Linkage of familial dilated cardiomyopathy to chromosome 9. Heart Muscle Disease Study Group.

Idiopathic dilated cardiomyopathy is a heart muscle disease of unknown etiology, characterized by impaired myocardial contractility and ventricular dilatation. The disorder is an important cause of morbidity and mortality and represents the chief indication for heart transplantation. Familial transmission is often recognized (familial dilated cardiomyopathy, or FDC), mostly with autosomal dominant inheritance. In order to understand the molecular genetic basis of the disease, a large six-generation kindred with autosomal dominant FDC was studied for linkage analysis. A genome-wide search was undertaken after a large series of candidate genes were excluded and was then extended to two other families with autosomal dominant pattern of transmission and identical clinical features. Coinheritance of the disease gene was excluded for > 95% of the genome, after 251 polymorphic markers were analyzed. Linkage was found for chromosome 9q13-q22, with a maximum multipoint lod score of 4.2. There was no evidence of heterogeneity. The FDC locus was placed in the interval between loci D9S153 and D9S152. Several candidate genes for causing dilated cardiomyopathy map in this region.     

Mutational and haplotype analyses of families with familial partial lipodystrophy (Dunnigan variety) reveal recurrent missense mutations in the globular C-terminal domain of lamin A/C

Familial partial lipodystrophy (FPLD), Dunnigan variety, is an autosomal dominant disorder characterized by marked loss of subcutaneous adipose tissue from the extremities and trunk but by excess fat deposition in the head and neck. The disease is frequently associated with profound insulin resistance, dyslipidemia, and diabetes. We have localized a gene for FPLD to chromosome 1q21-q23, and it has recently been proposed that nuclear lamin A/C is altered in FPLD, on the basis of a novel missense mutation (R482Q) in five Canadian probands. This gene had previously been shown to be altered in autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD-AD) and in dilated cardiomyopathy and conduction-system disease. We examined 15 families with FPLD for mutations in lamin A/C. Five families harbored the R482Q alteration that segregated with the disease phenotype. Seven families harbored an R482W alteration, and one family harbored a G465D alteration. All these mutations lie within exon 8 of the lamin A/C gene-an exon that has also been shown to harbor different missense mutations that are responsible for EDMD-AD. Mutations could not be detected in lamin A/C in one FPLD family in which there was linkage to chromosome 1q21-q23. One family with atypical FPLD harbored an R582H alteration in exon 11 of lamin A. This exon does not comprise part of the lamin C coding region. All mutations in FPLD affect the globular C-terminal domain of the lamin A/C protein. In contrast, mutations responsible for dilated cardiomyopathy and conduction-system disease are observed in the rod domain of the protein. The FPLD mutations R482Q and R482W occurred on different haplotypes, indicating that they are likely to have arisen more than once.     

The role of the nuclear envelope in Emery–Dreifuss muscular dystrophy

The X-linked form of Emery-Dreifuss muscular dystrophy (X-EDMD) is caused by absence, or greatly reduced amounts, of the inner nuclear-membrane protein, emerin. The autosomal dominant form (AD-EDMD) is caused by missense mutations in lamins A and C, two components of the nuclear lamina that interact directly with emerin. Lamin A/C mutations also cause one form of dilated cardiomyopathy (CMD1A) and one form of limb-girdle muscular dystrophy (LGMD1B), both of which have clinical features in common with EDMD, as well as a rare, unrelated form of lipodystrophy (FPLD). Evidence is now emerging that defective assembly of the nuclear lamina is a feature of all these diseases, although not necessarily the direct cause. Why only heart and skeletal muscle, and possibly connective tissue, are affected in EDMD and why expression of the disease is so extremely variable between individuals remains to be explained.     

Delineating the role of alterations in lipid metabolism to the pathogenesis of inherited skeletal and cardiac muscle disorders: Thematic Review Series: Genetics of Human Lipid Diseases

As the specific composition of lipids is essential for the maintenance of membrane integrity, enzyme function, ion channels, and membrane receptors, an alteration in lipid composition or metabolism may be one of the crucial changes occurring during skeletal and cardiac myopathies. Although the inheritance (autosomal dominant, autosomal recessive, and X-linked traits) and underlying/defining mutations causing these myopathies are known, the contribution of lipid homeostasis in the progression of these diseases needs to be established. The purpose of this review is to present the current knowledge relating to lipid changes in inherited skeletal muscle disorders, such as Duchenne/Becker muscular dystrophy, myotonic muscular dystrophy, limb-girdle myopathic dystrophies, desminopathies, rostrocaudal muscular dystrophy, and Dunnigan-type familial lipodystrophy. The lipid modifications in familial hypertrophic and dilated cardiomyopathies, as well as Barth syndrome and several other cardiac disorders associated with abnormal lipid storage, are discussed. Information on lipid alterations occurring in these myopathies will aid in the design of improved methods of screening and therapy in children and young adults with or without a family history of genetic diseases.     

Reactivation of autophagy ameliorates LMNA cardiomyopathy

Mutations in the LMNA gene, which encodes lamin A and C (lamin A/C), cause a diverse spectrum of tissue-selective diseases termed laminopathies. The most prevalent form affects striated muscles as dilated cardiomyopathy with variable skeletal muscle involvement, which includes autosomal Emery-Dreifuss muscular dystrophy. Mechanisms underlying the disease pathogenesis are beginning to be understood and they point toward defects in cell signaling. We therefore assessed putative signaling defects in a mouse model carrying a point mutation in Lmna (Lmna^H222P/H222P) that faithfully recapitulates human Emery-Dreifuss muscular dystrophy. We found that AKT-mechanistic target of rapamycin (MTOR) signaling was hyperactivated in hearts of Lmna^H222P/H222P mice and that reducing MTOR activity by pharmacological intervention ameliorated cardiomyopathy. Given the central role of MTOR in regulating autophagy, we assessed fasting-induced autophagic responses and found that they were impaired in hearts of these mice. Moreover, the improved heart function associated with pharmacological blockade of MTOR was correlated with enhanced autophagy. These findings demonstrated that signaling defects that impair autophagy underlie pathogenesis of dilated cardiomyopathy arising from LMNA mutation.     

A novel mutation of the LMNA gene in a family with dilated cardiomyopathy, conduction system disease, and sudden cardiac death of young females

The LMNA gene, which encodes the nuclear envelope protein lamin A/C, is considered to be the most common autosomal disease gene associated with familial dilated cardiomyopathy. To date, each mutation of the LMNA gene has been associated with a specific disease phenotype. Clinical data, family histories, and blood samples were collected from 27 biological members of a family with dilated cardiomyopathy, prominently occurring as heart failure and conduction system disease with a high incidence of sudden cardiac death in young females. Twelve exons of the LMNA gene were screened for nucleotide alterations. A novel insertion mutation (nucleotide 1526insA, amino acid T510Y) in exon nine of the LMNA gene was identified in seven subjects (7/27, 25.9 %). This reveals that the LMNA gene insertion mutation (T510Y frameshift mutation) can cause dilated cardiomyopathy, conduction system disease, and sudden cardiac death without skeletal myopathy, clinically manifested with early onset, severe symptoms, and poor prognosis.     

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 novel locus for autosomal-dominant dilated cardiomyopathy maps to chromosome 7q22.3-31.1

Inherited dilated cardiomyopathy (DCM) is a genetically and phenotypically very heterogeneous disease. DCM is caused by mutations in multiple genes encoding proteins that are involved in force generation, force transmission, energy production and several signalling pathways. Thus, the pathophysiology of heart failure is complex and not yet fully understood. Familial forms of DCM let the way to identify new key proteins by positional cloning and to study respective pathomechanisms that are critical for normal cardiac function, but may not have been correlated with heart disease before. Here we report a three-generation pedigree including 16 individuals affected by dilated cardiomyopathy without additional phenotypes. The pedigree is consistent with autosomal-dominant inheritance and age-related penetrance. A genome-wide linkage analysis excluded linkage to all known DCM genes and loci, whereas several close markers on chromosome 7q22.3-31.1 segregated with the disease (maximum logarithm of odds score, 4.20 at D7S471 and D7S501). The disease causing mutation lies in a 9.73 Mb interval between markers D7S2545 and D7S2554 that contains no known cytoskeletal genes. Coding exons of the candidate genes LAMB1, LAMB4 and PIK3CG were screened but no mutations were identified.Jost Schönberger, Leif Kühler, and Elisabete Martins authors contributed equally to the work     

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.     

Genetic linkage of Bietti crystallin corneoretinal dystrophy to chromosome 4q35

Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive retinal degeneration characterized by multiple glistening intraretinal dots scattered over the fundus, degeneration of the retina, and sclerosis of the choroidal vessels, ultimately resulting in progressive night blindness and constriction of the visual field. Although BCD has been associated with abnormalities in fatty-acid metabolism and absence of fatty-acid binding by two cytosolic proteins, the genetic basis of BCD is unknown. We report linkage of the BCD locus to D4S426 (maximum LOD score [Z(max)] 4.81; recombination fraction [straight theta] 0), D4S2688 (Zmax=3.97; straight theta=0), and D4S2299 (Zmax=5.31; straight theta=0), on chromosome 4q35-4qtel. Multipoint analysis confirmed linkage to the region telomeric of D4S1652 with a Z(max) of 5.3 located 4 cM telomeric of marker D4S2930.     

Dysferlin deficiency confers increased susceptibility to coxsackievirus‐induced cardiomyopathy

Coxsackievirus infection can lead to viral myocarditis and its sequela, dilated cardiomyopathy, which represent major causes of cardiovascular mortality worldwide in children. Yet, the host genetic susceptible factors and the underlying mechanisms by which viral infection damages cardiac function remain to be fully resolved. Dysferlin is a transmembrane protein highly expressed in skeletal and cardiac muscles. In humans, mutations in the dysferlin gene can cause limb‐girdle muscular dystrophy type 2B and Miyoshi myopathy. Dysferlin deficiency has also been linked to cardiomyopathy. Defective muscle membrane repair has been suggested to be an important mechanism responsible for muscle degeneration in dysferlin‐deficient patients and animals. Using both naturally occurring and genetically engineered dysferlin‐deficient mice, we demonstrated that loss of dysferlin confers increased susceptibility to coxsackievirus infection and myocardial damage. More interestingly, we found that dysferlin is cleaved following coxsackieviral infection through the proteolytic activity of virally encoded proteinases, suggesting an important mechanism underlying virus‐induced cardiac dysfunction. Our results in this study not only identify dysferlin deficiency as a novel host risk factor for viral myocarditis but also reveal a key mechanism by which coxsackievirus infection impairs cardiac function, leading to the development of dilated cardiomyopathy.     

Limb-girdle muscular dystrophy and Miyoshi myopathy in an aboriginal Canadian kindred map to LGMD2B and segregate with the same haplotype.

We report the results of our investigations of a large, inbred, aboriginal Canadian kindred with nine muscular dystrophy patients. The ancestry of all but two of the carrier parents could be traced to a founder couple, seven generations back. Seven patients presented with proximal myopathy consistent with limb girdle-type muscular dystrophy (LGMD), whereas two patients manifested predominantly distal wasting and weakness consistent with Miyoshi myopathy (distal autosomal recessive muscular dystrophy) (MM). Age at onset of symptoms, degree of creatine kinase elevation, and muscle histology were similar in both phenotypes. Segregation of LGMD/MM is consistent with autosomal recessive inheritance, and the putative locus is significantly linked (LOD scores >3.0) to six marker loci that span the region of the LGMD2B locus on chromosome 2p. Our initial hypothesis that the affected patients would all be homozygous by descent for microsatellite markers surrounding the disease locus was rejected. Rather, two different core haplotypes, encompassing a 4-cM region spanned by D2S291-D2S145-D2S286, segregated with the disease, indicating that there are two mutant alleles of independent origin in this kindred. There was no association, however, between the two different haplotypes and clinical variability; they do not distinguish between the LGMD and MM phenotypes. Thus, we conclude that LGMD and MM in our population are caused by the same mutation in LGMD2B and that additional factors, both genetic and nongenetic, must contribute to the clinical phenotype.     

Elevated MTORC1 signaling and impaired autophagy

A-type lamins, generated from the LMNA gene by differential splicing, are type V intermediate filament proteins that polymerize to form part of the nuclear lamina, and are of considerable medical interest because missense mutations in LMNA give rise to a wide range of dystrophic and progeroid syndromes. Among these are dilated cardiomyopathy and two forms of muscular dystrophy (limb-girdle and Emery-Dreifuss), which are modeled in lmna^?/? mice and mice engineered to express human disease mutations. Our recent study demonstrates that cardiac and skeletal muscle pathology in lmna^?/? mice can be attributed to elevated MTORC1 signaling leading to impairment of autophagic flux. An accompanying paper from another laboratory shows similar impairments in mice engineered to express the LMNA H222P associated with dilated cardiomyopathy in humans and also in left ventricular tissue from human subjects. MTORC1 inhibition with rapalogs restores autophagic flux and improves cardiac function in both mouse models, and extends survival in the lmna^?/? mice. These findings elaborate a potential treatment option for dilated cardiomyopathy and muscular dystrophy associated with LMNA mutation and supplement growing evidence linking impaired autophagy to human disease.     

The laminin alpha2 expressed by dystrophic dy(2J) mice is defective in its ability to form polymers

Mutations in LAMA2 cause severe congenital muscular dystrophy accompanied by nervous system defects [1]. Mice homozygous for the dy(2J) allele of LAMA2 express a laminin alpha2 subunit that has a deletion in the amino-terminal domain VI, providing an animal model for study of the molecular basis of congenital muscular dystrophy [2] [3]. Domain VI is predicted to be involved in laminin polymerization, along with amino-terminal domains from laminin beta and gamma chains [4]. In a solution-polymerization assay, we found that purified dy(2J) laminin assembled poorly and formed little polymer, in contrast to wild-type muscle laminin. Furthermore, dissolution of the collagen IV network caused dy(2J) laminin to be released into solution, indicating that laminin polymers within the skeletal muscle basement membrane were defective. In addition to loss of polymerization, dy(2J) laminin had a reduced affinity for heparin. Finally, recombinant laminin engineered with the dy(2J) deletion was more sensitive to proteolysis and was readily cleaved near the junction of domains V and VI. Thus, the dy(2J) deletion selectively disrupts polymer formation, reduces affinity for heparin, and destabilizes domain VI. These are the first specific functional defects to be identified in a muscular dystrophy laminin, and it is likely that these defects contribute to the abnormalities seen in dy(2J)/dy(2J) muscle and nerve.     

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.     

The Cardiomyopathy of Muscular Dystrophy: Report of Two Cases with a Review of the Literature

About 50% of patients with progressive muscular dystrophy have a cardiomyopathy, manifested commonly by tachycardia, but also by arrythmias, refractory congestive heart failure and sudden death. Studies from the literature report manifold but nonspecific electrocardiographic changes in 41% to 85% of patients with progressive muscular dystrophy. The principal lesion is a diffuse myocardial fibrosis with minor degenerative changes in myocardial fibres unaccompanied by significant inflammation. The heart is enlarged and has a prominent deposit of epicardial fat. The myocardium is pale, coarse, flabby and friable, often showing gross evidence of scarring. The dilated chambers often contain mural thrombus.     

CD90-positive cells, an additional cell population, produce laminin alpha2 upon transplantation to dy(3k)/dy(3k) mice

Laminin alpha2 is a component of skeletal and cardiac muscle basal lamina. A defect of the laminin alpha2 chain leads to severe congenital muscular dystrophy (MDC1A) in humans and dy/dy mice. Myogenic cells including myoblasts, myotubes, and myofibers in skeletal muscle are a possible source of the laminin alpha2 chain, and myogenic cells are thus proposed as a cell source for congenital muscular dystrophy therapy. However, we observed production of laminin alpha2 in non-myogenic cells of normal mice, and we could enrich these laminin alpha2-producing cells in CD90(+) cell fractions. Intriguingly, the number of CD90(+) cells increased dramatically during skeletal muscle regeneration in mice. This fraction did not include myogenic cells but exhibited a fibroblast-like phenotype. Moreover, these cells were resident in skeletal muscle, not derived from bone marrow. Finally, the production of laminin alpha2 in CD90(+) cells was not dependent on fusion with myogenic cells. Thus, CD90(+) cells are a newly identified additional cell fraction that increased during skeletal muscle regeneration in vivo and could be another cell source for therapy for lama2-deficient muscular dystrophy.     

Gender dimorphism influences extracellular matrix expression and regeneration of muscular tissue in mdx dystrophic mice

Mdx mouse, the animal model of Duchenne muscular dystrophy, lacks dystrophin and develops an X-linked recessive inflammatory myopathy characterized by degeneration of skeletal muscle fibers and connective tissue replacement. The present work aimed to assess whether gender dimorphism in mdx mice would influence skeletal muscle pathology at ages corresponding to main histological changes in the microenvironment of muscular tissue: myonecrosis, regeneration, and fibrosis. At the height of myonecrosis (6 weeks postnatal), skeletal muscles of male mdx mice showed increased sarcolemmal permeability, numerous inflammatory foci, and marked deposition of the extracellular matrix components (ECM) type I collagen and laminin. In contrast, age-matched mdx females showed mild ECM deposition, discrete myonecrosis, but increased numbers of regenerating fibers expressing the satellite cell marker NCAM. In contrast ovariectomized mdx females showed decreased numbers of regenerating fibers. Older (24 and 48 weeks postnatal) mdx females showed extensive fibrosis with increased sarcolemmal permeability and marked deposition of ECM components than corresponding males. These results suggest a role for female hormones in the control of myonecrosis probably by promoting regeneration of muscular tissue and mitigating inflammation especially at ages under the critical influence of sex hormones.     

Ultrastructural changes in the cardiomyopathy of dystrophic hamsters and mice

Changes in the ultrastructure of the cardiac muscle cells have been followed in dystrophic mice and hamsters (22-40 weeks of age) and in both species a severe cardiomyopathy accompanies the cellular damage of the skeletal muscle. The degradative changes of the myofilament apparatus of the heart cells and the specific changes in mitochondrial ultrastructure (including swelling, septation and apparent division) are characteristic of the cellular damage of both the dystrophic skeletal muscle and of normal cardiac muscle in which [Ca]i has been experimentally raised, confirming the suggestions that (i) the same gene is responsible for the myopathy of skeletal and cardiac muscle in animal dystrophy and (ii) that changes in [Ca]i are implicated in the degradative changes of muscle cells.     

The nuclear envelope in muscular dystrophy and cardiovascular diseases

Considerable interest has been focused on the nuclear envelope in recent years following the realization that several human diseases are linked to defects in genes encoding nuclear envelope specific proteins, most notably A-type lamins and emerin. These disorders, described as laminopathies or nuclear envelopathies, include both X-linked and autosomal dominant forms of Emery–Dreifuss muscular dystrophy, dilated cardiomyopathy with conduction system defects, limb girdle muscular dystrophy 1B with atrioventricular conduction disturbances, and Dunnigan-type familial partial lipodystrophy. Certain of these diseases are associated with nuclear structural abnormalities that can be seen in a variety of cells and tissues. These observations clearly demonstrate that A-type lamins in particular play a central role, not only in the maintenance of nuclear envelope integrity but also in the large-scale organization of nuclear architecture. What is not obvious, however, is why defects in nuclear envelope proteins that are found in most adult cell types should give rise to pathologies associated predominantly with skeletal and cardiac muscle and adipocytes. The recognition of these various disorders now raises the novel possibility that the nuclear envelope may have functions that go beyond housekeeping and which impact upon cell-type specific nuclear processes.