Nuclear Intermediate Filament Proteins Research Articles

Abstract Mo098: LMNA R190W Mutation Associated With Dilated Cardiomyopathy Causes Hypercontractility in iPSC-derived Cardiomyocytes and Engineered Heart Muscles

Familial dilated cardiomyopathy (DCM) caused by mutations in sarcomeric proteins have been extensively studied in vitro , however less is known about DCM caused by alterations in non-sarcomeric genes like Lamin A (LMNA). LMNA is a ubiquitously expressed nuclear intermediate filament protein that regulates nuclear structure, chromatin access and gene expression. LMNA mutations account for 6% of DCM, and affected patients develop drug-therapy resistant severe heart failure and lethal arrythmias that are not amenable to currently available therapies. Most identified LMNA-cardiomyopathy mutations have been associated with severely depressed cardiac systolic function in patients and cardiomyocyte hypocontractility in vitro . However, the underlying mechanisms are incompletely understood. This project examines the mechanisms by which a patient-derived pathogenic mutation, LMNA R190W, leads to cardiac dysfunction at the cellular and tissue levels. We hypothesize that LMNA R190W mutation causes dysregulated expression of calcium handling genes, eventually leading to altered contractility. First, we investigated early disease pathogenesis using iPSC-derived cardiomyocytes. Intriguingly, single cardiomyocyte traction force microscopy studies showed cardiomyocyte hypercontractility, and calcium flux assays revealed prolonged calcium transients. Second, in human engineered heart muscle tissues, LMNA CMs were also consistently hypercontractile. Through gene expression studies, we identified upregulation of a cardiac-specific sarcomeric calcium regulator, calsequestrin 2 (CASQ2), which modulates intracellular calcium availability and localization, and thereby indirectly impacts calcium-dependent contraction and electrophysiology. siRNA-mediated knockdown of CASQ2 in iPSC-CMs decreased cellular calcium content, suggesting Casq2 is necessary for increased calcium and contractility in LMNA R190W cells. These findings suggest that in the initial stages of LMNA R190W-associated cardiomyopathy, cardiomyocyte contractility is not diminished, but actually augmented, and this may be related to aberrant calcium handling. We posit that chronic inappropriate calcium overload may eventually lead to cell death, and predispose to cardiomyopathy. Understanding the initial processes driving the development of LMNA cardiomyopathy will inform more accurate mechanism-based subclassification of mutations, and the rational development of precision therapies.

Heterologous expression of Dictyostelium discoideum NE81 in mouse embryo fibroblasts reveals conserved mechanoprotective roles of lamins.

Lamins are nuclear intermediate filament proteins that are ubiquitously found in metazoan cells, where they contribute to nuclear morphology, stability, and gene expression. Lamin-like sequences have recently been identified in distantly related eukaryotes, but it remains unclear whether these proteins share conserved functions with the lamins found in metazoans. Here, we investigate conserved features between metazoan and amoebozoan lamins using a genetic complementation system to express the Dictyostelium discoideum lamin-like protein NE81 in mammalian cells lacking either specific lamins or all endogenous lamins. We report that NE81 localizes to the nucleus in cells lacking Lamin A/C, and that NE81 expression improves nuclear circularity, reduces nuclear deformability, and prevents nuclear envelope rupture in these cells. However, NE81 did not completely rescue loss of Lamin A/C, and was unable to restore normal distribution of metazoan lamin interactors, such as emerin and nuclear pore complexes, which are frequently displaced in Lamin A/C deficient cells. Collectively, our results indicate that the ability of lamins to modulate the morphology and mechanical properties of nuclei may have been a feature present in the common ancestor of Dictyostelium and animals, whereas other, more specialized interactions may have evolved more recently in metazoan lineages.

Native lamin A/C proteomes and novel partners from heart and skeletal muscle in a mouse chronic inflammation model of human frailty.

Clinical frailty affects ∼10% of people over age 65 and is studied in a chronically inflamed (Interleukin-10 knockout; "IL10-KO") mouse model. Frailty phenotypes overlap the spectrum of diseases ("laminopathies") caused by mutations in LMNA. LMNA encodes nuclear intermediate filament proteins lamin A and lamin C ("lamin A/C"), important for tissue-specific signaling, metabolism and chromatin regulation. We hypothesized that wildtype lamin A/C associations with tissue-specific partners are perturbed by chronic inflammation, potentially contributing to dysfunction in frailty. To test this idea we immunoprecipitated native lamin A/C and associated proteins from skeletal muscle, hearts and brains of old (21-22months) IL10-KO versus control C57Bl/6 female mice, and labeled with Tandem Mass Tags for identification and quantitation by mass spectrometry. We identified 502 candidate lamin-binding proteins from skeletal muscle, and 340 from heart, including 62 proteins identified in both tissues. Candidates included frailty phenotype-relevant proteins Perm1 and Fam210a, and nuclear membrane protein Tmem38a, required for muscle-specific genome organization. These and most other candidates were unaffected by IL10-KO, but still important as potential lamin A/C-binding proteins in native heart or muscle. A subset of candidates (21 in skeletal muscle, 30 in heart) showed significantly different lamin A/C-association in an IL10-KO tissue (p < 0.05), including AldoA and Gins3 affected in heart, and Lmcd1 and Fabp4 affected in skeletal muscle. To screen for binding, eleven candidates plus prelamin A and emerin controls were arrayed as synthetic 20-mer peptides (7-residue stagger) and incubated with recombinant purified lamin A "tail" residues 385-646 under relatively stringent conditions. We detected strong lamin A binding to peptides solvent exposed in Lmcd1, AldoA, Perm1, and Tmem38a, and plausible binding to Csrp3 (muscle LIM protein). These results validated both proteomes as sources for native lamin A/C-binding proteins in heart and muscle, identified four candidate genes for Emery-Dreifuss muscular dystrophy (CSRP3, LMCD1, ALDOA, and PERM1), support a lamin A-interactive molecular role for Tmem38A, and supported the hypothesis that lamin A/C interactions with at least two partners (AldoA in heart, transcription factor Lmcd1 in muscle) are altered in the IL10-KO model of frailty.

The lamin A/C Ig-fold undergoes cell density-dependent changes that alter epitope binding

ABSTRACT Lamins A/C are nuclear intermediate filament proteins that are involved in diverse cellular mechanical and biochemical functions. Here, we report that recognition of Lamins A/C by a commonly used antibody (JOL-2) that binds the Lamin A/C Ig-fold and other antibodies targeting similar epitopes is highly dependent on cell density, even though Lamin A/Clevels do not change. We propose that the effect is caused by partial unfolding or masking of the C’E and/or EF loops of the Ig-fold in response to cell spreading. Surprisingly, JOL-2 antibody labeling was insensitive to disruption of cytoskeletal filaments or the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Furthermore, neither nuclear stiffness nor nucleo-cytoskeletal force transmission changed with cell density. These findings are important for the interpretation of immunofluorescence data for Lamin A/C and also raise the intriguing prospect that the conformational changes may play a role in Lamin A/C mediated cellular function.

P.149 Differential expression of intermediate filament proteins: lamins A/C and desmin within and between adult skeletal muscles

The cell cytoskeleton is composed of three main components: microfilaments, microtubules, and intermediate filaments. Intermediate filaments consist of intermediate filament proteins that are widely distributed in cells and tissues, either in the cytoplasm or the nucleus. Desmin is a tissue-specific cytoplasmic intermediate filament protein, while A-type lamins including lamin A/C are ubiquitously expressed nuclear intermediate filament proteins. Both play major roles in skeletal muscle functions. Mutations in the <i>DES</i> gene, encoding Desmin, and <i>LMNA</i> gene, encoding A-type lamins, lead to a group of rare disorders called desminopathies and laminopathies, respectively. The most frequent pathologies are those involving skeletal muscles. Despite the universal expression of lamins A/C in almost all cells and tissues, different phenotypes associated with laminopathies reflect a high degree of tissue-specificity, the reasons for which are not completely understood. It has also been shown that specific muscles/muscle groups are affected earlier than others in these myopathies. We hypothesized that desmin and lamins A/C are not uniformly expressed in different skeletal muscles and or muscle parts. Accordingly, we have investigated the relative expression of desmin, lamin A & lamin C in 9 skeletal muscles, of which 7 were divided into belly and myotendinous junction (MTJ). Our preliminary results revealed that there is no significant difference in the relative expression of desmin and lamins A/C between bellies and MTJs within a single muscle. We also observed that the relative expression of lamins A & C is correlated with myogenic markers; MyoD and myogenin, while desmin is not. Strikingly, FDB muscle showed the highest expression of lamins A/C compared to other muscles. We are currently investigating desmin, lamin A and lamin C protein levels by western blot and immunohistochemistry and the effect of age and exercise on the expression of these intermediate filament proteins.

A heterozygous p.S143P mutation in LMNA associates with proteasome dysfunction and enhanced autophagy-mediated degradation of mutant lamins A and C.

Lamins A and C are nuclear intermediate filament proteins that form a proteinaceous meshwork called lamina beneath the inner nuclear membrane. Mutations in the LMNA gene encoding lamins A and C cause a heterogenous group of inherited degenerative diseases known as laminopathies. Previous studies have revealed altered cell signaling pathways in lamin-mutant patient cells, but little is known about the fate of mutant lamins A and C within the cells. Here, we analyzed the turnover of lamins A and C in cells derived from a dilated cardiomyopathy patient with a heterozygous p.S143P mutation in LMNA. We found that transcriptional activation and mRNA levels of LMNA are increased in the primary patient fibroblasts, but the protein levels of lamins A and C remain equal in control and patient cells because of a meticulous interplay between autophagy and the ubiquitin-proteasome system (UPS). Both endogenous and ectopic expression of p.S143P lamins A and C cause significantly reduced activity of UPS and an accumulation of K48-ubiquitin chains in the nucleus. Furthermore, K48-ubiquitinated lamins A and C are degraded by compensatory enhanced autophagy, as shown by increased autophagosome formation and binding of lamins A and C to microtubule-associated protein 1A/1B-light chain 3. Finally, chaperone 4-PBA augmented protein degradation by restoring UPS activity as well as autophagy in the patient cells. In summary, our results suggest that the p.S143P-mutant lamins A and C have overloading and deleterious effects on protein degradation machinery and pharmacological interventions with compounds enhancing protein degradation may be beneficial for cell homeostasis.

Skeletal and Cardiac Muscle Disorders Caused by Mutations in Genes Encoding Intermediate Filament Proteins.

Intermediate filaments are major components of the cytoskeleton. Desmin and synemin, cytoplasmic intermediate filament proteins and A-type lamins, nuclear intermediate filament proteins, play key roles in skeletal and cardiac muscle. Desmin, encoded by the DES gene (OMIM *125660) and A-type lamins by the LMNA gene (OMIM *150330), have been involved in striated muscle disorders. Diseases include desmin-related myopathy and cardiomyopathy (desminopathy), which can be manifested with dilated, restrictive, hypertrophic, arrhythmogenic, or even left ventricular non-compaction cardiomyopathy, Emery–Dreifuss Muscular Dystrophy (EDMD2 and EDMD3, due to LMNA mutations), LMNA-related congenital Muscular Dystrophy (L-CMD) and LMNA-linked dilated cardiomyopathy with conduction system defects (CMD1A). Recently, mutations in synemin (SYNM gene, OMIM *606087) have been linked to cardiomyopathy. This review will summarize clinical and molecular aspects of desmin-, lamin- and synemin-related striated muscle disorders with focus on LMNA and DES-associated clinical entities and will suggest pathogenetic hypotheses based on the interplay of desmin and lamin A/C. In healthy muscle, such interplay is responsible for the involvement of this network in mechanosignaling, nuclear positioning and mitochondrial homeostasis, while in disease it is disturbed, leading to myocyte death and activation of inflammation and the associated secretome alterations.

Beta-strand-mediated dimeric formation of the Ig-like domains of human lamin A/C and B1

Lamins are nuclear intermediate filament proteins that play an essential role in maintaining the nuclear structure by forming a 3-D meshwork. Lamins consist of the N-terminal unstructured head, the coiled-coil rod domain, and the C-terminal tail, which is mostly unstructured except for the Ig-like domain. To date, the Ig-like domain has been characterized as a monomeric structure. Here, we determined the crystal structures of human lamin A/C, including the Ig-like domain and its N- and C-terminal flanking sequences. Interestingly, the structures showed a homodimer formed by beta-strand interactions between the N- and C-terminal flanking sequences. This interaction also provides a molecular implication for the creation of a 3-D meshwork between the 3.5-nm-thick filaments. Furthermore, we determined the crystal structure of the corresponding region of lamin B1. The structure showed a similar dimeric assembly, also formed by beta-strand interactions, albeit the intersubunit distance was much shorter. Since the Ig-like domain contains many genetic hotspots causing lamin-related diseases in lamin A/C, our findings will help understand the detailed assembly of lamins in a 3-D meshwork structure and lamin-related diseases at the molecular level.

Physical evidence on desmin-lamin B interaction.

Desmin is a muscle specific intermediate filament protein located in cytoplasm. Lamin B, on the other hand, is a nuclear intermediate filament protein. There are studies suggesting a possible interaction between desmin and lamin B yet there is no physical evidence. In the present study, we have shown for the first time a physical interaction between desmin and lamin B via reciprocal co-immunoprecipitation from muscle tissue of wild type AB zebrafish (Danio rerio, Hamilton). The interaction between desmin and lamin B might be a lead on a novel nucleocytoplasmic communication network.

Cardiovascular Involvement in Pediatric Laminopathies. Report of Six Patients and Literature Revision.

Lamin A/C (LMNA) encodes for two nuclear intermediate filament proteins. Mutations in LMNA cause a highly heterogeneous group of diseases predominantly leading to muscular or cardiac disease, lipodystrophy syndromes, peripheral neuropathy, and accelerated aging disorders. Cardiac involvement includes progressive arrhythmias (brady/tachyarrhythmias, sudden cardiac death). Furthermore, cardiomyocyte damage often progresses into dilated cardiomyopathy (DCM), rarely described in the pediatric age group. Neuromuscular manifestations are even rarer in children. We report on six pediatric patients with LMNA mutations: patient 1 was operated on for aortic coarctation, non-compact left ventricle, atrial fibrillation (AF) preceding the diagnosis of DCM; patient 2 was operated on for ventricular septal defect (VSD), developed after years malignant arrhythmias preceding the progression to DCM (left ventricular non-compaction with LV dysfunction); patient 3 had ectopic atrial tachycardia as first manifestation of a DCM; patients 4 and 5 had no major arrhythmic events but only dilated ascending aorta, mildly dilated LV with mild hypertrabeculation of the lateral wall and a normally functioning but dilated left ventricle, respectively; patient 6 showed aortic coarctation, supraventricular tachycardia. Paroxysmal AF occurred in patients 1, 2, and 3 (50% of cases). Our series highlight the coexistence of congenital heart defects (CHDs) and aortic involvement with laminopathies in four of our patients: consisting of aortic coarctation (two patients), aortic root dilatation (one patient), and VSD (one patient). Aortic changes in laminopathies have been reported only once in an adult patient. This is the first report in the pediatric setting, and no associations with CHD have been previously described.

Prelamin A mediates myocardial inflammation in dilated and HIV-associated cardiomyopathies.

Cardiomyopathies are complex heart muscle diseases that can be inherited or acquired. Dilated cardiomyopathy can result from mutations in LMNA, encoding the nuclear intermediate filament proteins lamin A/C. Some LMNA mutations lead to accumulation of the lamin A precursor, prelamin A, which is disease causing in a number of tissues, yet its impact upon the heart is unknown. Here, we discovered myocardial prelamin A accumulation occurred in a case of dilated cardiomyopathy, and we show that a potentially novel mouse model of cardiac-specific prelamin A accumulation exhibited a phenotype consistent with inflammatory cardiomyopathy, which we observed to be similar to HIV-associated cardiomyopathy, an acquired disease state. Numerous HIV protease therapies are known to inhibit ZMPSTE24, the enzyme responsible for prelamin A processing, and we confirmed that accumulation of prelamin A occurred in HIV+ patient cardiac biopsies. These findings (a) confirm a unifying pathological role for prelamin A common to genetic and acquired cardiomyopathies; (b) have implications for the management of HIV patients with cardiac disease, suggesting protease inhibitors should be replaced with alternative therapies (i.e., nonnucleoside reverse transcriptase inhibitors); and (c) suggest that targeting inflammation may be a useful treatment strategy for certain forms of inherited cardiomyopathy.

Lamin A molecular compression and sliding as mechanisms behind nucleoskeleton elasticity

Lamin A is a nuclear intermediate filament protein critical for nuclear architecture and mechanics and mutated in a wide range of human diseases. Yet little is known about the molecular architecture of lamins and mechanisms of their assembly. Here we use SILAC cross-linking mass spectrometry to determine interactions within lamin dimers and between dimers in higher-order polymers. We find evidence for a compression mechanism where coiled coils in the lamin A rod can slide onto each other to contract rod length, likely driven by a wide range of electrostatic interactions with the flexible linkers between coiled coils. Similar interactions occur with unstructured regions flanking the rod domain during oligomeric assembly. Mutations linked to human disease block these interactions, suggesting that this spring-like contraction can explain in part the dynamic mechanical stretch and flexibility properties of the lamin polymer and other intermediate filament networks.

Lamin-B1 is a senescence-associated biomarker in clear-cell renal cell carcinoma.

Clear-cell renal cell carcinoma (ccRCC) is a von-Hippel-Lindau gene (VHL) associated tumor disease. In addition to activating the hypoxia inducible factor (HIF) dependent oxygen-sensing pathway, VHL loss also has an impact on a HIF-independent senescence program which functions as a tumorigenesis barrier. Lamin-B1 is a nuclear intermediate filament protein that exhibits effects on chromatin structure and gene expression and acts as a senescence effector. In the present study, the expression and prognostic relevance of Lamin-B1 in a large cohort of ccRCC patients was examined and the report presents initial functional data on possible therapeutic implications. The expression of Lamin-B1 was measured by immunohistochemistry using a tissue microarray containing tumor tissue samples from 763 ccRCC patients. Chi-squared tests, Kaplan-Meier curves and Cox regression models were used to investigate the possible association between Lamin-B1 expression, clinical and pathological characteristics and patient survival. High Lamin-B1 expression was associated with poor clinical outcomes and multivariate Cox regression analyses revealed that Lamin-B1 was an independent prognostic factor for cancer-specific survival. Furthermore in vitro data suggested that Lamin-B1 acted as a functional downstream senescence effector in RCC cell lines. In conclusion, patients affected by ccRCC with high Lamin-B1 expression exhibit poor prognosis. Lamin-B1 may serve as a tissue-based biomarker for new therapeutic agents targeting therapy-induced senescence.

Intermediate filaments in cardiomyopathy.

Intermediate filament (IF) proteins are critical regulators in health and disease. The discovery of hundreds of mutations in IF genes and posttranslational modifications has been linked to a plethora of human diseases, including, among others, cardiomyopathies, muscular dystrophies, progeria, blistering diseases of the epidermis, and neurodegenerative diseases. The major IF proteins that have been linked to cardiomyopathies and heart failure are the muscle-specific cytoskeletal IF protein desmin and the nuclear IF protein lamin, as a subgroup of the known desminopathies and laminopathies, respectively. The studies so far, both with healthy and diseased heart, have demonstrated the importance of these IF protein networks in intracellular and intercellular integration of structure and function, mechanotransduction and gene activation, cardiomyocyte differentiation and survival, mitochondrial homeostasis, and regulation of metabolism. The high coordination of all these processes is obviously of great importance for the maintenance of proper, life-lasting, and continuous contraction of this highly organized cardiac striated muscle and consequently a healthy heart. In this review, we will cover most known information on the role of IFs in the above processes and how their deficiency or disruption leads to cardiomyopathy and heart failure.

Elevated dual specificity protein phosphatase 4 in cardiomyopathy caused by lamin A/C gene mutation is primarily ERK1/2-dependent and its depletion improves cardiac function and survival.

Mutations in the lamin A/C gene (LMNA) encoding the nuclear intermediate filament proteins lamins A and C cause a group of tissue-selective diseases, the most common of which is dilated cardiomyopathy (herein referred to as LMNA cardiomyopathy) with variable skeletal muscle involvement. We previously showed that cardiomyocyte-specific overexpression of dual specificity protein phosphatase 4 (DUSP4) is involved in the pathogenesis of LMNA cardiomyopathy. However, how mutations in LMNA activate Dusp4 expression and whether it is necessary for the development of LMNA cardiomyopathy are currently unknown. We now show that female LmnaH222P/H222P mice, a model for LMNA cardiomyopathy, have increased Dusp4 expression and hyperactivation of extracellular signal-regulated kinase (ERK) 1/2 with delayed kinetics relative to male mice, consistent with the sex-dependent delay in the onset and progression of disease. Mechanistically, we show that the H222P amino acid substitution in lamin A enhances its binding to ERK1/2 and increases sequestration at the nuclear envelope. Finally, we show that genetic deletion of Dusp4 has beneficial effects on heart function and prolongs survival in LmnaH222P/H222P mice. These results further establish Dusp4 as a key contributor to the pathogenesis of LMNA cardiomyopathy and a potential target for drug therapy.

Inactivation of DNase1L2 and DNase2 in keratinocytes suppresses DNA degradation during epidermal cornification and results in constitutive parakeratosis

The stratum corneum of the epidermis constitutes the mammalian skin barrier to the environment. It is formed by cornification of keratinocytes, a process which involves the removal of nuclear DNA. Here, we investigated the mechanism of cornification-associated DNA degradation by generating mouse models deficient of candidate DNA-degrading enzymes and characterizing their epidermal phenotypes. In contrast to Dnase1l2−/− mice and keratinocyte-specific DNase2 knockout mice (Dnase2Δep), Dnase1l2−/−Dnase2Δep mice aberrantly retained nuclear DNA in the stratum corneum, a phenomenon commonly referred to as parakeratosis. The DNA within DNase1L2/DNase2-deficient corneocytes was partially degraded in a DNase1-independent manner. Isolation of corneocytes, i.e. the cornified cell components of the stratum corneum, and labelling of DNA demonstrated that corneocytes of Dnase1l2−/−Dnase2Δep mice contained DNA in a nucleus-shaped compartment that also contained nucleosomal histones but lacked the nuclear intermediate filament protein lamin A/C. Parakeratosis was not associated with altered corneocyte resistance to mechanical stress, changes in transepidermal water loss, or inflammatory infiltrates in Dnase1l2−/−Dnase2Δep mice. The results of this study suggest that cornification of epidermal keratinocytes depends on the cooperation of DNase1L2 and DNase2 and indicate that parakeratosis per se does not suffice to cause skin pathologies.

Hepatocyte-Specific Deletion of Mouse Lamin A/C Leads to Male-Selective Steatohepatitis.

Background & AimsLamins are nuclear intermediate filament proteins that comprise the major components of the nuclear lamina. Mutations in LMNA, which encodes lamins A/C, cause laminopathies, including lipodystrophy, cardiomyopathy, and premature aging syndromes. However, the role of lamins in the liver is unknown, and it is unclear whether laminopathy-associated liver disease is caused by primary hepatocyte defects or systemic alterations.MethodsTo address these questions, we generated mice carrying a hepatocyte-specific deletion of Lmna (knockout [KO] mice) and characterized the KO liver and primary hepatocyte phenotypes by immunoblotting, immunohistochemistry, microarray analysis, quantitative real-time polymerase chain reaction, and Oil Red O and Picrosirius red staining.ResultsKO hepatocytes manifested abnormal nuclear morphology, and KO mice showed reduced body mass. KO mice developed spontaneous male-selective hepatosteatosis with increased susceptibility to high-fat diet–induced steatohepatitis and fibrosis. The hepatosteatosis was associated with up-regulated transcription of genes encoding lipid transporters, lipid biosynthetic enzymes, lipid droplet-associated proteins, and interferon-regulated genes. Hepatic Lmna deficiency led to enhanced signal transducer and activator of transcription 1 (Stat1) expression and blocked growth hormone–mediated Janus kinase 2 (Jak2), signal transducer and activator of transcription 5 (Stat5), and extracellular signal–regulated kinase (Erk) signaling.ConclusionsLamin A/C acts cell-autonomously to maintain hepatocyte homeostasis and nuclear shape and buffers against male-selective steatohepatitis by positively regulating growth hormone signaling and negatively regulating Stat1 expression. Lamins are potential genetic modifiers for predisposition to steatohepatitis and liver fibrosis. The microarray data can be found in the Gene Expression Omnibus repository (accession number: GSE93643).

LMNA Sequences of 60,706 Unrelated Individuals Reveal 132 Novel Missense Variants in A-Type Lamins and Suggest a Link between Variant p.G602S and Type 2 Diabetes.

Mutations in LMNA, encoding nuclear intermediate filament proteins lamins A and C, cause multiple diseases (‘laminopathies’) including muscular dystrophy, dilated cardiomyopathy, familial partial lipodystrophy (FPLD2), insulin resistance syndrome and progeria. To assess the prevalence of LMNA missense mutations (‘variants’) in a broad, ethnically diverse population, we compared missense alleles found among 60,706 unrelated individuals in the ExAC cohort to those identified in 1,404 individuals in the laminopathy database (UMD-LMNA). We identified 169 variants in the ExAC cohort, of which 37 (∼22%) are disease-associated including p.I299V (allele frequency 0.0402%), p.G602S (allele frequency 0.0262%) and p.R644C (allele frequency 0.124%), suggesting certain LMNA mutations are more common than previously recognized. Independent analysis of LMNA variants via the type 2 diabetes (T2D) Knowledge Portal showed that variant p.G602S associated significantly with type 2 diabetes (p = 0.02; odds ratio = 4.58), and was more frequent in African Americans (allele frequency 0.297%). The FPLD2-associated variant I299V was most prevalent in Latinos (allele frequency 0.347%). The ExAC cohort also revealed 132 novel LMNA missense variants including p.K108E (limited to individuals with psychiatric disease; predicted to perturb coil-1B), p.R397C and p.R427C (predicted to perturb filament biogenesis), p.G638R and p.N660D (predicted to perturb prelamin A processing), and numerous Ig-fold variants predicted to perturb phenotypically characteristic protein–protein interactions. Overall, this two-pronged strategy— mining a large database for missense variants in a single gene (LMNA), coupled to knowledge about the structure, biogenesis and functions of A-type lamins— revealed an unexpected number of LMNA variants, including novel variants predicted to perturb lamin assembly or function. Interestingly, this study also correlated novel variant p.K108E with psychiatric disease, identified known variant p.I299V as a potential risk factor for metabolic disease in Latinos, linked variant p.G602 with type 2 diabetes, and identified p.G602S as a predictor of diabetes risk in African Americans.

Distinct Fiber Type Signature in Mouse Muscles Expressing a Mutant Lamin A Responsible for Congenital Muscular Dystrophy in a Patient.

Specific mutations in LMNA, which encodes nuclear intermediate filament proteins lamins A/C, affect skeletal muscle tissues. Early-onset LMNA myopathies reveal different alterations of muscle fibers, including fiber type disproportion or prominent dystrophic and/or inflammatory changes. Recently, we identified the p.R388P LMNA mutation as responsible for congenital muscular dystrophy (L-CMD) and lipodystrophy. Here, we asked whether viral-mediated expression of mutant lamin A in murine skeletal muscles would be a pertinent model to reveal specific muscle alterations. We found that the total amount and size of muscle fibers as well as the extent of either inflammation or muscle regeneration were similar to wildtype or mutant lamin A. In contrast, the amount of fast oxidative muscle fibers containing myosin heavy chain IIA was lower upon expression of mutant lamin A, in correlation with lower expression of genes encoding transcription factors MEF2C and MyoD. These data validate this in vivo model for highlighting distinct muscle phenotypes associated with different lamin contexts. Additionally, the data suggest that alteration of muscle fiber type identity may contribute to the mechanisms underlying physiopathology of L-CMD related to R388P mutant lamin A.

Up-Regulation of Lamin A/C Expression in Epstein-Barr Virus Immortalized B Cells and Burkitt Lymphoma Cell Lines of Activated B Cell Phenotype

Lamin A, B and C, the nuclear intermediate-filament proteins, play a role in epigenetic regulation. Lamin B could be detected in all nucleated cells studied, whereas the lamin A and lamin C isoforms (lamin A/C) encoded by the LMNA gene are co-expressed in most somatic cell types except mature B lymphocytes. Since Epstein-Barr virus (EBV), a human gammaherpesvirus, is associated with tumorigenic processes and is known to alter the epigenotype of its host cells, we studied the expression of the LMNA gene and its epigenetic marks in EBV-carrying human lymphoid cell lines. We observed a high lamin A/C mRNA expression in EBV-immortalized B lymphoblastoid cell lines (LCLs) and in a subset of Burkitt lymphoma (BL) lines characterized by an activated B cell phenotype and a unique latent EBV gene expression pattern (latency III). In these cells the first exon of LMNA was hypomethylated and associated with activating histone marks. In contrast, we observed a low level of lamin A/C mRNA expression in EBV negative BL lines and BL lines with a restricted expression of latent EBV products (latency I). Low LMNA promoter activity was associated with hypermethylation of the LMNA first exon. These data suggest a role for EBV latency products in switching on or upregulating the LMNA promoter (LMNAp) in EBV-infected activated B cells in vitro. Lamin A/C may contribute to the establishment of the activated B cell phenotype. Our data also imply a role of LMNA first exon methylation in the silencing of LMNAp.