas P Torian UM Viles KD Dancel R Tim RW Taivainen A Bartoloni L Gilchrist JM Stajich JM Gaskell PC Gilbert JR Vance JM Pericak-Vance MA Carpen O Westbrook CA Speer MC Myotilin is mutated in limb girdle muscular dystrophy 1A.Hum Mol Genet. 2000; 9: 2141-2147Crossref PubMed Scopus (238) Google Scholar; Selcen and Engel Selcen and Engel, 2004aSelcen D Engel AG Mutations in myotilin cause myofibrillar myopathy.Neurology. 2004; 62: 1363-1371Crossref PubMed Scopus (229) Google Scholar), Z-band alternatively spliced PDZ motif-containing protein (LDB3 [MIM 605906]) (Selcen and Engel Selcen and Engel, 2005Selcen D Engel AG Mutations in ZASP define a novel form of muscular dystrophy in humans.Ann Neurol. 2005; 57: 269-276Crossref PubMed Scopus (218) Google Scholar), and αB-crystallin (CRYAB [MIM 123590]) (Vicart et al. Vicart et al., 1998Vicart P Caron A Guicheney P Li Z Prevost MC Faure A Chateau D Chapon F Tome F Dupret JM Paulin D Fardeau M A missense mutation in the αB-crystallin chaperone gene causes a desmin-related myopathy.Nat Genet. 1998; 20: 92-95Crossref PubMed Scopus (935) Google Scholar). However, in the majority of patients with MFM, the presence of mutations in these genes was excluded (Selcen and Engel Selcen and Engel, 2005Selcen D Engel AG Mutations in ZASP define a novel form of muscular dystrophy in humans.Ann Neurol. 2005; 57: 269-276Crossref PubMed Scopus (218) Google Scholar), indicating that mutations in other genes are also causative of this disease. In the present study, we have identified an extended family of German origin with MFM, and we have excluded the involvement of the DES, TTID, and CRYAB genes by haplotype analysis (data not shown). Informed consent was obtained from all subjects (with approval of the ethics committee of Ruhr-University Bochum [#2221]). A total of 17 individuals (12 females and 5 males) were clinically affected. All patients presented with slowly progressive skeletal-muscle weakness, beginning in the lower extremities, which is compatible with the clinical signs of limb-girdle muscular dystrophy (LGMD). Eight of these patients (five females and three males) had been examined clinically (table 1). Their weakness started between the ages of 37 years and 57 years and was more prominent proximally in seven patients, whereas one patient (IV:16) had distal weakness of the calf muscles only. Serum creatine kinase levels were moderately elevated, up to 8-fold higher than the normal upper limit. Four of the patients had respiratory symptoms, and, in two of them (III:14 and III:16), nocturnal ventilation was required. One affected individual (IV:3) revealed findings compatible with cardiac involvement; three of the eight patients had evidence of an additional peripheral neuropathy (table 1). Individual IV:18 carries the FLNC mutation but appears to be presymptomatic as a result of his young age (28 years).Table 1Clinical Data of Eight Affected Individuals in a Pedigree with a Proven FLNC W2710X MutationPatientSexAge at Onset of MFM (years)Age at Examination (years)Initial SymptomsDistribution of Weakness and Muscle AtrophyEvidence of Peripheral Nerve/Eye/Cardiac InvolvementCreatine Kinase IncreaseIII:14F3764Weakness when climbing stairs, back painProximal > distal, lower > upper extremities; respiratory insufficiencyNo2-foldIII:16M4562Weakness when climbing stairs, lower back painProximal > distal, lower > upper extremities; respiratory insufficiencyNo6-foldIII:18M4558Waddling gait, chronic lumbar back painProximal > distal, lower > upper extremities; respiratory insufficiencyNo8-foldIII:19F4954Waddling gait, lower back painProximal in lower extremitiesPeripheral nerve3-foldIII:20F4448Weakness in legs, lower back painProximal in upper extremities; proximal > distal in lower extremitiesNo2-foldIV:3M5760Weakness when climbing stairs or walking uphillProximal > distal, lower > upper extremities; respiratory insufficiencyPeripheral nerve/incomplete right bundle branch block, ejection fraction 66%2-foldIV:16F4350Unsteady gaitDistal in lower extremitiesNo4-foldIV:17F4648Waddling gait, weakness when climbing stairs, lower back painProximal-dorsal and distal-anterior in lower extremitiesPeripheral nerve2-fold Open table in a new tab In patients III:18 and IV:3, the histological findings of muscle biopsies from a clinically affected muscle were typical of MFM. Many fibers showed structural changes harboring amorphous, granular, or hyaline deposits best recognized in trichrome-stained frozen sections (fig. 1A), whereas some fibers showed vacuoles. Oxidative enzyme activities (nicotinamide adenine dinucleotide dehydrogenase, succinic dehydrogenase, and cytochrome oxidase) were sharply reduced in these abnormal fiber regions. In each biopsy, increased internal nuclei, fiber splitting, and isolated necrotic fibers were present. Serial transverse cryostat sections (4 μm) of skeletal-muscle biopsies from these patients were studied by immunofluorescence assays with the use of monoclonal antibodies against filamin c (clone RR90; dilution 1;2) (van der Ven et al. van der Ven et al., 2000avan der Ven PFM Obermann WMJ Lemke B Gautel M Weber K Fürst DO Characterization of muscle filamin isoforms suggests a possible role of γ-filamin/ABP-L in sarcomeric Z-disc formation.Cell Motil Cytoskeleton. 2000; 45: 149-162Crossref PubMed Scopus (113) Google Scholar), desmin (D33; 1;500 [DAKO]), myotilin (RSO34; 1;20), dystrophin (Dy8/6C5; 1;20), α-, β-, γ-, and δ-sarcoglycan (Ad1/20A6, βSarc/SB1, 35DAG/21B5, δSarc3/12C1; 1;100 [Novocastra]), and isotype-specific secondary antibodies conjugated with fluorescein isothiocyanate or Cy2 (Southern Biotech). In each patient, many abnormal fibers revealed a marked accumulation of desmin, the histopathological hallmark of MFM, as well as filamin c in massive aggregates. These aggregates also showed strong immunoreactivity for the filamin c–binding Z-disk protein myotilin, for the sarcolemma-associated protein dystrophin, and for all sarcoglycans (fig. 1A). Ultrastructural analysis of skeletal muscle from patient IV:3 showed myofibrillar changes, including Z-disk streaming and extensive nemaline-rod formation (data not shown). As in MFMs that are due to heterozygous desmin mutations (Schröder et al. Schröder et al., 2003Schröder R Goudeau B Casteras Simon M Fischer D Eggermann T Clemen CS Li Z Reimann J Xue Z Rudnik-Schöneborn S Zerres K van der Ven PFM Fürst DO Kunz WS Vicart P On noxious desmin: functional effects of a novel desmin insertion mutation on the extrasarcomeric desmin cytoskeleton and mitochondria.Hum Mol Genet. 2003; 12: 657-669Crossref PubMed Scopus (80) Google Scholar; Goldfarb et al. Goldfarb et al., 2004Goldfarb LG Vicart P Goebel HH Dalakas MC Desmin myopathy.Brain. 2004; 127: 723-734Crossref PubMed Scopus (191) Google Scholar; Selcen and Engel Selcen and Engel, 2004bSelcen D Engel AG Myofibrillar myopathy.in: Engel AG Franzini-Armstrong C Myology. McGraw-Hill, New York2004: 1187-1202Google Scholar; Bär et al. Bär et al., 2005Bär H Fischer D Goudeau B Kley RA Clemen CS Vicart P Herrmann H Vorgerd M Schröder R Pathogenic effects of a novel heterozygous R350P desmin mutation on the assembly of desmin intermediate filaments in vivo and in vitro.Hum Mol Genet. 2005; 14: 1251-1260Crossref PubMed Scopus (66) Google Scholar), multiple fibers displayed intermyofibrillar and subsarcolemmal granulofilamentous protein aggregates (fig. 1B). The dramatic mislocalization of filamin c in the muscle fibers of our patients prompted us to perform haplotype analysis, by use of microsatellite markers of the FLNC gene region, and subsequently to screen the family with MFM for mutations within the FLNC gene (MIM 102565). For haplotype and mutational analyses, genomic DNA was isolated from peripheral blood lymphocytes in accordance with standard procedures. Haplotype analysis of the candidate FLNC gene on chromosome 7q32.1 was performed with markers included in the deCODE genetic map. Primer pairs were designed with the OLIGO software (primer sequences are available on request). The primers were labeled with one of four fluorophores: FAM, VIC, PET, or NED. PCR amplifications were performed in a T3 thermal cycler (Biometra), and amplicons were separated on an ABI 3100 Genetic Analyzer (Applied Biosystems). The pedigree was drawn with Cyrillic software, version 2.1 (Cherwell Scientific Publishing), and haplotype analysis was performed manually. Two-point LOD score calculations were performed using the LINKAGE program package, with the help of the LINKRUN program (T. F. Wienker, personal communication), under the assumption of autosomal dominant inheritance, equal male and female recombination rates, full penetrance, and a disease-allele frequency of 0.0001. The National Center for Biotechnology Information (NCBI) Map Viewer and Ensembl Genome Browser databases were used for localization of the microsatellite marker loci and identification of transcripts in the candidate region. The GDB Human Genome Database was used for information about microsatellite markers and their primer sequences. Indeed, haplotype analyses established linkage of the disease to the FLNC locus (fig. 2A), with a maximum LOD score of 4.879 (θ=0) at marker D7S635 (table 2).Table 2Two-Point LOD Scores between Chromosome 7 Markers and the FLNC LocusLOD at θ =Marker.000.001.010.050.100.150.200.300.400D7S4872.4582.4532.4072.2071.9641.7271.4921.014.509D7S2527.299.306.355.490.546.533.479.307.124D7S18732.3102.3062.2742.1171.8941.6551.410.931.470D7S18222.4172.4152.3932.2622.0501.8071.5481.020.505D7S6802.8712.8652.8082.5492.2181.8851.553.914.360D7S5142.5012.4952.4442.2131.9221.6291.340.787.317D7S6354.8794.8704.7864.4083.9203.4162.8971.827.767D7S25012.7982.7932.7502.5492.2741.9831.6821.075.499D7S5043.0223.0152.9542.6822.3432.0071.6781.050.483D7S18754.2864.2784.2033.8653.4342.9942.5471.640.741D7S5301.8221.8171.7711.5751.3521.143.941.550.219D7S25442.9022.8942.8292.5492.2201.9071.6041.003.419D7S25191.4601.4581.4371.3381.2021.057.906.597.294D7S2531.294.296.312.359.375.358.315.186.056D7S640−2.0901.0471.9882.4172.3662.1781.9261.317.639 Open table in a new tab Sequence analysis of FLNC included all 48 exons and was performed on genomic DNA from individuals IV:3 and IV:16 of the present family, as well as on DNA from four sporadic cases with similar clinical and morphological features pointing to LGMD or MFM. Sequencing was performed with an ABI 3100 Genetic Analyzer (Applied Biosystems) by use of the BigDye Terminator v 1.1 Cycle Sequencing kit (Applied Biosystems). Although no FLNC mutations were found in the sporadic cases, a single heterozygous nucleotide substitution (8130G→A), resulting in a substitution of tryptophan at position 2710 by a stop codon (W2710X) (human FLNC [GenBank accession number AF252549], human chromosome 7 clones containing FLNC [GenBank accession numbers AC025594 and AC024952], and FLNC [accession number Q14315]), was identified in exon 48 in patients IV:3 and IV:16 from the present family (fig. 2C). To confirm cosegregation of the mutation with the MFM disease phenotype, all available family members and 110 control individuals were investigated for the 8130G→A mutation by RFLP analysis and/or direct sequencing. Since the mutation did not delete or introduce a restriction site, a new AluI restriction site was created in the mutant PCR product by use of a lower primer with a single-nucleotide mismatch at the penultimate 3′ position (primer sequences are available on request). For restriction-enzyme digestion, the PCR product was incubated with AluI, and the cleaved fragments were separated on a 2% agarose gel and were visualized by ethidium bromide staining (for details, see the legend to fig. 2B). The 8130G→A mutation was detected in all affected family members and was confirmed by direct sequencing (fig. 2B). It was not observed in 220 control chromosomes. Recently, a large five-generation Spanish family with LGMD, with clinical features similar to those seen in the German family, was described (Gamez et al. Gamez et al., 2001Gamez J Navarro C Andreu AL Fernandez JM Palenzuela L Tejeira S Fernandez-Hojas R Schwartz S Karadimas C DiMauro S Hirano M Cervera C Autosomal dominant limb-girdle muscular dystrophy: a large kindred with evidence for anticipation.Neurology. 2001; 56: 450-454Crossref PubMed Scopus (36) Google Scholar), and, subsequently, the disorder was referred to as “LGMD1F” (MIM 608423) and was mapped to chromosome 7q32, which includes the FLNC locus. However, in this family, FLNC was excluded as a candidate gene (Palenzuela et al. Palenzuela et al., 2003Palenzuela L Andreu AL Gamez J Vila MR Kunimatsu T Meseguer A Cervera C Fernandez Cadenas I van der Ven PFM Nygaard TG Bonilla E Hirano M A novel autosomal dominant limb-girdle muscular dystrophy (LGMD 1F) maps to 7q32.1-32.2.Neurology. 2003; 61: 404-406Crossref PubMed Scopus (45) Google Scholar). In the Spanish family, skeletal-muscle weakness, predominantly involving the pelvic and shoulder girdle, started between the ages of 1 year and 58 years, whereas, in the German family, no patient with a juvenile-onset form was observed. In the Spanish family, the histochemical and immunohistochemical findings were compatible with LGMD; desmin immunostaining was not reported. In contrast, the massive protein aggregates in the German family clearly indicated MFM. Despite substantial overlap in the clinical phenotype, patients in the German family are distinct—not only genetically but also morphologically—from patients with LGMD1F. Because of the protein defect, we suggest classification of the disease found in the German family as “autosomal dominant filaminopathy,” a novel form of MFM. Filamins are a family of actin-binding proteins involved in reshaping the actin cytoskeleton and its associated structures (Stossel et al. Stossel et al., 2001Stossel TP Condeelis J Cooley L Hartwig JH Noegel A Schleicher M Shapiro SS Filamins as integrators of cell mechanics and signalling.Nat Rev Mol Cell Biol. 2001; 2: 138-145Crossref PubMed Scopus (767) Google Scholar; van der Flier and Sonnenberg van der Flier and Sonnenberg, 2001van der Flier A Sonnenberg A Structural and functional aspects of filamins.Biochim Biophys Acta. 2001; 1538: 99-117Crossref PubMed Scopus (313) Google Scholar). Actin binding is conferred by a pair of amino-terminal CH domains. For their actin filament–bundling and cross-linking activities, the filamins depend on their C-terminal region—more specifically, the immunoglobulin-like domain 24, which is responsible for dimer formation (Himmel et al. Himmel et al., 2003Himmel M van der Ven PFM Stöcklein W Fürst DO The limits of promiscuity: isoform-specific dimerization of filamins.Biochemistry. 2003; 42: 430-439Crossref PubMed Scopus (51) Google Scholar). Furthermore, filamins interact with a plethora of cellular proteins of great functional diversity, indicating that they are multifunctional signaling adapter proteins (van der Flier and Sonnenberg van der Flier and Sonnenberg, 2001van der Flier A Sonnenberg A Structural and functional aspects of filamins.Biochim Biophys Acta. 2001; 1538: 99-117Crossref PubMed Scopus (313) Google Scholar). Although mutations in the ubiquitously expressed genes encoding filamin a (Fox et al. Fox et al., 1998Fox JW Lamperti ED Eksioglu YZ Hong SE Feng Y Graham DA Scheffer IE Dobyns WB Hirsch BA Radtke RA Berkovic SF Huttenlocher PR Walsh CA Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia.Neuron. 1998; 21: 1315-1325Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar; Robertson et al. Robertson et al., 2003Robertson SP Twigg SR Sutherland-Smith AJ Biancalana V Gorlin RJ Horn D Kenwrick SJ Kim CA Morava E Newbury-Ecob R Orstavik KH Quarrell OW Schwartz CE Shears DJ Suri M Kendrick-Jones J Wilkie AO Localized mutations in the gene encoding the cytoskeletal protein filamin A cause diverse malformations in humans.Nat Genet. 2003; 33: 487-491Crossref PubMed Scopus (315) Google Scholar) and filamin b (Krakow et al. Krakow et al., 2004Krakow D Robertson SP King LM Morgan T Sebald ET Bertolotto C Wachsmann-Hogiu S Acuna D Shapiro SS Takafuta T Aftimos S Kim CA Firth H Steiner CE Cormier-Daire V Superti-Furga A Bonafe L Graham Jr, JM Grix A Bacino CA Allanson J Bialer MG Lachman RS Rimoin DL Cohn DH Mutations in the gene encoding filamin B disrupt vertebral segmentation, joint formation and skeletogenesis.Nat Genet. 2004; 36: 405-410Crossref PubMed Scopus (198) Google Scholar) are causative for a strikingly wide range of human diseases (Feng and Walsh Feng and Walsh, 2004Feng Y Walsh CA The many faces of filamin: a versatile molecular scaffold for cell motility and signalling.Nat Cell Biol. 2004; 6: 1034-1038Crossref PubMed Scopus (396) Google Scholar), up to the present, no disease-causing mutations had been found in FLNC, the gene encoding the muscle-specific filamin isoform. Its up-regulation during initial stages of myocyte differentiation, its localization predominantly at the periphery of Z-disks (van der Ven et al. van der Ven et al., 2000avan der Ven PFM Obermann WMJ Lemke B Gautel M Weber K Fürst DO Characterization of muscle filamin isoforms suggests a possible role of γ-filamin/ABP-L in sarcomeric Z-disc formation.Cell Motil Cytoskeleton. 2000; 45: 149-162Crossref PubMed Scopus (113) Google Scholar), and its direct interaction with myotilin (van der Ven et al. van der Ven et al., 2000bvan der Ven PFM Wiesner S Salmikangas P Auerbach D Himmel M Kempa S Hayess K Pacholsky D Taivainen A Schröder R Carpén O Fürst DO Indications for a novel muscular dystrophy pathway: γ-filamin, the muscle-specific filamin isoform, interacts with myotilin.J Cell Biol. 2000; 151: 235-248Crossref PubMed Scopus (159) Google Scholar) imply an important role for this filamin isoform during myofibrillogenesis. Since filamin c also binds γ- and δ-sarcoglycan at the sarcolemma (Thompson et al. Thompson et al., 2000Thompson TG Chan YM Hack AA Brosius M Rajala M Lidov HG McNally EM Watkins S Kunkel LM Filamin 2 (FLN2): a muscle-specific sarcoglycan interacting protein.J Cell Biol. 2000; 148: 115-126Crossref PubMed Scopus (224) Google Scholar), it was hypothesized that filamin c is involved in signaling pathways from the sarcolemma to the myofibril (Thompson et al. Thompson et al., 2000Thompson TG Chan YM Hack AA Brosius M Rajala M Lidov HG McNally EM Watkins S Kunkel LM Filamin 2 (FLN2): a muscle-specific sarcoglycan interacting protein.J Cell Biol. 2000; 148: 115-126Crossref PubMed Scopus (224) Google Scholar; van der Ven et al. van der Ven et al., 2000bvan der Ven PFM Wiesner S Salmikangas P Auerbach D Himmel M Kempa S Hayess K Pacholsky D Taivainen A Schröder R Carpén O Fürst DO Indications for a novel muscular dystrophy pathway: γ-filamin, the muscle-specific filamin isoform, interacts with myotilin.J Cell Biol. 2000; 151: 235-248Crossref PubMed Scopus (159) Google Scholar). The W2710X mutation in the reported family with MFM leads to a truncation of the filamin c immunoglobulin domain that is responsible for dimerization (Himmel et al. Himmel et al., 2003Himmel M van der Ven PFM Stöcklein W Fürst DO The limits of promiscuity: isoform-specific dimerization of filamins.Biochemistry. 2003; 42: 430-439Crossref PubMed Scopus (51) Google Scholar). To assess the pathogenic potential of the mutation, we examined its effects on the secondary structure of the mutant domain 24 and on its ability to dimerize. cDNA fragments encoding the wild-type and mutant domain 24 were cloned in pET23-T7 (Obermann et al. Obermann et al., 1998Obermann WMJ van der Ven PFM Steiner F Weber K Fürst DO Mapping of a myosin-binding domain and a regulatory phosphorylation site in M-protein, a structural protein of the sarcomeric M band.Mol Biol Cell. 1998; 9: 829-840Crossref PubMed Scopus (61) Google Scholar), and recombinant proteins were expressed in and purified from Escherichia coli BL21-CodonPlus-RP cells by use of standard procedures (Himmel et al. Himmel et al., 2003Himmel M van der Ven PFM Stöcklein W Fürst DO The limits of promiscuity: isoform-specific dimerization of filamins.Biochemistry. 2003; 42: 430-439Crossref PubMed Scopus (51) Google Scholar). Circular dichroism spectra were recorded from both proteins on a Jasco J-715 spectropolarimeter. Whereas analysis of the wild-type domain resulted in the typical β-sheet secondary structure that was previously reported for this domain (Himmel et al. Himmel et al., 2003Himmel M van der Ven PFM Stöcklein W Fürst DO The limits of promiscuity: isoform-specific dimerization of filamins.Biochemistry. 2003; 42: 430-439Crossref PubMed Scopus (51) Google Scholar; Pudas et al. Pudas et al., 2005Pudas R Kiema TR Butler PJ Stewart M Ylanne J Structural basis for vertebrate filamin dimerization.Structure. 2005; 13: 111-119Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), analysis of the truncated, mutated domain resulted in significantly lower signals, suggesting improper folding (fig. 3A). We next examined dimerization of mutant filamin c by chemical cross-linking experiments, essentially using previously established conditions for the wild-type protein (Himmel et al. Himmel et al., 2003Himmel M van der Ven PFM Stöcklein W Fürst DO The limits of promiscuity: isoform-specific dimerization of filamins.Biochemistry. 2003; 42: 430-439Crossref PubMed Scopus (51) Google Scholar), except that PBS was used instead of cross-linking buffer. Whereas the dimerization of the wild-type variant was reproduced under these conditions, cross-linking of the truncated domain 24 did not result in the detection of dimers. Instead, high–molecular mass aggregates of the mutated filamin fragment were observed on the SDS-polyacrylamide gel (fig. 3B) and on immunoblots (not shown). Although the precise structural changes that occur in the mutated filamin c are currently unknown, the formation of aggregates in the cross-linking assay and the tendency to precipitate in solution (not shown) imply a weaker stability of mutated filamin c in comparison with the wild-type protein and indicate a strong tendency for uncontrolled aggregation rather than defined dimerization. We have provided evidence of an altered distribution of myotilin and the dystrophin-sarcoglycan complex in affected individuals. We hypothesize that the fine balance of filamin c and its binding partners in both the Z-disk and the cell membrane may be disturbed. Therefore, defects in this protein may weaken myofibrils and, at the same time, destabilize the muscle cell membrane. This may ultimately result in clinical and molecular features resembling both MFM and LGMD. In summary, we describe here the first disease-related mutation within the FLNC gene and also the first mutation in a dimerization domain in the filamin family. The mutation results in the inability of the mutant protein to dimerize and leads to the generation of large filamin c–containing aggregates within the skeletal-muscle fibers. Biochemical experiments provide an explanation for this observation and show that dimer formation is essential for the biological function of filamin. We thank the family members for participation in this study; A. Hartmann-Schreiner, M. Fey, P. Mitzscherling, and G. Nürnberg, for technical assistance; and A. Freiberg and R. Seckler, for help with the circular dichroism spectrometry and for helpful discussions. M.V. and R.A.K. are supported by the Scientific Committee of the Kliniken Bergmannsheil Bochum and by the FoRUM program of the Ruhr-University Bochum. A.H., V.B., and K.K. were supported by the MeDDrive program of the Medical Faculty of Technical University Dresden. M.V., H.L., D.O.F., and A.H. are members of the German Network on Muscular Dystrophies (MD-NET), which is supported by the Bundesministerium für Forschung und Bildung (01GM0302).