"Laminopathies": a wide spectrum of human diseases

Mutations in genes encoding the intermediate filament nuclear lamins and associated proteins cause a wide spectrum of diseases sometimes called "laminopathies." Diseases caused by mutations in LMNA encoding A-type lamins include autosomal dominant Emery-Dreifuss muscular dystrophy and related myopathies, Dunnigan-type familial partial lipodystrophy, Charcot-Marie-Tooth disease type 2B1 and developmental and accelerated aging disorders. Duplication in LMNB1 encoding lamin B1 causes autosomal dominant leukodystrophy and mutations in LMNB2 encoding lamin B2 are associated with acquired partial lipodystrophy. Disorders caused by mutations in genes encoding lamin-associated integral inner nuclear membrane proteins include X-linked Emery-Dreifuss muscular dystrophy, sclerosing bone dysplasias, HEM/Greenberg skeletal dysplasia and Pelger-Huet anomaly. While mutations and clinical phenotypes of "laminopathies" have been carefully described, data explaining pathogenic mechanisms are only emerging. Future investigations will likely identify new "laminopathies" and a combination of basic and clinical research will lead to a better understanding of pathophysiology and the development of therapies.     

The laminopathies: nuclear structure meets disease

Most inherited diseases are associated with mutations in a specific gene. Sometimes, mutations in two or more different genes result in diseases with a similar phenotype. Rarely do different mutations in the same gene result in a multitude of seemingly different and unrelated diseases. In the past three years, different mutations in LMNA, the gene encoding the A-type lamins, have been shown to be associated with at least six different diseases. These diseases and at least two others caused by mutations in other proteins associated with the nuclear lamina are collectively called the laminopathies. How different tissue-specific diseases arise from unique mutations in the LMNA gene, encoding almost ubiquitously expressed nuclear proteins, are providing tantalizing insights into the structural organization of the nucleus, its relation to nuclear function in different tissues and the involvement of the nuclear envelope in the development of disease.     

Analysis of genetic variations of lamin A/C gene (LMNA) by denaturing high-performance liquid chromatography

The human LMNA gene, when mutated, has been shown to cause at least 7 human diseases: dilated cardiomyopathy, Emery Dreifuss muscular dystrophy, limb girdle muscular dystrophy, familial partial lipodystrophy, Charcot Marie tooth disease type II, mandibuloacral dysplasia, and Hutchinson-Gilford Progeria (OMIM #176670). This article describes a high-throughput method for screening the human lamin A/C (LMNA) gene for genetic mutations and sequence variation using denaturing high-performance liquid chromatography (DHPLC). In the present study, 76 patients with dilated cardiomyopathy were screened for mutations using DHPLC and sequence analysis. Abnormal elution profiles were identified and sequenced on an ABI 377 automatic sequencer. Heterozygous LMNA mutations were detected in 8% of the affected patients. In addition, a number of intronic and exonic single nucleotide polymorphisms were identified. LMNA mutations are clinically relevant in at least 6 human diseases. This study provides a protocol for high-throughput LMNA analysis applicable both in the research and in the clinical diagnostic setting.     

Unexpectedly many extinct hominins

Recent studies indicate that Neanderthal and Denisova hominins may have been separate species, while debate continues on the status of Homo floresiensis. The decade-long debate between "splitters," who recognize over 20 hominin species, and "lumpers," who maintain that all these fossils belong to just a few lineages, illustrates that we do not know how many extinct hominin species to expect. Here, we present probability distributions for the number of speciation events and the number of contemporary species along a branch of a phylogeny. With estimates of hominin speciation and extincton rates, we then show that the expected total number of extinct hominin species is 8, but may be as high as 27. We also show that it is highly unlikely that three very recent species disappeared due to natural, background extinction. This may indicate that human-like remains are too easily considered distinct species. Otherwise, the evidence suggesting that Neanderthal and the Denisova hominin represent distinct species implies a recent wave of extinctions, ostensibly driven by the only survivor, H. sapiens.     

Protein profiling reveals energy metabolism and cytoskeletal protein alterations in LMNA mutation carriers

Nuclear envelope-related muscular dystrophies, in particular those referred to as laminopathies, are relatively novel and unclear diseases, also considering the increasing number of mutations identified so far in genes of the nuclear envelope. As regard LMNA gene, only tentative relations between phenotype, type and localization of the mutations have been established in striated muscle diseases, while laminopathies affecting adipose tissue, peripheral nerves or progerioid syndromes could be linked to specific genetic variants. This study describes the biochemical phenotype of neuromuscular laminopathies in samples derived from LMNA mutant patients. Since it has been reported that nuclear alterations, due to LMNA defects, are present also in fibroblasts from Emery-Dreifuss muscular dystrophy and familial partial lipodystrophy patients, we analyzed 2D-maps of skin fibroblasts of patients carrying 12 different LMNA mutations spread along the entire gene. To recognize distinctive proteins underlying affected biochemical pathways, we compared them with fibroblasts from healthy controls and, more importantly, fibroblasts from patients with non-lamin related neuromuscular disorders. We found less abundance of cytoskeletal/structural proteins, confirming a dominant role for Lamin A/C in structural support of nuclear architecture. Interestingly, we also established significant changes in the expression of proteins involved in cellular energy production and oxidative stress response. To our knowledge, this is the first report where proteomics was applied to characterize ex-vivo cells from LMNA patients, suggesting that this may represent a new approach to better understand the molecular mechanisms of these rare diseases and facilitate the development of novel therapeutic treatments.     

Serum lipidomics meets cardiac magnetic resonance imaging: profiling of subjects at risk of dilated cardiomyopathy

Dilated cardiomyopathy (DCM), characterized by left ventricular dilatation and systolic dysfunction, constitutes a significant cause for heart failure, sudden cardiac death or need for heart transplantation. Lamin A/C gene (LMNA) on chromosome 1p12 is the most significant disease gene causing DCM and has been reported to cause 7-9% of DCM leading to cardiac transplantation. We have previously performed cardiac magnetic resonance imaging (MRI) to LMNA carriers to describe the early phenotype. Clinically, early recognition of subjects at risk of developing DCM would be important but is often difficult. Thus we have earlier used the MRI findings of these LMNA carriers for creating a model by which LMNA carriers could be identified from the controls at an asymptomatic stage. Some LMNA mutations may cause lipodystrophy. To characterize possible effects of LMNA mutations on lipid profile, we set out to apply global serum lipidomics using Ultra Performance Liquid Chromatography coupled to mass spectrometry in the same LMNA carriers, DCM patients without LMNA mutation and controls. All DCM patients, with or without LMNA mutation, differed from controls in regard to distinct serum lipidomic profile dominated by diminished odd-chain triglycerides and lipid ratios related to desaturation. Furthermore, we introduce a novel approach to identify associations between the molecular lipids from serum and the MR images from the LMNA carriers. The association analysis using dependency network and regression approaches also helped us to obtain novel insights into how the affected lipids might relate to cardiac shape and volume changes. Our study provides a framework for linking serum derived molecular markers not only with clinical endpoints, but also with the more subtle intermediate phenotypes, as derived from medical imaging, of potential pathophysiological relevance.     

利用CRISPR/Cas9敲除人源细胞系中LMNA基因的研究

LMNA基因编码A型和C型核纤层蛋白,参与细胞核核膜的组织,影响基因组稳定性并对细胞分化产生影响。人类肿瘤中LMNA表达异常普遍存在,其突变造成多种核纤层蛋白病,如Emery-Dreifuss肌营养不良症(Emery-Dreifussmusculardystrophy,EDMD)、扩张型心肌病(dilatedcardiomyopathy,DCM)和儿童早老症(Hutchinson-Glifordprogeriasyndrome,HGPS)等。为进一步研究LMNA在细胞内的功能,本研究利用CRISPR/Cas9技术对体外培养的293T与HepG2细胞株的LMNA基因进行编辑,获得两株LMNA基因敲除(LMNA KO)的稳定细胞系。与野生型相比,LMNAKO细胞系增殖能力相对减弱,凋亡增加。同时,细胞形态上也发生显著改变,核膜凹凸不平。本研究首次报道了LMNA KO永生细胞系构建和形态研究结果,为后续LMNA基因功能研究和致病突变体研究奠定基础。     

Lamins A and C are differentially dysfunctional in autosomal dominant Emery-Dreifuss muscular dystrophy

Mutations in the LMNA gene, which encodes nuclear lamins A and C by alternative splicing, can give rise to Emery-Dreifuss muscular dystrophy. The mechanism by which lamins A and C separately contribute to this molecular phenotype is unknown. To address this question we examined ten LMNA mutations exogenously expressed as lamins A and C in COS-7 cells. Eight of the mutations when expressed in lamin A, exhibited a range of nuclear mislocalisation patterns. However, two mutations (T150P and delQ355) almost completely relocated exogenous lamin A from the nuclear envelope to the cytoplasm, disrupted nuclear envelope reassembly following cell division and altered the protein composition of the mid-body. In contrast, exogenously expressed DsRed2-tagged mutant lamin C constructs were only inserted into the nuclear lamina if co-expressed with any EGFP-tagged lamin A construct, except with one carrying the T150P mutation. The T150P, R527P and L530P mutations reduced the ability of lamin A, but not lamin C from binding to emerin. These data identify specific functional roles for the emerin-lamin C- and emerin-lamin A- containing protein complexes and is the first report to suggest that the A-type lamin mutations may be differentially dysfunctional for the same LMNA mutation.     

The role of lamins and mutations of LMNA gene in physiological and premature aging

Lamins belong to type V intermediate filaments superfamily. They are the main structural constituencies of the nuclear lamina but they also influence on chromatin structure, regulation of gene expression, localization and probably protein degradation. Because lamins play many different roles within the cell, mutations in their genes can results in variety of pathological phenotypes. Mutations in LMNA gene are the cause of many different diseases, called laminopathies. Among laminopathies are muscle tissue diseases, adipose tissue diseases and also progerias, the premature aging syndromes. One of the progerias, which results from mutation in LMNA gene, is Hutchinson-Gilford progeria syndrome (HGPS). It seems that the same molecular mechanisms which are responsible for premature aging of cells of HGPS patients, are involved in physiological aging.     

Different mutations in the LMNA gene cause autosomal dominant and autosomal recessive Emery-Dreifuss muscular dystrophy

Emery-Dreifuss muscular dystrophy (EMD) is a condition characterized by the clinical triad of early-onset contractures, progressive weakness in humeroperoneal muscles, and cardiomyopathy with conduction block. The disease was described for the first time as an X-linked muscular dystrophy, but autosomal dominant and autosomal recessive forms were reported. The genes for X-linked EMD and autosomal dominant EMD (AD-EMD) were identified. We report here that heterozygote mutations in LMNA, the gene for AD-EMD, may cause diverse phenotypes ranging from typical EMD to no phenotypic effect. Our results show that LMNA mutations are also responsible for the recessive form of the disease. Our results give further support to the notion that different genetic forms of EMD have a common pathophysiological background. The distribution of the mutations in AD-EMD patients (in the tail and in the 2A rod domain) suggests that unique interactions between lamin A/C and other nuclear components exist that have an important role in cardiac and skeletal muscle function.     

Human lipodystrophies linked to mutations in A-type lamins and to HIV protease inhibitor therapy are both associated with prelamin A accumulation, oxidative stress and premature cellular senescence

Lipodystrophic syndromes associated with mutations in LMNA, encoding A-type lamins, and with HIV antiretroviral treatments share several clinical characteristics. Nuclear alterations and prelamin A accumulation have been reported in fibroblasts from patients with LMNA mutations and adipocytes exposed to protease inhibitors (PI). As genetically altered lamin A maturation also results in premature ageing syndromes with lipodystrophy, we studied prelamin A expression and senescence markers in cultured human fibroblasts bearing six different LMNA mutations or treated with PIs. As compared to control cells, fibroblasts with LMNA mutations or treated with PIs had nuclear shape abnormalities and reduced proliferative activity that worsened with increasing cellular passages. They exhibited prelamin A accumulation, increased oxidative stress, decreased expression of mitochondrial respiratory chain proteins and premature cellular senescence. Inhibition of prelamin A farnesylation prevented cellular senescence and oxidative stress. Adipose tissue samples from patients with LMNA mutations or treated with PIs also showed retention of prelamin A, overexpression of the cell cycle checkpoint inhibitor p16 and altered mitochondrial markers. Thus, both LMNA mutations and PI treatment result in accumulation of farnesylated prelamin A and oxidative stress that trigger premature cellular senescence. These alterations could participate in the pathophysiology of lipodystrophic syndromes and lead to premature ageing complications.     

Mandibuloacral dysplasia is caused by a mutation in LMNA-encoding lamin A/C

Mandibuloacral dysplasia (MAD) is a rare autosomal recessive disorder, characterized by postnatal growth retardation, craniofacial anomalies, skeletal malformations, and mottled cutaneous pigmentation. The LMNA gene encoding two nuclear envelope proteins (lamins A and C [lamin A/C]) maps to chromosome 1q21 and has been associated with five distinct pathologies, including Dunnigan-type familial partial lipodystrophy, a condition that is characterized by subcutaneous fat loss and is invariably associated with insulin resistance and diabetes. Since patients with MAD frequently have partial lipodystrophy and insulin resistance, we hypothesized that the disease may be caused by mutations in the LMNA gene. We analyzed five consanguineous Italian families and demonstrated linkage of MAD to chromosome 1q21, by use of homozygosity mapping. We then sequenced the LMNA gene and identified a homozygous missense mutation (R527H) that was shared by all affected patients. Patient skin fibroblasts showed nuclei that presented abnormal lamin A/C distribution and a dysmorphic envelope, thus demonstrating the pathogenic effect of the R527H LMNA mutation.     

The nuclear lamina. Both a structural framework and a platform for genome organization

The inner face of the nuclear envelope of metazoan cells is covered by a thin lamina consisting of a one-layered network of intermediate filaments interconnecting with a complex set of transmembrane proteins and chromatin associating factors. The constituent proteins, the lamins, have recently gained tremendous recognition, because mutations in the lamin A gene, LMNA, are the cause of a complex group of at least 10 different diseases in human, including the Hutchinson-Gilford progeria syndrome. The analysis of these disease entities has made it clear that besides cytoskeletal functions, the lamina has an important role in the "behaviour" of the genome and is, probably as a consequence of this function, intimately involved in cell fate decisions. Furthermore, these functions are related to the involvement of lamins in organizing the position and functional state of interphase chromosomes as well as to the occurrence of lamins and lamina-associated proteins within the nucleoplasm. However, the structural features of these lamins and the nature of the factors that assist them in genome organization present an exciting challenge to modern biochemistry and cell biology.     

Mouse models of the laminopathies

The A and B type lamins are nuclear intermediate filament proteins that comprise the bulk of the nuclear lamina, a thin proteinaceous structure underlying the inner nuclear membrane. The A type lamins are encoded by the lamin A gene (LMNA). Mutations in this gene have been linked to at least nine diseases, including the progeroid diseases Hutchinson-Gilford progeria and atypical Werner's syndromes, striated muscle diseases including muscular dystrophies and dilated cardiomyopathies, lipodystrophies affecting adipose tissue deposition, diseases affecting skeletal development, and a peripheral neuropathy. To understand how different diseases arise from different mutations in the same gene, mouse lines carrying some of the same mutations found in the human diseases have been established. We, and others have generated mice with different mutations that result in progeria, muscular dystrophy, and dilated cardiomyopathy. To further our understanding of the functions of the lamins, we also created mice lacking lamin B1, as well as mice expressing only one of the A type lamins. These mouse lines are providing insights into the functions of the lamina and how changes to the lamina affect the mechanical integrity of the nucleus as well as signaling pathways that, when disrupted, may contribute to the disease.     

A G613A missense in the Hutchinson’s progeria lamin A/C gene causes a lone,autosomal dominant atrioventricular block

Background

LMNA/C mutations have been linked to the premature aging syndrome Hutchinson’s progeria, dilated cardiomyopathy 1A, skeletal myopathies (such as the autosomal dominant variant of Emery-Dreifuss muscular dystrophy and limb-girdle muscular dystrophy), Charcot-Marie-Tooth disorder type 2B1, mandibuloacral dysplasia, autosomal dominant partial lipodystrophy, and axonal neuropathy. Atrioventricular block (AVB) can be associated with several cardiac disorders and it can also be a highly heritable, primitive disease.One of the most common pathologies associated with AVB is dilated cardiomyopathy (DCM), which is characterized by cardiac dilatation and reduced systolic function. In this case, onset has been correlated with several mutations in genes essential for the proper maturation of cardiomyocytes, such as the gene for lamin A/C. However, no clear genotype–phenotype relationship has been reported to date between LMNA/C mutations and cardiomyopathies.

Results

DNA and medical histories were collected from (n?=?11) members of different generations of one family, the proband of which was implanted with a pacemaker for lone, type II AVB. Exome sequencing analysis was performed on three relatives with AVB, and the mutations therein identified validated in a further three AVB-affected family members.In the initial three AVB family members, we identified 10 shared nonsynonymous single-nucleotide variations with a rare or unreported allele frequency in the 1000 Genomes Project database. Follow-up genetic screening in the additional three affected relatives disclosed a correlation between the lone AVB phenotype and the single-nucleotide polymorphism rs56816490, which generates an E317K change in lamin A/C. Although this mutation has already been described by others in a DCM-affected proband with familiarity for AVB and sudden death, the absence of DCM in our large, AVB-affected family is indicative of genotype–phenotype correlation between rs56816490 and a familial, autosomal dominant form of lone AVB.

Conclusions

Screening for G613A in LMNA/C in patients with lone AVB and their relatives might prevent sudden death in families affected by AVB but without familiarity for DCM. Lone AVB is an age-related disease caused by mutations in LMN A/C gene rather than a complication of DCM.

     

Lamin A/C mutations alter differentiation potential of mesenchymal stem cells

Mutations in the lamin A/C gene (LMNA) lead to severe disorders collectively called laminopathies. The mechanisms by which lamin mutations cause the diseases are not clear. Since the mesenchymal lineages, adipose tissue in particular, are mostly affected in laminopathies, the aim of the study was to estimate the effect of LMNA mutations on differentiation of mesenchymal stem cells, adipose tissue stromal cells (ATSCs), into adipose lineages. ATSCs transduced with lentiviral vectors carrying LMNA gene mutations associated with various syndromes (myodystrophy, cardiomyopathy, lipodystrophy, progeroid syndrome) were induced to adipose differentiate. It was found that introduction of genetic constructions with LMNA gene point mutations G465D, R482L, and R527C promote adipogenic differentiation compared to wild-type lamin gene; mutation R471C reduced the differentiation. Introduction of R471C or R527C lamin mutations profoundly increased the expression of adipogenesis markers PPARG, SREBP1, and adipsin. Mutations in A/C lamin gene strongly and variously affect the differentiation of mesenchymal stem cells that probably underlie the pathogenic changes in patients with laminopathies.     

A novel <Emphasis Type="Italic">TPM2</Emphasis> gene splice-site mutation causes severe congenital myopathy with arthrogryposis and dysmorphic features

To date, only two splice-site mutations within the TPM2 gene have been shown to be causative for congenital myopathies. While the majority of TPM2 gene mutations are causative for nemaline myopathy, cap disease or distal arthrogryposis, some mutations in this gene have been found to be associated with non-specific congenital myopathy. We report on a patient with such an unspecified congenital myopathy associated with distinctive facial dysmorphic features and distal arthrogryposis. Using the whole exome sequencing (WES) approach we were able to identify a novel heterozygous splice-site mutation within the TPM2 gene, showing the utility of WES in molecular diagnostics of congenital myopathies without recognizable morphological hallmarks.