Megan J. Puckelwartz

Disruption of nesprin-1 produces an Emery Dreifuss muscular dystrophy-like phenotype in mice

Mutations in the gene encoding the inner nuclear membrane proteins lamins A and C produce cardiac and skeletal muscle dysfunction referred to as Emery Dreifuss muscular dystrophy. Lamins A and C participate in the LINC complex that, along with the nesprin and SUN proteins, LInk the Nucleoskeleton with the Cytoskeleton. Nesprins 1 and 2 are giant spectrin-repeat containing proteins that have large and small forms. The nesprins contain a transmembrane anchor that tethers to the nuclear membrane followed by a short domain that resides within the lumen between the inner and outer nuclear membrane. Nesprins luminal domain binds directly to SUN proteins. We generated mice where the C-terminus of nesprin-1 was deleted. This strategy produced a protein lacking the transmembrane and luminal domains that together are referred to as the KASH domain. Mice homozygous for this mutation exhibit lethality with approximately half dying at or near birth from respiratory failure. Surviving mice display hindlimb weakness and an abnormal gait. With increasing age, kyphoscoliosis, muscle pathology and cardiac conduction defects develop. The protein components of the LINC complex, including mutant nesprin-1alpha, lamin A/C and SUN2, are localized at the nuclear membrane in this model. However, the LINC components do not normally associate since coimmunoprecipitation experiments with SUN2 and nesprin reveal that mutant nesprin-1 protein no longer interacts with SUN2. These findings demonstrate the role of the LINC complex, and nesprin-1, in neuromuscular and cardiac disease.

Mutation of SYNE-1, encoding an essential component of the nuclear lamina, is responsible for autosomal recessive arthrogryposis

Arthrogryposis multiplex congenita (AMC) is a group of disorders characterized by congenital joint contractures caused by reduced fetal movements. AMC has an incidence of 1 in 3000 newborns and is genetically heterogeneous. We describe an autosomal recessive form of myogenic AMC in a large consanguineous family. The disease is characterized by bilateral clubfoot, decreased fetal movements, delay in motor milestones, then progressive motor decline after the first decade. Genome-wide linkage analysis revealed a single locus on chromosome 6q25 with Z(max) = 3.55 at theta = 0.0 and homozygosity of the polymorphic markers at this locus in patients. Homozygous A to G nucleotide substitution of the conserved AG splice acceptor site at the junction of intron 136 and exon 137 of the SYNE-1 gene was found in patients. This mutation results in an aberrant retention of intron 136 of SYNE-1 RNA leading to premature stop codons and the lack of the C-terminal transmembrane domain KASH of nesprin-1, the SYNE-1 gene product. Mice lacking the KASH domain of nesprin-1 display a myopathic phenotype similar to that observed in patients. Altogether, these data strongly suggest that the splice site mutation of SYNE-1 gene found in the family is responsible for AMC. Recent reports have shown that mutations of the SYNE-1 gene might be responsible for autosomal recessive adult onset cerebellar ataxia. These data indicate that mutations of nesprin-1 which interacts with lamin A/C may lead to at least two distinct human disease phenotypes, myopathic or neurological, a feature similar to that found in laminopathies.

Nesprin-1 mutations in human and murine cardiomyopathy

Mutations in LMNA, the gene encoding the nuclear membrane proteins, lamins A and C, produce cardiac and muscle disease. In the heart, these autosomal dominant LMNA mutations lead to cardiomyopathy frequently associated with cardiac conduction system disease. Herein, we describe a patient with the R374H missense variant in nesprin-1alpha, a protein that binds lamin A/C. This individual developed dilated cardiomyopathy requiring cardiac transplantation. Fibroblasts from this individual had increased expression of nesprin-1alpha and lamins A and C, indicating changes in the nuclear membrane complex. We characterized mice lacking the carboxy-terminus of nesprin-1 since this model expresses nesprin-1 without its carboxy-terminal KASH domain. These Delta/DeltaKASH mice have a normally assembled but dysfunctional nuclear membrane complex and provide a model for nesprin-1 mutations. We found that Delta/DeltaKASH mice develop cardiomyopathy with associated cardiac conduction system disease. Older mutant animals were found to have elongated P wave duration, elevated atrial and ventricular effective refractory periods indicating conduction defects in the myocardium, and reduced fractional shortening. Cardiomyocyte nuclei were found to be elongated with reduced heterochromatin in the Delta/DeltaKASH hearts. These findings mirror what has been described from lamin A/C gene mutations and reinforce the importance of an intact nuclear membrane complex for a normally functioning heart.

Population-Based Variation in Cardiomyopathy Genes

Background—Hypertrophic cardiomyopathy and dilated cardiomyopathy arise from mutations in genes encoding sarcomere proteins including MYH7, MYBPC3, and TTN. Genetic diagnosis of cardiomyopathy relies on complete sequencing of the gene coding regions, and most pathogenic variation is rare. The 1000 Genomes Project is an ongoing consortium designed to deliver whole genome sequence information from an ethnically diverse population and, therefore, is a rich source to determine both common and rare genetic variants. Methods and Results—We queried the 1000 Genomes Project database of 1092 individuals for exonic variants within 3 sarcomere genes MHY7, MYBPC3, and TTN. We focused our analysis on protein-altering variation, including nonsynonymous single nucleotide polymorphisms, insertion/deletion polymorphisms, or splice site altering variants. We identified known and predicted pathogenic variation in MYBPC3 and MYH7 at a higher frequency than what would be expected based on the known prevalence of cardiomyopathy. We also found substantial variation, including protein-disrupting sequences, in TTN. Conclusions—Cardiomyopathy is a genetically heterogeneous disorder caused by mutations in multiple genes. The frequency of predicted pathogenic protein-altering variation in cardiomyopathy genes suggests that many of these variants may be insufficient to cause disease on their own but may modify phenotype in a genetically susceptible host. This is suggested by the high prevalence of TTN insertion/deletions in the 1000 Genomes Project cohort. Given the possibility of additional genetic variants that modify the phenotype of a primary driver mutation, broad-based genetic testing should be employed.

Altered chromosomal positioning, compaction, and gene expression with a lamin A/C gene mutation.

Background Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression. Methods/Findings To investigate the hypothesis that mutant lamin A/C changes the laminas ability to interact with chromatin, we studied gene misexpression resulting from the cardiomyopathic LMNA E161K mutation and correlated this with changes in chromosome positioning. We identified clusters of misexpressed genes and examined the nuclear positioning of two such genomic clusters, each harboring genes relevant to striated muscle disease including LMO7 and MBNL2. Both gene clusters were found to be more centrally positioned in LMNA-mutant nuclei. Additionally, these loci were less compacted. In LMNA mutant heart and fibroblasts, we found that chromosome 13 had a disproportionately high fraction of misexpressed genes. Using three-dimensional fluorescence in situ hybridization we found that the entire territory of chromosome 13 was displaced towards the center of the nucleus in LMNA mutant fibroblasts. Additional cardiomyopathic LMNA gene mutations were also shown to have abnormal positioning of chromosome 13, although in the opposite direction. Conclusions These data support a model in which LMNA mutations perturb the intranuclear positioning and compaction of chromosomal domains and provide a mechanism by which gene expression may be altered.

Nesprins, but not sun proteins, switch isoforms at the nuclear envelope during muscle development

Nesprins are a family of nuclear transmembrane proteins anchored via Sun proteins to the nuclear membrane. Analysis of nesprins during human muscle development revealed an increase in nesprin‐1‐giant during early myogenesis in vitro. During the transition from immature to mature muscle fibres in vivo, nesprin‐2 partly replaced nesprin‐1 at the nuclear envelope and short nesprin isoforms became dominant. Sun1 and Sun2 proteins remained unchanged during this fibre maturation. In emerin‐negative skin fibroblasts, nesprin‐2‐giant was relocated from the nuclear envelope to the cytoplasm, not to the endoplasmic reticulum, while nesprin‐1 remained at the nuclear envelope. In emerin‐negative keratinocytes lacking nesprin‐1, nesprin‐2 remained at the nuclear envelope. HeLa cell nuclear envelopes lacked nesprin‐1, which was the dominant form in myoblasts, while a novel 130‐kD nesprin‐2 isoform dominated Ntera‐2 cells. The results suggest the possibility of isoform‐specific and tissue‐specific roles for nesprins in nuclear positioning. Developmental Dynamics 239:998–1009, 2010.

The Genetic Landscape of Cardiomyopathy and Its Role in Heart Failure

Heart failure is highly influenced by heritability, and nearly 100 genes link to familial cardiomyopathy. Despite the marked genetic diversity that underlies these complex cardiovascular phenotypes, several key genes and pathways have emerged. Hypertrophic cardiomyopathy is characterized by increased contractility and a greater energetic cost of cardiac output. Dilated cardiomyopathy is often triggered by mutations that disrupt the giant protein titin. The energetic consequences of these mutations offer molecular targets and opportunities for new drug development and gene correction therapies.

Targeted Analysis of Whole Genome Sequence Data to Diagnose Genetic Cardiomyopathy

Background—Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of >50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated

Gene expression, chromosome position and lamin A/C mutations

1000 genome, it is expected that genetic testing will shift toward comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy. Methods and Results—Whole genome sequencing with an average of 37× coverage was combined with targeted analysis focused on 204 genes linked to cardiomyopathy. Genetic variants were scored using multiple prediction algorithms combined with frequency data from public databases. This pipeline yielded 1 to 14 potentially pathogenic variants per individual. Variants were further analyzed using clinical criteria and segregation analysis, where available. Three of 3 previously identified primary mutations were detected by this analysis. In 6 subjects for whom the primary mutation was previously unknown, we identified mutations that segregated with disease, had clinical correlates, and had additional pathological correlation to provide evidence for causality. For 2 subjects with previously known primary mutations, we identified additional variants that may act as modifiers of disease severity. In total, we identified the likely pathological mutation in 9 of 11 (82%) subjects. Conclusions—These pilot data demonstrate that ≈30 to 40× coverage whole genome sequencing combined with targeted analysis is feasible and sensitive to identify rare variants in cardiomyopathy-associated genes.

Supercomputing for the parallelization of whole genome analysis.

The nuclear lamina is increasingly being appreciated for its epigenetic role in regulating gene expression. The nuclear lamina underlies the inner nuclear membrane and, in post mitotic cells, is composed of a latticework primarily formed by the intermediate filament protein, lamin A/C. Although not well defined, lamin-associated domains have been described, and these domains are determined by DNA sequence and chromatin conformation. Lamin-associated domains are positioned to mediate the interaction with the nuclear membrane, where they contribute to transcriptional regulation. Although lamin-associated domains are primarily considered to be repressive in nature, those nearer to nuclear pores may actually promote transcription. Mutations in LMNA, the gene encoding lamins A and C, are a relatively common cause of inherited cardiomyopathy. As substantial data supports a role for the lamina in its interaction with chromatin and gene regulation, we examined the role of a genetically disrupted lamina and the consequences thereof. A dominant LMNA mutation, E161K, that causes inherited cardiomyopathy was studied. Gene expression changes were profiled in a human cardiomyopathic E161K heart, and it was found that chromosome 13 had a high percentage of misexpressed genes. Chromosome 13 was also found to be less tightly associated with the nuclear membrane in E161K mutant cells, thereby linking abnormal gene expression and intranuclear position. These and other studies support a role for the nuclear membrane as an active regulator of gene expression and provide additional support that disrupting this regulation is a mechanism of human disease.