Mutations In Lamin Research Articles

Dysfunction of Lamin B and Physiological Aging Cause Telomere Instability in Drosophila Germline

Chromatin spatial organization in the nucleus is essential for the genome functioning and regulation of gene activity. The nuclear lamina and lamina-associated proteins, lamins, play a key role in this process. Lamin dysfunction leads to the decompaction and transcriptional activation of heterochromatin, which is associated with the premature aging syndrome. In many cell types, telomeres are located at the nuclear periphery, where their replication and stability are ensured by the nuclear lamina. Moreover, diseases associated with defects in lamins and telomeres have similar manifestations and resemble physiological aging. Understanding molecular changes associated with aging at the organismal level is especially important. In this study, we compared the effects caused by the mutation in lamin B and physiological aging in the germline of the model organism Drosophila melanogaster. We have shown that the impaired localization of lamin B leads to the heterochromatin decompaction and transcriptional activation of some transposable elements and telomeric repeats. Both DNA damage and activation of homologous recombination in the telomeres were observed in the germ cells of lamin B mutants. The instability of repeat-enriched heterochromatin can be directly related to the genome destabilization, germ cell death, and sterility observed in lamin B mutants. Similar processes were observed in Drosophila germline in the course of physiological aging, which indicates a close link between the maintenance of the heterochromatin stability at the nuclear periphery and mechanisms of aging.

Aberrant interaction between TEAD1 and Lamin A/C causes cardiomyopathy

Abstract Background Mutations in the LMNA gene encoding Lamin A/C, a major component of the nuclear lamina, cause laminopathies including dilated cardiomyopathy (DCM). DCM patients with LMNA mutations have particularly severe clinical courses such as heart transplantation and death due to heart failure. However, underlying mechanisms of LMNA-induced DCM remains elusive. Methods and results We identified LMNA Q353R mutation in a DCM family with severe heart failure. We generated Q353R heterozygous knock-in mice, which showed sarcomere dysplasia and perinatal lethality. Integrative single-cell analyses of the fetal murine hearts and patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSCMs) revealed that transcriptional regulation of cardiomyocyte maturation/development genes governed by TEAD1 was impaired in LMNA mutant cardiomyocytes. Protein array and immunostaining uncovered increased binding of TEAD1 to mutant Lamin A/C protein and abnormal localization of TEAD1 at the nuclear periphery. Furthermore, TT-10, a Hippo pathway inhibitor, rescued the dysregulation of cardiac developmental genes in LMNA mutant cardiomyocytes. Single-cell RNA-seq of cardiac tissues from DCM patients with the LMNA Q353R mutation confirmed the dysregulated expression of TEAD1 and its target genes. These results demonstrated abnormal interaction between TEAD1 and mutant Lamin A/C impairs structural maturation of cardiomyocytes and suggests that LMNA Q353R-related DCM can be treated through intervention in the Hippo pathway. Conclusion TEAD1 trapped by mutant Lamin A/C protein at the nuclear membrane perturbs transcriptional maturation in LMNA Q353R-related DCM. Funding Acknowledgement Type of funding sources: None.

VP.70 OPALE: a patient registry for laminopathies and emerinopathies in France

Laminopathies and emerinopathies are rare disorders due to mutations in A-type lamins (<i>LMNA</i>) and emerin (<i>EMD</i>) genes. Among them, disorders affecting striated muscle and fat tissues are the most frequent, with cardiac disease being a major cause of death. Clinico-genetic spectrum of these diseases have been extensively described since the 1990's. Precise phenotype/genotype relations remain elusive but several clues have been identified. Apart from symptomatic treatments, first specific treatments to prevent or slow down disease progression are undergoing therapeutic trials. Since 2013, we developed opale, an approved multicenter web-based registry dedicated to laminopathies and emerinopathies French patients. The main inclusion criterion is the presence of a proven pathogenic (or likely pathogenic=ACMG class 4) <i>LMNA</i> or <i>EMD</i> gene mutation. OPALE's objectives are to provide detailed capture of patient genetic data, neuro-cardio-endocrinological and respiratory assessments, in order to allow <i>i)</i> precise characterization of disease natural history, <i>ii)</i> estimation of the prevalence of various complications and <i>iii)</i> identification of prognostic factors. Currently, opale gathers 28 centers including neurological and/or cardiological and/or endocrinological departments. A total of 766 patients (749 <i>LMNA</i> and 17 <i>EMD</i> mutated) have been already included and 230 additional patients (141 <i>LMNA</i>, 89 <i>EMD</i>) are currently screened for a future inclusion. The clinical spectrum of the included patients comprises 108 asymptomatic mutation carriers (102 <i>LMNA</i>, 6 <i>EMD</i> mutated), 232 with isolated cardiac disease (228 <i>LMNA</i>, 4 <i>EMD</i> mutated), 267 with myopathy with or without cardiac disease (260 <i>LMNA</i>, 7 <i>EMD</i> mutated), 88 <i>LMNA</i> mutated with lipodystrophy, 7 <i>LMNA</i> mutated with peripheral nerve disease, 16 <i>LMNA</i> mutated with progeria or progeroid phenotypes and 42 <i>LMNA</i> mutated with diverse muscle-heart-fat-nerve-progeroid overlapping features. The remaining patients are undergoing clinical characterization. We also set up a prospective quantitative muscle evaluation study to follow muscle involvement natural history and a serum biobanking to explore blood biomarkers. OPALE registry is currently the largest registry dedicated to laminopathies and emerinopathies. It allows a better understanding of the clinico-genetic spectrum.

Combined alteration of lamin and nuclear morphology influences the localization of the tumor-associated factor AKTIP

BackgroundLamins, key nuclear lamina components, have been proposed as candidate risk biomarkers in different types of cancer but their accuracy is still debated. AKTIP is a telomeric protein with the property of being enriched at the nuclear lamina. AKTIP has similarity with the tumor susceptibility gene TSG101. AKTIP deficiency generates genome instability and, in p53−/− mice, the reduction of the mouse counterpart of AKTIP induces the exacerbation of lymphomas. Here, we asked whether the distribution of AKTIP is altered in cancer cells and whether this is associated with alterations of lamins.MethodsWe performed super-resolution imaging, quantification of lamin expression and nuclear morphology on HeLa, MCF7, and A549 tumor cells, and on non-transformed fibroblasts from healthy donor and HGPS (LMNA c.1824C > T p.Gly608Gly) and EDMD2 (LMNA c.775 T > G) patients. As proof of principle model combining a defined lamin alteration with a tumor cell setting, we produced HeLa cells exogenously expressing the HGPS lamin mutant progerin that alters nuclear morphology.ResultsIn HeLa cells, AKTIP locates at less than 0.5 µm from the nuclear rim and co-localizes with lamin A/C. As compared to HeLa, there is a reduced co-localization of AKTIP with lamin A/C in both MCF7 and A549. Additionally, MCF7 display lower amounts of AKTIP at the rim. The analyses in non-transformed fibroblasts show that AKTIP mislocalizes in HGPS cells but not in EDMD2. The integrated analysis of lamin expression, nuclear morphology, and AKTIP topology shows that positioning of AKTIP is influenced not only by lamin expression, but also by nuclear morphology. This conclusion is validated by progerin-expressing HeLa cells in which nuclei are morphologically altered and AKTIP is mislocalized.ConclusionsOur data show that the combined alteration of lamin and nuclear morphology influences the localization of the tumor-associated factor AKTIP. The results also point to the fact that lamin alterations per se are not predictive of AKTIP mislocalization, in both non-transformed and tumor cells. In more general terms, this study supports the thesis that a combined analytical approach should be preferred to predict lamin-associated changes in tumor cells. This paves the way of next translational evaluation to validate the use of this combined analytical approach as risk biomarker.

Effects of mutant lamins on nucleo-cytoskeletal coupling in Drosophila models of LMNA muscular dystrophy.

The nuclei of multinucleated skeletal muscles experience substantial external force during development and muscle contraction. Protection from such forces is partly provided by lamins, intermediate filaments that form a scaffold lining the inner nuclear membrane. Lamins play a myriad of roles, including maintenance of nuclear shape and stability, mediation of nuclear mechanoresponses, and nucleo-cytoskeletal coupling. Herein, we investigate how disease-causing mutant lamins alter myonuclear properties in response to mechanical force. This was accomplished via a novel application of a micropipette harpooning assay applied to larval body wall muscles of Drosophila models of lamin-associated muscular dystrophy. The assay enables the measurement of both nuclear deformability and intracellular force transmission between the cytoskeleton and nuclear interior in intact muscle fibers. Our studies revealed that specific mutant lamins increase nuclear deformability while other mutant lamins cause nucleo-cytoskeletal coupling defects, which were associated with loss of microtubular nuclear caging. We found that microtubule caging of the nucleus depended on Msp300, a KASH domain protein that is a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. Taken together, these findings identified residues in lamins required for connecting the nucleus to the cytoskeleton and suggest that not all muscle disease-causing mutant lamins produce similar defects in subcellular mechanics.

A method to map the interaction network of the nuclear lamina with genetically encoded photo-crosslinkers in vivo.

Lamins are intermediate filaments that assemble in a meshwork at the inner nuclear periphery of metazoan cells. The nuclear periphery fulfils important functions by providing stability to the nuclear membrane, connecting the cytoskeleton with chromatin, and participating in signal transduction. Mutations in lamins interfere with these functions and cause severe, phenotypically diverse diseases collectively referred to as laminopathies. The molecular consequences of these mutations are largely unclear but likely include alterations in lamin-protein and lamin-chromatin interactions. These interactions are challenging to study biochemically mainly because the lamina is resistant to high salt and detergent concentrations and co-immunoprecipitation are susceptible to artefacts. Here, we used genetic code expansion to install photo-activated crosslinkers to capture direct lamin-protein interactions in vivo. Mapping the Ig-fold of laminC for interactions, we identified laminC-crosslink products with laminB1, LAP2, and TRIM28. We observed significant changes in the crosslink intensities between laminC mutants mimicking different phosphorylation states. Similarly, we found variations in laminC crosslink product intensities comparing asynchronous cells and cells synchronized in prophase. This method can be extended to other laminC domains or other lamins to reveal changes in their interactome as a result of mutations or cell cycle stages.

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.

A novel deletion mutation accompanied by a point mutation in Lamin A/C gene: Screened from a dilated cardiomyopathy family.

There are 30%-40% of patients with dilated cardiomyopathy (DCM) having genetic causes, among which Lamin A and C gene (LMNA) mutation is the second most frequent DCM-related mutation, and Lamin A/C may be involved in the pathogenesis of DCM through the regulation of gene transcription or the direct effect of cell structure. Methods: Echocardiography and electrocardiogram were used to diagnose DCM and arrhythmia in a DCM family. Then, linked mutations on LMNA were screened out by high-throughput sequencing and verified by Sanger sequencing in all research individuals. Meanwhile, Human Genome Variation Society (HGVS) and Integrative Genomics Viewer (IGV) were used to analyse the characteristics of the mutated Lamin A/C protein. Finally, mutated-type and wild-type LMNA plasmid was transfected into AC-16 cardiomyocytes with the form of a lentivirus vector, and its effect on nucleus and actin was studied by immunofluorescence detection. In this study, we found a new frame-shifted mutation of LMNA (p.Ser414Alafs*66) linked with another point mutation from a DCM family by using High-throughput sequencing, and this deletion mutation led to a truncation of Lamin A/C. By analysing the clinical characteristics of this DCM family, we found that all DCM patients with arrhythmia were carriers of this co-segregation mutation. In the cytological experiment, we found that the mutated-type transfections showed weaker fluorescent intensities on both actin and cell nucleus. A co-segregation mutation of LMNA (Point mutation chr1 156107548 c.1712 G>A and truncated frame-shifted mutation chr1 156106086 c.1240delA) was found from a DCM family, and this type of mutation could participate in the pathogenesis of DCM by affecting the expression of actin.

Familial atrial myopathy in a large multigenerational heart-hand syndrome pedigree carrying an LMNA missense variant in rod 2B domain (p.R335W).

The literature on laminopathy with ventricular phenotype is extensive. However, the pathogenicity of LMNA variations in atrial lesions still lacks research. The purpose of this study was to characterize the atrial phenotypes and possible mechanisms in a large Chinese family with heart-hand syndrome carrying a LMNA missense variant in rod 2B domain (c.1003C>T p.R335W). Clinical characteristics were collected on the basis of the pedigree investigation. Comprehensive functional analyses, including molecular dynamic (MD) simulation, cellular, and animal functional assays, determined the pathogenicity in atrial myopathy. In the pedigree investigation, 6 of 13 of the mutation carriers showed heterogeneous cardiac phenotypes and 8 carriers also had brachydactyly. In silico molecular dynamics simulations predicted increased binding energy of the R335W mutant lamin A. Atrial cardiomyocytes (HL-1, human induced pluripotent stem cell-derived atrial cardiomyocytes) expressing R335W showed abnormal nuclear morphology, compromised DNA repair, and dysfunctional contraction. Adult zebrafish expressing mutant lamin A showed increased P wave duration in the electrocardiogram, decreased peak A wave velocity in echocardiography, and atrial lesions under the transmission electron microscope. LMNA p.R335W mutation leads to familial heart-hand syndrome characterized by an overlapping phenotype of prominent atrial lesions and brachydactyly. The unstable lamin dimerization and impaired DNA repair are possible mechanisms underlying cardiac phenotypes. Our findings consolidated the genetic role in the course of atrial arrhythmias and cardiac aging,which is helpful in the diagnosis and treatment of cardiac laminopathy.

Regulation of Lipid Metabolism by Lamin in Mutation-Related Diseases

Nuclear lamins, known as type 5 intermediate fibers, are composed of lamin A, lamin C, lamin B1, and lamin B2, which are encoded by LMNA and LMNB genes, respectively. Importantly, mutations in nuclear lamins not only participate in lipid disorders but also in the human diseases, such as lipodystrophy, metabolic-associated fatty liver disease, and dilated cardiomyopathy. Among those diseases, the mechanism of lamin has been widely discussed. Thereby, this review mainly focuses on the regulatory mechanism of the mutations in the lamin gene in lipid alterations and the human diseases. Considering the protean actions, targeting nuclear lamins may be a potent therapeutic avenue for lipid metabolic disorders and human diseases in the future.

Nuclear lamins: Structure and function in mechanobiology.

Nuclear lamins are type V intermediate filament proteins that polymerize into complex filamentous meshworks at the nuclear periphery and in less structured forms throughout the nucleoplasm. Lamins interact with a wide range of nuclear proteins and are involved in numerous nuclear and cellular functions. Within the nucleus, they play roles in chromatin organization and gene regulation, nuclear shape, size, and mechanics, and the organization and anchorage of nuclear pore complexes. At the whole cell level, they are involved in the organization of the cytoskeleton, cell motility, and mechanotransduction. The expression of different lamin isoforms has been associated with developmental progression, differentiation, and tissue-specific functions. Mutations in lamins and their binding proteins result in over 15 distinct human diseases, referred to as laminopathies. The laminopathies include muscular (e.g., Emery–Dreifuss muscular dystrophy and dilated cardiomyopathy), neurological (e.g., microcephaly), and metabolic (e.g., familial partial lipodystrophy) disorders as well as premature aging diseases (e.g., Hutchinson–Gilford Progeria and Werner syndromes). How lamins contribute to the etiology of laminopathies is still unknown. In this review article, we summarize major recent findings on the structure, organization, and multiple functions of lamins in nuclear and more global cellular processes.

Progerin-expressing endothelial cells are unable to adapt to shear stress

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disease caused by a single-point mutation in the lamin A gene, resulting in a truncated and farnesylated form of lamin A. This mutant lamin A protein, known as progerin, accumulates at the periphery of the nuclear lamina, resulting in both an abnormal nuclear morphology and nuclear stiffening. Patients with HGPS experience rapid onset of atherosclerosis, with death from heart attack or stroke as teenagers. Progerin expression has been shown to cause dysfunction in both vascular smooth muscle cells and endothelial cells (ECs). In this study, we examined how progerin-expressing endothelial cells adapt to fluid shear stress, the principal mechanical force from blood flow. We compared the response to shear stress for progerin-expressing, wild-type lamin A overexpressing, and control endothelial cells to physiological levels of fluid shear stress. Additionally, we also knocked down ZMPSTE24 in endothelial cells, which results in increased farnesylation of lamin A and similar phenotypes to HGPS. Our results showed that endothelial cells either overexpressing progerin or with ZMPSTE24 knockdown were unable to adapt to shear stress, experiencing significant cell loss at a longer duration of exposure to shear stress (3 days). Endothelial cells overexpressing wild-type lamin A also exhibited similar impairments in adaptation to shear stress, including similar levels of cell loss. Quantification of nuclear morphology showed that progerin-expressing endothelial cells had similar nuclear abnormalities in both static and shear conditions. Treatment of progerin-expressing cells and ZMPSTE24 KD cells with lonafarnib and methystat, drugs previously shown to improve HGPS nuclear morphology, resulted in improvements in adaptation to shear stress. Additionally, the prealignment of cells to shear stress before progerin-expression prevented cell loss. Our results demonstrate that changes in nuclear lamins can affect the ability of endothelial cells to properly adapt to shear stress.

Monitoring of Chromatin Organization at the Nuclear Pore Complex, Inner Nuclear Membrane, and Nuclear Interior in Live Cells by Fluorescence Ratiometric Imaging of Chromatin (FRIC).

The image analysis tool FRIC (Fluorescence Ratiometric Imaging of Chromatin) quantitatively monitors dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As a proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation as well as expression of the mutant lamin A protein "progerin," which causes Hutchinson-Gilford Progeria Syndrome. In summary FRIC is versatile, unbiased, robust, requires a minimum of experimental steps and is suitable for screening purposes.

Hutchinson - Gilford syndrome (Progeria): Clinical and Postmortem Findings

Hutchinson-Gilford Progeria syndrome, a rare genetic syndrome is being reviewed in this communication. It has striking features resembling premature aging. In most patients, Hutchinson-Gilford Progeria syndrome is caused by new genetic changes that occur randomly for unknown reasons. These changes (mutations) are transmitted as an autosomal dominant trait. Involving aberrant splicing of the LMNAgene, resulting in the production of a disease-causing mutant lamin Aprotein called progerin. Because neither parent carries or expresses the mutation, each case is believed to represent a sporadic, new mutation that happens most notably in a single sperm or egg immediately prior to conception. Children with progeria usually have a normal appearance in early infancy. At approximately six months of age, there are profound growth delays, resulting in low weight, premature bodily aging (progeria) and dwarfism. They have a birdlike, “wizened old man” facial appearance. These individuals die because of atherosclerosis and myocardial infarction. In this review article, we have reviewed several studies and reports regarding the genetic basis, diagnoses with characteristics and development of Hutchinson - Gilford syndrome (HGPS) or progéria. This review summarizes the clinical characteristics of this disease, the underlying mutation in the lamin A (LMNA) gene that results in this phenotype and PM findings through articles found in the databases: PubMed Central, Scielo, BVS, Bireme, Scientific Electronic Library Online etc.

Modulation of muscle redox and protein aggregation rescues lethality caused by mutant lamins

Mutations in the human LMNA gene cause a collection of diseases called laminopathies, which includes muscular dystrophy and dilated cardiomyopathy. The LMNA gene encodes lamins, filamentous proteins that form a meshwork on the inner side of the nuclear envelope. How mutant lamins cause muscle disease is not well understood, and treatment options are currently limited. To understand the pathological functions of mutant lamins so that therapies can be developed, we generated new Drosophila models and human iPS cell-derived cardiomyocytes. In the Drosophila models, muscle-specific expression of the mutant lamins caused nuclear envelope defects, cytoplasmic protein aggregation, activation of the Nrf2/Keap1 redox pathway, and reductive stress. These defects reduced larval motility and caused death at the pupal stage. Patient-derived cardiomyocytes expressing mutant lamins showed nuclear envelope deformations. The Drosophila models allowed for genetic and pharmacological manipulations at the organismal level. Genetic interventions to increase autophagy, decrease Nrf2/Keap1 signaling, or lower reducing equivalents partially suppressed the lethality caused by mutant lamins. Moreover, treatment of flies with pamoic acid, a compound that inhibits the NADPH-producing malic enzyme, partially suppressed lethality. Taken together, these studies have identified multiple new factors as potential therapeutic targets for LMNA-associated muscular dystrophy.

Abstract 10732: Systemic Administration of Telomerase RNA Reverses Vascular Senescence and Extends Lifespan in Progeria Mice

Introduction: Hutchinson-Gilford Progeria Syndrome (HGPS) is an accelerated aging syndrome associated with premature vascular disease and death due to heart attack and stroke. In HGPS, a mutation in lamin A (progerin) alters nuclear morphology and gene expression. iPSCs derived endothelium from progeria patients show impairment in their morphology and function including replication arrest, DNA damage, increased expression of inflammatory markers, and telomere erosion, compared to iPSCs derived endothelial cells from their unaffected parents. We have shown that hTERT mRNA therapy rescues their senescent phenotypes and restores normal function and gene expression in-vitro. To validate our finding in-vivo, we have used lentivirus harboring mouse telomerase to treat mice model of progeria and investigated the beneficial effects on mice vasculature in different organs. Hypothesis: Gene therapy with mRNA encoding mice telomerase improves survival and reversing cell senescence in a mice model of HGPS. Methods: HGPS mice were injected twice with a lentivirus construct encoding mice telomerase via tail vein. Mice were monitored for survival; endothelial cells were isolated at the time of euthanasia and concentrations of cytokines, chemokines, and growth factors were analyzed using Elisa, PCR and immunofluorescence staining. For clinical translation we developed a lipid nanoparticle formulation to deliver mRNA encoding mTERT to mice, validating its function using mRNA encoding Luciferase to demonstrate biodistribution and safety profile. Results: Two treatments with lentiviral mTERT increased the lifespan of HGPS mice by 20% and reduced mediators of inflammation and thrombosis in HGPS such as thrombomodulin. HGPS mice treated with mTERT had lower DNA damage markers and increased SIRT1 in their vasculature and other organs. A Lipid nanoparticle encapsulating mTERT RNA and shows a favorable safety profile and the capacity to systemically and safely deliver luciferase mRNA, generating active luciferase, documenting a predominantly hepatic biodistribution. Conclusions: Vascular rejuvenation using telomerase mRNA is a promising approach for progeria and other age-related diseases. Further development in systemic delivery is thus underway

Abstract 12946: Identification of a LMNA Synonymous Variant Associated With Severe Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) is one of the most common cardiac phenotypes caused by mutations of lamin A/C (LMNA) gene in humans. Almost 50% of LMNA variants reported in ClinVar as pathogenic or likely pathogenic are due to frameshift, nonsense, or splicing variants, predictive of haploinsufficiency for LMNA protein. In contrast, synonymous variants that do not immediately suggest a haploinsufficiency mechanism are much harder to interpret. We evaluated a cohort of 57 patients who underwent heart transplant for dilated cardiomyopathy for LMNA variants and identified a novel synonymous variant c.936G&gt;A in the last nucleotide of exon 5 of LMNA in a DCM family. Clinically, LMNA c.936G&gt;A carriers presented with severe DCM, conduction disease and high creatine-kinase levels. Sanger sequencing showed the presence of LMNA c.936G&gt;A in the genomic DNA but not in the cDNA derived from one family member’s heart tissue. RT-PCR using RNA from the patient’s heart tissue was performed with primers that amplified LMNA exons 4-6. DNA fragments amplified by RT-PCR were then cloned into pcDNAII vector and 40 clones were picked and sequenced. An aberrantly spliced mRNA containing the variant and a premature stop codon was identified in 1 of the 40 clones. Real-time quantitative PCR showed significantly lower LMNA mRNA levels in the patient’s heart compared to the controls. Our findings suggest that the c.936G&gt;A LMNA variant resulted in aberrant RNA splicing, which possibly triggers nonsense-mediated RNA decay, leading to reduced mRNA and likely protein expression. These results expand the understanding on the association between synonymous LMNA variants and the molecular pathogenesis of LMNA-DCM.

Abstract 11821: Telomerase Therapy Reverses Vascular Senescence and Extends Lifespan in Progeria Mice

Background: Hutchinson-Gilford Progeria Syndrome (HGPS) is an accelerated aging syndrome associated with premature vascular disease and death due to heart attack and stroke. In HGPS a mutation in lamin A (progerin), alters nuclear morphology and gene expression. Current therapy increases the lifespan of these children only modestly. Thus, greater understanding of the underlying mechanisms of HGPS is required to improve therapy. Endothelial cells (ECs) differentiated from induced pluripotent stem cells (iPSCs) derived from these patients exhibit hallmarks of senescence including replication arrest, DNA damage, increased expression of inflammatory markers and telomere erosion. We hypothesized that correction of shortened telomeres may reverse these measures of vascular aging. Methods: We generated ECs from iPSCs belong to children with HGPS and their unaffected parents. Telomerase mRNA (hTERT) was used to treat HGPS ECs. Endothelial morphology and functions were assessed, as well as proteomic and transcriptional profiles with attention to inflammatory markers, DNA damage and EC identity genes. In a mouse model of HGPS, we assessed the effects of lentiviral transfection of mTERT on measures of senescence, focusing on the EC phenotype in various organs. Results: hTERT treatment of human HGPS ECs improved replicative capacity; restored endothelial functions such as nitric oxide generation, acLDL uptake and angiogenesis; and reduced the elaboration of inflammatory cytokines. In addition, hTERT treatment improved cellular and nuclear morphology, in association with a normalization of the transcriptional profile, effects which may be mediated in part by a reduction in progerin expression and an increase in SIRT1. Progeria mice treated with mTERT Lentivirus manifested similar improvements, with a reduction in inflammatory and DNA damage markers and increased SIRT1 in their vasculature and other organs. Furthermore, mTERT therapy increased the lifespan of HGPS mice. Conclusion: Vascular rejuvenation using telomerase mRNA is a promising approach for progeria and other age-related diseases.

Pro-inflammatory cytokines as emerging molecular determinants in cardiolaminopathies.

Mutations in Lamin A/C gene (lmna) cause a wide spectrum of cardiolaminopathies strictly associated with significant deterioration of the electrical and contractile function of the heart. Despite the continuous flow of biomedical evidence, linking cardiac inflammation to heart remodelling in patients harbouring lmna mutations is puzzling. Therefore, we profiled 30 serum cytokines/chemokines in patients belonging to four different families carrying pathogenic lmna mutations segregating with cardiac phenotypes at different stages of severity (n = 19) and in healthy subjects (n = 11). Regardless lmna mutation subtype, high levels of circulating granulocyte colony‐stimulating factor (G‐CSF) and interleukin 6 (IL‐6) were found in all affected patients’ sera. In addition, elevated levels of Interleukins (IL) IL‐1Ra, IL‐1β IL‐4, IL‐5 and IL‐8 and the granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) were measured in a large subset of patients associated with more aggressive clinical manifestations. Finally, the expression of the pro‐inflammatory 70 kDa heat shock protein (Hsp70) was significantly increased in serum exosomes of patients harbouring the lmna mutation associated with the more severe phenotype. Overall, the identification of patient subsets with overactive or dysregulated myocardial inflammatory responses could represent an innovative diagnostic, prognostic and therapeutic tool against Lamin A/C cardiomyopathies.

In Silico and In Vivo Analysis of Amino Acid Substitutions That Cause Laminopathies.

Mutations in the LMNA gene cause diseases called laminopathies. LMNA encodes lamins A and C, intermediate filaments with multiple roles at the nuclear envelope. LMNA mutations are frequently single base changes that cause diverse disease phenotypes affecting muscles, nerves, and fat. Disease-associated amino acid substitutions were mapped in silico onto three-dimensional structures of lamin A/C, revealing no apparent genotype–phenotype connections. In silico analyses revealed that seven of nine predicted partner protein binding pockets in the Ig-like fold domain correspond to sites of disease-associated amino acid substitutions. Different amino acid substitutions at the same position within lamin A/C cause distinct diseases, raising the question of whether the nature of the amino acid replacement or genetic background differences contribute to disease phenotypes. Substitutions at R249 in the rod domain cause muscular dystrophies with varying severity. To address this variability, we modeled R249Q and R249W in Drosophila Lamin C, an orthologue of LMNA. Larval body wall muscles expressing mutant Lamin C caused abnormal nuclear morphology and premature death. When expressed in indirect flight muscles, R249W caused a greater number of adults with wing posturing defects than R249Q, consistent with observations that R249W and R249Q cause distinct muscular dystrophies, with R249W more severe. In this case, the nature of the amino acid replacement appears to dictate muscle disease severity. Together, our findings illustrate the utility of Drosophila for predicting muscle disease severity and pathogenicity of variants of unknown significance.