Clinical and genetic heterogeneity in laminopathies

Mutations in the LMNA gene encoding lamins A/C are responsible for more than ten different disorders called laminopathies which affect various tissues in an isolated (striated muscle, adipose tissue or peripheral nerve) or systemic (premature aging syndromes) fashion. Overlapping phenotypes are also observed. Associated with this wide clinical variability, there is also a large genetic heterogeneity, with 408 different mutations being reported to date. Whereas a few hotspot mutations emerge for some types of laminopathies, relationships between genotypes and phenotypes remain poor for laminopathies affecting the striated muscles. In addition, there is important intrafamilial variability, explained only in a few cases by digenism, thus suggesting an additional contribution from modifier genes. In this regard, a chromosomal region linked to the variability in the age at onset of myopathic symptoms in striated muscle laminopathies has recently been identified. This locus is currently under investigation to identify modifier variants responsible for this variability.     

Phenomics and lamins: from disease to therapy

Systematic correlation of phenotype with genotype is a key goal of the emerging field of phenomics, which is expected to help define complex diseases. Careful evaluation of phenotype-genotype associations in monogenic disorders, such as laminopathies, might provide new hypotheses to be tested with molecular and cellular studies and might also suggest potential new intervention strategies. For instance, evaluation of the clinical features of carriers of mutant LMNA in kindreds with familial partial lipodystrophy suggests rational, staged intervention using established pharmaceutical agents to prevent cardiovascular complications not just for patients with lipodystrophy but by extension for patients with the common metabolic syndrome. Careful non-invasive imaging shows phenotypic differences between partial lipodystrophy due to mutant LMNA and not due to mutant LMNA. Furthermore, hierarchical cluster analysis detects systematic relationships between organ involvement in laminopathies and mutation position in the LMNA genomic sequence. However, sometimes the same LMNA mutation can underlie markedly different clinical phenotypes; cellular and molecular experiments can help to explain the mechanistic basis for such differences. Finally, promising novel treatment modalities for laminopathies, such as farnesyl transferase inhibition and gene-based therapies, might help not only to illuminate mechanisms that link genotype to phenotype, but also to provide hope for patients suffering with laminopathies, since these treatments are designed to modulate key early or proximal steps in the pathogenesis of these disorders.     

"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.     

Progeroide Laminopathien zwischen Hutchinson-Gilford-Syndrom, restriktiver Dermopathie und mandibuloakraler Dysplasie

There is a group of primary and secondary laminopathies that show progeroid phenotypes. They are associated with pleiotropic mutations in the LMNA or ZMPSTE24 genes and are clinically associated with Hutchinson-Gilford progeria syndrome, restrictive dermopathy and mandibuloacral dysplasia. Besides these clinically distinct entities, there is also an increasing number of atypical progeroid laminopathies??also known as atypical Werner syndrome??that cannot be categorized clinically as they show features of several laminopathies. As an example, two atypical primary and secondary progeroid laminopathies that show overlapping features of Hutchinson-Gilford progeria syndrome, restrictive dermopathy, dysplasia and even muscular laminopathies are presented.     

Characterization of lamin mutation phenotypes in Drosophila and comparison to human laminopathies

Lamins are intermediate filament proteins that make up the nuclear lamina, a matrix underlying the nuclear membrane in all metazoan cells that is important for nuclear form and function. Vertebrate A-type lamins are expressed in differentiating cells, while B-type lamins are expressed ubiquitously. Drosophila has two lamin genes that are expressed in A- and B-type patterns, and it is assumed that similarly expressed lamins perform similar functions. However, Drosophila and vertebrate lamins are not orthologous, and their expression patterns evolved independently. It is therefore of interest to examine the effects of mutations in lamin genes. Mutations in the mammalian lamin A/C gene cause a range of diseases, collectively called laminopathies, that include muscular dystrophies and premature aging disorders. We compared the sequences of lamin genes from different species, and we have characterized larval and adult phenotypes in Drosophila bearing mutations in the lam gene that is expressed in the B-type pattern. Larvae move less and show subtle muscle defects, and surviving lam adults are flightless and walk like aged wild-type flies, suggesting that lam phenotypes might result from neuromuscular defects, premature aging, or both. The resemblance of Drosophila lam phenotypes to human laminopathies suggests that some lamin functions may be performed by differently expressed genes in flies and mammals. Such still-unknown functions thus would not be dependent on lamin gene expression pattern, suggesting the presence of other lamin functions that are expression dependent. Our results illustrate a complex interplay between lamin gene expression and function through evolution.     

Diseases of the Nuclear Envelope

In the past decade, a wide range of fascinating monogenic diseases have been linked to mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins of the nuclear envelope. These diseases include dilated cardiomyopathy with variable muscular dystrophy, Dunnigan-type familial partial lipodystrophy, a Charcot-Marie-Tooth type 2 disease, mandibuloacral dysplasia, and Hutchinson-Gilford progeria syndrome. Several diseases are also caused by mutations in genes encoding B-type lamins and proteins that associate with the nuclear lamina. Studies of these so-called laminopathies or nuclear envelopathies, some of which phenocopy common human disorders, are providing clues about functions of the nuclear envelope and insights into disease pathogenesis and human aging.Mutations in LMNA encoding the A-type lamins cause a group of human disorders often collectively called laminopathies. The major A-type lamins, lamin A and lamin C, arise by alternative splicing of the LMNA pre-mRNA and are expressed in virtually all differentiated somatic cells. Although the A-type lamins are widely expressed, LMNA mutations are responsible for at least a dozen different clinically defined disorders with tissue-selective abnormalities. Mutations in genes encoding B-type lamins and lamin-associated proteins, most of which are similarly expressed in almost all somatic cells, also cause tissue-selective diseases.Research on the laminopathies has provided novel clues about nuclear envelope function. Recent studies have begun to shed light on how alterations in the nuclear envelope could explain disease pathogenesis. Along with basic research on nuclear structure, the nuclear lamins, and lamina-associated proteins, clinical research on the laminopathies will contribute to a complete understanding of the functions of the nuclear envelope in normal physiology and in human pathology.     

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.     

Embryonic senescence and laminopathies in a progeroid zebrafish model

Background

Mutations that disrupt the conversion of prelamin A to mature lamin A cause the rare genetic disorder Hutchinson-Gilford progeria syndrome and a group of laminopathies. Our understanding of how A-type lamins function in vivo during early vertebrate development through aging remains limited, and would benefit from a suitable experimental model. The zebrafish has proven to be a tractable model organism for studying both development and aging at the molecular genetic level. Zebrafish show an array of senescence symptoms resembling those in humans, which can be targeted to specific aging pathways conserved in vertebrates. However, no zebrafish models bearing human premature senescence currently exist.

Principal Findings

We describe the induction of embryonic senescence and laminopathies in zebrafish harboring disturbed expressions of the lamin A gene (LMNA). Impairments in these fish arise in the skin, muscle and adipose tissue, and sometimes in the cartilage. Reduced function of lamin A/C by translational blocking of the LMNA gene induced apoptosis, cell-cycle arrest, and craniofacial abnormalities/cartilage defects. By contrast, induced cryptic splicing of LMNA, which generates the deletion of 8 amino acid residues lamin A (zlamin A-Δ8), showed embryonic senescence and S-phase accumulation/arrest. Interestingly, the abnormal muscle and lipodystrophic phenotypes were common in both cases. Hence, both decrease-of-function of lamin A/C and gain-of-function of aberrant lamin A protein induced laminopathies that are associated with mesenchymal cell lineages during zebrafish early development. Visualization of individual cells expressing zebrafish progerin (zProgerin/zlamin A-Δ37) fused to green fluorescent protein further revealed misshapen nuclear membrane. A farnesyltransferase inhibitor reduced these nuclear abnormalities and significantly prevented embryonic senescence and muscle fiber damage induced by zProgerin. Importantly, the adult Progerin fish survived and remained fertile with relatively mild phenotypes only, but had shortened lifespan with obvious distortion of body shape.

Conclusion

We generated new zebrafish models for a human premature aging disorder, and further demonstrated the utility for studying laminopathies. Premature aging could also be modeled in zebrafish embryos. This genetic model may thus provide a new platform for future drug screening as well as genetic analyses aimed at identifying modifier genes that influence not only progeria and laminopathies but also other age-associated human diseases common in vertebrates.     

Mouse models of laminopathies

The A‐ and B‐type lamins are nuclear intermediate filament proteins in eukaryotic cells with a broad range of functions, including the organization of nuclear architecture and interaction with proteins in many cellular functions. Over 180 disease‐causing mutations, termed ‘laminopathies,’ have been mapped throughout LMNA, the gene for A‐type lamins in humans. Laminopathies can range from muscular dystrophies, cardiomyopathy, to Hutchinson–Gilford progeria syndrome. A number of mouse lines carrying some of the same mutations as those resulting in human diseases have been established. These LMNA‐related mouse models have provided valuable insights into the functions of lamin A biogenesis and the roles of individual A‐type lamins during tissue development. This review groups these LMNA‐related mouse models into three categories: null mutants, point mutants, and progeroid mutants. We compare their phenotypes and discuss their potential implications in laminopathies and aging.     

Tethering by lamin A stabilizes and targets the ING1 tumour suppressor

ING proteins interact with core histones through their plant homeodomains (PHDs) and with histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes to alter chromatin structure. Here we identify a lamin interaction domain (LID) found only in ING proteins, through which they bind to and colocalize with lamin A. Lamin knockout (LMNA(-/-)) cells show reduced levels of ING1 that mislocalize. Ectopic lamin A expression increases ING1 levels and re-targets it to the nucleus to act as an epigenetic regulator. ING1 lacking the LID does not interact with lamin A or affect apoptosis. In LMNA(-/-) cells, apoptosis is not affected by ING1. Mutation of lamin A results in several laminopathies, including Hutchinson-Gilford progeria syndrome (HGPS), a severe premature ageing disorder. HGPS cells have reduced ING1 levels that mislocalize. Expression of LID peptides to block lamin A-ING1 interaction induces phenotypes reminiscent of laminopathies including HGPS. These data show that targeting of ING1 to the nucleus by lamin A maintains ING1 levels and biological function. Known roles for ING proteins in regulating apoptosis and chromatin structure indicate that loss of lamin A-ING interaction may be an effector of lamin A loss, contributing to the HGPS phenotype.     

Inner nuclear membrane proteins: impact on human disease

In the past decade, the inner nuclear membrane has become a focus of research on inherited diseases. A heterogeneous group of genetic disorders known as laminopathies have been described that result from mutations in genes encoding nuclear lamins, intermediate filament proteins associated with the inner nuclear membrane. Mutations in genes encoding integral inner nuclear membrane proteins, many of which bind to nuclear lamins, also cause diseases that sometimes are very similar to those caused by lamin gene mutations. The pathogenic mechanisms that underlie these diseases, which often selectively affect different tissues or organ systems despite the near-ubiquitous expression of the proteins, are only beginning to be elucidated. The unfolding story of the laminopathies provides a remarkable example of how research in basic cell biology has impacted upon medicine and human health.     

A‐type lamins and signaling: The PI 3‐kinase/Akt pathway moves forward

Lamin A/C is a nuclear lamina constituent mutated in a number of human inherited disorders collectively referred to as laminopathies. The occurrence and significance of lamin A/C interplay with signaling molecules is an old question, suggested by pioneer studies performed in vitro. However, this relevant question has remained substantially unanswered, until data obtained in cellular and organismal models of laminopathies have indicated two main aspects of lamin A function. The first aspect is that lamins establish functional interactions with different protein platforms, the second aspect is that lamin A/C activity and altered function may elicit different effects in different cells and tissue types and even in different districts of the same tissue. Both these observations strongly suggest that signaling mechanisms targeting lamin A/C or its binding partners may regulate such a plastic behavior. A number of very recent data show involvement of kinases, as Akt and Erk, or phosphatases, as PP1 and PP2, in lamin A‐linked cellular mechanisms. Moreover, altered activation of signaling in laminopathies and rescue of the pathological phenotype in animal models by inhibitors of signaling pathways, strongly suggest that signaling effectors related to lamin A/C may be implicated in the pathogenesis of laminopathies and may represent targets of therapeutic intervention. In face of such an open perspective of basic and applied research, we review current evidence of lamin A/C interplay with signaling molecules, with particular emphasis on the lamin A‐Akt interaction and on the biological significance of their relationship. J. Cell. Physiol. 220: 553–561, 2009. © 2009 Wiley‐Liss, Inc.     

Tissue-specific defects are caused by loss of the Drosophila MAN1 LEM domain protein

The nuclear lamina represents a protein network required for nuclear structure and function. One family of lamina proteins is defined by an approximately 40-aa LAP2, Emerin, and MAN1 (LEM) domain (LEM-D) that binds the nonspecific DNA-binding protein, barrier-to-autointegration factor (BAF). Through interactions with BAF, LEM-D proteins serve as a bridge between chromosomes and the nuclear envelope. Mutations in genes encoding LEM-D proteins cause human laminopathies that are associated with tissue-restricted pathologies. Drosophila has five genes that encode proteins with LEM homology. Using yeast two-hybrid analyses, we demonstrate that four encode proteins that bind Drosophila (d)BAF. In addition to dBAF, dMAN1 associates with lamins, the LEM-D protein Bocksbeutel, and the receptor-regulated Smads, demonstrating parallel protein interactions with vertebrate homologs. P-element mobilization was used to generate null dMAN1 alleles. These mutants showed decreased viability, with surviving adults displaying male sterility, decreased female fertility, wing patterning and positioning defects, flightlessness, and locomotion difficulties that became more severe with age. Increased phospho-Smad staining in dMAN1 mutant wing discs is consistent with a role in transforming growth factor (TGF)-beta/bone morphogenic protein (BMP) signaling. The tissue-specific, age-enhanced dMAN1 mutant phenotypes are reminiscent of human laminopathies, suggesting that studies in Drosophila will provide insights into lamina dysfunction associated with disease.     

Specific contribution of lamin A and lamin C in the development of laminopathies

Mutations in the lamin A/C gene are involved in multiple human disorders for which the pathophysiological mechanisms are partially understood. Conflicting results prevail regarding the organization of lamin A and C mutants within the nuclear envelope (NE) and on the interactions of each lamin to its counterpart. We over-expressed various lamin A and C mutants both independently and together in COS7 cells. When expressed alone, lamin A with cardiac/muscular disorder mutations forms abnormal aggregates inside the NE and not inside the nucleoplasm. Conversely, the equivalent lamin C organizes as intranucleoplasmic aggregates that never connect to the NE as opposed to wild type lamin C. Interestingly, the lamin C molecules present within these aggregates exhibit an abnormal increased mobility. When co-expressed, the complex formed by lamin A/C aggregates in the NE. Lamin A and C mutants for lipodystrophy behave similarly to the wild type. These findings reveal that lamins A and C may be differentially affected depending on the mutation. This results in multiple possible physiological consequences which likely contribute in the phenotypic variability of laminopathies. The inability of lamin C mutants to join the nuclear rim in the absence of lamin A is a potential pathophysiological mechanism for laminopathies.     

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.     

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.