Functional characterization of the novel DES mutation p.L136P ...

Journal of Molecular and Cellular Cardiology 91 (2016) 207–214

Contents lists available at ScienceDirect

Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc

Original article

Functional characterization of the novel DES mutation p.L136P associated with dilated cardiomyopathy reveals a dominant filament assembly defect Andreas Brodehl a,b,⁎, Mareike Dieding c, Niklas Biere c, Andreas Unger d, Bärbel Klauke a, Volker Walhorn c, Jan Gummert a, Uwe Schulz a, Wolfgang A. Linke d, Brenda Gerull b, Matthias Vorgert e, Dario Anselmetti c, Hendrik Milting a,⁎⁎ a

Erich and Hanna Klessmann Institute for Cardiovascular Research & Development (EHKI), Heart and Diabetes Center NRW, Ruhr University Bochum, D-32545 Bad Oeynhausen, Germany Libin Cardiovascular Institute of Alberta, Department of Cardiac Sciences, University of Calgary, 3280 Hospital Drive NW, T2N4Z6 Calgary, AB, Canada c Experimental Biophysics and Applied Nanoscience, Faculty of Physics and Bielefeld Institute for Biophysics and Nanoscience (BINAS), Bielefeld University, D-33615 Bielefeld, Germany d Department of Cardiovascular Physiology, Ruhr University Bochum, D-44780 Bochum, Germany e Neurologische Klinik und Poliklinik, Universitätsklinikum Bergmannsheil, Buerkle-de-la-Camp-Platz 1, D-44789 Bochum, Germany b

a r t i c l e

i n f o

Article history: Received 17 May 2015 Received in revised form 11 December 2015 Accepted 19 December 2015 Available online 23 December 2015 Keywords: Desmin Intermediate filaments Dilated cardiomyopathy Myofibrillar myopathy Desmosomes

a b s t r a c t Background: Dilated cardiomyopathy (DCM) could be caused by mutations in more than 40 different genes. However, the pathogenic impact of specific mutations is in most cases unknown complicating the genetic counseling of affected families. Therefore, functional studies could contribute to distinguish pathogenic mutations and benign variants. Here, we present a novel heterozygous DES missense variant (c.407C N T; p.L136P) identified by next generation sequencing in a DCM patient. DES encodes the cardiac intermediate filament protein desmin, which has important functions in mechanical stabilization and linkage of the cell structures in cardiomyocytes. Methods and results: Cell transfection experiments and assembly assays of recombinant desmin in combination with atomic force microscopy were used to investigate the impact of this novel DES variant on filament formation. Desmin-p.L136P forms cytoplasmic aggregates indicating a severe intrinsic filament assembly defect of this mutant. Co-transfection experiments of wild-type and mutant desmin conjugated to different fluorescence proteins revealed a dominant affect of this mutant on filament assembly. These experiments were complemented by apertureless scanning near-field optical microscopy. Conclusion: In vitro analysis demonstrated that desmin-p.L136P is unable to form regular filaments and accumulate instead within the cytoplasm. Therefore, we classified DES-p.L136P as a likely pathogenic mutation. In conclusion, the functional characterization of DES-p.L136P might have relevance for the genetic counseling of affected families with similar DES mutations and could contribute to distinguish pathogenic mutations from benign rare variants. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Dilated cardiomyopathy (DCM) has a heterogeneous etiology and is the most frequent cause for heart transplantation (HTx). Since the next generation sequencing revolution there is increasing evidence that genetic factors cause different cardiomyopathies [1,2]. ⁎ Correspondence to: A. Brodehl, Libin Cardiovascular Institute of Alberta, Department of Cardiac Sciences, University of Calgary, 3280 Hospital Drive NW, T2N4Z6 Calgary, AB, Canada. ⁎⁎ Correspondence to: H. Milting, E. & H. Klessmann Institute for Cardiovascular Research & Development (EHKI), Heart and Diabetes Center NRW, Ruhr-University Bochum, D32545 Bad Oeynhausen, Germany. E-mail addresses: [email protected] (A. Brodehl), [email protected] (H. Milting).

http://dx.doi.org/10.1016/j.yjmcc.2015.12.015 0022-2828/© 2016 Elsevier Ltd. All rights reserved.

Mutations in genes encoding mainly for sarcomeric or cytoskeletal proteins were identified in DCM patients (for a review see [3]). Recently, a prominent role of the TTN-locus was found in approximately 25% of the DCM patients [4,5]. Nevertheless, the high number of additional DCM-causing genes complicates routine genetic diagnostics. In addition, the analysis of whole exome data revealed that a significant portion of previously reported mutations are indeed not pathogenic [6]. However, functional data on specific mutations, which might support genetic counseling are lacking in most cases. Thus, the interpretation of novel and known variants for their pathogenic impact is currently challenging and more functional insights are necessary to distinguish pathogenic mutations from benign rare genetic variants. In this study, we identified with a next generation sequencing (NGS) panel a novel DES variant (c.407C N T, p.L136P) in a DCM-patient and

208

A. Brodehl et al. / Journal of Molecular and Cellular Cardiology 91 (2016) 207–214

characterized in vitro the deleterious effect of this mutant on desmin filament assembly. DES encodes the intermediate filament protein desmin, which is expressed in cardiac and skeletal muscles. Desmin filaments have stabilizing functions and form scaffolds to connect the cytoskeleton to different cellular structures like desmosomes [7], Z-bands [8], mitochondria [9], costameres [10] and presumably also to the nuclei [11]. The Des knock-out mice are viable and after birth develop DCM characterized by a disarray of contractile filaments [12], abnormal localization of desmin associated proteins [13] and apoptosis leading to the degeneration of cardiomyocytes [14,15]. Recently, Capetanaki et al. described that the complement system is activated in the Des knock-out mice [16]. The Des-p.R349P knock-in mouse develops also DCM associated with arrhythmias and conduction defects [17]. However, in human DES mutation carriers the clinical phenotypes are highly variable even within the same family [18–21]. Here, we identified and characterized a novel DES variant in a DCM patient. The in vitro analysis using cell transfection experiments and atomic force microscopy (AFM) demonstrated that mutant desminp.L136P cannot form extended filaments. These experiments were supported by molecular modeling and immunohistochemistry of explanted human heart tissue of the patient. Furthermore, apertureless scanning near-field optical microscopy (aSNOM) and confocal microscopy were used to investigate the co-assembly of mutant and wild-type recombinant desmin simulating the heterozygous genotype. 2. Materials and methods 2.1. Clinical description of the patient The Caucasian patient (Fig. 1A, III:2) was transplanted due to DCM aged 48 years. Coronary artery disease was excluded by angiography. He had at the time of heart transplantation (HTx) biventricular dilatation with global akinesia (left/right ventricular end-diastolic diameter, LVEDD/RVEDD: 65/45 mm, left ventricular ejection fraction (LVEF) of 36%). There were no signs of a skeletal muscle involvement in patient III:2. He reported no specific neuromuscular symptoms before his HTx. At the age of 59 years the neurological examination revealed no generalized or focal skeletal muscle atrophies or muscle weakness. The mild sensory abnormalities of a reduced vibration sense in his feet were attributed to a mild diabetic neuropathy. CK value was within normal limits. He received his first cardiac diagnosis aged 36 years while being in military service. At that time the patient had signs of a sick sinus syndrome and a mildly dilated LV (LVEDD = 56 mm) and borderline RV. The patient reported that two cousins of his mother died due to sudden cardiac deaths (SCD), one of those during a soccer game (Fig. 1A, II:7 and II:9). Since in these SCD victims no cardiac disease was known before death and the onset of symptoms were reported to be below one hour, these cousins fulfilled the criteria of SCD [50]. The mother (Fig. 1A, II:2) of the index patient was cardiologically examined by echocardiography and electrocardiogram (ECG). She had a tricuspid and mitral valve insufficiency grade II but apparently no signs of a cardiomyopathy. The son of the index patient (IV:1) was followed-up and echocardiography excluded a dilated phenotype in this proband.

washington.edu/EVS/, May-08-2015) and with Exome Aggregation Consortium (ExAC), Cambridge, USA (ref. http://exac.broadinstitute.org, May-08-2015). The consent of all probands was obtained and the study was approved by the local ethics committees (Ruhr University Bochum, Bad Oeynhausen, Germany, Reg.-No. 27.1/2011). 2.3. Molecular modeling Recently, the atomic structure of the coil 1 fragment of the homologous protein vimentin was described [22]. Since the desmin subdomain containing p.L136P is highly homologous to the vimentin sequence, we used publicly available structural data of vimentin coil 1 (PDB ID: 3S4R; ref. http://www.rcsb.org/pdb/) as a template to model the desmin mutation. ‘PyMOL Molecular Graphics System, version 1.3 Schrödinger, LLC’ was used to replace the different amino acids in the vimentin sequence against the corresponding amino acids of desmin [23]. 2.4. Plasmid generation The plasmids pET100D-Desmin-WT and pEYFP-N1-Desmin-WT were described previously [24,25]. The ‘QuickChange Lightning Kit’ (Agilent Technologies, Santa Clara, USA) was used to insert the variants into these plasmids using appropriate primers. PCR was used to fuse XhoI and AgeI to the 5′ or 3′ ends of the mRuby cDNA. Afterwards it was cloned by TOPO-TA-cloning into pCRII-TOPO vector (Life Technologies) and the EYFP sequence was replaced via XhoI and AgeI against the mRuby cDNA. All plasmids were sequenced. 2.5. Cell culture SW-13, HEK293, HeLa, H9c2 and C2C12 cells were cultured according to the information of ATCC (ref. www.atcc.org). The HL-1 cells were cultured in Claycomb medium (Sigma-Aldrich, St. Louis, USA) supplemented with 10% FCS, 2 mmol/L L-Glutamine, 100 nmol/L Norepinephrine and Penicillin/Streptomycin [26]. Lipofectamin 2000 (Life Technologies) was used to transfect the cells. For co-transfection experiments of wild-type and mutant desmin the same amount of plasmids was used. The used expression constructs are based on the same promoter for wild-type and mutant desmin. 2.6. Immunohistochemistry Myocardial left ventricular heart slides (5 μm) were deparaffinized and rehydrated as previously described [27]. The samples were blocked with 5% bovine serum albumin for 1 h at room temperature. Afterwards the samples were incubated with rabbit anti-desmin antibodies over night at 4 °C (1:200, ab15200, Abcam, Cambridge, UK). After washing with phosphate buffered saline (PBS) the slides were incubated with donkey anti-rabbit-IgG conjugated with Alexa Fluor 555 for 1 h at room temperature (1:100, Life Technologies) and embedded in ProLong Gold antifade reagent (Life Technologies). 2.7. Confocal fluorescence microscopy

2.2. DNA sequencing The illustra blood genomicPrep Mini Spin Kit (GE Healthcare, Chalfont St. Giles, UK) was used to isolate and purify the genomic DNA of the patients. For sequencing the cardiac NGS panel TruSight™ Cardiomyopathy (Illumina Inc., San Diego, USA, 46 genes) was used. Verification of NGS data was done by conventional Sanger sequencing using the BigDye® Terminator v1.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, USA) and an ABI PRISM® 310 Genetic Analyzer (Life Technologies). The allele frequency of this DES variant was compared with the NHLBI Exome Sequencing Project (ESP, ref. http://evs.gs.

The cells were grown and transfected on coverslips. 24 h after transfection the cells were washed three times with PBS, fixed with methanol (−20 °C, 5 min) and embedded with ‘ProLong Gold antifade reagent’. The ‘LSM 5 Exciter’ (Carl Zeiss Microscopy, Oberkochen, Germany) was used for the confocal analysis. 4′,6-diamidino-2-phenylindole (DAPI) was excited at 405 nm and the emission was detected in a range between 420 and 480 nm. EYFP was excited at 488 nm and the emission was detected at 505–550 nm. The red fluorescence protein mRuby and Alexa Fluor 555 were excited at a wavelength of 543 nm and the emission was detected in the range between 560 and 615 nm.


209

Fig. 1. Identification and verification of the DES mutation c.407 T N C, p.L136P. (A) Pedigree of the family. Squares represent males and circles females. Deceased individuals are indicated by slashes. The index patient is marked with an arrow. Genotypes are shown by present (+) or by absent (−) of the heterozygous DES mutation. (B) Electropherogram showing the heterozygous mutation c.407 T N C, p.L136P. Converted codons lead to the amino acid exchange p.L136P. (C) Alignment of the DES sequences from different species. The heptad sequence is a typical feature of intermediate filaments proteins. Hydrophobic amino acids at the ‘a’ and ‘d’ positions (heptad sequence) form a hydrophobic seam. The heptad sequence is highlighted in yellow and the position of the novel mutation p.L136P is marked in red. (D–F) Molecular modeling of the DES-mutation p.L136P. The introduction of a proline residue is predicted to disrupt the alpha-helix of the desmin monomer, since proline is not able to form the axial hydrogen bond.

2.8. Desmin expression, purification and assembly The expression and purification of recombinant desmin was done as described earlier [28]. A stepwise dialysis was used to decrease the urea

concentration. Filament formation of recombinant desmin was initiated by addition of sodium chloride buffer (200 mmol/L NaCl, 45 mmol/L Tris–HCl, pH 7.0) and subsequent heating to 37 °C for 1 h as previously described [29].

210


2.9. Atomic force microscopy and apertureless scanning near-field optical microscopy AFM and aSNOM are scanning probe techniques. For aSNOM measurements recombinant desmin-p.L136P molecules were conjugated with Atto740 fluorescent dyes as reported recently [30]. Notably, the degree of labeling (DOL) of desmin monomers should be low to ensure unhindered filament assembly. For this reason and to allow discrimination between both desmin species labeled desmin-p.L136P was coassembled with wild-type desmin at a 1:10 M ratio. For AFM imaging both mutant and wild-type desmin were additionally assembled separately. The assembled desmin species (150 μg/mL) were immobilized on freshly cleaved mica substrates (Plano, Wetzlar, Germany) in case of AFM imaging and on etched and cleaned glass cover slips for aSNOM measurements. Subsequently, the samples were rinsed with deionized water and dried under a gentle flow of nitrogen. Topographic AFM imaging was done with a Multimode AFM and Nanoscope IIIa controller (Bruker, Santa Barbara, USA) whereas aSNOM measurements were performed on a self-made setup as previously reported [30]. All experiments were done under ambient conditions. 2.10. Transmission electron microscopy (TEM) Human heart samples were fixed in PBS supplemented with 4% paraformaldehyde, 1% glutaraldehyde, and 15% picric acid, pH 7.4, at 4 °C overnight. Tissue blocks were cut into 50 μm longitudinal sections using a VT 1000S Leica vibratome (Mannheim, Germany), before rinsing twice in PBS. Following treatment with 0.5% OsO4 for 45 min and several washing steps in 100 mM PBS, samples were counterstained with uranyl acetate, dehydrated via ethanol series, and embedded in Durcupan ACM epoxy resin (Fluka, Switzerland). Ultrathin sections were prepared from resin blocks using a Leica Ultracut S (Mannheim, Germany) and adsorbed to glow-discharged formvar-carbon-coated copper single slot grids. Electron micrographs were recorded using a Zeiss LEO 910 electron microscope; images were taken with a TRS sharpeye CCD camera (Troendle, Moorenwies, Germany). 3. Results 3.1. Genetic analysis Using a cardiac specific NGS gene panel (see supplement for gene lists) we sequenced several genes and identified DES c.407C N T, p.L136P in a German patient (III:2, Fig. 1A, B). Other pathogenic mutations in the sequenced genes could be excluded based on the minor allele frequency of these variants in healthy controls (see Table S1 in the supplements).

The genetic analysis within the German family revealed that the unaffected son (IV:1, Fig. 1A) was wild-type for DES. In the history of the mother's (II:2, Fig. 1A) family two male cousins with an unknown genotype (II:7, II:9, Fig. 1A) died from SCD. The DES mutation p.L136P is highly conserved within several species (Fig. 1C). p.L136 is even conserved in the intermediate filament protein lamin of hydra vulgaris (Fig. 1C). It is localized within the α-helix of the coil-1 region (Fig. S1). We used seven different bioinformatics tools to predict the impact of this missense mutation (see Table S1). All bioinformatics algorithms predicted that DES p.L136P is probably damaging. The exchange of c.407 T N C causes the incorporation of a proline at position 136. This proline at the d-position within the heptad repeat (Fig. 1C) is predicted to destabilize the desmin molecule by the loss of a stabilizing hydrogen bond within the backbone of the α-helix (Fig. 1D–F).

3.2. Functional characterization of desmin-p.L136P To prove the hypothesis that the protein folding and the assembly into intermediate filaments of desmin are affected by p.L136P we expressed and purified recombinant wild-type and mutant desmin and analyzed the filament assembly by AFM. These experiments revealed that the recombinant desmin-p.L136P accumulated as small protofilaments (Fig. 2A). Also a mixture of wild-type and mutant desmin demonstrated this filament assembly defect (Fig. 2B). As expected, the recombinant wild-type desmin formed in comparison regular intermediate filaments of different lengths (Fig. 2C). Furthermore, we expressed EYFP-tagged versions of desmin in several different transiently transfected cell lines. In all cell lines ranging from SW-13 cells to HL-1 cardiomyocytes the wild-type desmin formed filamentous networks (Fig. 3). In comparison, the mutant desmin-p.L136P accumulated within the cytoplasm (Fig. 3). Because of the heterozygosity of the patient we addressed the question if desmin-p.L136P had a dominant negative effect on filament assembly. Therefore, we performed co-transfection experiments fusing wild-type and mutant desmin to different fluorescent proteins. In addition, we used aSNOM to analyze the co-assembly of recombinant wildtype and mutant desmin. The cell culture experiments as well as the aSNOM analysis of recombinant desmin molecules revealed coaccumulation of mutant and wild-type desmin indicating a dominant impact on the in vitro filament assembly (Fig. 4). The immunohistochemistry of left ventricular myocardial tissue demonstrated also an irregular desmin staining of the heterozygous DES-p.L136P patient (III:2) at the Z-bands in comparison to the control samples (Fig. 5). The Z-bands were disorganized and partially degraded in comparison to the control samples (Fig. 5). The intercalated disk localization of desmin was not affected. Furthermore, we verified these disruptive effects in explanted human left ventricular tissue of the index patient by transmission electron microscopy (Fig. S3).

Fig. 2. Filament formation of recombinant mutant desmin. Representative atomic force microscopy topographic images of desmin-p.L136P (A), desmin wild-type:desmin-p.L136P coassembled in a molar ratio of 10:1 (B) and desmin wild-type (C). Recombinant desmin molecules were purified and assembled in vitro by addition of sodium chloride. Of note, in comparison to the control experiment with wild-type desmin, the mutant desmin-p.L136P does not form filaments and inhibited even the co-assembly of both forms indicating an intrinsic protein folding defect.


211

4. Discussion

Fig. 3. Impairment of filament formation by desmin mutants in transfected cells. Representative fluorescence images of transfected HEK293, HeLa, SW13, C2C12, H9c2 and HL-1 cells expressing desmin-eYFP constructs (yellow). The nuclei were stained with DAPI (blue). Scale bars represent 10 μm.

During the last 15 years it was shown that several DES mutations cause different cardiac and skeletal myopathies [21,24,31–33]. Most of the described patients present a combination of cardiac and skeletal phenotypes [20]. More then 20% of the patients with a DES mutation present isolated cardiac symptoms without involvement of the skeletal muscle [20]. However, the observed phenotypes could vary even within the same family [18] and include DCM [34], hypertrophic cardiomyopathy (HCM, [35]), restrictive cardiomyopathy (RCM, [36]), arrhythmogenic cardiomyopathy (AC, [24,37]) and overlapping borderline cardiomyopathies [21]. The molecular reasons for the development of different phenotypes in the heart and skeletal muscle and the different expressivity even within members of the same family are unknown. However, because desmin filaments connect in addition to costameres and Z-band in the cardiomyocytes the desmosomes with the cytoskeleton it could be speculated if these structural differences between cardiac and skeletal myocytes contribute to the different effects of DES mutations in both organs. Furthermore, muscle satellite cells are present in skeletal muscle renewing the damaged skeletal muscle [52]. In contrast the regeneration capacity of mammalian cardiac tissue is pretty low [53] which could explain that the described proband carrying DES-p.L136P had a cardiac phenotype without involvement of the skeletal muscle. Especially, in the age of NGS the genetic analysis becomes an increasingly powerful tool for the clinical routine diagnosis of cardiomyopathies [2]. Nevertheless, the abundance of different genes involved in the pathomechanisms of cardiomyopathies complicates the interpretation and analysis of specific variants. In addition, Andreasen et al. demonstrated convincingly that a significant portion of earlier reported variants are more likely rare benign single nucleotide polymorphisms (SNP) rather than pathogenic mutations [6]. Recently, Kostareva et al. demonstrated that DES-p.A213V represents such a rare benign SNP [38]. In this context, functional analysis which is lacking for the vast majority of putative pathogenic genetic variants might contribute to their classification and interpretation. Here, we identified and characterized the novel DES variant p.L136P, which is absent in healthy control people. DES-p.L136P was neither reported in the ESP [13.006 control chromosomes] nor in the ExAC samples [122.972 control chromosomes]. The genetic family analysis revealed that the son (IV:1) of the index patient, who has no cardiac phenotype, is wild-type for the DES gene. However, in the family branch of the mother, two male cousins (II:7 and II:9) died from SCD. Surprisingly, the mother of the patient, who is also a carrier of DES-p.L136P, did not present an apparent cardiac phenotype suggesting an incomplete penetrance. This is in good agreement with earlier reports describing also an incomplete penetrance and heterogeneous expressivity of other DES mutations [39,40]. However, it remains unclear which genetic, epigenetic or environmental modifiers contribute to the incomplete penetrance in desminopathies. Recently, Meyer et al. reported that gender effects might have an impact on the development and progression of cardiomyopathies in general [41] and it could be speculated that this is also the case for this DES mutation. Desmin is a member of the intermediate filament protein family and mutations in the homologous genes LMNA and GFAP are known to cause also cardiac or neurological diseases. Interestingly, McPherson et al. published a DCM associated missense mutation (p.L59R) in the LMNA gene at the corresponding position [42]. Furthermore, Meins et al. and Li et al. identified at the corresponding position in GFAP encoding the glial fibrillary acidic protein the missense mutation p.L97P leading to Alexander disease [43,44]. During the preparation of this manuscript Pugh et al. reported also a DES missense variant c.407 T N A leading to the amino acid exchange p.L136H [2]. Because of the lack of functional data the authors classified DES-p.L136H as a GVUS [2]. These reports indicate the importance of this leucine residue for the intermediate

212


Fig. 4. Co-transfection and co-assembly experiments of wild-type desmin and desmin-p.L136P. (A–C) Representative fluorescence images of co-transfected H9c2 cells expressing (A) desmin-WT-mRuby and (B) desmin-p.L136P-eYFP and the corresponding overlay (C). For these co-transfection experiments the same amount of wild-type and mutant desmin expression constructs was used. Scale bars represent 10 μm. (D) Histogram of the fluorescence intensities along the red line in image (C, asterisk). (E–G) Representative aSNOM data derived from fluorescence labeled desmin-p.L136P co-assembled with wild-type desmin in a 1:10 molar ratio. The topography data (E) exposes short (b1 μm) fibrils and protofilaments (color scale 30 nm). (F) Super resolution fluorescence data shows sporadically distributed sharp fluorescence peaks that are derived from raw fluorescence data (color scale 2000 counts s−1). (G) Superposition of topography and fluorescence reveals the strict co-localization of fluorescence peaks with desmin filaments and accumulated desmin indicating the co-assembly of desmin-p.L136P (arrowheads). The size of aSNOM images =10 × 10 μm2. (H) Height and (unfiltered) fluorescence intensity cross sections extracted from (G, asterisk) showing two well separated fluorescence peaks (red) that are congruent with the desmin topography (black). The width of either peak is in the range of 50 nm (full width at half maximum). Of note, the cell culture experiments as well as the co-assembly experiments using recombinant wild-type and mutant desmin demonstrate the dominant effect on filament assembly even if the relative concentrations of mutant desmin were low.

Fig. 5. Immunohistochemical analysis of left ventricular myocardial tissue. Three randomly selected areas from the heterozygous DES-p.L136P patient (A–C) and of a control sample (D–F) were shown. Disorganized and blurry desmin staining at the Z-bands was prominent in the DES-p.L136P patient but not in the control sample. The localization of desmin at the intercalated disk seems comparable in both samples.


filament assembly in general and are in good agreement with our results specific for DES-p.L136P. The variant c.407 T N C introduces a proline residue at position 136 within the α-helix of coil 1 of desmin. Proline introduces an imino group decreasing the stability of α-helices by preventing hydrogen bonding within the protein's backbone (see Fig. 1D–F). Several other pathogenic missense mutations leading to an incorporation of a proline residue were previously described in the coil-2 region of desmin [34,45, 46]. However, our report demonstrates that also proline mutations within coil 1 could cause a severe filament assembly defect. To further establish the pathogenic impact of both DES variants, we performed in vitro cell culture experiments and AFM analysis of recombinant proteins. We used HL-1, H9c2 and C2C12 cell lines, because these cell lines express endogenous desmin. Furthermore, we used HEK293 and HeLa cells because these cells do not express endogenous desmin but vimentin — a homologous cytoplasmic intermediate filament protein. SW13 cells were used, because this cell line does not express any cytoplasmic intermediate filament protein. Desmin wild-type forms filaments and filamentous networks, independently of the cell line. In contrast, mutant desmin-p.L136P accumulates in the cytoplasm similar to other desmin mutants [21,28]. We proved by Western blot analysis that the recombinant mutant and wild-type desmin are expressed in the transfected cells at a comparable level (Fig. S2). Therefore, we could exclude that a higher expression level of the mutant desmin is the cause for the observed protein aggregation. Clemen et al. demonstrated recently that the expression level of another in-frame heterozygous DES deletion mutation in different human patients can be highly complex and variable [51]. The high inter-individual variability could be an explanation for the absence or different severity of the phenotypes in different mutation carriers. The analysis of recombinant desmin molecules by AFM verified this observation indicating also a severe intrinsic assembly defect of desmin-p.L136P. Most of the described DES mutations are heterozygous missense mutations. Only some homozygous or compound heterozygous DES mutations are currently described [31,47,48]. This raises the question whether the heterozygous mutation p.L136P has a dominant negative impact on desmin filament assembly. Therefore, we performed co-transfection experiments using two different fluorescence tags. In addition, we assembled in vitro mutant desmin-p.L136P conjugated with a fluorescence dye together with unconjugated wild-type desmin and performed an aSNOM analysis. The cell culture experiments as well as the aSNOM experiments revealed that wild-type desmin and mutant desmin-p.L136P co-aggregate indicating a dominant effect on filament assembly of desmin-p.L136P as earlier described for desmin-p.E114del and partially for -p.N116S [28]. In summary, we present here functional data which justify the classification of DES-p.L136P according to the criteria of the ‘American College of Medical Genetics and Genomis’ (ACMG, 49) as a ‘likely pathogenic’ DCM associated mutation. The functional characterization might have relevance for the genetic counseling of families with DES mutations.

5. Limitations and genetic classification Because of the complexity of human cardiomyopathies we cannot completely exclude other genetic, epigenetic or environmental cofactors contributing to the clinical phenotype in our patient. However, according to guidelines and standards for the classification and interpretation of human genetic sequence variations of the ‘American College of Medical Genetics and Genomics’ [49] DES-p.L136P fulfills clearly three criteria for evidence of pathogenicity but no criteria for benign impact justifying the classification as ‘likely pathogenic’. • PS3: Our functional studies support a damaging effect of DES-p.L136P (strong criterion). • PM2: DES-p.L136P is absent in controls (moderate criterion)

213

(1) ExAc Browser (http://exac.broadinstitute.org, absent in 60,706 control people, Dec.-01-2015). (2) NHLBI Exome Sequencing Project (ESP), Exome Variant Server (http://evs.gs.washington.edu/EVS/ absent in 6503 control people, Dec.-01-2015). • PP3: Computational evidence supports the deleterious effect of DESp.L136P (supportive criterion).

Disclosure None. Funding sources This study was kindly funded by a grant from the Deutsche Forschungsgemeinschaft MI 1146/2-1 to D.A. & H.M. and the Erich and Hanna Klessmann Foundation, Gütersloh, Germany to H.M. Acknowledgments We would like to thank all participating patients for their continuous help and support of this research project. The authors are grateful to Ramona Cebulla and Désireé Gerdes for excellent technical assistance. In addition, we thank Dr. William Claycomb (New Orleans, USA) for providing the HL-1 cardiomyocytes and Dr. Gerd Ulrich Nienhaus (Karlsruhe Institute of Technology, Germany) for providing the cDNA of mRuby. The authors would like to thank the Exome Aggregation Consortium and the groups that provided exome variant data for comparison. A full list of contributing groups can be found at http://exac. broadinstitute.org/about. Furthermore, we thank Dr. Vanessa French (University of Calgary, Canada) for critical reading and discussion of the manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.yjmcc.2015.12.015. References [1] B. Gerull, M. Gramlich, J. Atherton, M. McNabb, K. Trombitás, S. Sasse-Klaassen, J.G. Seidman, C. Seidman, H. Granzier, S. Labeit, et al., Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy, Nat. Genet. 30 (2002) 201–204. [2] T.J. Pugh, M.A. Kelly, S. Gowrisankar, E. Hynes, M.A. Seidman, S.M. Baxter, M. Bowser, B. Harrison, D. Aaron, L.M. Mahanta, et al., The landscape of genetic variation in dilated cardiomyopathy as surveyed by clinical DNA sequencing, Genet. Med. (2014). [3] J.A. Towbin, Inherited cardiomyopathies, Circ. J. 78 (2014) 2347–2356. [4] D.S. Herman, L. Lam, M.R.G. Taylor, L. Wang, P. Teekakirikul, D. Christodoulou, L. Conner, S.R. DePalma, B. McDonough, E. Sparks, et al., Truncations of titin causing dilated cardiomyopathy, N. Engl. J. Med. 366 (2012) 619–628. [5] N. Norton, D. Li, E. Rampersaud, A. Morales, E.R. Martin, S. Zuchner, S. Guo, M. Gonzalez, D.J. Hedges, P.D. Robertson, et al., Exome sequencing and genome-wide linkage analysis in 17 families illustrate the complex contribution of TTN truncating variants to dilated cardiomyopathy, Circ. Cardiovasc. Genet. 6 (2013) 144–153. [6] C. Andreasen, J.B. Nielsen, L. Refsgaard, A.G. Holst, A.H. Christensen, L. Andreasen, A. Sajadieh, S. Haunsø, J.H. Svendsen, M.S. Olesen, New population-based exome data are questioning the pathogenicity of previously cardiomyopathy-associated genetic variants, Eur. J. Hum. Genet. 21 (2013) 918–928. [7] J. Kartenbeck, W.W. Franke, J.G. Moser, U. Stoffels, Specific attachment of desmin filaments to desmosomal plaques in cardiac myocytes, EMBO J. 2 (1983) 735–742. [8] H.E. Osinska, L.F. Lemanski, Immunofluorescent localization of desmin and vimentin in developing cardiac muscle of Syrian hamster, Anat. Rec. 223 (1989) 406–413. [9] Y. Capetanaki, Desmin cytoskeleton: a potential regulator of muscle mitochondrial behavior and function, Trends Cardiovasc. Med. 12 (2002) 339–348. [10] P. Konieczny, P. Fuchs, S. Reipert, W.S. Kunz, A. Zeöld, I. Fischer, D. Paulin, R. Schröder, G. Wiche, Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms, J. Cell Biol. 181 (2008) 667–681. [11] C.S. Mermelstein, L.R. Andrade, D.M. Portilho, M.L. Costa, Desmin filaments are stably associated with the outer nuclear surface in chick myoblasts, Cell Tissue Res. 323 (2006) 351–357.

214


[12] Y. Capetanaki, D.J. Milner, G. Weitzer, Desmin in muscle formation and maintenance: knockouts and consequences, Cell Struct. Funct. 22 (1997) 103–116. [13] L. Carlsson, Z.L. Li, D. Paulin, M.G. Price, J. Breckler, R.M. Robson, G. Wiche, L.E. Thornell, Differences in the distribution of synemin, paranemin, and plectin in skeletal muscles of wild-type and desmin knock-out mice, Histochem. Cell Biol. 114 (2000) 39–47. [14] M. Lindén, Z. Li, D. Paulin, T. Gotow, J.F. Leterrier, Effects of desmin gene knockout on mice heart mitochondria, J. Bioenerg. Biomembr. 33 (2001) 333–341. [15] A.M. Sprinkart, W. Block, F. Träber, R. Meyer, D. Paulin, C.S. Clemen, R. Schröder, J. Gieseke, H. Schild, D. Thomas, Characterization of the failing murine heart in a desmin knock-out model using a clinical 3 T MRI scanner, Int. J. Card. Imaging 28 (2012) 1699–1705. [16] M. Mavroidis, C.H. Davos, S. Psarras, A.C. Varela, N. Athanasiadis, M. Katsimpoulas, I. Kostavasili, C. Maasch, A. Vater, J.P. van Tintelen, et al., Complement system modulation as a target for treatment of arrhythmogenic cardiomyopathy, Basic Res. Cardiol. 110 (2015) 485. [17] C.S. Clemen, F. Stöckigt, K.-H. Strucksberg, F. Chevessier, L. Winter, J. Schütz, R. Bauer, J.-M. Thorweihe, D. Wenzel, U. Schlötzer-Schrehardt, et al., The toxic effect of R350P mutant desmin in striated muscle of man and mouse, Acta Neuropathol. (2014). [18] J.E.H. Bergman, H.E. Veenstra-Knol, A.J. van Essen, C.M.A. van Ravenswaaij, W.F.A. den Dunnen, A. van den Wijngaard, J.P. van Tintelen, Two related Dutch families with a clinically variable presentation of cardioskeletal myopathy caused by a novel S13F mutation in the desmin gene, Eur. J. Med. Genet. 50 (2007) 355–366. [19] E. Otten, A. Asimaki, A. Maass, I.M. van Langen, A. van der Wal, N.d. Jonge, M.P. van den Berg, J.E. Saffitz, A.A.M. Wilde, J.D.H. Jongbloed, et al., Desmin mutations as a cause of right ventricular heart failure affect the intercalated disks, Heart Rhythm. 7 (2010) 1058–1064. [20] K.Y. van Spaendonck-Zwarts, L. van Hessem, J.D.H. Jongbloed, H.E.K. de Walle, Y. Capetanaki, A.J. van der Kooi, I.M. van Langen, M.P. van den Berg, J.P. van Tintelen, Desmin-related myopathy, Clin. Genet. 80 (2011) 354–366. [21] A. Brodehl, M. Dieding, B. Klauke, E. Dec, S. Madaan, T. Huang, J. Gargus, A. Fatima, T. Saric, H. Cakar, et al., The novel Desmin mutant p.A120D impairs filament formation, prevents intercalated disk localization and causes sudden cardiac death, Circ. Cardiovasc. Genet. (2013). [22] A.A. Chernyatina, S. Nicolet, U. Aebi, H. Herrmann, S.V. Strelkov, Atomic structure of the vimentin central α-helical domain and its implications for intermediate filament assembly, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 13620–13625. [23] E. Bramucci, A. Paiardini, F. Bossa, S. Pascarella, PyMod: sequence similarity searches, multiple sequence-structure alignments, and homology modeling within PyMOL, BMC Bioinf. 13 (Suppl. 4) (2012) S2. [24] B. Klauke, S. Kossmann, A. Gaertner, K. Brand, I. Stork, A. Brodehl, M. Dieding, V. Walhorn, D. Anselmetti, D. Gerdes, et al., De novo desmin-mutation N116S is associated with arrhythmogenic right ventricular cardiomyopathy, Hum. Mol. Genet. 19 (2010) 4595–4607. [25] A. Brodehl, M. Dieding, H. Cakar, B. Klauke, V. Walhorn, J. Gummert, D. Anselmetti, H. Milting, Functional characterization of desmin mutant p.P419S, Eur. J. Hum. Genet. 21 (2013) 589–590. [26] W.C. Claycomb, N.A. Lanson, B.S. Stallworth, D.B. Egeland, J.B. Delcarpio, A. Bahinski, N.J. Izzo, HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 2979–2984. [27] B. Gerull, F. Kirchner, J.X. Chong, J. Tagoe, K. Chandrasekharan, O. Strohm, D. Waggoner, C. Ober, H.J. Duff, Homozygous founder mutation in desmocollin-2 (DSC2) causes arrhythmogenic cardiomyopathy in the Hutterite population, Circ. Cardiovasc. Genet. 6 (2013) 327–336. [28] A. Brodehl, P.N. Hedde, M. Dieding, A. Fatima, V. Walhorn, S. Gayda, T. Šarić, B. Klauke, J. Gummert, D. Anselmetti, et al., Dual color photoactivation localization microscopy of cardiomyopathy-associated desmin mutants, J. Biol. Chem. 287 (2012) 16047–16057. [29] L. Kreplak, H. Bär, J.F. Leterrier, H. Herrmann, U. Aebi, Exploring the mechanical behavior of single intermediate filaments, J. Mol. Biol. 354 (2005) 569–577. [30] A. Harder, M. Dieding, V. Walhorn, S. Degenhard, A. Brodehl, C. Wege, H. Milting, D. Anselmetti, Apertureless scanning near-field optical microscopy of sparsely labeled tobacco mosaic viruses and the intermediate filament desmin, Beilstein J. Nanotechnol. 4 (2013) 510–516. [31] L.G. Goldfarb, K.Y. Park, L. Cervenáková, S. Gorokhova, H.S. Lee, O. Vasconcelos, J.W. Nagle, C. Semino-Mora, K. Sivakumar, M.C. Dalakas, Missense mutations in desmin associated with familial cardiac and skeletal myopathy, Nat. Genet. 19 (1998) 402–403. [32] A.M. Muñoz-Mármol, G. Strasser, M. Isamat, P.A. Coulombe, Y. Yang, X. Roca, E. Vela, J.L. Mate, J. Coll, M.T. Fernández-Figueras, et al., A dysfunctional desmin mutation in a patient with severe generalized myopathy, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 11312–11317.

[33] A. Nalini, N. Gayathri, P. Richard, A.-M. Cobo, J.A. Urtizberea, New mutation of the desmin gene identified in an extended Indian pedigree presenting with distal myopathy and cardiac disease, Neurol. India 61 (2013) 622–626. [34] M.R.G. Taylor, D. Slavov, L. Ku, A. Di Lenarda, G. Sinagra, E. Carniel, K. Haubold, M.M. Boucek, D. Ferguson, S.L. Graw, et al., Prevalence of desmin mutations in dilated cardiomyopathy, Circulation 115 (2007) 1244–1251. [35] M. Olivé, J. Armstrong, F. Miralles, A. Pou, M. Fardeau, L. Gonzalez, F. Martínez, D. Fischer, M. Matos, J. Antonio, A. Shatunov, et al., Phenotypic patterns of desminopathy associated with three novel mutations in the desmin gene, Neuromuscul. Disord. 17 (2007) 443–450. [36] S. Hager, H. Mahrholdt, L.G. Goldfarb, H.H. Goebel, U. Sechtem, Images in cardiovascular medicine. Giant right atrium in the setting of desmin-related restrictive cardiomyopathy, Circulation 113 (2006) e53–e55. [37] J.P. van Tintelen, I.C. van Gelder, A. Asimaki, A.J.H. Suurmeijer, A.C.P. Wiesfeld, J.D.H. Jongbloed, A. van den Wijngaard, J.B.M. Kuks, K.Y. van Spaendonck-Zwarts, N. Notermans, et al., Severe cardiac phenotype with right ventricular predominance in a large cohort of patients with a single missense mutation in the DES gene, Heart Rhythm. 6 (2009) 1574–1583. [38] A. Kostareva, G. Sjoberg, A. Gudkova, N. Smolina, E. Semernin, E. Shlyakhto, T. Sejersen, Desmin A213V substitution represents a rare polymorphism but not a mutation and is more prevalent in patients with heart dilation of various origins, Acta Myol. 30 (2011) 42–45. [39] D. Li, T. Tapscoft, O. Gonzalez, P.E. Burch, M.A. Quiñones, W.A. Zoghbi, R. Hill, L.L. Bachinski, D.L. Mann, R. Roberts, Desmin mutation responsible for idiopathic dilated cardiomyopathy, Circulation 100 (1999) 461–464. [40] H. Bär, M. Schopferer, S. Sharma, B. Hochstein, N. Mücke, H. Herrmann, N. Willenbacher, Mutations in desmin's carboxy-terminal “tail” domain severely modify filament and network mechanics, J. Mol. Biol. 397 (2010) 1188–1198. [41] S. Meyer, P. van der Meer, J.P. van Tintelen, M.P. van den Berg, Sex differences in cardiomyopathies, Eur. J. Heart Fail. (2014). [42] E. McPherson, L. Turner, I. Zador, K. Reynolds, D. Macgregor, P.F. Giampietro, Ovarian failure and dilated cardiomyopathy due to a novel lamin mutation, Am. J. Med. Genet. A 149A (2009) 567–572. [43] M. Meins, K. Brockmann, S. Yadav, M. Haupt, J. Sperner, U. Stephani, F. Hanefeld, Infantile Alexander disease: a GFAP mutation in monozygotic twins and novel mutations in two other patients, Neuropediatrics 33 (2002) 194–198. [44] R. Li, A.B. Johnson, G. Salomons, J.E. Goldman, S. Naidu, R. Quinlan, B. Cree, S.Z. Ruyle, B. Banwell, M. D'Hooghe, et al., Glial fibrillary acidic protein mutations in infantile, juvenile, and adult forms of Alexander disease, Ann. Neurol. 57 (2005) 310–326. [45] H. Bär, D. Fischer, B. Goudeau, R.A. Kley, C.S. Clemen, P. Vicart, H. Herrmann, M. Vorgerd, R. Schröder, Pathogenic effects of a novel heterozygous R350P desmin mutation on the assembly of desmin intermediate filaments in vivo and in vitro, Hum. Mol. Genet. 14 (2005) 1251–1260. [46] F. Ochsner, J. Lobrinus, X. Jeanrenaud, S. Rudnik-Schoenborn, T. Eggermann, M. Dunand, T. Kuntzer, C.P.4.03 Right ventricular arrhythmic cardiomyopathy with an autosomal dominant R355P DES gene mutation, Neuromuscul. Disord. 17 (2007) 879. [47] G. Piñol-Ripoll, A. Shatunov, A. Cabello, P. Larrodé, I. de la Puerta, J. Pelegrín, F.J. Ramos, M. Olivé, L.G. Goldfarb, Severe infantile-onset cardiomyopathy associated with a homozygous deletion in desmin, Neuromuscul. Disord. 19 (2009) 418–422. [48] N. Cetin, B. Balci-Hayta, H. Gundesli, P. Korkusuz, N. Purali, B. Talim, E. Tan, D. Selcen, S. Erdem-Ozdamar, P. Dincer, A novel desmin mutation leading to autosomal recessive limb-girdle muscular dystrophy: distinct histopathological outcomes compared with desminopathies, J. Med. Genet. 50 (2013) 437–443. [49] S. Richards, N. Aziz, S. Bale, D. Bick, S. Das, J. Gastier-Foster, W.W. Grody, M. Hegde, E. Lyon, E. Spector, et al., Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology, Genet. Med. 17 (2015) 405–424. [50] Z.-J. Zheng, J.B. Croft, W.H. Giles, G.A. Mensah, Sudden cardiac death in the United States, 1989 to 1998, Circulation 104 (2001) 2158–2163. [51] C.S. Clemen, D. Fischer, J. Reimann, L. Eichinger, C.R. Müller, H.D. Müller, H.H. Goebel, R. Schröder, How much mutant protein is needed to cause a protein aggregate myopathy in vivo? Lessons from an exceptional desminopathy, Hum. Mutat. 30 (2009) E490–E499. [52] N. Motohashi, A. Asakura, Muscle satellite cell heterogeneity and self-renewal, Front. Cell Dev. Biol. 2 (2014) 1. [53] M. Leone, A. Magadum, F.B. Engel, Cardiomyocyte proliferation in cardiac development and regeneration: a guide to methodologies and interpretations, Am. J. Physiol. Heart Circ. Physiol. 309 (2015) H1237–H1250.