A recurrent homozygous LMNA missense variant p.Thr528Met causes atypical progeroid syndrome characterized by mandibuloacral dysostosis, severe muscular dystrophy, and skeletal deformities.

Atypical progeroid syndromes (APS) are premature aging syndromes caused by pathogenic LMNA missense variants, associated with unaltered expression levels of lamins A and C, without accumulation of wild-type or deleted prelamin A isoforms, as observed in Hutchinson-Gilford progeria syndrome (HGPS) or HGPS-like syndromes. A specific LMNA missense variant, (p.Thr528Met), was previously identified in a compound heterozygous state in patients affected by APS and severe familial partial lipodystrophy, whereas heterozygosity was recently identified in patients affected by Type 2 familial partial lipodystrophy. Here, we report four unrelated boys harboring homozygosity for the p.Thr528Met, variant who presented with strikingly homogeneous APS clinical features, including osteolysis of mandibles, distal clavicles and phalanges, congenital muscular dystrophy with elevated creatine kinase levels, and major skeletal deformities. Immunofluorescence analyses of patient-derived primary fibroblasts showed a high percentage of dysmorphic nuclei with nuclear blebs and typical honeycomb patterns devoid of lamin B1. Interestingly, in some protrusions emerin or LAP2α formed aberrant aggregates, suggesting pathophysiology-associated clues. These four cases further confirm that a specific LMNA variant can lead to the development of strikingly homogeneous clinical phenotypes, in these particular cases a premature aging phenotype with major musculoskeletal involvement linked to the homozygous p.Thr528Met variant.

Random walk with restart on multiplex and heterogeneous biological networks.

Motivation: Recent years have witnessed an exponential growth in the number of identified interactions between biological molecules. These interactions are usually represented as large and complex networks, calling for the development of appropriated tools to exploit the functional information they contain. Random walk with restart (RWR) is the state-of-the-art guilt-by-association approach. It explores the network vicinity of gene/protein seeds to study their functions, based on the premise that nodes related to similar functions tend to lie close to each other in the networks. Results: In this study, we extended the RWR algorithm to multiplex and heterogeneous networks. The walk can now explore different layers of physical and functional interactions between genes and proteins, such as protein-protein interactions and co-expression associations. In addition, the walk can also jump to a network containing different sets of edges and nodes, such as phenotype similarities between diseases. We devised a leave-one-out cross-validation strategy to evaluate the algorithms abilities to predict disease-associated genes. We demonstrate the increased performances of the multiplex-heterogeneous RWR as compared to several random walks on monoplex or heterogeneous networks. Overall, our framework is able to leverage the different interaction sources to outperform current approaches. Finally, we applied the algorithm to predict candidate genes for the Wiedemann-Rautenstrauch syndrome, and to explore the network vicinity of the SHORT syndrome. Availability and implementation: The source code is available on GitHub at: https://github.com/alberto-valdeolivas/RWR-MH. In addition, an R package is freely available through Bioconductor at: http://bioconductor.org/packages/RandomWalkRestartMH/. Supplementary information: Supplementary data are available at Bioinformatics online.

Nuclear matrix, nuclear envelope and premature aging syndromes in a translational research perspective.

Lamin A-related progeroid syndromes are genetically determined, extremely rare and severe. In the past ten years, our knowledge and perspectives for these diseases has widely progressed, through the progressive dissection of their pathophysiological mechanisms leading to precocious and accelerated aging, from the genes mutations discovery until therapeutic trials in affected children. A-type lamins are major actors in several structural and functional activities at the nuclear periphery, as they are major components of the nuclear lamina. However, while this is usually poorly considered, they also play a key role within the rest of the nucleoplasm, whose defects are related to cell senescence. Although nuclear shape and nuclear envelope deformities are obvious and visible events, nuclear matrix disorganization and abnormal composition certainly represent the most important causes of cell defects with dramatic pathological consequences. Therefore, lamin-associated diseases should be better referred as laminopathies instead of envelopathies, this later being too restrictive, considering neither the key structural and functional roles of soluble lamins in the entire nucleoplasm, nor the nuclear matrix contribution to the pathophysiology of lamin-associated disorders and in particular in defective lamin A processing-associated aging diseases. Based on both our understanding of pathophysiological mechanisms and the biological and clinical consequences of progeria and related diseases, therapeutic trials have been conducted in patients and were terminated less than 10 years after the gene discovery, a quite fast issue for a genetic disease. Pharmacological drugs have been repurposed and used to decrease the toxicity of the accumulated, unprocessed and truncated prelaminA in progeria. To date, none of them may be considered as a cure for progeria and these clinical strategies were essentially designed toward reducing a subset of the most dramatic and morbid features associated to progeria. New therapeutic strategies under study, in particular targeting the protein expression pathway at the mRNA level, have shown a remarkable efficacy both in vitro in cells and in vivo in mice models. Strategies intending to clear the toxic accumulated proteins from the nucleus are also under evaluation. However, although exceedingly rare, improving our knowledge of genetic progeroid syndromes and searching for innovative and efficient therapies in these syndromes is of paramount importance as, even before they can be used to save lives, they may significantly (i) expand the affected childrens' lifespan and preserve their quality of life; (ii) improve our understanding of aging-related disorders and other more common diseases; and (iii) expand our fundamental knowledge of physiological aging and its links with major physiological processes such as those involved in oncogenesis.

WITHDRAWN: Nuclear matrix, nuclear envelope and premature aging syndromes in a translational research perspective.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.semcdb.2014.03.022. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

A conserved splicing mechanism of the LMNA gene controls premature aging.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder phenotypically characterized by many features of premature aging. Most cases of HGPS are due to a heterozygous silent mutation (c.1824C>T; p.Gly608Gly) that enhances the use of an internal 5' splice site (5'SS) in exon 11 of the LMNA pre-mRNA and leads to the production of a truncated protein (progerin) with a dominant negative effect. Here we show that HGPS mutation changes the accessibility of the 5'SS of LMNA exon 11 which is sequestered in a conserved RNA structure. Our results also reveal a regulatory role of a subset of serine-arginine (SR)-rich proteins, including serine-arginine rich splicing factor 1 (SRSF1) and SRSF6, on utilization of the 5'SS leading to lamin A or progerin production and a modulation of this regulation in the presence of the c.1824C>T mutation is shown directly on HGPS patient cells. Mutant mice carrying the equivalent mutation in the LMNA gene (c.1827C>T) also accumulate progerin and phenocopy the main cellular alterations and clinical defects of HGPS patients. RNAi-induced depletion of SRSF1 in the HGPS-like mouse embryonic fibroblasts (MEFs) allowed progerin reduction and dysmorphic nuclei phenotype correction, whereas SRSF6 depletion aggravated the HGPS-like MEF's phenotype. We demonstrate that changes in the splicing ratio between lamin A and progerin are key factors for lifespan since heterozygous mice harboring the mutation lived longer than homozygous littermates but less than the wild-type. Genetic and biochemical data together favor the view that physiological progerin production is under tight control of a conserved splicing mechanism to avoid precocious aging.

Absence of T and B lymphocytes modulates dystrophic features in dysferlin deficient animal model.

Dysferlin mutations cause muscular dystrophy (dysferlinopathy) characterized by adult onset muscle weakness, high serum creatine kinase levels, attenuation of muscle regeneration and a prominent inflammatory infiltrate. In order to verify the role of lymphocytes and immune cells on this disease, we generated the Scid/A/J transgenic mice and compared these animals with the age-matched A/J mice. The absence of T and B lymphocytes in this animal model of dysferlinopathy resulted in an improvement of the muscle regeneration. Scid/A/J mice showed increased specific force in the myosin heavy chain 2A-expressing fibers of the diaphragm and abdominal muscles. Moreover, a partial reduction in complement deposition was observed together with a diminution in pro-inflammatory M1 macrophages. Consistent with this model, T and B lymphocytes seem to have a role in the muscle damaging immune response. The knowledge of the involvement of immune system in the development of dysferlinopathies could represent an important tool for their rescuing. By studying Scid/blAJ mice, we showed that it could be possible to modulate the pathological symptoms of these diseases by interfering with different components of the immune system.

An inherited LMNA gene mutation in atypical Progeria syndrome.

Hutchinson-Gilford Progeria syndrome (HGPS) is a rare genetic disorder, characterized by several clinical features that begin in early childhood, recalling an accelerated aging process. The diagnosis of HGPS is based on the recognition of common clinical features and detection of the recurrent heterozygous c.1824C>T (p.Gly608Gly) mutation within exon 11 in the Lamin A/C encoding gene (LMNA). Besides "typical HGPS," several "atypical progeria" syndromes (APS) have been described, in a clinical spectrum ranging from mandibuloacral dysplasia to atypical Werner syndrome. These patients's clinical features include progeroid manifestations, such as short stature, prominent nose, premature graying of hair, partial alopecia, skin atrophy, lipodystrophy, skeletal anomalies, such as mandibular hypoplasia and acroosteolyses, and in some cases severe atherosclerosis with metabolic complications. APS are due in several cases to de novo heterozygous LMNA mutations other than the p.Gly608Gly, or due to homozygous BAFN1 mutations in Nestor-Guillermo Progeria syndrome (NGPS). We report here and discuss the observation of a non-consanguineous Moroccan patient presenting with atypical progeria. The molecular studies showed the heterozygous mutation c.412G>A (p.Glu138Lys) of the LMNA gene. This mutation, previously reported as a de novo mutation, was inherited from the apparently healthy father who showed a somatic cell mosaicism.

Heterozygous expression of Cre recombinase in podocytes has no impact on the anti-glomerular basement membrane glomerulonephritis model in C57BL/6J mice.

A recent article described a thickening of the glomerular basement membrane (GBM) along with changes in the expression of key components of the extracellular matrix in 6-month-old NPHS2-Cre transgenic mice, which express the Cre recombinase specifically in podocytes. This transgenic line has been widely used to characterize the implication of candidate genes in glomerular diseases in younger mice. Using a different mouse strain (C57BL/6J) than the previous report (129S6/SvEvTac), we sought to characterize 3- and 6-month-old NPHS2-Cre+/- mice in control and pathological conditions. At baseline, there was no difference in renal function and histology between control and NPHS2-Cre+/- mice. Notably, GBM thickness evaluated by transmission electron microscopy was similar between the two groups. We then induced an immune-mediated severe glomerular insult, the anti-glomerular basement membrane glomerulonephritis model (anti-GBM-GN) in 3-month-old control and NPHS2-Cre+/- mice. NPHS2-Cre+/- mice exhibited the same alterations in renal function and structure as control mice. In summary, our study strongly suggests that NPHS2-Cre+/- transgenic mice on a C57BL/6J background can be safely used for podocyte-specific gene inactivation in control conditions and in the anti-GBM-GN model.

Vulnerability of progeroid smooth muscle cells to biomechanical forces is mediated by MMP13.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disease in children that leads to early death. Smooth muscle cells (SMCs) are the most affected cells in HGPS individuals, although the reason for such vulnerability remains poorly understood. In this work, we develop a microfluidic chip formed by HGPS-SMCs generated from induced pluripotent stem cells (iPSCs), to study their vulnerability to flow shear stress. HGPS-iPSC SMCs cultured under arterial flow conditions detach from the chip after a few days of culture; this process is mediated by the upregulation of metalloprotease 13 (MMP13). Importantly, double-mutant LmnaG609G/G609GMmp13-/- mice or LmnaG609G/G609GMmp13+/+ mice treated with a MMP inhibitor show lower SMC loss in the aortic arch than controls. MMP13 upregulation appears to be mediated, at least in part, by the upregulation of glycocalyx. Our HGPS-SMCs chip represents a platform for developing treatments for HGPS individuals that may complement previous pre-clinical and clinical treatments.

HGPS and related premature aging disorders: from genomic identification to the first therapeutic approaches.

Progeroid syndromes are heritable human disorders displaying features that recall premature ageing. In these syndromes, premature aging is defined as "segmental" since only some of its features are accelerated. A number of cellular biological pathways have been linked to aging, including regulation of the insulin/growth hormone axis, pathways involving ROS metabolism, caloric restriction, and DNA repair. The number of identified genes associated with progeroid syndromes has increased in recent years, possibly shedding light as well on mechanisms underlying ageing in general. Among these, premature aging syndromes related to alterations of the LMNA gene have recently been identified. This review focuses on Hutchinson-Gilford Progeria syndrome and Restrictive Dermopathy, two well-characterized Lamin-associated premature aging syndromes, pointing out the current knowledge concerning their pathophysiology and the development of possible therapeutic approaches.

[A-type lamins and progeroïd syndromes : persistent farnesylation with dramatic effects]. / Lamines A et syndromes progéroïdes : une farnésylation persistante aux conséquences dramatiques.

Hutchinson-Gilford Progeria (HGPS), a rare and severe developmental disorder characterized by features recalling premature aging, and Restrictive Dermopathy (RD), a neonatal lethal genodermatosis, have recently been identified as being primary or secondary << Laminopathies >>. These heterogeneous disorders are caused by altered Lamin maturation pathway. In physiological conditions, mature Lamin A is obtained through a series of post-translational processing steps performed on a protein precursor, Prelamin A. The major pathophysiological mechanism involved in Progeria is an aberrant splicing due to a de novo heterozygous point mutation, leading to the accumulation of truncated Lamin A precursor. The same aberrant splicing mechanism was involved in RD, whereas the majority of RD cases are caused by ZMPSTE24/FACE1 inactivation, a key enzyme involved in the Lamin A maturation pathway. In functional terms, all these conditions share the same pathophysiological mechanism, i.e. the intranuclear accumulation of Lamin A precursors, which cannot be fully processed and exert a toxic effect on nuclear homeostasis. In this article, we review the structure and functions of A-type Lamins, focusing namely on HGPS, RD or MAD disorders, in relation to existing animal models and possible future therapeutic approaches.

Substrate Topography Modulates Cell Aging on a Progeria Cell Model.

Aging is characterized by a progressive accumulation of cellular damage, which leads to impaired function. Little is known whether substrates can influence cell aging. This is of utmost importance in the development of medical devices that are in contact with human tissue for long periods of time. To address this question, we have used an accelerated aging cell model derived from Hutchinson-Gilford Progeria Syndrome (HGPS) induced pluripotent stem cells (iPSCs). Our results show that HGPS-iPSC smooth muscle cells (SMCs) have an increased aging profile in substrates with specific micropatterns than in flat ones. This is characterized by an up-regulation in the expression of progerin, ß-galactosidase, annexin 3 and 5, and caspase 9. Signs of cell aging are also observed in SMCs without HGPS cultured in substrates with specific microtopographies. It is further showed that specific micropatterned substrates induce cell aging by triggering a DNA damage program likely by the disruption between cyto- and nucleoskeleton.

MG132-induced progerin clearance is mediated by autophagy activation and splicing regulation.

Hutchinson-Gilford progeria syndrome (HGPS) is a lethal premature and accelerated aging disease caused by a de novo point mutation in LMNA encoding A-type lamins. Progerin, a truncated and toxic prelamin A issued from aberrant splicing, accumulates in HGPS cells' nuclei and is a hallmark of the disease. Small amounts of progerin are also produced during normal aging. We show that progerin is sequestered into abnormally shaped promyelocytic nuclear bodies, identified as novel biomarkers in late passage HGPS cell lines. We found that the proteasome inhibitor MG132 induces progerin degradation through macroautophagy and strongly reduces progerin production through downregulation of SRSF-1 and SRSF-5 accumulation, controlling prelamin A mRNA aberrant splicing. MG132 treatment improves cellular HGPS phenotypes. MG132 injection in skeletal muscle of LmnaG609G/G609G mice locally reduces SRSF-1 expression and progerin levels. Altogether, we demonstrate progerin reduction based on MG132 dual action and shed light on a promising class of molecules toward a potential therapy for children with HGPS.

Antisense-Based Progerin Downregulation in HGPS-Like Patients' Cells.

Progeroid laminopathies, including Hutchinson-Gilford Progeria Syndrome (HGPS, OMIM #176670), are premature and accelerated aging diseases caused by defects in nuclear A-type Lamins. Most HGPS patients carry a de novo point mutation within exon 11 of the LMNA gene encoding A-type Lamins. This mutation activates a cryptic splice site leading to the deletion of 50 amino acids at its carboxy-terminal domain, resulting in a truncated and permanently farnesylated Prelamin A called Prelamin A Δ50 or Progerin. Some patients carry other LMNA mutations affecting exon 11 splicing and are named "HGPS-like" patients. They also produce Progerin and/or other truncated Prelamin A isoforms (Δ35 and Δ90) at the transcriptional and/or protein level. The results we present show that morpholino antisense oligonucleotides (AON) prevent pathogenic LMNA splicing, markedly reducing the accumulation of Progerin and/or other truncated Prelamin A isoforms (Prelamin A Δ35, Prelamin A Δ90) in HGPS-like patients' cells. Finally, a patient affected with Mandibuloacral Dysplasia type B (MAD-B, carrying a homozygous mutation in ZMPSTE24, encoding an enzyme involved in Prelamin A maturation, leading to accumulation of wild type farnesylated Prelamin A), was also included in this study. These results provide preclinical proof of principle for the use of a personalized antisense approach in HGPS-like and MAD-B patients, who may therefore be eligible for inclusion in a therapeutic trial based on this approach, together with classical HGPS patients.

Metformin decreases progerin expression and alleviates pathological defects of Hutchinson-Gilford progeria syndrome cells.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that causes systemic accelerated aging in children. This syndrome is due to a mutation in the LMNA gene that leads to the production of a truncated and toxic form of lamin A called progerin. Because the balance between the A-type lamins is controlled by the RNA-binding protein SRSF1, we have hypothesized that its inhibition may have therapeutic effects for HGPS. For this purpose, we evaluated the antidiabetic drug metformin and demonstrated that 48 h treatment with 5 mmol/l metformin decreases SRSF1 and progerin expression in mesenchymal stem cells derived from HGPS induced pluripotent stem cells (HGPS MSCs). The effect of metformin on progerin was then confirmed in several in vitro models of HGPS, i.e., human primary HGPS fibroblasts, LmnaG609G/G609G mouse fibroblasts and healthy MSCs previously treated with a PMO (phosphorodiamidate morpholino oligonucleotide) that induces progerin. This was accompanied by an improvement in two in vitro phenotypes associated with the disease: nuclear shape abnormalities and premature osteoblastic differentiation of HGPS MSCs. Overall, these results suggest a novel approach towards therapeutics for HGPS that can be added to the currently assayed treatments that target other molecular defects associated with the disease.

Truncated prelamin A expression in HGPS-like patients: a transcriptional study.

Premature aging syndromes are rare genetic disorders mimicking clinical and molecular features of aging. A recently identified group of premature aging syndromes is linked to mutation of the LMNA gene encoding lamins A and C, and is associated with nuclear deformation and dysfunction. Hutchinson-Gilford progeria syndrome (HGPS) was the first premature aging syndrome linked to LMNA mutation and its molecular bases have been deeply investigated. It is due to a recurrent de novo mutation leading to aberrant splicing and the production of a truncated and toxic nuclear lamin A precursor (prelamin AΔ50), also called progerin. In this work and based on the literature data, we propose to distinguish two main groups of premature aging laminopathies: (1) HGPS and HGP-like syndromes, which share clinical features due to hampered processing and intranuclear toxic accumulation of prelamin A isoforms; and (2) APS (atypical progeria syndromes), due to dominant or recessive missense mutations affecting lamins A and C. Among HGPS-like patients, several deleted prelamin A transcripts (prelamin AΔ50, AΔ35 and AΔ90) have been described. The purpose of this work was to characterize those transcripts in eight patients affected with HGP-like rare syndromes. When fibroblasts were available, the relationships between the presence and ratios of these transcripts and other parameters were studied, aiming to increase our understanding of genotype-phenotype relationships in HGPS-like patients. Altogether our results evidence that progerin accumulation is the major pathogenetic mechanism responsible for HGP-like syndromes due to mutations near the donor splice site of exon 11.

Induced pluripotent stem cells reveal functional differences between drugs currently investigated in patients with hutchinson-gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome is a rare congenital disease characterized by premature aging in children. Identification of the mutation and related molecular mechanisms has rapidly led to independent clinical trials testing different marketed drugs with a preclinically documented impact on those mechanisms. However, the extensive functional effects of those drugs remain essentially unexplored. We have undertaken a systematic comparative study of the three main treatments currently administered or proposed to progeria-affected children, namely, a farnesyltransferase inhibitor, the combination of an aminobisphosphonate and a statin (zoledronate and pravastatin), and the macrolide antibiotic rapamycin. This work was based on the assumption that mesodermal stem cells, which are derived from Hutchinson-Gilford progeria syndrome-induced pluripotent stem cells expressing major defects associated with the disease, may be instrumental to revealing such effects. Whereas all three treatments significantly improved misshapen cell nuclei typically associated with progeria, differences were observed in terms of functional improvement in prelamin A farnesylation, progerin expression, defective cell proliferation, premature osteogenic differentiation, and ATP production. Finally, we have evaluated the effect of the different drug combinations on this cellular model. This study revealed no additional benefit compared with single-drug treatments, whereas a cytostatic effect equivalent to that of a farnesyltransferase inhibitor alone was systematically observed. Altogether, these results reveal the complexity of the modes of action of different drugs, even when they have been selected on the basis of a similar mechanistic hypothesis, and underscore the use of induced pluripotent stem cell derivatives as a critical and powerful tool for standardized, comparative pharmacological studies.

New ZMPSTE24 (FACE1) mutations in patients affected with restrictive dermopathy or related progeroid syndromes and mutation update.

Restrictive dermopathy (RD) is a rare and extremely severe congenital genodermatosis, characterized by a tight rigid skin with erosions at flexure sites, multiple joint contractures, low bone density and pulmonary insufficiency generally leading to death in the perinatal period. RD is caused in most patients by compound heterozygous or homozygous ZMPSTE24 null mutations. This gene encodes a metalloprotease specifically involved in lamin A post-translational processing. Here, we report a total of 16 families for whom diagnosis and molecular defects were clearly established. Among them, we report seven new ZMPSTE24 mutations, identified in classical RD or Mandibulo-acral dysplasia (MAD) affected patients. We also report nine families with one or two affected children carrying the common, homozygous thymine insertion in exon 9 and demonstrate the lack of a founder effect. In addition, we describe several new ZMPSTE24 variants identified in unaffected controls or in patients affected with non-classical progeroid syndromes. In addition, this mutation update includes a comprehensive search of the literature on previously described ZMPSTE24 mutations and associated phenotypes. Our comprehensive analysis of the molecular pathology supported the general rule: complete loss-of-function of ZMPSTE24 leads to RD, whereas other less severe phenotypes are associated with at least one haploinsufficient allele.

Full-length dysferlin expression driven by engineered human dystrophic blood derived CD133+ stem cells.

The protein dysferlin is abundantly expressed in skeletal and cardiac muscles, where its main function is membrane repair. Mutations in the dysferlin gene are involved in two autosomal recessive muscular dystrophies: Miyoshi myopathy and limb-girdle muscular dystrophy type 2B. Development of effective therapies remains a great challenge. Strategies to repair the dysferlin gene by skipping mutated exons, using antisense oligonucleotides (AONs), may be suitable only for a subset of mutations, while cell and gene therapy can be extended to all mutations. AON-treated blood-derived CD133+ stem cells isolated from patients with Miyoshi myopathy led to partial dysferlin reconstitution in vitro but failed to express dysferlin after intramuscular transplantation into scid/blAJ dysferlin null mice. We thus extended these experiments producing the full-length dysferlin mediated by a lentiviral vector in blood-derived CD133+ stem cells isolated from the same patients. Transplantation of engineered blood-derived CD133+ stem cells into scid/blAJ mice resulted in sufficient dysferlin expression to correct functional deficits in skeletal muscle membrane repair. Our data suggest for the first time that lentivirus-mediated delivery of full-length dysferlin in stem cells isolated from Miyoshi myopathy patients could represent an alternative therapeutic approach for treatment of dysferlinopathies.