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Muscular Dystrophy-Associated SUN1 and SUN2 Variants Disrupt Nuclear-Cytoskeletal Connections and Myonuclear Organization

Muscular Dystrophy-Associated SUN1 and SUN2 Variants Disrupt Nuclear-Cytoskeletal Connections and... Proteins of the nuclear envelope (NE) are associated with a range of inherited disorders, most commonly involving muscular dystrophy and cardiomyopathy, as exemplified by Emery-Dreifuss muscular dystrophy (EDMD). EDMD is both genetically and phenotypically variable, and some evidence of modifier genes has been reported. Six genes have so far been linked to EDMD, four encoding proteins associated with the LINC complex that connects the nucleus to the cytoskeleton. However, 50% of patients have no identifiable mutations in these genes. Using a candidate approach, we have identified putative disease-causing variants in the SUN1 and SUN2 genes, also encoding LINC complex components, in patients with EDMD and related myopathies. Our data also suggest that SUN1 and SUN2 can act as disease modifier genes in individuals with co- segregating mutations in other EDMD genes. Five SUN1/SUN2 variants examined impaired rearward nuclear repositioning in fibroblasts, confirming defective LINC complex function in nuclear-cytoskeletal coupling. Furthermore, myotubes from a patient carrying compound heterozygous SUN1 mutations displayed gross defects in myonuclear organization. This was accompanied by loss of recruitment of centrosomal marker, pericentrin, to the NE and impaired microtubule nucleation at the NE, events that are required for correct myonuclear arrangement. These defects were recapitulated in C2C12 myotubes expressing exogenous SUN1 variants, demonstrating a direct link between SUN1 mutation and impairment of nuclear- microtubule coupling and myonuclear positioning. Our findings strongly support an important role for SUN1 and SUN2 in muscle disease pathogenesis and support the hypothesis that defects in the LINC complex contribute to disease pathology through disruption of nuclear-microtubule association, resulting in defective myonuclear positioning. Citation: Meinke P, Mattioli E, Haque F, Antoku S, Columbaro M, et al. (2014) Muscular Dystrophy-Associated SUN1 and SUN2 Variants Disrupt Nuclear- Cytoskeletal Connections and Myonuclear Organization. PLoS Genet 10(9): e1004605. doi:10.1371/journal.pgen.1004605 Editor: Gregory A. Cox, The Jackson Laboratory, United States of America Received October 2, 2013; Accepted July 16, 2014; Published September 11, 2014 Copyright:  2014 Meinke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funding from the Wellcome Trust (grant number WT087244MA) awarded to SS and MW; from the Fondo 5 per mille 2010 awarded to the Istituto Ortopedico Rizzoli and FIRB MIUR 2010 to the CNR Institute for Molecular Genetics and EU COST BM1002 to GL; and from the U.S. NIH (grant number NS059352) awarded to GGG and HJW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] ¤a Current address: The Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom ¤b Current address: Retired, Rostock, Germany . These authors contributed equally to this work. that plays a vital role in supporting the NE and maintaining Introduction nuclear integrity, whilst also contributing to chromatin organiza- The nuclear envelope (NE) is composed of the nuclear tion and regulation of gene expression (reviewed in [2]). membranes, nuclear lamina and nuclear pore complexes and Mutations in genes encoding NE proteins are associated with a encloses the chromatin in eukaryotic cells. Lamin intermediate range of tissue-restricted inherited disorders that can affect striated filament proteins are the major structural components of the NE muscle, bone, fat or neurons and in some cases cause premature and polymerize to form a fibrous meshwork that underlies the ageing syndromes [3]. Most strikingly, different mutations in one nucleoplasmic face of the inner nuclear membrane. This nuclear gene – the LMNA gene that encodes A-type nuclear lamins – can lamina is attached to the inner nuclear membrane through cause many diseases, which have collectively been termed interactions with multiple integral inner nuclear membrane (INM) laminopathies [4]. Diseases affecting striated muscle are the most proteins [1]. Together, these proteins form a structural network common of the laminopathies and include autosomal dominant PLOS Genetics | www.plosgenetics.org 1 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies network through interaction with plectin [26], whilst nesprin-4 is Author Summary specific to epithelial cells and connects the NE to microtubules via Emery-Dreifuss muscular dystrophy (EDMD) is an inherited the kinesin-1 motor protein [27]. disorder involving muscle wasting and weakness, accom- There are several proposed mechanisms to explain the tissue panied by cardiac defects. The disease is variable in its specificity of EDMD and other laminopathies, which centre severity and also in its genetic cause. So far, 6 genes have around the ‘‘gene expression’’ and ‘‘structural’’ hypotheses [28]. been linked to EDMD, most encoding proteins that form a Current evidence strongly supports the ‘‘structural hypothesis’’, structural network that supports the nucleus of the cell which suggests that muscle-associated laminopathies primarily and connects it to structural elements of the cytoplasm. result from weakening of the structural networks of the nuclear This network is particularly important in muscle cells, lamina and cytoskeleton and the LINC complex that connects providing resistance to mechanical strain. Weakening of these two networks [29]. Since myocytes are subject to recurrent this network is thought to contribute to development of mechanical strain from contractile forces, weakening of these muscle disease in these patients. Despite rigorous screen- structural networks renders the cells particularly susceptible to ing, at least 50% of patients with EDMD have no damage. However, the LINC complex is also vital for correct detectable mutation in the 6 known genes. We therefore myonuclear positioning [30–33] and defects in this process are undertook screening and identified mutations in two implicated in impaired muscle function [34,35]. additional genes that encode other components of the Despite the genetic studies so far carried out, causative nuclear structural network, SUN1 and SUN2. Our findings mutations have been identified in only approximately 50% of add to the genetic complexity of this disease since some EDMD and related muscle disease cases [10]. It is therefore highly individuals carry mutations in more than one gene. We likely that mutations in additional genes contribute to the disease. also show that the mutations disrupt connections between the nucleus and the structural elements of cytoplasm, Furthermore, there is significant heterogeneity in disease severity leading to mis-positioning and clustering of nuclei in even within families carrying the same gene mutation [36–40], muscle cells. This nuclear mis-positioning is likely to be which has led to the suggestion of modifier genes [41–43]. another factor contributing to pathogenesis of EDMD. Given that SUN1 and SUN2 interact with at least four of the known muscle disease-associated NE proteins and that these interactions can be perturbed by disease-causing LMNA and and recessive Emery-Dreifuss muscular dystrophy (EDMD2 and EMD mutations [44], we investigated whether the SUN1 and EDMD3, respectively; OMIM#181350), limb-girdle muscular SUN2 genes may also be mutated in some individuals. Screening dystrophy (LGMD) type 2B and dilated cardiomyopathy and of the SUN1 and SUN2 genes in a large cohort of patients with conduction system disease (CMD) type 1A [5–8]. These diseases EDMD and phenotypically related myopathies identified SUN1 share the common feature of cardiomyopathy, but EDMD and and/or SUN2 variants in several patients. Presence of SUN1 or LGMD also involve progressive muscle wasting and weakness. In SUN2 variants correlated with increased disease severity in all cases, premature sudden death can result from cardiac patients with EDMD carrying mutations in other genes, thus arrhythmia and conduction defects. identifying SUN1 and SUN2 as modifiers of the EDMD disease Striated muscle disease, in particular EDMD, can also be phenotype. We further provide evidence that these mutations caused by mutations in genes encoding other NE proteins. An X- disrupt nuclear-cytoskeletal connection and nuclear positioning, linked form of EDMD (EDMD1; OMIM#310300) is caused by supporting the hypothesis that muscular dystrophies arise from mutations in EMD, that encodes the integral INM protein emerin defective nuclear-cytoskeletal coupling. [9]. Together, mutations in LMNA and EMD account for around 40% of cases of EDMD [10]. Rare mutations in the genes Results encoding FHL1, TMEM43 (also named LUMA), nesprin-1 and Screening SUN1 and SUN2 genes in a cohort of patients nesprin-2 have also been reported [11–13]. Interestingly, A-type lamins, nesprins and emerin all interact with each other [14–16], with EDMD and related myopathies contributing to a network that connects the nuclear lamina to the We analyzed DNA from 175 unrelated patients with EDMD cytoskeleton, termed the LINC (Linker of Nucleoskeleton and and related myopathies, who had previously undergone screening Cytoskeleton) complex [17]. Furthermore, interactions are often for mutations in the LMNA, EMD, SYNE1/SYNE2 alpha and perturbed by muscle disease-causing mutations, indicating that this beta (encoding short isoforms of nesprin-1 and nesprin-2, network of interactions plays an important role in muscle function respectively) and FHL1 genes and in whom no causative mutation [12,18,19]. had been found. These included both sporadic cases and index The central components of the LINC complex in mammals are patients from familial cases. Furthermore, there have been several SUN and nesprin proteins that reside in the INM and outer reports of modifiers of the phenotype of LMNA-linked muscle nuclear membrane (ONM), respectively. The conserved SUN and diseases [41–43]. We therefore also screened EDMD patients KASH domains of the respective proteins interact in the carrying identified LMNA, SYNE1/SYNE2 alpha and beta and perinuclear space to form a bridge spanning the INM, perinuclear EMD mutations to determine whether mutation of SUN1 or space and ONM that connects the nuclear lamina to the SUN2 may influence disease phenotype. Most individuals were of cytoskeleton. The nucleoplasmic N-termini of the SUN proteins, Caucasian origin, except where otherwise stated. SUN1 and SUN2, interact with the nuclear lamina, anchoring the The 23 exons of the SUN1 gene (see Figure S1) and 19 exons of LINC complex at the NE [20–22]. In turn, the cytoplasmic the SUN2 gene (ENSG00000100242, ENST00000405510), domains of the nesprins connect to the cytoskeleton. There are 4 including intron/exon boundaries and promoter regions were nesprin isoforms encoded by different genes. Giant isoforms of analyzed. DNA was amplified using PCR and analyzed by direct nesprins-1 and -2 contain an N-terminal calponin homology Sanger sequencing. In total, we found 34 single nucleotide domain responsible for actin binding [23,24] and linkage to the polymorphisms within the coding regions of SUN1 and SUN2, centrosome through microtubules and their motor proteins [25]. 18 of which were classified as rare, non-synonymous changes Nesprin-3 connects to the cytoplasmic intermediate filament following analysis of their frequencies in sequenced genome PLOS Genetics | www.plosgenetics.org 2 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies Figure 1. SUN1 and SUN2 variants identified and associated family pedigrees. (A) Schematic diagram of the SUN1 and SUN2 protein domain organization and locations of disease-associated variants identified in our cohort. Mutation SUN1 M50T, indicated in purple, did not disrupt LINC complex function in migration assays and thus may not be truly disease-causing. The mapped lamin A/C (green) and emerin (orange) binding sites, located in the nucleoplasmic N-terminal domain, are indicated. Regions of high hydrophobicity and the transmembrane domain are shown in grey and black, respectively. Coiled-coil domains responsible for oligomerization (blue) and the highly conserved SUN domain (red), found within the luminal C-terminal domain, are also indicated. (B) Pedigree with recessive inheritance of compound heterozygous SUN1 variants. (C) Pedigrees where severely affected index cases carry SUN1 and/or SUN2 variants in combination with other gene mutations. Filled circles/squares indicate affected females/males. Circles containing a dot, in family 3, indicate unaffected female carriers of the X-linked EMD mutation. Arrows indicate index patients. doi:10.1371/journal.pgen.1004605.g001 databases (Table S1, Figure S3). Three of these variants did not had EDMD or related myopathy phenotypes (Table 1). Sporadic segregate with disease in the respective families (Figure S2). In nine patient MD-11 carried a single SUN2 p.R620C sequence unrelated families or sporadic cases, however, we identified 10 rare variation. We had no access to DNA from family members for non-synonymous variants in SUN1 and SUN2 for which we have this patient, but the high degree of evolutionary conservation of obtained evidence of pathogenic effects, as deduced from genetic, R620 is supportive of disease-association (Figure S3). Patient MD- phenotypic and/or functional data (Figure 1A). 1 carried compound heterozygous SUN1 p.G68D and p.G338S variants. For patient MD-1 we had access to DNA from family Putative disease-associated SUN1 and SUN2 variants in members and observed apparent recessive inheritance, with one patients with EDMD-like phenotypes mutation coming from each of the unaffected parents (Figure 1B). We identified 5 rare, non-synonymous SUN1 and/or SUN2 SUN1 p.G338S was also present in the reference population at variants in 3 individuals who lacked mutations in other genes but low frequency (see Table S1). These residues are located within the PLOS Genetics | www.plosgenetics.org 3 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies Table 1. Putative disease-causing variants in SUN1 and SUN2 in patients with EDMD-like phenotypes. Family Index case SUN1 variant SUN2 variant Other mutations Disease phenotype 1 MD-1 p.G68D p.G338S none none Male; age at onset 10 years; mild muscle weakness; rigid spine; serum creatine kinase elevation 6X; no cardiac involvement; last clinical examination at age 10 years; sporadic case. 11 MD-11 none p.R620C none Sporadic EDMD-related myopathy, no other clinical information available. 12 MD-12 p.W377C p.E438D none Heart rhythm disturbances at age 34 years; partial lipodystrophy on left lower leg; sporadic case doi:10.1371/journal.pgen.1004605.t001 poorly conserved N-terminal domain of the protein and, in this mutation was inherited from an unaffected parent. Again, the context, are moderately conserved (Figure S3), but functional data disease expression in both index patients was more severe than is presented below present compelling evidence of the involvement typical for EDMD [49,50], with early onset at age 1 and 4 years, of both mutations in disease causation. Sporadic patient MD-12 respectively, and early heart involvement including heart trans- carried heterozygous changes in both SUN1 and SUN2, encoding plantation before age 20 years (Table 2). SUN1 p.W377C and SUN2 p.E438D, respectively. E438 is We also identified two SUN2 variants, encoding variants conserved in mammals, whilst W377 is conserved across all species p.M50T and pV378I, in patient MD-2 who had hypertrophic examined (Figure S3). cardiomyopathy and also carried a mutation in MYBPC3 (p.G148R), which encodes a myosin binding protein. The same SUN1 and SUN2 variants with disease modifying effects in MYBPC3 mutation was previously reported in a Dutch family, where a severely affected index patient had compound heterozy- patients with co-segregating mutations in other genes Because patients with EDMD-like phenotypes exhibit variable gous mutations in MYCBP3 but other family members carrying only p.G148R were either asymptomatic or developed cardiomy- disease severity that could be explained by mutations or polymorphisms in additional genes, we screened for SUN1 and opathy late in life [51]. This MYBPC3 mutation was present in SUN2 variants in patients with known mutations in causative both patient MD-2 and his father (Figure 1C; S. Waldmueller, genes. SUN1 or SUN2 variants were indeed present in some personal communication). Patient MD-2 presented as a 6 month- patients from families with LMNA or X-linked EMD mutations old boy with hypertrophic cardiomyopathy and died at 16 years (Table 2; Figure 1C). These sequence changes correlated with from heart failure. His father, who carries the SUN2 p.M50T increased disease severity. In one example, a SUN1 p.A203V variant but not p.V378I, is asymptomatic. Thus, the SUN2 polymorphism in patient MD-3 co-segregated with a previously p.V378I variant appears to have a dramatic effect on disease reported EMD p.L84Pfs*6 mutation in two brothers with severity. Notably, this variant is present in the reference unusually severe EDMD (Figure 1C, Family 3) [39]. The EMD population at low frequency (see Table S1), suggesting that it p.L84Pfs*6 mutation, which abolishes emerin expression, has been may be a relatively common genetic modifier of inherited reported in an unrelated family, where the course of the disease cardiomyopathy. was significantly milder EDMD with later age of onset and no loss of ambulation [45]. Another unrelated patient carrying EMD SUN1 and SUN2 disease-associated variants disrupt p.L84Pfs*6 was included in this study but no SUN1 or SUN2 centrosome reorientation and nuclear movement in variants were found and their phenotype was similar to that NIH3T3 fibroblasts described by Manilal et al. [45]. In another case, the SUN1 Our genetic results suggest that mutations or polymorphisms in p.G76A mutation, when combined with EMD p.A56Pfs*9 in a SUN1 and SUN2 may cause muscular dystrophy and act as previously described Korean patient (MD-4), led to a very severe modifiers of EDMD and cardiomyopathy. To obtain additional clinical picture with complete atrioventricular block requiring pace evidence that these variants play a role in pathophysiology, we maker implantation at age 14 years [46]. Similarly, SUN1 examined the effects of several variants on a known function of the p.W377C was detected in combination with LMNA p.R453W LINC complex, namely centrosome orientation and nuclear in patient MD-5. This individual had severe disease and died early movement in migrating cells. SUN2, along with the nesprin-2G at the age of 34 years from heart failure. The patient’s son, isoform, assembles into transmembrane actin-associated nuclear carrying LMNA p.R453W only, did not show clinical signs of (TAN) lines that couple actin cables to the nucleus to move it contractures or muscular weakness at age 10 years. LMNA rearward and reorient the centrosome toward the leading edge in p.R453W is a common EDMD-associated LMNA mutation and is migrating NIH3T3 fibroblasts [52,53]. While SUN1 is not in TAN generally not associated with severe cardiac disease, suggesting lines, it also functions in connecting the nucleus to the cytoskeleton that, in patient MD-5, SUN1 p.W377C had a modifying effect to via the LINC complex. increase disease severity [47,48]. The same SUN1 p.W377C We expressed three myc-SUN1 and three myc-SUN2 variants in variant was detected in patient MD-12, who had an EDMD-like NIH3T3 fibroblasts at the edge of a wounded monolayer by DNA phenotype but did not have mutations in EMD or LMNA but microinjection and stimulated nuclear movement and centrosome carried a concurrent SUN2 p.E438D variant, as described above. reorientation with the serum factor, lysophosphatidic acid (LPA). We detected SUN2 mutations in combination with LMNA Upon stimulation, non-expressing NIH3T3 cells or NIH3T3 cells mutations in two additional index cases. Patient MD-6 carried LMNA p.T528K and SUN2 p.A56P, whilst patient MD-7 carried exogenously expressing wild-type (WT) SUN1 or SUN2, as well as LMNA p.R98P and SUN2 p.V378I (Table 2; Figure 1C). In both the variant SUN2 M50T, reoriented their centrosomes (Figure 2A– cases, the LMNA mutation had arisen de novo, whilst the SUN2 B). Notably, SUN2 p.M50T did not appear to influence disease PLOS Genetics | www.plosgenetics.org 4 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies Table 2. SUN1 and SUN2 variants with disease-modifying effects in patients with MYBPC3, EMD and LMNA mutations. Family Index case SUN1 variant SUN2 variant Other mutations Disease phenotype 2 MD-2 none p.M50T p.V378I MYBPC3 p.G148R Male; age at onset 6 months; hypertrophic cardiomyopathy; at age 9 years ECG showed cardiac arrhythmia, supraventricular extrasystols, Echocardiogram: right ventricular septum hypertrophy, first degree atrioventricular block; no muscular weakness or dystrophy; sinus tachycardia and bradycardia, mild left ventricular functional impairment; died at age16 years from heart failure; sporadic case. 3 MD-3 p.A203V none EMD p.L84Pfs*6 Male; age at onset 2 years; severe contractures of neck, thoracolumbar spine, elbows, and Achilles tendons; Achillotomia at age 6; loss of ambulation at age 15; moderate to severe muscle weakness; left anterior hemi-block and ventricular ectopy at age 23; ventricular dilation at age 33; X-linked EDMD [39]. 4 MD-4 p.G76A none EMD p.A56Pfs*9 Male; age of onset 1 year, wasting and weakness of shoulder girdle and limb-girdle muscles; at age 14 severe contractures of neck, elbow and Achilles tendons, tendon reflexes absent; at age 14 dilated right atrial and ventricular dilation, atrial fibrillation, complete AV block and junctional escape rhythm; pacemaker since age 14; CK elevation 4X; X-linked EDMD [46]. 5 MD-5 p.W377C none LMNA p.R453W Male; age of onset 8 years; slowly progressive humero-peroneal muscular weakness; since age 14 rigid spine, contractures of elbow and Achilles-tendons; at age 25 cardiac disturbances; AV- block III; heart pacemaker at age 31; died at age 34 of heart failure; autosomal dominant EDMD. 6 MD-6 none p.A56P LMNA p.T528K Male; age at onset 1 year; delayed early childhood developmental mile stones; later difficulties in climbing stairs, muscular weakness; at age 15 tachycardia, extrasystols; at age 17 intra- ventricular cardiac conduction defects, contractures of elbow and Achilles tendons; proximal humero-peroneal muscle atrophy, rigid spine, Gower’s maneuver; CK elevated 3–5X; de novo LMNA mutation leading to sporadic EDMD. 7 MD-7 none p.V378I LMNA p.R99P Female; age of onset 4 years, diffuse muscular weakness; later bilateral contractures of the elbows and ankles, dilated cardiomyopathy, first degree atrioventricular block; at age 14 heart pacemaker; histopathology showed fibrosis of the heart muscle; at age 15 heart transplantation; de novo LMNA mutation leading to sporadic EDMD. doi:10.1371/journal.pgen.1004605.t002 severity in family 2. In contrast, cells expressing the putative disease- expression of LINC complex components and known SUN1 binding causing SUN1 variants, G68D, G338S or W377C, inhibited partners in the myoblasts by immunofluorescence microscopy. Since centrosome reorientation by blocking rearward positioning of the nesprin-1 is not significantly expressed in myoblasts [54,55] (Figure nucleus (Figure 2A–B). Similarly, cells expressing SUN2 A56P or S4), we stained for nesprin-2, lamin A/C, emerin, SUN1 and SUN2. R620C failed to reorient their centrosome due to an inability to No obvious defects in localization of SUN1, SUN2 or their position their nuclei rearward of the cell centroid (Figure 2A–B). All interacting NE partners were observed, but expression of SUN1 of the expressed SUN1 and SUN2 variants had a normal nuclear and nesprin-2 at the NE was enhanced in the patient myoblasts localization similar to the wild-type proteins (Figure 2A). The (Figure 3A–B). Quantification of fluorescence intensity suggested that centrosome orientation defect in cells expressing the SUN variants their expression was increased approximately 2-fold. occurred due to defective rearward nuclear movement and not Since fluorescence intensity does not always provide an accurate disruption of positioning of the centrosome at the cell centroid reflection of total protein levels, we then examined total protein (Figure 2C). Hence, five putative disease-causing or disease-modify- expression level by Western blot and found that SUN1 levels were ing SUN variants blocked rearward nuclear movement in migrating elevated 8-fold in the patient versus control myoblasts (Figure 3C,E). NIH3T3 fibroblasts and it can be concluded that these mutants Consistent with the fact that SUN proteins form complexes with, and disrupt LINC complex function. are responsible for anchoring of nesprins in the ONM, we also observed a 4-fold increase in expression of the intermediate-sized Expression of LINC complex components is altered in muscle-enriched isoforms of nesprin-2 [55] in the patient myoblasts patient myoblasts with compound heterozygous SUN1 (Figure 3D–E). However, it remains possible that the bands observed are degradation products of nesprin-2 giant. In contrast, levels of mutations SUN2, lamin A/C and emerin were not significantly altered in the Having established that some of the variants identified in our patient cells (Figure 3C,F). To exclude the possibility that the observed patient cohort disrupted LINC complex function in fibroblasts, we changes were due to different levels of background differentiation in the next wished to examine their role in muscle cells. We were able to obtain primary myoblasts from patient MD-1, carrying compound control and patient cultures, we quantified expression of myogenin, an heterozygous SUN1 p.G68D/p.G338S variants. To gain initial early marker of myogenic differentiation, and found no detectable level insight into the cellular effects of these mutations, we examined in either culture (data not shown). PLOS Genetics | www.plosgenetics.org 5 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies Figure 2. SUN1 and SUN2 variants disrupt nuclear-cytoskeletal coupling in NIH3T3 fibroblasts. (A) Representative immunofluorescence micrographs of LPA-stimulated NIH3T3 fibroblasts expressing SUN1 or SUN2 wild-type (WT) or variant proteins. Cells were immunostained for myc (green), tubulin (red) and DAPI (blue). Location of centrosome (arrows) was determined by the center of microtubule array. Bar, 20 mm. (B) Quantification of centrosome reorientation in LPA-stimulated NIH3T3 fibroblasts expressing the indicated SUN variants. Significant differences are indicated by * with a,0.05 based on Student’s t-test when the sample is compared to non-expressing NIH3T3 fibroblasts. (C) Quantification of nucleus and centrosome position relative to the cell centroid in NIH3T3 fibroblasts expressing the indicated SUN variants. Positive values are toward the leading edge, negative values toward the cell rear. Data are from at least three independent experiments for each sample. Significant differences are indicated by * with a,0.05 based on Student’s t-test when the sample is compared to non-expressing NIH3T3 fibroblasts. doi:10.1371/journal.pgen.1004605.g002 To address the mechanism of SUN1 elevation in the patient due to weakening of the protein interaction network supporting myoblasts, we examined SUN1 mRNA levels by qPCR and found no the NE [28,56]. To determine whether muscle disease-associated significant increase in mRNA level compared to the control, SUN1 alterations disrupt interactions with other LINC complex indicating that the p.G68D/p.G338S SUN1 variants do not lead components, we performed immunoprecipitation with anti-SUN1 to increased mRNA levels (Figure S4). However, in analyzing mRNA antibodies on protein extracts from control and patient MD-1 levels of other LINC complex-associated proteins, we observed a myoblasts and detected co-precipitating proteins. We observed a statistically significant increase in expression of LMNA, SUN2, reproducible reduction in SUN1 interaction with emerin in the SYNE1 and SYNE2. In contrast, EMD (encoding emerin) expression patient myoblasts, whilst interaction with lamin A/C was not was decreased in the patient relative to control myoblasts (Figure S4). obviously perturbed (Figure 4A–B). Consistent with the enhanced recruitment of nesprin-2 to the NE, interaction of SUN1 with SUN1 interaction with emerin is impaired in MD-1 nesprin-2 was also maintained in the MD-1 myoblasts (Figure 4C). myoblasts Larger isoforms of nesprin-2 were enriched in the immunopre- One hypothesis to explain NE-associated muscle diseases is the cipitate compared to the initial lysates, indicating a preferential ‘‘structural hypothesis’’, which suggests that muscle damage occurs interaction of SUN1 with these less abundant isoforms. Thus, the PLOS Genetics | www.plosgenetics.org 6 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies Figure 3. Expression of LINC complex proteins is increased in patient MD-1 (SUN1 p.G68D/p.G338S) myoblasts. (A) Control and MD-1 myoblasts were fixed in methanol and analysed by immunofluorescence microscopy using SUN1, SUN2, emerin, nesprin-2G and lamin A/C antibodies, as indicated, together with DAPI staining of DNA. Scale bar, 22 mm. (B) Mean fluorescence intensity of SUN1, SUN2, emerin, nesprin 2 and PLOS Genetics | www.plosgenetics.org 7 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies ‘ ‘ lamin A/C was measured in individual DAPI-stained nuclei using an Olympus Scan R screening station and analysed using Scan R analysis software. The results are presented as mean 6 S.E. of 1000 cells taken from at least 3 independent experiments. **P#0.05. Significant P-value for SUN1 was P = 0.009. (C) Total protein extracts from control (C) and patient MD-1 myoblasts were Western blotted using antibodies against LINC complex- associated proteins, as indicated. (D) Samples prepared as in A were Western blotted using nesprin-2 (N2–N3) antibodies. (E–F) Protein expression was quantified by densitometric analysis of at least 3 independent experiments. The results are presented as mean 6 S.E. *P#0.05 and **P#0.01. Each significant P- values are as follows: SUN1 P = 0.05, a-tubulin P = 0.003, nesprin-2 P = 0.002. P- value for emerin was P = 0.06. doi:10.1371/journal.pgen.1004605.g003 SUN1 p.G68D/p.G338S variants in patient MD-1 appear to structs. SUN1 was immunoprecipitated using anti-myc antibodies specifically disrupt interaction with emerin. and co-precipitating GFP-emerin detected by Western blotting. We confirmed defective interactions with emerin in HEK293 We observed a significant reduction in interaction of emerin with cells transiently expressing GFP-emerin and myc-SUN1 con- both SUN1 G338S and W377C, whilst there was only a modest Figure 4. Emerin binding to p.G68D/p.G338S SUN1 is reduced in vivo. (A) SUN1 was immunoprecipitated from control (C) or MD-1 myoblast soluble lysates using 2383 SUN1 antibodies and samples Western blotted to detect co-immunopreciptated proteins. (B) Densitometric analysis of SUN1, lamin A/C and emerin bands from immunoprecipitated samples is plotted in arbitrary units (A.U.). (C) SUN1 was immunoprecipitated from control or MD-1 myoblasts and co-precipitated nesprin-2 was detected using N2–N3 antibody. Size markers (kDa) are indicated. Note that larger nesprin-2 isoforms are enriched in the immunoprecipitate compared to the lysate. (D) HEK293 cells were co-transfected with myc-SUN1 mutant and GFP-emerin plasmids and harvested after 48 hours. Co-precipitated GFP-emerin was detected by immunoblotting using GFP antibodies (myc-SUN1 IP). (E) Densitometric values of immunoblotted GFP-emerin bands are reported in arbitrary units (A.U.). (F) Human fibroblasts from an EDMD2 patient carrying the R401C LMNA mutation were transfected with SUN1-WT or SUN1-W377C cDNAs and fixed 48 hours after transfection. Lamin A/C was labelled using specific antibodies and revealed by FITC-conjugated secondary antibody (green). SUN1 was detected using Cy3-conjugated anti-myc antibody (red). Nuclei were counterstained with DAPI. Images show pairs of daughter cells from recent cell divisions. Increased dysmorphic nuclei with nuclear blebbing and honeycomb structures (arrows) are observed in double-mutant cells. The distance between daughter cells has been reduced using Photoshop 7 to allow magnification. (G) Percentage of dysmorphic nuclei in EDMD2 cells left untreated (untransfected), transfected with WT-SUN1 or transfected with W377C-SUN1 is reported in the graph as the mean of three independent experiments. Statistically significant differences (P,0.05) relative to controls or WT-transfected samples are indicated by asterisks. doi:10.1371/journal.pgen.1004605.g004 PLOS Genetics | www.plosgenetics.org 8 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies decrease in interaction with SUN1 G68D (Figure 4D–E). This that, whilst SUN1 polarization was normal (Figure S5), SUN2 correlates with the close proximity of G338 and W377 to the failed to polarize in clustered nuclei (Figure 5D,G). In keeping emerin binding site on SUN1 (see Figure 1A; [44]). with earlier observations in myoblasts, we also found that nesprin- 2 fluorescence intensity was significantly increased in all patient myotubes (Figure S6). Ultrastructural analysis showed that myo- SUN1 W377C increases the severity of nuclear defects in nuclear clustering occurred within single MD-1 myotubes EDMD2 fibroblasts (Figure 5H) and further demonstrated that enlarged highly Abnormalities in nuclear morphology and a honeycombed differentiated myotubes with misaligned nuclei were devoid of pattern of protein expression at the NE are commonly observed in detectable sarcomeric structures that were visible in controls EDMD fibroblasts obtained from patients with LMNA or EMD (Figure 5H, arrowheads). mutations [57–59]. We did not observe any obvious defects in nuclear morphology or protein localization in myoblasts from Pericentrin recruitment and microtubule nucleation at patient MD-1 (Figure 3A) or in cells transiently expressing a range the NE is defective in MD-1 myotubes of SUN1 or SUN2 mutants (for example, see Figure 2). However, During myotube formation, the microtubule network is we sought to determine whether SUN1 mutants have a modifying reorganized into a parallel array along the longitudinal axis of effect to increase the severity of nuclear defects when expressed in the myotube and is nucleated from the nuclear surface, which combination with mutations in LMNA or EMD. To achieve this, becomes the primary microtubule organizing centre (MTOC) of we expressed SUN1 W377C, found in combination with a LMNA the cell [64]. As part of this process, centrosomes undergo partial R453W mutation in patient MD-5, in fibroblasts obtained from an disassembly and centrosomal proteins, including c-tubulin, EDMD2 patient carrying LMNA R401C. Cells expressing SUN1 pericentrin and PCM-1, become concentrated at the nuclear W377C had a 4-fold increase in the level of nuclear dysmorphol- periphery [65,66]. We hypothesized that SUN proteins contribute ogy, in terms of increase in nuclear blebbing and formation of to centrosomal protein recruitment to the NE and that polariza- honeycomb structures (Figure 4F, arrows), compared to those tion of SUN proteins at the nuclear poles may promote linear expressing WT SUN1 (Figure 4F–G). Cells expressing WT SUN1 nuclear organization in myotubes. We therefore investigated the had a 2-fold reduction in nuclear abnormalities compared to recruitment of centrosomal proteins to the NE during myogenesis, untransfected cells, suggesting a protective effect of SUN1 over- using pericentrin as a marker. In control myotubes we found that expression. pericentrin was recruited to the NE and there was a suggestion that it concentrated at the poles of the nuclei, in a similar manner MD-1 myoblasts exhibit an enhanced rate of to SUN2 (Figure 6A). In contrast, pericentrin failed to accumulate differentiation but myonuclei are disorganized to any significant degree at the nuclear surface in MD-1 myotubes Exogenous expression of EDMD2-associated lamin A/C and was instead found in cytoplasmic foci. These findings support variants and disruption of endogenous lamin A/C, emerin or our hypothesis that SUN proteins are involved in the recruitment the LINC complex all affect myoblast differentiation [60–63]. of pericentrin to the NE in myotubes. Furthermore, studies in Drosophila indicate that the LINC We next investigated whether microtubule nucleation from the complex is required for correct myonuclear spacing [34]. We NE was disrupted in the patient myotubes by observing therefore determined whether the myoblasts carrying SUN1 microtubule regrowth following nocodazole-induced depolymer- variants had defects in their ability to differentiate to form mature, ization. In control cells, microtubules could be clearly observed multinuclear myotubes. In order to account for inherent emanating from around the nuclear surface after nocodazole differences in differentiation capacity between cell lines, we wash-out for 30 minutes (Figure S7). In contrast, the microtubule compared MD-1 cultures with a total of 5 control cultures network in patient cells was very disorganized and often did not independently established from different individuals. SUN1 appear to be attached to the NE, suggesting a defect in staining was clearly evident at the nuclear envelope of MD-1 microtubule nucleation, anchoring or organization. To investigate myotubes, as previously observed in myoblasts (Figure 5A). this further, we performed a short 5-minute nocodazole wash-out However, we found that the MD-1 myoblasts exhibited an to detect sites of microtubule nucleation. Microtubule asters increased rate of differentiation, both in terms of the number of regrowing from the nuclear envelope were observed in control myotubes and the number of nuclei per myotube. There was a myotubes (Figure 6B, panel a) and committed myoblasts (Fig- striking increase in the percentage of nuclei within myotubes ure 6B, panel b). Microtubule nucleation correlated with sites of (defined as muscle-specific caveolin3-positive cells containing at pericentrin concentration at the nuclear poles (arrows in least 3 nuclei) in patient cultures (2866% versus 5869% in Figure 6B). In MD-1 myotubes and committed myoblasts, control and MD-1 cultures, respectively; mean 6 sem) and a 5- microtubules were seen to nucleate mainly from multiple sites in fold increase in myotubes possessing more than 10 nuclei the cytoplasm, corresponding with the locations of cytoplasmic (Figure 5A–E). Strikingly, 45% of these myotubes displayed gross pericentrin foci (Figure 6B, arrowheads). Counting fifty myotubes nuclear misalignment and clustering, with up to 30 nuclei per per sample, we could demonstrate that the mean number of myotube in some instances (Figure 5D,F). microtubules nucleating from myotube nuclei was significantly Together with our earlier observation of defective nuclear reduced in MD-1 (Figure 6D). repositioning in fibroblasts expressing disease-associated variants These data suggested that centrosome attachment to the and in double-mutant EDMD2 fibroblasts, these findings suggest- nucleus may also be disrupted in myoblasts from this patient. ed that mutations in SUN1 and SUN2 disrupt connections with We therefore examined nuclear-centrosomal distance in MD-1 the cytoskeleton, thereby perturbing nuclear anchorage. Studies myoblasts and indeed observed a 2-fold increase in separation have previously shown that SUN1 and SUN2 become concen- between the nucleus and centrosomes in the patient myoblasts trated at the poles of the nucleus during human primary myoblast compared to controls (mean distance 4.34 mm versus 2.07 mm, differentiation and that this polarization is linked to correct respectively) (Figure 6C,E). myonuclear spacing [32]. We therefore examined SUN1 and To confirm that loss of pericentrin recruitment to the NE was a SUN2 polarization in patient MD-1 myotube nuclei and found direct consequence of SUN1 mutation, we observed pericentrin PLOS Genetics | www.plosgenetics.org 9 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies Figure 5. Enhanced rate of differentiation, nuclear misalignment and clustering in MD-1 myotubes. (A) Immunofluorescence staining of MD-1 myotubes using SUN1 (green) and desmin antibodies (red). Chromatin was stained with DAPI (blue). Scale bar, 30 mm. (B) Graphical representation of the percentage of nuclei within myotubes counted in MD-1 and in five independent control cultures. (C) Phase contrast image of living control and MD-1 cultured myotubes (arrows) showing myonuclear clustering in patient cells. Scale bar, 10 mm. (D) Immunofluorescence staining of control and MD-1 myotubes with SUN2 (green) and caveolin 3 (red) antibodies. Arrows indicate SUN2 polarization at the nuclear poles in control cells. (E–F) Graphical representation of the percentage of myotubes with more than 10 nuclei and the percentage of myotubes with myonuclear clustering in control and MD-1 cultures. Data are presented as mean values 6 S.D. of three independent experiments (50 myotubes per sample were counted). (G) Graphical representation of the percentage of committed myoblast and myotube nuclei with enrichment of SUN2 staining PLOS Genetics | www.plosgenetics.org 10 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies at the nuclear pole(s). Data are presented as mean values 6S.D. of 3 independent experiments (200 nuclei per sample). (H) Transmission electron microscopy analysis of control and MD-1 myotubes (see Materials and Methods for details). Sarcomeric structures are evident in control myotubes (arrowheads), whereas they are absent from MD-1 myotubes showing myonuclear clustering. Arrows indicate myonuclei. Scale bar, 10 mm. doi:10.1371/journal.pgen.1004605.g005 localization in C2C12 myotubes following transient transfection with Finally, to directly link the SUN1 mutants to the nuclear myc-SUN1 variants. Pericentrin was absent from the NE of clustering phenotype observed myotubes of patient MD-1, we myonuclei expressing SUN1 G68D, G338S and W377C variants, assessed the degree of nuclear clustering in myotubes expressing but exhibited clear nuclear rim staining in myonuclei expressing WT WT SUN1 and the G68D, G338S and W377C variants. Whilst SUN1 (Figure 7 A–B). Thus, all 3 mutants tested acted in a dominant 22% of myotubes expressing WT SUN1 displayed myonuclear manner in C2C12 cells to displace pericentrin from the NE. clustering, this value was increased by 2-fold in the cultures Figure 6. Impaired pericentrin localization and microtubule nucleation at the nuclear envelope in MD-1 myotubes. (A) Beta-tubulin (red) and pericentrin (green) double immunofluorescence staining of control and MD-1 myotubes. Chromatin was stained with DAPI (blue). Scale bar, 10 mm. Arrows indicate apparent pericentrin accumulation at the nuclear poles. (B) Control and MD-1 myotubes were treated with nocodazole followed by 5 min recovery in culture medium to allow microtubule regrowth. Samples were then fixed and stained as in panel A. Panels a and c show myotubes, whilst panels b and d show committed myoblasts from control and MD-1 cultures, respectively. Arrows in control cells indicate sites of microtubule regrowth at the nuclear poles, co-inciding with pericentrin localization. Arrowheads in patient cells indicate microtubule regrowth from cytoplasmic pericentrin foci. (C) Control and MD-1 myoblasts were fixed in methanol and subjected to immunofluorescence analysis using c- tubulin antibodies to stain the centrosome and DAPI to stain the DNA. Distances between centrosomes and the nuclear periphery (mm) are indicated. Scale bar, 10 mm. (D) The number of microtubules nucleating from individual myotube nuclei prepared in B was counted and is presented as the mean 6S.D (n = 50 nuclei per sample). * p,0.05 as calculated using the Student’s t-test. (E) Nucleus-centrosome distance was measured in 100 control and MD-1 myoblasts prepared in C, in two independent experiments, using Leica LAS AF Lite software and analysed using SPSS software. The median values (thick black lines) were 0.96 and 2.43 mm for control and MD-1 cells, respectively. P = 0.00012.# and * correspond to mild and extreme outliers, respectively. doi:10.1371/journal.pgen.1004605.g006 PLOS Genetics | www.plosgenetics.org 11 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies Figure 7. Exogenously expressed SUN1 mutants impair pericentrin recruitment to the nuclear envelope. (A) Differentiated C2C12 myotubes transfected with wild-type (WT) SUN1, or the indicated mutants, were labelled with anti-myc (red) and anti-pericentrin antibodies (green). Desmin antibody (violet) was used as a muscle differentiation marker. Nuclei were counterstained using DAPI. Samples were observed using a Nikon laser confocal microscope. Bar, 10 mm. (B–C) Transfected myotubes prepared as in A were quantified for the absence of pericentrin staining at the nuclear envelope (B) and myonuclear clustering (C). Thirty myotubes per sample were counted in two independent experiments. Differences for all mutants were statistically significant with respect to wild-type-transfected myotubes (P,0.01). doi:10.1371/journal.pgen.1004605.g007 expressing each of the 3 SUN1 variants (Figure 7C). These myopathy patients. Ten of these variants, identified in nine findings confirm that the SUN1 p.G68D and p.G338S mutations unrelated families, had putative pathogenic effects as deduced are the primary cause of the failure to recruit pericentrin to the NE from genetic and functional analyses. and the defective nuclear positioning in myotubes from patient MD-1 and further indicate that this is likely to be common to Multiple modes of inheritance of SUN1 and SUN2 muscle from patients carrying other SUN1 mutations, including mutations p.W377C. Our data add to an increasingly complex picture concerning the In summary, our data demonstrate that muscle disease-associated genetics of EDMD and related myopathies, where multiple genes, alterations in SUN proteins result in loss of nuclear connectivity to either alone or in combination, can cause or modify the disease the cytoskeleton. In myotubes, SUN1 mutations disrupt connec- phenotype. The SUN1 and SUN2 variants appear to be inherited tions with centrosomal components and the microtubule network, in highly variable manners, with or without the presence of a in particular impairing microtubule organization and nucleation mutation in a second gene. In 2 families (families 1 and 2), SUN1 from the NE. This in turn is likely to lead to impaired myonuclear or SUN2 variants were inherited from each of the unaffected positioning in multinuclear myotubes, which we propose may be an parents of the index patients, strongly supporting an autosomal important contributor to muscle dysfunction. recessive mode of inheritance in those families. One sporadic case carried heterozygous mutations in both the SUN1 and SUN2 Discussion genes, suggesting that mutations in the 2 genes could have additive We have identified a total of 11 SUN1 and 7 SUN2 rare, non- effects, as has been observed in Sun1/Sun2 knockout mice [30]. In synonymous variants in our cohort of EDMD and related other instances, the index case carried only one SUN1 or SUN2 PLOS Genetics | www.plosgenetics.org 12 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies variant and often represented a sporadic case, suggestive of either Despite our findings increasing the number of known EDMD- associated genes to 8, still almost 50% of patients in our cohort a dominant de novo mutation or presence of a concurrent mutation in another, as yet unidentified, gene. have no identified mutations in any of these genes. Most of these patients represent sporadic cases, with no family history of disease, We also identified SUN1 or SUN2 variants in individuals from making mutation screening difficult. Furthermore, since 6 of the 4 families harbouring known LMNA or EMD mutations. In all known genes each account for only a small percentage of cases, it cases, the SUN1/SUN2 mutation alone did not cause disease in is likely that there are multiple genes remaining to be identified. other family members. However, disease severity was significantly Proteins associated with the LINC complex are clearly very strong increased in the individuals carrying both mutations compared to candidates and there are several such proteins that should be family members, or unrelated individuals, carrying only the examined as a priority, including Samp1 [71,72]. LMNA or EMD mutation. Furthermore, their phenotype was on the severe end of the spectrum, as defined by Yates et al. [50]. These findings suggest that some SUN1/SUN2 variants act as Muscular dystrophy-associated SUN1 and SUN2 variants modifiers to increase disease severity. There has been much disrupt nuclear-cytoskeleton connection and nuclear speculation as to the existence of modifier genes in EDMD due to positioning high variability in disease phenotype between affected individuals Through the various nesprin isoforms expressed at the ONM, within families [37,38,41,47] and there is now some evidence to the LINC complex mediates attachment to all three cytoskeletal support this. In a family with X-linked EDMD caused by an EMD filament networks [17,73]. In this study, we have demonstrated, in p.Y105X mutation, disease severity was increased in one several different systems, that muscular dystrophy-associated individual due to a second mutation present in the LMNA gene mutations in SUN1 or SUN2 impair nuclear coupling to the [42]. Similarly, an individual with severe disease and carrying a both actin and microtubule networks and disrupt nuclear LMNA p.R644C mutation was found to carry a second mutation movement and positioning. in the gene encoding desmin [42]. In mouse NIH3T3 fibroblasts, five out of the six SUN1 and In our cohort, for some cases (such as patient MD-3) the SUN2 variants that we examined inhibited rearward movement of increased severity was expressed as clinically more severe muscular the nucleus, which has previously been shown to be achieved dystrophy [39]. In other cases, the additional presence of a SUN1/ through LINC complex attachment to actin cables closely SUN2 mutation was associated with more severe cardiac disease. associated with the nuclear surface [53]. Our data strongly For example, patient MD-5 from family 5, carrying both LMNA indicate that at least five of the variants identified in our patient p.R453W and SUN1 p.W377C mutations, developed cardiac cohort have a negative functional impact upon nuclear-cytoskel- disturbances at age 25 and died from heart failure at age 34, which etal connection via the LINC complex, which is likely to be a is much earlier than is typical for EDMD patients [50]. major contributor to muscle disease pathophysiology. One of the Furthermore, LMNA p.R453W is a relatively common mutation variants examined, SUN2 p.M50T, did not impair rearward that has been reported in at least 15 individuals with EDMD and is nuclear movement, suggesting that this variant is not disease- usually associated with mild disease [8,48,67–70]. Thus, it is likely causing and this is entirely possible given the complex genetics in that the SUN1 mutation carried by patient MD-5 contributed to the individual carrying this mutation (patient MD-2). In agree- their increased disease severity. We also identified SUN1 ment with our findings, EDMD-associated lamin A variants were p.W377C in combination with SUN2 p.E438D in a sporadic recently shown to cause a similar defect in nuclear movement in case, supporting the idea that a mutation in a second gene is NIH3T3 cells [52] and disrupted nuclear-cytoskeletal coupling required for disease causation in this instance. Further support for [74]. a modifying role for the p.W377C mutation came from the ability We also observed defects in nuclear positioning in differentiat- of this mutant to worsen nuclear dysmorphology when expressed ing myotubes derived from patient MD-1, carrying compound in LMNA R401C patient fibroblasts. heterozygous SUN1 p.G68D/p.G338S mutations, which is Individuals MD-6 and MD-7 (carrying a SUN2 variant in consistent with recent findings that proper SUN1 and SUN2 addition to LMNA T528K and R99P mutations, respectively) also recruitment to the NE is required for myonuclear spacing [32]. In had more severe disease than is typical for EDMD, with early strong support for a direct role of SUN proteins, nuclear onset at age 1 and 4 years, respectively, and unusually early positioning in skeletal muscle is disrupted in double Sun1/Sun2 requirement for heart transplantation. In each of these sporadic knockout mice or in mice with targeted disruptions of nesprin-1/ cases, the LMNA mutation arose de novo and so no comparisons nesprin-2 and this leads to clustering similar to that observed in can be made with family members, however, their clinical our patient myotubes [30,31,75]. Thus, the phenotype we phenotype is consistent with the suggestion that the SUN2 observed in patient MD-1 is consistent with a defect in the LINC variants contribute to increased disease severity. Whilst our genetic complex. Several recent studies have shown that myonuclear and cell-based data strongly support a modifying role for SUN position is controlled by nuclear attachment to the microtubule mutations in some patients, studies involving larger patient cohorts network and that this is mediated by the LINC complex will be necessary to prove this conclusively. [34,35,76–78]. At the onset of myoblast differentiation, proteins For 8 of the rare, non-synonymous variants identified in our involved in microtubule nucleation redistribute from the centro- cohort, there was a lack of compelling evidence of their disease some to the NE. Our observations of impaired pericentrin association. However, given the complex interplay between muta- recruitment and microtubule nucleation/organization at the NE tions in different genes, more investigation is required before entirely in the patient myotubes therefore support a model whereby ruling out their involvement. In particular, SUN1 p.V846I was found mutations in SUN proteins impair nuclear-microtubule connec- in an isolated sporadic case and thus no co-segregation analysis could tion and prevent correct positioning of myonuclei (Figure 8). It is be performed to support its disease association. Yet this is a mutation also well established in other systems that unanchored nuclei float of highly conserved residue (Figure S3) that lies within the SUN freely in the cytoplasm and tend to clump together, as observed in domain that is involved in nesprin binding. Thus it will be important MD-1 myotubes [34,77,79]. in the future to utilize functional studies to investigate the impact of It is currently not clear how the mutant SUN proteins, which such mutations on LINC complex interactions. are located at the INM, mediate disruption of microtubule PLOS Genetics | www.plosgenetics.org 13 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies Figure 8. Schematic model of nuclear positioning and microtubule connections during differentiation of normal and SUN1/2 mutant myoblasts. (A) In myoblasts, the close positioning of centrosomes (red) to the outer nuclear surface is disrupted by SUN1/2 mutants, which is likely to be accompanied by impaired microtubule (green) association with the NE. (B) Upon commitment to differentiation in normal myoblasts, pericentrin and other centrosomal proteins redistribute from the centrosome to the nuclear surface, which becomes the major site of microtubule nucleation. In mutant committed myoblasts, pericentrin fails to associate with the NE and there is impairment of microtubule nucleation from the nuclear surface. (C) After cell fusion to form myotubes, the microtubules reorganize into overlapping parallel arrays along the long axis of the cell. The myonuclei become positioned evenly along the length of the cell in a microtubule-dependent manner with the involvement of dynein and kinesin motor proteins. In mutant myotubes, nuclei are clumped in a disorganized fashion and we propose that this is due to an inability to interact with the microtubule network. doi:10.1371/journal.pgen.1004605.g008 attachment to the NE. Studies have indicated that nesprins are loss of myonuclear anchoring and impaired muscle function important for microtubule association with the NE, through their [34,35]. Myonuclear positioning defects have been observed at the H22P/H22P interaction with microtubule motor proteins [77,80]. However, we myotendinous junctions of Lmna and Lmna knock-out did not obtain any evidence that the central SUN1-nesprin-2 mice, and in EDMD patients with mutations in LMNA [33,84]. LINC complex interaction was perturbed in MD-1 myoblasts, However, to our knowledge, ours is the first observation of such a suggesting that the defect may lie elsewhere. Instead, SUN1 pronounced myonuclear mispositioning phenotype in humans. It interaction with emerin was disrupted and, consistent with this, will be important, in future studies, to demonstrate directly that uncoupling of the nucleus from the microtubule network through both emerin mRNA and protein levels were reduced in myoblasts from patient MD-1. Impairment of SUN1/SUN2 interaction with SUN mutation does lead to muscle disease in vivo with emerin has also been observed in cases of EDMD1 due to physiological expression levels of SUN mutants. mutations in emerin itself [44]. Furthermore, EDMD has been In summary, our data clearly implicate defects in pericentrin associated with defects in emerin interaction with lamin A/C and recruitment, microtubule nucleation/organization and nuclear- nesprins [18,19]. Interestingly, emerin has been shown to partially cytoskeletal attachment in NE-associated muscular dystrophy localize at the ONM, where it may contribute to centrosomal pathogenesis and are in agreement with the bulk of results attachment to the NE and, in agreement with our findings, others showing SUN1/SUN2 involvement in nuclear positioning and cell have observed increased centrosomal separation from the nucleus migration. It remains to be determined precisely how centrosomal in EDMD1 cells [81,82]. Thus, dysregulation of emerin may play components are recruited to the nuclear envelope in differentiating a role in disease causation. myotubes and how defects in this process result in misalignment of myonuclei in muscular dystrophy. Contribution of SUN mutations to muscle disease Materials and Methods pathophysiology To date, most studies have focused on the role of the nuclear Ethics statement lamina and LINC complex in cellular resistance to mechanical This study involved the use of human DNA samples and myoblasts strain in support of the ‘‘structural’’ hypothesis of laminopathy derived from muscle biopsies. These were obtained following disease causation. There is now strong evidence to indicate that informed consent using protocols and consent forms approved by defects in these structural networks make a significant contribution the Ethics Committee of Ernst-Moritz-Arndt University, Greifswald. to the pathophysiology of EDMD and related disorders [29]. Given our observations of defective interaction networks in SUN- Patients and controls mutated cells and uncoupling of nuclear-cytoskeletal connections, EDMD patients for this study were selected based on the results it is therefore likely that the variants we have discovered in SUN1 of a routine diagnostic mutational analysis of EMD, LMNA, FHL1, and SUN2 also impact upon cell mechanics. Nuclear dysmor- SYNE1 and SYNE2. 175 pseudo-anonymized patients negative for phology is a common feature of laminopathy cells and, whilst the mutations in these genes and 70 patients known to carry mutations exact cause and effect of this phenomenon is not understood, it is in the genes encoding the LINC components emerin, lamin A/C likely to reflect changes in the organization of the nuclear lamina and nesprin 1 or 2 alpha and beta were tested for mutations in and its interactions with the nuclear envelope [83], together with SUN1 and SUN2. The clinical features of these unrelated, increased susceptibility to mechanical deformation. The exacer- predominantly Caucasian index cases were within the diagnostic bation of nuclear dysmorphology, induced by SUN1 W377C criteria for EDMD [50] despite the variable clinical expression. expression in LMNA R401C fibroblasts, again highlights a role for SUN proteins in NE organization and integrity. Our findings in patient MD-1 myotubes further indicate that Mutation analysis defects in nuclear positioning may play a significant role in disease Primer pairs for all the coding exons and flanking intronic pathogenesis, particularly since a link has also been made between sequences of SUN1 (UNC84A, ENSG00000164828; see Fig. S1) PLOS Genetics | www.plosgenetics.org 14 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies and SUN2 (UNC84B, ENSG00000100242, ENST00000405510) CATG-39 from an oligo dT-primed reverse transcription of U2OS were designed using Primer-Blast (http://www.ncbi.nlm.nih.gov/ cell mRNA and cloned into the BglII-SalI sites of the initial tools/primer-blast/index.cgi; Table S2). To standardize the construct. EDMD-associated mutations were introduced using the sequencing reaction, all primers were tagged with an M13-tail QuikChange II site-directed mutagenesis kit (Stratagene), accord- (forward: 59-GTAAAACGACGGCCAGT-39 reverse: 59-CAG- ing to the manufacturer’s instructions. GAAACAGCTATGAC-39). Amplifications were performed in 25 ml volumes using Amplikon-Taq Polymerase (Biomol) under Antibodies the following thermal conditions: initial denaturation at 94u for Anti-human SUN1 2383 and anti-human SUN2 2853 antibod- 5 min followed by 35 cycles of denaturation (94uC for 15 sec), ies have been described previously [44]. Anti-SUN1 Atlas annealing at the appropriate temperature for 15 sec (see Table S2) antibody (HPA008346) was obtained from Sigma prestige and elongation (72uC for 1 min). A final elongation (72uC for antibodies. Anti-nesprin-2 (N2N3) antibody was kind gift from 7 min) preceded a 4uC cooling step Q. Zhang (King’s College London) and has been described Direct Sanger sequencing was used to analyse PCR products. previously [16]. Anti-nesprin-2G has been reported previously Excess dNTPs and primers were removed using ExoSAP-IT [53]. Anti-nesprin-2 monoclonal antibody (IQ562) was purchased (Affymetrix). Sequencing reactions were performed using ABI from Immuquest. Monoclonal anti-emerin antibody was a kind BigDye Terminator v3.1 Cycle Sequencing Kit with addition of gift from G. Morris (Center for Inherited Neuromuscular Disease, 5% DMSO to the reaction mix. M13-oligonucleotides were used Oswestry, UK). Anti-lamin A/C (sc-6215) and GFP antibodies as sequencing primers. The reactions were analysed on a 3130xl were purchased from Santa Cruz Biotechnologies. Anti- GAPDH GA DNA Sequencer (Applied Biosystems) according to the (MAM374) was obtained from Millipore. Anti-a-tubulin (T9026), manufacturer’s instructions. All DNA variations identified were anti-b-actin (A5441), anti-c-tubulin (T6557), anti-myc and anti- validated using a second independent DNA sample. desmin antibodies were purchased from Sigma. Anti-caveolin 3 monoclonal antibody (610420) was purchased from Transduction Analysis of the frequency of DNA variations Laboratories and anti-desmin polyclonal antibody (MONX10657) Unique and rare sequence variations were tested for their was purchased from Monosan. Anti-pericentrin polyclonal anti- frequency in 400 alleles of a Caucasian reference population. body (Ab4448) was obtained from Abcam. Additionally, sequence variations found in a patient of Turkish origin were tested in 138 alleles of a Turkish reference population. Cell culture and transfection Co-segregation of DNA variations with the disease was analysed in Myoblasts from patient MD-1 and controls were routinely patient families if available. For estimating the frequency of DNA cultured in high-glucose DMEM supplemented with 20% foetal variations found, restriction digestion and high resolution melting bovine serum plus antibiotics penicillin, streptomycin and (HRM) were performed using patient DNA as positive control. amphotericin B, at 37uC and 5% CO2, and were used between Restriction enzymes cutting specifically at the DNA variation were passages 3 and 7. Myoblasts at confluence were allowed to selected using NEB-cutter (http://tools.neb.com/NEBcutter2/). differentiate into myotubes in the same culture medium for 8–15 HRM products amplified with LightCycler 480 High Resolution days, replacing the medium every 5 days. HeLa cells were cultured Melting Master (Roche) were analysed on a LightCycler 480 II in DMEM supplemented with 10% FBS and antibiotics. For (Roche) according to the manufacturer’s instructions. Samples emerin co-immunoprecipitation experiments, HEK293 cells were showing abnormal signals were examined by restriction endonu- transfected with the appropriate pCMVTag3-SUN1 constructs clease digestion or direct sequencing. The frequency of changes together with GFP-emerin [85] using Fugene 6 (Promega), found in patients of different origin was estimated from online according to the manufacturer’s instructions. pCMVTag3-SUN1 accessible genome sequencing data (Table S1). constructs were transfected into C2C12 mouse myotubes using the Amaxa Nucleofector (Lonza), according to the manufacturer’s Real-time PCR instructions. Cultures were fixed 24 hours after transfection and RNA was extracted from patient MD-1 and control myoblasts processed for immunofluorescence analysis. using TRIzol (Invitrogen) according to the manufacturer’s instructions. Real-time PCR was performed using a RealTime Centrosome reorientation and nuclear movement assay ready custom panel and LightCycler 480 Probes Master (Roche), NIH3T3 fibroblasts were cultured in 10% calf serum in DMEM with primers as described in Supplementary Material Table S3, (Gibco) as previously described [86]. Following serum starvation and evaluated on a LightCycler 480 II (Roche), according to the for two days, confluent monolayers were ‘‘wounded’’ by removing manufacturer’s instructions. Values for each gene were normalised a strip of cells and nuclei of cells at the edge of the wound were to both actin and GAPDH. microinjected with the appropriate myc-tagged SUN1 or SUN2 DNA plasmids. After expression for 2 hr, cells were stimulated Plasmid constructs and site-directed mutagenesis with 10 mM LPA for 2 hr, fixed in 4% paraformaldehyde, For plasmid constructs, the pCMVTag3B vector (Stratagene) extracted with Triton X-100 and stained with antibodies to was used to fuse a myc tag to the N-terminus of SUN1. The 916 tyrosinated a-tubulin (rat monoclonal antibody at 1/40 of culture amino acid version of the SUN1 cDNA, lacking the ATG start supernatant), myc (mouse monoclonal antibody from clone 9E10, codon, was generated by PCR amplification in two stages. First, Roche) and DAPI (Sigma) followed by appropriate secondary codons 2–362 were amplified using primers 59-CACAGAATTC- antibodies. Stained samples were observed with a Nikon TE300 GATTTTTCTCGGCTTCACAT-39 and 59-CACAGTCGA- microscope using a 406 Plan Apo N.A. = 1.0 or 606 Plan-Apo CCTATCCGATCCTGCGCAAGATCTGC-39 with IMAGE N.A. = 1.4 objective and filter cubes optimized for DAPI, clone 40148216 as template and inserted into pCMVTag3B via fluorescein/GFP, and rhodamine. Images were acquired with the EcoRI and SalI sites. This introduced a BglII site via a silent CoolSNAP HQ camera (Photometrics) driven by Metamorph mutation at codon 356–358. Codons 352–916 were then amplified software (MDS Analytical Technologies) and further processed in using 59-TTACTTCTTGCTGCAGATCTTGCGCAGGAT- Image J. Centrosomes were considered oriented if they were CGG-39 and 59-GAGAGTCGACTCACTTGACAGGTTCGC- localized in the pie-shaped sector between the nuclear membrane PLOS Genetics | www.plosgenetics.org 15 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies the leading edge scored, as described [86,87]. Random orientation and were observed at 0u tilt angle with a Geol Jem 1011 is ,33% by this measure. Nuclear and centrosome position transmission electron microscope, operated at 100 kV. At least 30 relative to the cell centroid were determined as described [88]. myoblasts/myotubes per sample were observed. Data were plotted as % of the cell radius to normalize for differences in cell size. Statistical analysis In all cases, statistical analysis was performed using a Student’s Cell extracts, immunoprecipitation, and immunoblotting t-test to compare differences in values obtained for patient/mutant To prepare total cell extracts for immunoblotting, cells were versus control samples. scrapedinto cold16phosphate-buffered saline (PBS), pelleted by centrifuging at 2006g for 5 min and then pellets were resuspended in Supporting Information lysis buffer (10 mM HEPES [pH 7.4], 5 mM EDTA, 50 mM NaCl, Figure S1 Transcript variant of SUN1 used in this study. (A) 1% Triton X-100, 0.1% SDS) supplemented with 1 mM PMSF and The 23-exon SUN1 isoform used for our investigations contained protease inhibitor cocktail (Roche) and an equal volume of Laemmli exons 4 to 26 of ENST00000456758. The start codon used is the buffer was then added. For human myoblast immunoprecipitations, same used in isoform ENST00000405266. (B) The resulting cells were grown on 10 cm dishes and then immunoprecipitated as isoform encodes 916 residues and corresponds to the full length described previously (Haque et al., 2006) using 2 mg of SUN1 2383 mouse isoform of SUN1 that predominates in most tissues [89]. antibody. 5% of the initial lysate was retained for immunoblot analysis. All samples were boiled in an equal volume of 26Laemmli Alternating exons are indicated in black and blue. Residues spanning splice sites are indicated in red. buffer, resolved on 6% or 7.5% or 10% polyacrylamide gels, followed by semidry transfer onto nitrocellulose membrane. Membranes were (TIF) probed using the appropriate primary antibodies and dilutions: Figure S2 Pedigrees of MD families with index patients carrying hSUN1 ATLAS (1:400), hSUN2 2853 (1:500), lamin A/C (1:2000), heterozygous SUN1 or SUN2 variants that do not co-segregate with emerin (1:1500), nesprin-2 N2N3 (1:1500), a-tubulin (1: 10,000), b- disease. Index cases are indicated by arrows. There was no evidence of actin (1:20,000), GAPDH (1: 10,000). Primary antibodies were increased disease severity in the index cases carrying the SUN1 detected using horseradish peroxidase-conjugated secondary antibod- variants. ies (Sigma), and visualization was performed using ECL reagents (PDF) (Geneflow). Figure S3 Evolutionary conservation of SUN1 and SUN2 mutated residues. All rare, non-synonymous variants identified Indirect immunofluorescence microscopy in SUN1 and SUN2 are shown. Those for which there is strong Myoblasts and myotubes grown on glass coverslips were fixed in genetic and/or functional evidence of disease-association are methanol at 220uC and processed for indirect immunofluorescence indicated in red. The mutated residues and their equivalents in microscopy as previously described (Haque et al., 2006). For SUN1 other species are highlighted in beige. staining, cells were instead fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 at room temperature for (PDF) 5 min. Cells were washed in PBS and incubated with antibodies Figure S4 SUN1 mRNA levels are not altered in MD-1 diluted in PBS–3% bovine serum albumin, using hSUN1 2383 (1:150), myoblasts. Expression level of the indicated genes was assessed hSUN2 2853 (1:100), lamin A/C (1:400), emerin (1:500), nesprin-2G by quantitative real-time PCR using total RNA isolated from (1:300), c-tubulin (1:500) pericentrin (1:50), caveolin 3 (1:30) and control and MD-1 myoblasts. Values are expressed relative to two desmin (1:100) antibodies. Secondary antibodies were goat anti-rabbit control genes, ACTB and GAPDH, and show the average of 2 AlexaFluor 488, donkey anti-mouse AlexaFluor 594 and donkey anti- independent experiments performed in duplicate 6S.E. Signifi- goat AlexaFluor 594 (Molecular Probes Inc.). DNA was stained with cant P-values are as follows: SUN2 P = 0.019, LMNA P = 0.009, 50 mg/ml 49,6-diamidino-2-phenylindole (DAPI; Sigma). Coverslips SYNE1 P = 0.0016, SYNE2 P = 0.01. were mounted in 80% glycerol–3% n-propyl gallate (in PBS) or (PDF) ProLong gold antifading reagent (Invitrogen). Fluorescence microscopy Figure S5 SUN1 can polarize in MD-1 myotubes. SUN1 (green) was performed with a Nikon TE300 inverted microscope with an ORCA-R charge-couple device camera (Hamamatsu) and Volocity and caveolin (red) immunofluorescence staining in control and software (PerkinElmer). Where required fluorescence microscopy was patient MD-1 myotubes, along with DAPI (blue) staining of DNA. also performed with Leica TCS SP5 confocal laser scanning Arrowheads indicate nuclei in which SUN1 is enriched at the microscope and Leica LAS AF software. Images were processed with poles. Adobe Photoshop (Adobe Systems). Quantification of fluorescence (TIF) intensity was performed using an Olympus Scan Rmicroscope witha Figure S6 Nesprin-2 expression is elevated in patient MD-1 206 objective. Approximately 1000 nuclei from 3 independent myotubes. (A) Nesprin-2 staining in myotubes from control and experiments were randomly selected by their DAPI signal, and the patient MD-1. Immunofluorescence labeling was performed with intensity of SUN1, SUN2, emerin, lamin A/C and nesprin-2 was nesprin-2 monoclonal (red) and desmin (green) antibodies. Desmin measured within the DAPI-stained region. was used as a muscle cell marker. (B) Nesprin-2 fluorescence intensity was measured using the NIS software analysis system and Electron microscopy 50 myotubes per sample were analysed. Data are presented as Myotubes (at passage 2–3) from patient and age-matched mean value 6S.D. Significant P-value for patient MD-1 was controls were fixed in 2.5% glutaraldehyde-0.1 M cacodylate 0.042. Scale bar, 10 mm. buffer pH 7.4 for 3 h at 4uC. After post-fixation with 1% osmium (TIF) tetroxide (OsO4) in cacodylate buffer for 2 h, samples were dehydrated in an ethanol series, infiltrated with propylene oxide Figure S7 The number of microtubules nucleating from the and embedded in Epon resin. Ultrathin sections (60 nm thick) nuclear envelope is reduced in MD-1 myotubes. Beta-tubulin (red) were stained with uranyl acetate and lead citrate (10 min each) and pericentrin (green) double immunofluorescence staining in PLOS Genetics | www.plosgenetics.org 16 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies untreated control and MD-1 myotubes, or following nocodazole Acknowledgments treatment and 30 min recovery in culture medium. Chromatin We would like to thank the patients and their families for taking part in this was stained with DAPI (blue). Scale bar, 10 mm. Higher study. For providing clinical data, we acknowledge the contributions of magnification (36) of nuclear envelopes in nocodazole-treated Drs. M. Hoeltzenbein, W. Ko¨hler, U. Kordass, T. Lunke, A. Madej, W. cells is shown on the right of each picture. Mu¨ller-Felber, M. Munteanu, A. Petschaelis, G. Ramb, U. Reuner, C. (TIF) Vesely, S. Vielhaber, T. Voit, M. Vorgert, and M. Walter. We thank Muscle Tissue Culture Collection (MTCC) for providing myoblast Table S1 Single nucleotide changes found in coding regions of samples. The MTCC is part of the German network on muscular SUN1 and SUN2 and their frequencies in sequenced genome dystrophies (MD-NET, service structure S1, 01GM0601), funded by the databases. Rare, non-synonymous variants are highlighted in bold, German ministry of education and research (BMBF, Bonn, Germany), and with blue shading. *Patient MD-1 was of Turkish origin, therefore is a partner of EuroBioBank (www.eurobiobank.org) and TREAT-NMD 150 alleles from ethnically matched controls were also screened for (www.treatnmd.eu). We are also grateful to A. Fry (University of Leicester, UK) for helpful discussions and for critical reading of the manuscript. mutations p.G68D and p.G338S. (PDF) Author Contributions Table S2 Primer sequences and annealing temperatures for Conceived and designed the experiments: SS MW GL GGG HJW PM EM genomic amplification of SUN1 and SUN2 exons. FH SA. Performed the experiments: PM EM FH MC SA KRS. Analyzed (PDF) the data: PM MW EM GL FH KRS SS SA HJW GGG. Contributed Table S3 Primers used for real-time PCR. reagents/materials/analysis tools: SS GL MW GGG KRS. Wrote the (PDF) paper: SS GL MW GGG HJW. References 1. 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(2006) individual mammalian Sun1 isoforms depends on the cell type. Commun Integr Lamin A/C and emerin are critical for skeletal muscle satellite cell Biol 4: 440–442. differentiation. Genes Dev 20: 486–500. PLOS Genetics | www.plosgenetics.org 18 September 2014 | Volume 10 | Issue 9 | e1004605 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS Genetics Public Library of Science (PLoS) Journal

Muscular Dystrophy-Associated SUN1 and SUN2 Variants Disrupt Nuclear-Cytoskeletal Connections and Myonuclear Organization

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Copyright: © 2014 Meinke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funding from the Wellcome Trust (grant number WT087244MA) awarded to SS and MW; from the Fondo 5 per mille 2010 awarded to the Istituto Ortopedico Rizzoli and FIRB MIUR 2010 to the CNR Institute for Molecular Genetics and EU COST BM1002 to GL; and from the U.S. NIH (grant number NS059352) awarded to GGG and HJW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.
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1553-7390
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1553-7404
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10.1371/journal.pgen.1004605
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Abstract

Proteins of the nuclear envelope (NE) are associated with a range of inherited disorders, most commonly involving muscular dystrophy and cardiomyopathy, as exemplified by Emery-Dreifuss muscular dystrophy (EDMD). EDMD is both genetically and phenotypically variable, and some evidence of modifier genes has been reported. Six genes have so far been linked to EDMD, four encoding proteins associated with the LINC complex that connects the nucleus to the cytoskeleton. However, 50% of patients have no identifiable mutations in these genes. Using a candidate approach, we have identified putative disease-causing variants in the SUN1 and SUN2 genes, also encoding LINC complex components, in patients with EDMD and related myopathies. Our data also suggest that SUN1 and SUN2 can act as disease modifier genes in individuals with co- segregating mutations in other EDMD genes. Five SUN1/SUN2 variants examined impaired rearward nuclear repositioning in fibroblasts, confirming defective LINC complex function in nuclear-cytoskeletal coupling. Furthermore, myotubes from a patient carrying compound heterozygous SUN1 mutations displayed gross defects in myonuclear organization. This was accompanied by loss of recruitment of centrosomal marker, pericentrin, to the NE and impaired microtubule nucleation at the NE, events that are required for correct myonuclear arrangement. These defects were recapitulated in C2C12 myotubes expressing exogenous SUN1 variants, demonstrating a direct link between SUN1 mutation and impairment of nuclear- microtubule coupling and myonuclear positioning. Our findings strongly support an important role for SUN1 and SUN2 in muscle disease pathogenesis and support the hypothesis that defects in the LINC complex contribute to disease pathology through disruption of nuclear-microtubule association, resulting in defective myonuclear positioning. Citation: Meinke P, Mattioli E, Haque F, Antoku S, Columbaro M, et al. (2014) Muscular Dystrophy-Associated SUN1 and SUN2 Variants Disrupt Nuclear- Cytoskeletal Connections and Myonuclear Organization. PLoS Genet 10(9): e1004605. doi:10.1371/journal.pgen.1004605 Editor: Gregory A. Cox, The Jackson Laboratory, United States of America Received October 2, 2013; Accepted July 16, 2014; Published September 11, 2014 Copyright:  2014 Meinke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funding from the Wellcome Trust (grant number WT087244MA) awarded to SS and MW; from the Fondo 5 per mille 2010 awarded to the Istituto Ortopedico Rizzoli and FIRB MIUR 2010 to the CNR Institute for Molecular Genetics and EU COST BM1002 to GL; and from the U.S. NIH (grant number NS059352) awarded to GGG and HJW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] ¤a Current address: The Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom ¤b Current address: Retired, Rostock, Germany . These authors contributed equally to this work. that plays a vital role in supporting the NE and maintaining Introduction nuclear integrity, whilst also contributing to chromatin organiza- The nuclear envelope (NE) is composed of the nuclear tion and regulation of gene expression (reviewed in [2]). membranes, nuclear lamina and nuclear pore complexes and Mutations in genes encoding NE proteins are associated with a encloses the chromatin in eukaryotic cells. Lamin intermediate range of tissue-restricted inherited disorders that can affect striated filament proteins are the major structural components of the NE muscle, bone, fat or neurons and in some cases cause premature and polymerize to form a fibrous meshwork that underlies the ageing syndromes [3]. Most strikingly, different mutations in one nucleoplasmic face of the inner nuclear membrane. This nuclear gene – the LMNA gene that encodes A-type nuclear lamins – can lamina is attached to the inner nuclear membrane through cause many diseases, which have collectively been termed interactions with multiple integral inner nuclear membrane (INM) laminopathies [4]. Diseases affecting striated muscle are the most proteins [1]. Together, these proteins form a structural network common of the laminopathies and include autosomal dominant PLOS Genetics | www.plosgenetics.org 1 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies network through interaction with plectin [26], whilst nesprin-4 is Author Summary specific to epithelial cells and connects the NE to microtubules via Emery-Dreifuss muscular dystrophy (EDMD) is an inherited the kinesin-1 motor protein [27]. disorder involving muscle wasting and weakness, accom- There are several proposed mechanisms to explain the tissue panied by cardiac defects. The disease is variable in its specificity of EDMD and other laminopathies, which centre severity and also in its genetic cause. So far, 6 genes have around the ‘‘gene expression’’ and ‘‘structural’’ hypotheses [28]. been linked to EDMD, most encoding proteins that form a Current evidence strongly supports the ‘‘structural hypothesis’’, structural network that supports the nucleus of the cell which suggests that muscle-associated laminopathies primarily and connects it to structural elements of the cytoplasm. result from weakening of the structural networks of the nuclear This network is particularly important in muscle cells, lamina and cytoskeleton and the LINC complex that connects providing resistance to mechanical strain. Weakening of these two networks [29]. Since myocytes are subject to recurrent this network is thought to contribute to development of mechanical strain from contractile forces, weakening of these muscle disease in these patients. Despite rigorous screen- structural networks renders the cells particularly susceptible to ing, at least 50% of patients with EDMD have no damage. However, the LINC complex is also vital for correct detectable mutation in the 6 known genes. We therefore myonuclear positioning [30–33] and defects in this process are undertook screening and identified mutations in two implicated in impaired muscle function [34,35]. additional genes that encode other components of the Despite the genetic studies so far carried out, causative nuclear structural network, SUN1 and SUN2. Our findings mutations have been identified in only approximately 50% of add to the genetic complexity of this disease since some EDMD and related muscle disease cases [10]. It is therefore highly individuals carry mutations in more than one gene. We likely that mutations in additional genes contribute to the disease. also show that the mutations disrupt connections between the nucleus and the structural elements of cytoplasm, Furthermore, there is significant heterogeneity in disease severity leading to mis-positioning and clustering of nuclei in even within families carrying the same gene mutation [36–40], muscle cells. This nuclear mis-positioning is likely to be which has led to the suggestion of modifier genes [41–43]. another factor contributing to pathogenesis of EDMD. Given that SUN1 and SUN2 interact with at least four of the known muscle disease-associated NE proteins and that these interactions can be perturbed by disease-causing LMNA and and recessive Emery-Dreifuss muscular dystrophy (EDMD2 and EMD mutations [44], we investigated whether the SUN1 and EDMD3, respectively; OMIM#181350), limb-girdle muscular SUN2 genes may also be mutated in some individuals. Screening dystrophy (LGMD) type 2B and dilated cardiomyopathy and of the SUN1 and SUN2 genes in a large cohort of patients with conduction system disease (CMD) type 1A [5–8]. These diseases EDMD and phenotypically related myopathies identified SUN1 share the common feature of cardiomyopathy, but EDMD and and/or SUN2 variants in several patients. Presence of SUN1 or LGMD also involve progressive muscle wasting and weakness. In SUN2 variants correlated with increased disease severity in all cases, premature sudden death can result from cardiac patients with EDMD carrying mutations in other genes, thus arrhythmia and conduction defects. identifying SUN1 and SUN2 as modifiers of the EDMD disease Striated muscle disease, in particular EDMD, can also be phenotype. We further provide evidence that these mutations caused by mutations in genes encoding other NE proteins. An X- disrupt nuclear-cytoskeletal connection and nuclear positioning, linked form of EDMD (EDMD1; OMIM#310300) is caused by supporting the hypothesis that muscular dystrophies arise from mutations in EMD, that encodes the integral INM protein emerin defective nuclear-cytoskeletal coupling. [9]. Together, mutations in LMNA and EMD account for around 40% of cases of EDMD [10]. Rare mutations in the genes Results encoding FHL1, TMEM43 (also named LUMA), nesprin-1 and Screening SUN1 and SUN2 genes in a cohort of patients nesprin-2 have also been reported [11–13]. Interestingly, A-type lamins, nesprins and emerin all interact with each other [14–16], with EDMD and related myopathies contributing to a network that connects the nuclear lamina to the We analyzed DNA from 175 unrelated patients with EDMD cytoskeleton, termed the LINC (Linker of Nucleoskeleton and and related myopathies, who had previously undergone screening Cytoskeleton) complex [17]. Furthermore, interactions are often for mutations in the LMNA, EMD, SYNE1/SYNE2 alpha and perturbed by muscle disease-causing mutations, indicating that this beta (encoding short isoforms of nesprin-1 and nesprin-2, network of interactions plays an important role in muscle function respectively) and FHL1 genes and in whom no causative mutation [12,18,19]. had been found. These included both sporadic cases and index The central components of the LINC complex in mammals are patients from familial cases. Furthermore, there have been several SUN and nesprin proteins that reside in the INM and outer reports of modifiers of the phenotype of LMNA-linked muscle nuclear membrane (ONM), respectively. The conserved SUN and diseases [41–43]. We therefore also screened EDMD patients KASH domains of the respective proteins interact in the carrying identified LMNA, SYNE1/SYNE2 alpha and beta and perinuclear space to form a bridge spanning the INM, perinuclear EMD mutations to determine whether mutation of SUN1 or space and ONM that connects the nuclear lamina to the SUN2 may influence disease phenotype. Most individuals were of cytoskeleton. The nucleoplasmic N-termini of the SUN proteins, Caucasian origin, except where otherwise stated. SUN1 and SUN2, interact with the nuclear lamina, anchoring the The 23 exons of the SUN1 gene (see Figure S1) and 19 exons of LINC complex at the NE [20–22]. In turn, the cytoplasmic the SUN2 gene (ENSG00000100242, ENST00000405510), domains of the nesprins connect to the cytoskeleton. There are 4 including intron/exon boundaries and promoter regions were nesprin isoforms encoded by different genes. Giant isoforms of analyzed. DNA was amplified using PCR and analyzed by direct nesprins-1 and -2 contain an N-terminal calponin homology Sanger sequencing. In total, we found 34 single nucleotide domain responsible for actin binding [23,24] and linkage to the polymorphisms within the coding regions of SUN1 and SUN2, centrosome through microtubules and their motor proteins [25]. 18 of which were classified as rare, non-synonymous changes Nesprin-3 connects to the cytoplasmic intermediate filament following analysis of their frequencies in sequenced genome PLOS Genetics | www.plosgenetics.org 2 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies Figure 1. SUN1 and SUN2 variants identified and associated family pedigrees. (A) Schematic diagram of the SUN1 and SUN2 protein domain organization and locations of disease-associated variants identified in our cohort. Mutation SUN1 M50T, indicated in purple, did not disrupt LINC complex function in migration assays and thus may not be truly disease-causing. The mapped lamin A/C (green) and emerin (orange) binding sites, located in the nucleoplasmic N-terminal domain, are indicated. Regions of high hydrophobicity and the transmembrane domain are shown in grey and black, respectively. Coiled-coil domains responsible for oligomerization (blue) and the highly conserved SUN domain (red), found within the luminal C-terminal domain, are also indicated. (B) Pedigree with recessive inheritance of compound heterozygous SUN1 variants. (C) Pedigrees where severely affected index cases carry SUN1 and/or SUN2 variants in combination with other gene mutations. Filled circles/squares indicate affected females/males. Circles containing a dot, in family 3, indicate unaffected female carriers of the X-linked EMD mutation. Arrows indicate index patients. doi:10.1371/journal.pgen.1004605.g001 databases (Table S1, Figure S3). Three of these variants did not had EDMD or related myopathy phenotypes (Table 1). Sporadic segregate with disease in the respective families (Figure S2). In nine patient MD-11 carried a single SUN2 p.R620C sequence unrelated families or sporadic cases, however, we identified 10 rare variation. We had no access to DNA from family members for non-synonymous variants in SUN1 and SUN2 for which we have this patient, but the high degree of evolutionary conservation of obtained evidence of pathogenic effects, as deduced from genetic, R620 is supportive of disease-association (Figure S3). Patient MD- phenotypic and/or functional data (Figure 1A). 1 carried compound heterozygous SUN1 p.G68D and p.G338S variants. For patient MD-1 we had access to DNA from family Putative disease-associated SUN1 and SUN2 variants in members and observed apparent recessive inheritance, with one patients with EDMD-like phenotypes mutation coming from each of the unaffected parents (Figure 1B). We identified 5 rare, non-synonymous SUN1 and/or SUN2 SUN1 p.G338S was also present in the reference population at variants in 3 individuals who lacked mutations in other genes but low frequency (see Table S1). These residues are located within the PLOS Genetics | www.plosgenetics.org 3 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies Table 1. Putative disease-causing variants in SUN1 and SUN2 in patients with EDMD-like phenotypes. Family Index case SUN1 variant SUN2 variant Other mutations Disease phenotype 1 MD-1 p.G68D p.G338S none none Male; age at onset 10 years; mild muscle weakness; rigid spine; serum creatine kinase elevation 6X; no cardiac involvement; last clinical examination at age 10 years; sporadic case. 11 MD-11 none p.R620C none Sporadic EDMD-related myopathy, no other clinical information available. 12 MD-12 p.W377C p.E438D none Heart rhythm disturbances at age 34 years; partial lipodystrophy on left lower leg; sporadic case doi:10.1371/journal.pgen.1004605.t001 poorly conserved N-terminal domain of the protein and, in this mutation was inherited from an unaffected parent. Again, the context, are moderately conserved (Figure S3), but functional data disease expression in both index patients was more severe than is presented below present compelling evidence of the involvement typical for EDMD [49,50], with early onset at age 1 and 4 years, of both mutations in disease causation. Sporadic patient MD-12 respectively, and early heart involvement including heart trans- carried heterozygous changes in both SUN1 and SUN2, encoding plantation before age 20 years (Table 2). SUN1 p.W377C and SUN2 p.E438D, respectively. E438 is We also identified two SUN2 variants, encoding variants conserved in mammals, whilst W377 is conserved across all species p.M50T and pV378I, in patient MD-2 who had hypertrophic examined (Figure S3). cardiomyopathy and also carried a mutation in MYBPC3 (p.G148R), which encodes a myosin binding protein. The same SUN1 and SUN2 variants with disease modifying effects in MYBPC3 mutation was previously reported in a Dutch family, where a severely affected index patient had compound heterozy- patients with co-segregating mutations in other genes Because patients with EDMD-like phenotypes exhibit variable gous mutations in MYCBP3 but other family members carrying only p.G148R were either asymptomatic or developed cardiomy- disease severity that could be explained by mutations or polymorphisms in additional genes, we screened for SUN1 and opathy late in life [51]. This MYBPC3 mutation was present in SUN2 variants in patients with known mutations in causative both patient MD-2 and his father (Figure 1C; S. Waldmueller, genes. SUN1 or SUN2 variants were indeed present in some personal communication). Patient MD-2 presented as a 6 month- patients from families with LMNA or X-linked EMD mutations old boy with hypertrophic cardiomyopathy and died at 16 years (Table 2; Figure 1C). These sequence changes correlated with from heart failure. His father, who carries the SUN2 p.M50T increased disease severity. In one example, a SUN1 p.A203V variant but not p.V378I, is asymptomatic. Thus, the SUN2 polymorphism in patient MD-3 co-segregated with a previously p.V378I variant appears to have a dramatic effect on disease reported EMD p.L84Pfs*6 mutation in two brothers with severity. Notably, this variant is present in the reference unusually severe EDMD (Figure 1C, Family 3) [39]. The EMD population at low frequency (see Table S1), suggesting that it p.L84Pfs*6 mutation, which abolishes emerin expression, has been may be a relatively common genetic modifier of inherited reported in an unrelated family, where the course of the disease cardiomyopathy. was significantly milder EDMD with later age of onset and no loss of ambulation [45]. Another unrelated patient carrying EMD SUN1 and SUN2 disease-associated variants disrupt p.L84Pfs*6 was included in this study but no SUN1 or SUN2 centrosome reorientation and nuclear movement in variants were found and their phenotype was similar to that NIH3T3 fibroblasts described by Manilal et al. [45]. In another case, the SUN1 Our genetic results suggest that mutations or polymorphisms in p.G76A mutation, when combined with EMD p.A56Pfs*9 in a SUN1 and SUN2 may cause muscular dystrophy and act as previously described Korean patient (MD-4), led to a very severe modifiers of EDMD and cardiomyopathy. To obtain additional clinical picture with complete atrioventricular block requiring pace evidence that these variants play a role in pathophysiology, we maker implantation at age 14 years [46]. Similarly, SUN1 examined the effects of several variants on a known function of the p.W377C was detected in combination with LMNA p.R453W LINC complex, namely centrosome orientation and nuclear in patient MD-5. This individual had severe disease and died early movement in migrating cells. SUN2, along with the nesprin-2G at the age of 34 years from heart failure. The patient’s son, isoform, assembles into transmembrane actin-associated nuclear carrying LMNA p.R453W only, did not show clinical signs of (TAN) lines that couple actin cables to the nucleus to move it contractures or muscular weakness at age 10 years. LMNA rearward and reorient the centrosome toward the leading edge in p.R453W is a common EDMD-associated LMNA mutation and is migrating NIH3T3 fibroblasts [52,53]. While SUN1 is not in TAN generally not associated with severe cardiac disease, suggesting lines, it also functions in connecting the nucleus to the cytoskeleton that, in patient MD-5, SUN1 p.W377C had a modifying effect to via the LINC complex. increase disease severity [47,48]. The same SUN1 p.W377C We expressed three myc-SUN1 and three myc-SUN2 variants in variant was detected in patient MD-12, who had an EDMD-like NIH3T3 fibroblasts at the edge of a wounded monolayer by DNA phenotype but did not have mutations in EMD or LMNA but microinjection and stimulated nuclear movement and centrosome carried a concurrent SUN2 p.E438D variant, as described above. reorientation with the serum factor, lysophosphatidic acid (LPA). We detected SUN2 mutations in combination with LMNA Upon stimulation, non-expressing NIH3T3 cells or NIH3T3 cells mutations in two additional index cases. Patient MD-6 carried LMNA p.T528K and SUN2 p.A56P, whilst patient MD-7 carried exogenously expressing wild-type (WT) SUN1 or SUN2, as well as LMNA p.R98P and SUN2 p.V378I (Table 2; Figure 1C). In both the variant SUN2 M50T, reoriented their centrosomes (Figure 2A– cases, the LMNA mutation had arisen de novo, whilst the SUN2 B). Notably, SUN2 p.M50T did not appear to influence disease PLOS Genetics | www.plosgenetics.org 4 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies Table 2. SUN1 and SUN2 variants with disease-modifying effects in patients with MYBPC3, EMD and LMNA mutations. Family Index case SUN1 variant SUN2 variant Other mutations Disease phenotype 2 MD-2 none p.M50T p.V378I MYBPC3 p.G148R Male; age at onset 6 months; hypertrophic cardiomyopathy; at age 9 years ECG showed cardiac arrhythmia, supraventricular extrasystols, Echocardiogram: right ventricular septum hypertrophy, first degree atrioventricular block; no muscular weakness or dystrophy; sinus tachycardia and bradycardia, mild left ventricular functional impairment; died at age16 years from heart failure; sporadic case. 3 MD-3 p.A203V none EMD p.L84Pfs*6 Male; age at onset 2 years; severe contractures of neck, thoracolumbar spine, elbows, and Achilles tendons; Achillotomia at age 6; loss of ambulation at age 15; moderate to severe muscle weakness; left anterior hemi-block and ventricular ectopy at age 23; ventricular dilation at age 33; X-linked EDMD [39]. 4 MD-4 p.G76A none EMD p.A56Pfs*9 Male; age of onset 1 year, wasting and weakness of shoulder girdle and limb-girdle muscles; at age 14 severe contractures of neck, elbow and Achilles tendons, tendon reflexes absent; at age 14 dilated right atrial and ventricular dilation, atrial fibrillation, complete AV block and junctional escape rhythm; pacemaker since age 14; CK elevation 4X; X-linked EDMD [46]. 5 MD-5 p.W377C none LMNA p.R453W Male; age of onset 8 years; slowly progressive humero-peroneal muscular weakness; since age 14 rigid spine, contractures of elbow and Achilles-tendons; at age 25 cardiac disturbances; AV- block III; heart pacemaker at age 31; died at age 34 of heart failure; autosomal dominant EDMD. 6 MD-6 none p.A56P LMNA p.T528K Male; age at onset 1 year; delayed early childhood developmental mile stones; later difficulties in climbing stairs, muscular weakness; at age 15 tachycardia, extrasystols; at age 17 intra- ventricular cardiac conduction defects, contractures of elbow and Achilles tendons; proximal humero-peroneal muscle atrophy, rigid spine, Gower’s maneuver; CK elevated 3–5X; de novo LMNA mutation leading to sporadic EDMD. 7 MD-7 none p.V378I LMNA p.R99P Female; age of onset 4 years, diffuse muscular weakness; later bilateral contractures of the elbows and ankles, dilated cardiomyopathy, first degree atrioventricular block; at age 14 heart pacemaker; histopathology showed fibrosis of the heart muscle; at age 15 heart transplantation; de novo LMNA mutation leading to sporadic EDMD. doi:10.1371/journal.pgen.1004605.t002 severity in family 2. In contrast, cells expressing the putative disease- expression of LINC complex components and known SUN1 binding causing SUN1 variants, G68D, G338S or W377C, inhibited partners in the myoblasts by immunofluorescence microscopy. Since centrosome reorientation by blocking rearward positioning of the nesprin-1 is not significantly expressed in myoblasts [54,55] (Figure nucleus (Figure 2A–B). Similarly, cells expressing SUN2 A56P or S4), we stained for nesprin-2, lamin A/C, emerin, SUN1 and SUN2. R620C failed to reorient their centrosome due to an inability to No obvious defects in localization of SUN1, SUN2 or their position their nuclei rearward of the cell centroid (Figure 2A–B). All interacting NE partners were observed, but expression of SUN1 of the expressed SUN1 and SUN2 variants had a normal nuclear and nesprin-2 at the NE was enhanced in the patient myoblasts localization similar to the wild-type proteins (Figure 2A). The (Figure 3A–B). Quantification of fluorescence intensity suggested that centrosome orientation defect in cells expressing the SUN variants their expression was increased approximately 2-fold. occurred due to defective rearward nuclear movement and not Since fluorescence intensity does not always provide an accurate disruption of positioning of the centrosome at the cell centroid reflection of total protein levels, we then examined total protein (Figure 2C). Hence, five putative disease-causing or disease-modify- expression level by Western blot and found that SUN1 levels were ing SUN variants blocked rearward nuclear movement in migrating elevated 8-fold in the patient versus control myoblasts (Figure 3C,E). NIH3T3 fibroblasts and it can be concluded that these mutants Consistent with the fact that SUN proteins form complexes with, and disrupt LINC complex function. are responsible for anchoring of nesprins in the ONM, we also observed a 4-fold increase in expression of the intermediate-sized Expression of LINC complex components is altered in muscle-enriched isoforms of nesprin-2 [55] in the patient myoblasts patient myoblasts with compound heterozygous SUN1 (Figure 3D–E). However, it remains possible that the bands observed are degradation products of nesprin-2 giant. In contrast, levels of mutations SUN2, lamin A/C and emerin were not significantly altered in the Having established that some of the variants identified in our patient cells (Figure 3C,F). To exclude the possibility that the observed patient cohort disrupted LINC complex function in fibroblasts, we changes were due to different levels of background differentiation in the next wished to examine their role in muscle cells. We were able to obtain primary myoblasts from patient MD-1, carrying compound control and patient cultures, we quantified expression of myogenin, an heterozygous SUN1 p.G68D/p.G338S variants. To gain initial early marker of myogenic differentiation, and found no detectable level insight into the cellular effects of these mutations, we examined in either culture (data not shown). PLOS Genetics | www.plosgenetics.org 5 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies Figure 2. SUN1 and SUN2 variants disrupt nuclear-cytoskeletal coupling in NIH3T3 fibroblasts. (A) Representative immunofluorescence micrographs of LPA-stimulated NIH3T3 fibroblasts expressing SUN1 or SUN2 wild-type (WT) or variant proteins. Cells were immunostained for myc (green), tubulin (red) and DAPI (blue). Location of centrosome (arrows) was determined by the center of microtubule array. Bar, 20 mm. (B) Quantification of centrosome reorientation in LPA-stimulated NIH3T3 fibroblasts expressing the indicated SUN variants. Significant differences are indicated by * with a,0.05 based on Student’s t-test when the sample is compared to non-expressing NIH3T3 fibroblasts. (C) Quantification of nucleus and centrosome position relative to the cell centroid in NIH3T3 fibroblasts expressing the indicated SUN variants. Positive values are toward the leading edge, negative values toward the cell rear. Data are from at least three independent experiments for each sample. Significant differences are indicated by * with a,0.05 based on Student’s t-test when the sample is compared to non-expressing NIH3T3 fibroblasts. doi:10.1371/journal.pgen.1004605.g002 To address the mechanism of SUN1 elevation in the patient due to weakening of the protein interaction network supporting myoblasts, we examined SUN1 mRNA levels by qPCR and found no the NE [28,56]. To determine whether muscle disease-associated significant increase in mRNA level compared to the control, SUN1 alterations disrupt interactions with other LINC complex indicating that the p.G68D/p.G338S SUN1 variants do not lead components, we performed immunoprecipitation with anti-SUN1 to increased mRNA levels (Figure S4). However, in analyzing mRNA antibodies on protein extracts from control and patient MD-1 levels of other LINC complex-associated proteins, we observed a myoblasts and detected co-precipitating proteins. We observed a statistically significant increase in expression of LMNA, SUN2, reproducible reduction in SUN1 interaction with emerin in the SYNE1 and SYNE2. In contrast, EMD (encoding emerin) expression patient myoblasts, whilst interaction with lamin A/C was not was decreased in the patient relative to control myoblasts (Figure S4). obviously perturbed (Figure 4A–B). Consistent with the enhanced recruitment of nesprin-2 to the NE, interaction of SUN1 with SUN1 interaction with emerin is impaired in MD-1 nesprin-2 was also maintained in the MD-1 myoblasts (Figure 4C). myoblasts Larger isoforms of nesprin-2 were enriched in the immunopre- One hypothesis to explain NE-associated muscle diseases is the cipitate compared to the initial lysates, indicating a preferential ‘‘structural hypothesis’’, which suggests that muscle damage occurs interaction of SUN1 with these less abundant isoforms. Thus, the PLOS Genetics | www.plosgenetics.org 6 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies Figure 3. Expression of LINC complex proteins is increased in patient MD-1 (SUN1 p.G68D/p.G338S) myoblasts. (A) Control and MD-1 myoblasts were fixed in methanol and analysed by immunofluorescence microscopy using SUN1, SUN2, emerin, nesprin-2G and lamin A/C antibodies, as indicated, together with DAPI staining of DNA. Scale bar, 22 mm. (B) Mean fluorescence intensity of SUN1, SUN2, emerin, nesprin 2 and PLOS Genetics | www.plosgenetics.org 7 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies ‘ ‘ lamin A/C was measured in individual DAPI-stained nuclei using an Olympus Scan R screening station and analysed using Scan R analysis software. The results are presented as mean 6 S.E. of 1000 cells taken from at least 3 independent experiments. **P#0.05. Significant P-value for SUN1 was P = 0.009. (C) Total protein extracts from control (C) and patient MD-1 myoblasts were Western blotted using antibodies against LINC complex- associated proteins, as indicated. (D) Samples prepared as in A were Western blotted using nesprin-2 (N2–N3) antibodies. (E–F) Protein expression was quantified by densitometric analysis of at least 3 independent experiments. The results are presented as mean 6 S.E. *P#0.05 and **P#0.01. Each significant P- values are as follows: SUN1 P = 0.05, a-tubulin P = 0.003, nesprin-2 P = 0.002. P- value for emerin was P = 0.06. doi:10.1371/journal.pgen.1004605.g003 SUN1 p.G68D/p.G338S variants in patient MD-1 appear to structs. SUN1 was immunoprecipitated using anti-myc antibodies specifically disrupt interaction with emerin. and co-precipitating GFP-emerin detected by Western blotting. We confirmed defective interactions with emerin in HEK293 We observed a significant reduction in interaction of emerin with cells transiently expressing GFP-emerin and myc-SUN1 con- both SUN1 G338S and W377C, whilst there was only a modest Figure 4. Emerin binding to p.G68D/p.G338S SUN1 is reduced in vivo. (A) SUN1 was immunoprecipitated from control (C) or MD-1 myoblast soluble lysates using 2383 SUN1 antibodies and samples Western blotted to detect co-immunopreciptated proteins. (B) Densitometric analysis of SUN1, lamin A/C and emerin bands from immunoprecipitated samples is plotted in arbitrary units (A.U.). (C) SUN1 was immunoprecipitated from control or MD-1 myoblasts and co-precipitated nesprin-2 was detected using N2–N3 antibody. Size markers (kDa) are indicated. Note that larger nesprin-2 isoforms are enriched in the immunoprecipitate compared to the lysate. (D) HEK293 cells were co-transfected with myc-SUN1 mutant and GFP-emerin plasmids and harvested after 48 hours. Co-precipitated GFP-emerin was detected by immunoblotting using GFP antibodies (myc-SUN1 IP). (E) Densitometric values of immunoblotted GFP-emerin bands are reported in arbitrary units (A.U.). (F) Human fibroblasts from an EDMD2 patient carrying the R401C LMNA mutation were transfected with SUN1-WT or SUN1-W377C cDNAs and fixed 48 hours after transfection. Lamin A/C was labelled using specific antibodies and revealed by FITC-conjugated secondary antibody (green). SUN1 was detected using Cy3-conjugated anti-myc antibody (red). Nuclei were counterstained with DAPI. Images show pairs of daughter cells from recent cell divisions. Increased dysmorphic nuclei with nuclear blebbing and honeycomb structures (arrows) are observed in double-mutant cells. The distance between daughter cells has been reduced using Photoshop 7 to allow magnification. (G) Percentage of dysmorphic nuclei in EDMD2 cells left untreated (untransfected), transfected with WT-SUN1 or transfected with W377C-SUN1 is reported in the graph as the mean of three independent experiments. Statistically significant differences (P,0.05) relative to controls or WT-transfected samples are indicated by asterisks. doi:10.1371/journal.pgen.1004605.g004 PLOS Genetics | www.plosgenetics.org 8 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies decrease in interaction with SUN1 G68D (Figure 4D–E). This that, whilst SUN1 polarization was normal (Figure S5), SUN2 correlates with the close proximity of G338 and W377 to the failed to polarize in clustered nuclei (Figure 5D,G). In keeping emerin binding site on SUN1 (see Figure 1A; [44]). with earlier observations in myoblasts, we also found that nesprin- 2 fluorescence intensity was significantly increased in all patient myotubes (Figure S6). Ultrastructural analysis showed that myo- SUN1 W377C increases the severity of nuclear defects in nuclear clustering occurred within single MD-1 myotubes EDMD2 fibroblasts (Figure 5H) and further demonstrated that enlarged highly Abnormalities in nuclear morphology and a honeycombed differentiated myotubes with misaligned nuclei were devoid of pattern of protein expression at the NE are commonly observed in detectable sarcomeric structures that were visible in controls EDMD fibroblasts obtained from patients with LMNA or EMD (Figure 5H, arrowheads). mutations [57–59]. We did not observe any obvious defects in nuclear morphology or protein localization in myoblasts from Pericentrin recruitment and microtubule nucleation at patient MD-1 (Figure 3A) or in cells transiently expressing a range the NE is defective in MD-1 myotubes of SUN1 or SUN2 mutants (for example, see Figure 2). However, During myotube formation, the microtubule network is we sought to determine whether SUN1 mutants have a modifying reorganized into a parallel array along the longitudinal axis of effect to increase the severity of nuclear defects when expressed in the myotube and is nucleated from the nuclear surface, which combination with mutations in LMNA or EMD. To achieve this, becomes the primary microtubule organizing centre (MTOC) of we expressed SUN1 W377C, found in combination with a LMNA the cell [64]. As part of this process, centrosomes undergo partial R453W mutation in patient MD-5, in fibroblasts obtained from an disassembly and centrosomal proteins, including c-tubulin, EDMD2 patient carrying LMNA R401C. Cells expressing SUN1 pericentrin and PCM-1, become concentrated at the nuclear W377C had a 4-fold increase in the level of nuclear dysmorphol- periphery [65,66]. We hypothesized that SUN proteins contribute ogy, in terms of increase in nuclear blebbing and formation of to centrosomal protein recruitment to the NE and that polariza- honeycomb structures (Figure 4F, arrows), compared to those tion of SUN proteins at the nuclear poles may promote linear expressing WT SUN1 (Figure 4F–G). Cells expressing WT SUN1 nuclear organization in myotubes. We therefore investigated the had a 2-fold reduction in nuclear abnormalities compared to recruitment of centrosomal proteins to the NE during myogenesis, untransfected cells, suggesting a protective effect of SUN1 over- using pericentrin as a marker. In control myotubes we found that expression. pericentrin was recruited to the NE and there was a suggestion that it concentrated at the poles of the nuclei, in a similar manner MD-1 myoblasts exhibit an enhanced rate of to SUN2 (Figure 6A). In contrast, pericentrin failed to accumulate differentiation but myonuclei are disorganized to any significant degree at the nuclear surface in MD-1 myotubes Exogenous expression of EDMD2-associated lamin A/C and was instead found in cytoplasmic foci. These findings support variants and disruption of endogenous lamin A/C, emerin or our hypothesis that SUN proteins are involved in the recruitment the LINC complex all affect myoblast differentiation [60–63]. of pericentrin to the NE in myotubes. Furthermore, studies in Drosophila indicate that the LINC We next investigated whether microtubule nucleation from the complex is required for correct myonuclear spacing [34]. We NE was disrupted in the patient myotubes by observing therefore determined whether the myoblasts carrying SUN1 microtubule regrowth following nocodazole-induced depolymer- variants had defects in their ability to differentiate to form mature, ization. In control cells, microtubules could be clearly observed multinuclear myotubes. In order to account for inherent emanating from around the nuclear surface after nocodazole differences in differentiation capacity between cell lines, we wash-out for 30 minutes (Figure S7). In contrast, the microtubule compared MD-1 cultures with a total of 5 control cultures network in patient cells was very disorganized and often did not independently established from different individuals. SUN1 appear to be attached to the NE, suggesting a defect in staining was clearly evident at the nuclear envelope of MD-1 microtubule nucleation, anchoring or organization. To investigate myotubes, as previously observed in myoblasts (Figure 5A). this further, we performed a short 5-minute nocodazole wash-out However, we found that the MD-1 myoblasts exhibited an to detect sites of microtubule nucleation. Microtubule asters increased rate of differentiation, both in terms of the number of regrowing from the nuclear envelope were observed in control myotubes and the number of nuclei per myotube. There was a myotubes (Figure 6B, panel a) and committed myoblasts (Fig- striking increase in the percentage of nuclei within myotubes ure 6B, panel b). Microtubule nucleation correlated with sites of (defined as muscle-specific caveolin3-positive cells containing at pericentrin concentration at the nuclear poles (arrows in least 3 nuclei) in patient cultures (2866% versus 5869% in Figure 6B). In MD-1 myotubes and committed myoblasts, control and MD-1 cultures, respectively; mean 6 sem) and a 5- microtubules were seen to nucleate mainly from multiple sites in fold increase in myotubes possessing more than 10 nuclei the cytoplasm, corresponding with the locations of cytoplasmic (Figure 5A–E). Strikingly, 45% of these myotubes displayed gross pericentrin foci (Figure 6B, arrowheads). Counting fifty myotubes nuclear misalignment and clustering, with up to 30 nuclei per per sample, we could demonstrate that the mean number of myotube in some instances (Figure 5D,F). microtubules nucleating from myotube nuclei was significantly Together with our earlier observation of defective nuclear reduced in MD-1 (Figure 6D). repositioning in fibroblasts expressing disease-associated variants These data suggested that centrosome attachment to the and in double-mutant EDMD2 fibroblasts, these findings suggest- nucleus may also be disrupted in myoblasts from this patient. ed that mutations in SUN1 and SUN2 disrupt connections with We therefore examined nuclear-centrosomal distance in MD-1 the cytoskeleton, thereby perturbing nuclear anchorage. Studies myoblasts and indeed observed a 2-fold increase in separation have previously shown that SUN1 and SUN2 become concen- between the nucleus and centrosomes in the patient myoblasts trated at the poles of the nucleus during human primary myoblast compared to controls (mean distance 4.34 mm versus 2.07 mm, differentiation and that this polarization is linked to correct respectively) (Figure 6C,E). myonuclear spacing [32]. We therefore examined SUN1 and To confirm that loss of pericentrin recruitment to the NE was a SUN2 polarization in patient MD-1 myotube nuclei and found direct consequence of SUN1 mutation, we observed pericentrin PLOS Genetics | www.plosgenetics.org 9 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies Figure 5. Enhanced rate of differentiation, nuclear misalignment and clustering in MD-1 myotubes. (A) Immunofluorescence staining of MD-1 myotubes using SUN1 (green) and desmin antibodies (red). Chromatin was stained with DAPI (blue). Scale bar, 30 mm. (B) Graphical representation of the percentage of nuclei within myotubes counted in MD-1 and in five independent control cultures. (C) Phase contrast image of living control and MD-1 cultured myotubes (arrows) showing myonuclear clustering in patient cells. Scale bar, 10 mm. (D) Immunofluorescence staining of control and MD-1 myotubes with SUN2 (green) and caveolin 3 (red) antibodies. Arrows indicate SUN2 polarization at the nuclear poles in control cells. (E–F) Graphical representation of the percentage of myotubes with more than 10 nuclei and the percentage of myotubes with myonuclear clustering in control and MD-1 cultures. Data are presented as mean values 6 S.D. of three independent experiments (50 myotubes per sample were counted). (G) Graphical representation of the percentage of committed myoblast and myotube nuclei with enrichment of SUN2 staining PLOS Genetics | www.plosgenetics.org 10 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies at the nuclear pole(s). Data are presented as mean values 6S.D. of 3 independent experiments (200 nuclei per sample). (H) Transmission electron microscopy analysis of control and MD-1 myotubes (see Materials and Methods for details). Sarcomeric structures are evident in control myotubes (arrowheads), whereas they are absent from MD-1 myotubes showing myonuclear clustering. Arrows indicate myonuclei. Scale bar, 10 mm. doi:10.1371/journal.pgen.1004605.g005 localization in C2C12 myotubes following transient transfection with Finally, to directly link the SUN1 mutants to the nuclear myc-SUN1 variants. Pericentrin was absent from the NE of clustering phenotype observed myotubes of patient MD-1, we myonuclei expressing SUN1 G68D, G338S and W377C variants, assessed the degree of nuclear clustering in myotubes expressing but exhibited clear nuclear rim staining in myonuclei expressing WT WT SUN1 and the G68D, G338S and W377C variants. Whilst SUN1 (Figure 7 A–B). Thus, all 3 mutants tested acted in a dominant 22% of myotubes expressing WT SUN1 displayed myonuclear manner in C2C12 cells to displace pericentrin from the NE. clustering, this value was increased by 2-fold in the cultures Figure 6. Impaired pericentrin localization and microtubule nucleation at the nuclear envelope in MD-1 myotubes. (A) Beta-tubulin (red) and pericentrin (green) double immunofluorescence staining of control and MD-1 myotubes. Chromatin was stained with DAPI (blue). Scale bar, 10 mm. Arrows indicate apparent pericentrin accumulation at the nuclear poles. (B) Control and MD-1 myotubes were treated with nocodazole followed by 5 min recovery in culture medium to allow microtubule regrowth. Samples were then fixed and stained as in panel A. Panels a and c show myotubes, whilst panels b and d show committed myoblasts from control and MD-1 cultures, respectively. Arrows in control cells indicate sites of microtubule regrowth at the nuclear poles, co-inciding with pericentrin localization. Arrowheads in patient cells indicate microtubule regrowth from cytoplasmic pericentrin foci. (C) Control and MD-1 myoblasts were fixed in methanol and subjected to immunofluorescence analysis using c- tubulin antibodies to stain the centrosome and DAPI to stain the DNA. Distances between centrosomes and the nuclear periphery (mm) are indicated. Scale bar, 10 mm. (D) The number of microtubules nucleating from individual myotube nuclei prepared in B was counted and is presented as the mean 6S.D (n = 50 nuclei per sample). * p,0.05 as calculated using the Student’s t-test. (E) Nucleus-centrosome distance was measured in 100 control and MD-1 myoblasts prepared in C, in two independent experiments, using Leica LAS AF Lite software and analysed using SPSS software. The median values (thick black lines) were 0.96 and 2.43 mm for control and MD-1 cells, respectively. P = 0.00012.# and * correspond to mild and extreme outliers, respectively. doi:10.1371/journal.pgen.1004605.g006 PLOS Genetics | www.plosgenetics.org 11 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies Figure 7. Exogenously expressed SUN1 mutants impair pericentrin recruitment to the nuclear envelope. (A) Differentiated C2C12 myotubes transfected with wild-type (WT) SUN1, or the indicated mutants, were labelled with anti-myc (red) and anti-pericentrin antibodies (green). Desmin antibody (violet) was used as a muscle differentiation marker. Nuclei were counterstained using DAPI. Samples were observed using a Nikon laser confocal microscope. Bar, 10 mm. (B–C) Transfected myotubes prepared as in A were quantified for the absence of pericentrin staining at the nuclear envelope (B) and myonuclear clustering (C). Thirty myotubes per sample were counted in two independent experiments. Differences for all mutants were statistically significant with respect to wild-type-transfected myotubes (P,0.01). doi:10.1371/journal.pgen.1004605.g007 expressing each of the 3 SUN1 variants (Figure 7C). These myopathy patients. Ten of these variants, identified in nine findings confirm that the SUN1 p.G68D and p.G338S mutations unrelated families, had putative pathogenic effects as deduced are the primary cause of the failure to recruit pericentrin to the NE from genetic and functional analyses. and the defective nuclear positioning in myotubes from patient MD-1 and further indicate that this is likely to be common to Multiple modes of inheritance of SUN1 and SUN2 muscle from patients carrying other SUN1 mutations, including mutations p.W377C. Our data add to an increasingly complex picture concerning the In summary, our data demonstrate that muscle disease-associated genetics of EDMD and related myopathies, where multiple genes, alterations in SUN proteins result in loss of nuclear connectivity to either alone or in combination, can cause or modify the disease the cytoskeleton. In myotubes, SUN1 mutations disrupt connec- phenotype. The SUN1 and SUN2 variants appear to be inherited tions with centrosomal components and the microtubule network, in highly variable manners, with or without the presence of a in particular impairing microtubule organization and nucleation mutation in a second gene. In 2 families (families 1 and 2), SUN1 from the NE. This in turn is likely to lead to impaired myonuclear or SUN2 variants were inherited from each of the unaffected positioning in multinuclear myotubes, which we propose may be an parents of the index patients, strongly supporting an autosomal important contributor to muscle dysfunction. recessive mode of inheritance in those families. One sporadic case carried heterozygous mutations in both the SUN1 and SUN2 Discussion genes, suggesting that mutations in the 2 genes could have additive We have identified a total of 11 SUN1 and 7 SUN2 rare, non- effects, as has been observed in Sun1/Sun2 knockout mice [30]. In synonymous variants in our cohort of EDMD and related other instances, the index case carried only one SUN1 or SUN2 PLOS Genetics | www.plosgenetics.org 12 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies variant and often represented a sporadic case, suggestive of either Despite our findings increasing the number of known EDMD- associated genes to 8, still almost 50% of patients in our cohort a dominant de novo mutation or presence of a concurrent mutation in another, as yet unidentified, gene. have no identified mutations in any of these genes. Most of these patients represent sporadic cases, with no family history of disease, We also identified SUN1 or SUN2 variants in individuals from making mutation screening difficult. Furthermore, since 6 of the 4 families harbouring known LMNA or EMD mutations. In all known genes each account for only a small percentage of cases, it cases, the SUN1/SUN2 mutation alone did not cause disease in is likely that there are multiple genes remaining to be identified. other family members. However, disease severity was significantly Proteins associated with the LINC complex are clearly very strong increased in the individuals carrying both mutations compared to candidates and there are several such proteins that should be family members, or unrelated individuals, carrying only the examined as a priority, including Samp1 [71,72]. LMNA or EMD mutation. Furthermore, their phenotype was on the severe end of the spectrum, as defined by Yates et al. [50]. These findings suggest that some SUN1/SUN2 variants act as Muscular dystrophy-associated SUN1 and SUN2 variants modifiers to increase disease severity. There has been much disrupt nuclear-cytoskeleton connection and nuclear speculation as to the existence of modifier genes in EDMD due to positioning high variability in disease phenotype between affected individuals Through the various nesprin isoforms expressed at the ONM, within families [37,38,41,47] and there is now some evidence to the LINC complex mediates attachment to all three cytoskeletal support this. In a family with X-linked EDMD caused by an EMD filament networks [17,73]. In this study, we have demonstrated, in p.Y105X mutation, disease severity was increased in one several different systems, that muscular dystrophy-associated individual due to a second mutation present in the LMNA gene mutations in SUN1 or SUN2 impair nuclear coupling to the [42]. Similarly, an individual with severe disease and carrying a both actin and microtubule networks and disrupt nuclear LMNA p.R644C mutation was found to carry a second mutation movement and positioning. in the gene encoding desmin [42]. In mouse NIH3T3 fibroblasts, five out of the six SUN1 and In our cohort, for some cases (such as patient MD-3) the SUN2 variants that we examined inhibited rearward movement of increased severity was expressed as clinically more severe muscular the nucleus, which has previously been shown to be achieved dystrophy [39]. In other cases, the additional presence of a SUN1/ through LINC complex attachment to actin cables closely SUN2 mutation was associated with more severe cardiac disease. associated with the nuclear surface [53]. Our data strongly For example, patient MD-5 from family 5, carrying both LMNA indicate that at least five of the variants identified in our patient p.R453W and SUN1 p.W377C mutations, developed cardiac cohort have a negative functional impact upon nuclear-cytoskel- disturbances at age 25 and died from heart failure at age 34, which etal connection via the LINC complex, which is likely to be a is much earlier than is typical for EDMD patients [50]. major contributor to muscle disease pathophysiology. One of the Furthermore, LMNA p.R453W is a relatively common mutation variants examined, SUN2 p.M50T, did not impair rearward that has been reported in at least 15 individuals with EDMD and is nuclear movement, suggesting that this variant is not disease- usually associated with mild disease [8,48,67–70]. Thus, it is likely causing and this is entirely possible given the complex genetics in that the SUN1 mutation carried by patient MD-5 contributed to the individual carrying this mutation (patient MD-2). In agree- their increased disease severity. We also identified SUN1 ment with our findings, EDMD-associated lamin A variants were p.W377C in combination with SUN2 p.E438D in a sporadic recently shown to cause a similar defect in nuclear movement in case, supporting the idea that a mutation in a second gene is NIH3T3 cells [52] and disrupted nuclear-cytoskeletal coupling required for disease causation in this instance. Further support for [74]. a modifying role for the p.W377C mutation came from the ability We also observed defects in nuclear positioning in differentiat- of this mutant to worsen nuclear dysmorphology when expressed ing myotubes derived from patient MD-1, carrying compound in LMNA R401C patient fibroblasts. heterozygous SUN1 p.G68D/p.G338S mutations, which is Individuals MD-6 and MD-7 (carrying a SUN2 variant in consistent with recent findings that proper SUN1 and SUN2 addition to LMNA T528K and R99P mutations, respectively) also recruitment to the NE is required for myonuclear spacing [32]. In had more severe disease than is typical for EDMD, with early strong support for a direct role of SUN proteins, nuclear onset at age 1 and 4 years, respectively, and unusually early positioning in skeletal muscle is disrupted in double Sun1/Sun2 requirement for heart transplantation. In each of these sporadic knockout mice or in mice with targeted disruptions of nesprin-1/ cases, the LMNA mutation arose de novo and so no comparisons nesprin-2 and this leads to clustering similar to that observed in can be made with family members, however, their clinical our patient myotubes [30,31,75]. Thus, the phenotype we phenotype is consistent with the suggestion that the SUN2 observed in patient MD-1 is consistent with a defect in the LINC variants contribute to increased disease severity. Whilst our genetic complex. Several recent studies have shown that myonuclear and cell-based data strongly support a modifying role for SUN position is controlled by nuclear attachment to the microtubule mutations in some patients, studies involving larger patient cohorts network and that this is mediated by the LINC complex will be necessary to prove this conclusively. [34,35,76–78]. At the onset of myoblast differentiation, proteins For 8 of the rare, non-synonymous variants identified in our involved in microtubule nucleation redistribute from the centro- cohort, there was a lack of compelling evidence of their disease some to the NE. Our observations of impaired pericentrin association. However, given the complex interplay between muta- recruitment and microtubule nucleation/organization at the NE tions in different genes, more investigation is required before entirely in the patient myotubes therefore support a model whereby ruling out their involvement. In particular, SUN1 p.V846I was found mutations in SUN proteins impair nuclear-microtubule connec- in an isolated sporadic case and thus no co-segregation analysis could tion and prevent correct positioning of myonuclei (Figure 8). It is be performed to support its disease association. Yet this is a mutation also well established in other systems that unanchored nuclei float of highly conserved residue (Figure S3) that lies within the SUN freely in the cytoplasm and tend to clump together, as observed in domain that is involved in nesprin binding. Thus it will be important MD-1 myotubes [34,77,79]. in the future to utilize functional studies to investigate the impact of It is currently not clear how the mutant SUN proteins, which such mutations on LINC complex interactions. are located at the INM, mediate disruption of microtubule PLOS Genetics | www.plosgenetics.org 13 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies Figure 8. Schematic model of nuclear positioning and microtubule connections during differentiation of normal and SUN1/2 mutant myoblasts. (A) In myoblasts, the close positioning of centrosomes (red) to the outer nuclear surface is disrupted by SUN1/2 mutants, which is likely to be accompanied by impaired microtubule (green) association with the NE. (B) Upon commitment to differentiation in normal myoblasts, pericentrin and other centrosomal proteins redistribute from the centrosome to the nuclear surface, which becomes the major site of microtubule nucleation. In mutant committed myoblasts, pericentrin fails to associate with the NE and there is impairment of microtubule nucleation from the nuclear surface. (C) After cell fusion to form myotubes, the microtubules reorganize into overlapping parallel arrays along the long axis of the cell. The myonuclei become positioned evenly along the length of the cell in a microtubule-dependent manner with the involvement of dynein and kinesin motor proteins. In mutant myotubes, nuclei are clumped in a disorganized fashion and we propose that this is due to an inability to interact with the microtubule network. doi:10.1371/journal.pgen.1004605.g008 attachment to the NE. Studies have indicated that nesprins are loss of myonuclear anchoring and impaired muscle function important for microtubule association with the NE, through their [34,35]. Myonuclear positioning defects have been observed at the H22P/H22P interaction with microtubule motor proteins [77,80]. However, we myotendinous junctions of Lmna and Lmna knock-out did not obtain any evidence that the central SUN1-nesprin-2 mice, and in EDMD patients with mutations in LMNA [33,84]. LINC complex interaction was perturbed in MD-1 myoblasts, However, to our knowledge, ours is the first observation of such a suggesting that the defect may lie elsewhere. Instead, SUN1 pronounced myonuclear mispositioning phenotype in humans. It interaction with emerin was disrupted and, consistent with this, will be important, in future studies, to demonstrate directly that uncoupling of the nucleus from the microtubule network through both emerin mRNA and protein levels were reduced in myoblasts from patient MD-1. Impairment of SUN1/SUN2 interaction with SUN mutation does lead to muscle disease in vivo with emerin has also been observed in cases of EDMD1 due to physiological expression levels of SUN mutants. mutations in emerin itself [44]. Furthermore, EDMD has been In summary, our data clearly implicate defects in pericentrin associated with defects in emerin interaction with lamin A/C and recruitment, microtubule nucleation/organization and nuclear- nesprins [18,19]. Interestingly, emerin has been shown to partially cytoskeletal attachment in NE-associated muscular dystrophy localize at the ONM, where it may contribute to centrosomal pathogenesis and are in agreement with the bulk of results attachment to the NE and, in agreement with our findings, others showing SUN1/SUN2 involvement in nuclear positioning and cell have observed increased centrosomal separation from the nucleus migration. It remains to be determined precisely how centrosomal in EDMD1 cells [81,82]. Thus, dysregulation of emerin may play components are recruited to the nuclear envelope in differentiating a role in disease causation. myotubes and how defects in this process result in misalignment of myonuclei in muscular dystrophy. Contribution of SUN mutations to muscle disease Materials and Methods pathophysiology To date, most studies have focused on the role of the nuclear Ethics statement lamina and LINC complex in cellular resistance to mechanical This study involved the use of human DNA samples and myoblasts strain in support of the ‘‘structural’’ hypothesis of laminopathy derived from muscle biopsies. These were obtained following disease causation. There is now strong evidence to indicate that informed consent using protocols and consent forms approved by defects in these structural networks make a significant contribution the Ethics Committee of Ernst-Moritz-Arndt University, Greifswald. to the pathophysiology of EDMD and related disorders [29]. Given our observations of defective interaction networks in SUN- Patients and controls mutated cells and uncoupling of nuclear-cytoskeletal connections, EDMD patients for this study were selected based on the results it is therefore likely that the variants we have discovered in SUN1 of a routine diagnostic mutational analysis of EMD, LMNA, FHL1, and SUN2 also impact upon cell mechanics. Nuclear dysmor- SYNE1 and SYNE2. 175 pseudo-anonymized patients negative for phology is a common feature of laminopathy cells and, whilst the mutations in these genes and 70 patients known to carry mutations exact cause and effect of this phenomenon is not understood, it is in the genes encoding the LINC components emerin, lamin A/C likely to reflect changes in the organization of the nuclear lamina and nesprin 1 or 2 alpha and beta were tested for mutations in and its interactions with the nuclear envelope [83], together with SUN1 and SUN2. The clinical features of these unrelated, increased susceptibility to mechanical deformation. The exacer- predominantly Caucasian index cases were within the diagnostic bation of nuclear dysmorphology, induced by SUN1 W377C criteria for EDMD [50] despite the variable clinical expression. expression in LMNA R401C fibroblasts, again highlights a role for SUN proteins in NE organization and integrity. Our findings in patient MD-1 myotubes further indicate that Mutation analysis defects in nuclear positioning may play a significant role in disease Primer pairs for all the coding exons and flanking intronic pathogenesis, particularly since a link has also been made between sequences of SUN1 (UNC84A, ENSG00000164828; see Fig. S1) PLOS Genetics | www.plosgenetics.org 14 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies and SUN2 (UNC84B, ENSG00000100242, ENST00000405510) CATG-39 from an oligo dT-primed reverse transcription of U2OS were designed using Primer-Blast (http://www.ncbi.nlm.nih.gov/ cell mRNA and cloned into the BglII-SalI sites of the initial tools/primer-blast/index.cgi; Table S2). To standardize the construct. EDMD-associated mutations were introduced using the sequencing reaction, all primers were tagged with an M13-tail QuikChange II site-directed mutagenesis kit (Stratagene), accord- (forward: 59-GTAAAACGACGGCCAGT-39 reverse: 59-CAG- ing to the manufacturer’s instructions. GAAACAGCTATGAC-39). Amplifications were performed in 25 ml volumes using Amplikon-Taq Polymerase (Biomol) under Antibodies the following thermal conditions: initial denaturation at 94u for Anti-human SUN1 2383 and anti-human SUN2 2853 antibod- 5 min followed by 35 cycles of denaturation (94uC for 15 sec), ies have been described previously [44]. Anti-SUN1 Atlas annealing at the appropriate temperature for 15 sec (see Table S2) antibody (HPA008346) was obtained from Sigma prestige and elongation (72uC for 1 min). A final elongation (72uC for antibodies. Anti-nesprin-2 (N2N3) antibody was kind gift from 7 min) preceded a 4uC cooling step Q. Zhang (King’s College London) and has been described Direct Sanger sequencing was used to analyse PCR products. previously [16]. Anti-nesprin-2G has been reported previously Excess dNTPs and primers were removed using ExoSAP-IT [53]. Anti-nesprin-2 monoclonal antibody (IQ562) was purchased (Affymetrix). Sequencing reactions were performed using ABI from Immuquest. Monoclonal anti-emerin antibody was a kind BigDye Terminator v3.1 Cycle Sequencing Kit with addition of gift from G. Morris (Center for Inherited Neuromuscular Disease, 5% DMSO to the reaction mix. M13-oligonucleotides were used Oswestry, UK). Anti-lamin A/C (sc-6215) and GFP antibodies as sequencing primers. The reactions were analysed on a 3130xl were purchased from Santa Cruz Biotechnologies. Anti- GAPDH GA DNA Sequencer (Applied Biosystems) according to the (MAM374) was obtained from Millipore. Anti-a-tubulin (T9026), manufacturer’s instructions. All DNA variations identified were anti-b-actin (A5441), anti-c-tubulin (T6557), anti-myc and anti- validated using a second independent DNA sample. desmin antibodies were purchased from Sigma. Anti-caveolin 3 monoclonal antibody (610420) was purchased from Transduction Analysis of the frequency of DNA variations Laboratories and anti-desmin polyclonal antibody (MONX10657) Unique and rare sequence variations were tested for their was purchased from Monosan. Anti-pericentrin polyclonal anti- frequency in 400 alleles of a Caucasian reference population. body (Ab4448) was obtained from Abcam. Additionally, sequence variations found in a patient of Turkish origin were tested in 138 alleles of a Turkish reference population. Cell culture and transfection Co-segregation of DNA variations with the disease was analysed in Myoblasts from patient MD-1 and controls were routinely patient families if available. For estimating the frequency of DNA cultured in high-glucose DMEM supplemented with 20% foetal variations found, restriction digestion and high resolution melting bovine serum plus antibiotics penicillin, streptomycin and (HRM) were performed using patient DNA as positive control. amphotericin B, at 37uC and 5% CO2, and were used between Restriction enzymes cutting specifically at the DNA variation were passages 3 and 7. Myoblasts at confluence were allowed to selected using NEB-cutter (http://tools.neb.com/NEBcutter2/). differentiate into myotubes in the same culture medium for 8–15 HRM products amplified with LightCycler 480 High Resolution days, replacing the medium every 5 days. HeLa cells were cultured Melting Master (Roche) were analysed on a LightCycler 480 II in DMEM supplemented with 10% FBS and antibiotics. For (Roche) according to the manufacturer’s instructions. Samples emerin co-immunoprecipitation experiments, HEK293 cells were showing abnormal signals were examined by restriction endonu- transfected with the appropriate pCMVTag3-SUN1 constructs clease digestion or direct sequencing. The frequency of changes together with GFP-emerin [85] using Fugene 6 (Promega), found in patients of different origin was estimated from online according to the manufacturer’s instructions. pCMVTag3-SUN1 accessible genome sequencing data (Table S1). constructs were transfected into C2C12 mouse myotubes using the Amaxa Nucleofector (Lonza), according to the manufacturer’s Real-time PCR instructions. Cultures were fixed 24 hours after transfection and RNA was extracted from patient MD-1 and control myoblasts processed for immunofluorescence analysis. using TRIzol (Invitrogen) according to the manufacturer’s instructions. Real-time PCR was performed using a RealTime Centrosome reorientation and nuclear movement assay ready custom panel and LightCycler 480 Probes Master (Roche), NIH3T3 fibroblasts were cultured in 10% calf serum in DMEM with primers as described in Supplementary Material Table S3, (Gibco) as previously described [86]. Following serum starvation and evaluated on a LightCycler 480 II (Roche), according to the for two days, confluent monolayers were ‘‘wounded’’ by removing manufacturer’s instructions. Values for each gene were normalised a strip of cells and nuclei of cells at the edge of the wound were to both actin and GAPDH. microinjected with the appropriate myc-tagged SUN1 or SUN2 DNA plasmids. After expression for 2 hr, cells were stimulated Plasmid constructs and site-directed mutagenesis with 10 mM LPA for 2 hr, fixed in 4% paraformaldehyde, For plasmid constructs, the pCMVTag3B vector (Stratagene) extracted with Triton X-100 and stained with antibodies to was used to fuse a myc tag to the N-terminus of SUN1. The 916 tyrosinated a-tubulin (rat monoclonal antibody at 1/40 of culture amino acid version of the SUN1 cDNA, lacking the ATG start supernatant), myc (mouse monoclonal antibody from clone 9E10, codon, was generated by PCR amplification in two stages. First, Roche) and DAPI (Sigma) followed by appropriate secondary codons 2–362 were amplified using primers 59-CACAGAATTC- antibodies. Stained samples were observed with a Nikon TE300 GATTTTTCTCGGCTTCACAT-39 and 59-CACAGTCGA- microscope using a 406 Plan Apo N.A. = 1.0 or 606 Plan-Apo CCTATCCGATCCTGCGCAAGATCTGC-39 with IMAGE N.A. = 1.4 objective and filter cubes optimized for DAPI, clone 40148216 as template and inserted into pCMVTag3B via fluorescein/GFP, and rhodamine. Images were acquired with the EcoRI and SalI sites. This introduced a BglII site via a silent CoolSNAP HQ camera (Photometrics) driven by Metamorph mutation at codon 356–358. Codons 352–916 were then amplified software (MDS Analytical Technologies) and further processed in using 59-TTACTTCTTGCTGCAGATCTTGCGCAGGAT- Image J. Centrosomes were considered oriented if they were CGG-39 and 59-GAGAGTCGACTCACTTGACAGGTTCGC- localized in the pie-shaped sector between the nuclear membrane PLOS Genetics | www.plosgenetics.org 15 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies the leading edge scored, as described [86,87]. Random orientation and were observed at 0u tilt angle with a Geol Jem 1011 is ,33% by this measure. Nuclear and centrosome position transmission electron microscope, operated at 100 kV. At least 30 relative to the cell centroid were determined as described [88]. myoblasts/myotubes per sample were observed. Data were plotted as % of the cell radius to normalize for differences in cell size. Statistical analysis In all cases, statistical analysis was performed using a Student’s Cell extracts, immunoprecipitation, and immunoblotting t-test to compare differences in values obtained for patient/mutant To prepare total cell extracts for immunoblotting, cells were versus control samples. scrapedinto cold16phosphate-buffered saline (PBS), pelleted by centrifuging at 2006g for 5 min and then pellets were resuspended in Supporting Information lysis buffer (10 mM HEPES [pH 7.4], 5 mM EDTA, 50 mM NaCl, Figure S1 Transcript variant of SUN1 used in this study. (A) 1% Triton X-100, 0.1% SDS) supplemented with 1 mM PMSF and The 23-exon SUN1 isoform used for our investigations contained protease inhibitor cocktail (Roche) and an equal volume of Laemmli exons 4 to 26 of ENST00000456758. The start codon used is the buffer was then added. For human myoblast immunoprecipitations, same used in isoform ENST00000405266. (B) The resulting cells were grown on 10 cm dishes and then immunoprecipitated as isoform encodes 916 residues and corresponds to the full length described previously (Haque et al., 2006) using 2 mg of SUN1 2383 mouse isoform of SUN1 that predominates in most tissues [89]. antibody. 5% of the initial lysate was retained for immunoblot analysis. All samples were boiled in an equal volume of 26Laemmli Alternating exons are indicated in black and blue. Residues spanning splice sites are indicated in red. buffer, resolved on 6% or 7.5% or 10% polyacrylamide gels, followed by semidry transfer onto nitrocellulose membrane. Membranes were (TIF) probed using the appropriate primary antibodies and dilutions: Figure S2 Pedigrees of MD families with index patients carrying hSUN1 ATLAS (1:400), hSUN2 2853 (1:500), lamin A/C (1:2000), heterozygous SUN1 or SUN2 variants that do not co-segregate with emerin (1:1500), nesprin-2 N2N3 (1:1500), a-tubulin (1: 10,000), b- disease. Index cases are indicated by arrows. There was no evidence of actin (1:20,000), GAPDH (1: 10,000). Primary antibodies were increased disease severity in the index cases carrying the SUN1 detected using horseradish peroxidase-conjugated secondary antibod- variants. ies (Sigma), and visualization was performed using ECL reagents (PDF) (Geneflow). Figure S3 Evolutionary conservation of SUN1 and SUN2 mutated residues. All rare, non-synonymous variants identified Indirect immunofluorescence microscopy in SUN1 and SUN2 are shown. Those for which there is strong Myoblasts and myotubes grown on glass coverslips were fixed in genetic and/or functional evidence of disease-association are methanol at 220uC and processed for indirect immunofluorescence indicated in red. The mutated residues and their equivalents in microscopy as previously described (Haque et al., 2006). For SUN1 other species are highlighted in beige. staining, cells were instead fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 at room temperature for (PDF) 5 min. Cells were washed in PBS and incubated with antibodies Figure S4 SUN1 mRNA levels are not altered in MD-1 diluted in PBS–3% bovine serum albumin, using hSUN1 2383 (1:150), myoblasts. Expression level of the indicated genes was assessed hSUN2 2853 (1:100), lamin A/C (1:400), emerin (1:500), nesprin-2G by quantitative real-time PCR using total RNA isolated from (1:300), c-tubulin (1:500) pericentrin (1:50), caveolin 3 (1:30) and control and MD-1 myoblasts. Values are expressed relative to two desmin (1:100) antibodies. Secondary antibodies were goat anti-rabbit control genes, ACTB and GAPDH, and show the average of 2 AlexaFluor 488, donkey anti-mouse AlexaFluor 594 and donkey anti- independent experiments performed in duplicate 6S.E. Signifi- goat AlexaFluor 594 (Molecular Probes Inc.). DNA was stained with cant P-values are as follows: SUN2 P = 0.019, LMNA P = 0.009, 50 mg/ml 49,6-diamidino-2-phenylindole (DAPI; Sigma). Coverslips SYNE1 P = 0.0016, SYNE2 P = 0.01. were mounted in 80% glycerol–3% n-propyl gallate (in PBS) or (PDF) ProLong gold antifading reagent (Invitrogen). Fluorescence microscopy Figure S5 SUN1 can polarize in MD-1 myotubes. SUN1 (green) was performed with a Nikon TE300 inverted microscope with an ORCA-R charge-couple device camera (Hamamatsu) and Volocity and caveolin (red) immunofluorescence staining in control and software (PerkinElmer). Where required fluorescence microscopy was patient MD-1 myotubes, along with DAPI (blue) staining of DNA. also performed with Leica TCS SP5 confocal laser scanning Arrowheads indicate nuclei in which SUN1 is enriched at the microscope and Leica LAS AF software. Images were processed with poles. Adobe Photoshop (Adobe Systems). Quantification of fluorescence (TIF) intensity was performed using an Olympus Scan Rmicroscope witha Figure S6 Nesprin-2 expression is elevated in patient MD-1 206 objective. Approximately 1000 nuclei from 3 independent myotubes. (A) Nesprin-2 staining in myotubes from control and experiments were randomly selected by their DAPI signal, and the patient MD-1. Immunofluorescence labeling was performed with intensity of SUN1, SUN2, emerin, lamin A/C and nesprin-2 was nesprin-2 monoclonal (red) and desmin (green) antibodies. Desmin measured within the DAPI-stained region. was used as a muscle cell marker. (B) Nesprin-2 fluorescence intensity was measured using the NIS software analysis system and Electron microscopy 50 myotubes per sample were analysed. Data are presented as Myotubes (at passage 2–3) from patient and age-matched mean value 6S.D. Significant P-value for patient MD-1 was controls were fixed in 2.5% glutaraldehyde-0.1 M cacodylate 0.042. Scale bar, 10 mm. buffer pH 7.4 for 3 h at 4uC. After post-fixation with 1% osmium (TIF) tetroxide (OsO4) in cacodylate buffer for 2 h, samples were dehydrated in an ethanol series, infiltrated with propylene oxide Figure S7 The number of microtubules nucleating from the and embedded in Epon resin. Ultrathin sections (60 nm thick) nuclear envelope is reduced in MD-1 myotubes. Beta-tubulin (red) were stained with uranyl acetate and lead citrate (10 min each) and pericentrin (green) double immunofluorescence staining in PLOS Genetics | www.plosgenetics.org 16 September 2014 | Volume 10 | Issue 9 | e1004605 SUN1 and SUN2 Variants in Muscular Dystrophies untreated control and MD-1 myotubes, or following nocodazole Acknowledgments treatment and 30 min recovery in culture medium. Chromatin We would like to thank the patients and their families for taking part in this was stained with DAPI (blue). Scale bar, 10 mm. Higher study. For providing clinical data, we acknowledge the contributions of magnification (36) of nuclear envelopes in nocodazole-treated Drs. M. Hoeltzenbein, W. Ko¨hler, U. Kordass, T. Lunke, A. Madej, W. cells is shown on the right of each picture. Mu¨ller-Felber, M. Munteanu, A. Petschaelis, G. Ramb, U. Reuner, C. (TIF) Vesely, S. Vielhaber, T. Voit, M. Vorgert, and M. Walter. We thank Muscle Tissue Culture Collection (MTCC) for providing myoblast Table S1 Single nucleotide changes found in coding regions of samples. The MTCC is part of the German network on muscular SUN1 and SUN2 and their frequencies in sequenced genome dystrophies (MD-NET, service structure S1, 01GM0601), funded by the databases. Rare, non-synonymous variants are highlighted in bold, German ministry of education and research (BMBF, Bonn, Germany), and with blue shading. *Patient MD-1 was of Turkish origin, therefore is a partner of EuroBioBank (www.eurobiobank.org) and TREAT-NMD 150 alleles from ethnically matched controls were also screened for (www.treatnmd.eu). We are also grateful to A. Fry (University of Leicester, UK) for helpful discussions and for critical reading of the manuscript. mutations p.G68D and p.G338S. (PDF) Author Contributions Table S2 Primer sequences and annealing temperatures for Conceived and designed the experiments: SS MW GL GGG HJW PM EM genomic amplification of SUN1 and SUN2 exons. FH SA. Performed the experiments: PM EM FH MC SA KRS. Analyzed (PDF) the data: PM MW EM GL FH KRS SS SA HJW GGG. Contributed Table S3 Primers used for real-time PCR. reagents/materials/analysis tools: SS GL MW GGG KRS. Wrote the (PDF) paper: SS GL MW GGG HJW. References 1. 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(2006) individual mammalian Sun1 isoforms depends on the cell type. Commun Integr Lamin A/C and emerin are critical for skeletal muscle satellite cell Biol 4: 440–442. differentiation. Genes Dev 20: 486–500. PLOS Genetics | www.plosgenetics.org 18 September 2014 | Volume 10 | Issue 9 | e1004605

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