Phenotypic Diversity of the LMNA Mutations

The LMNA gene, encoding the Lamin A (LMNA) protein, is a fascinating gene and likely unique in the human genome for the diversity of its phenotypic expression caused by the mutations (mutation is defined in a clinical term as a pathogenic variant whose causality in the phenotype is well-established). At least a dozen diseases have been linked to mutations in the LMNA gene, which are collectively referred to as laminopathies (Table 1). Cardiac involvement, typically characterized by a composite phenotype of dilated cardiomyopathy (DCM), conduction defects, bradyarrhythmia, atrial fibrillation, and ventricular arrhythmias predominates in a subset of laminopathies that mainly involve the striated muscles. (1) Given the compound nature of cardiac phenotype in laminopathies, the disease is best identified with the gene name and referred to as cardiomyopathy, i.e., LMNA-cardiomyopathy, rather than DCM alone. (2)

Table I.

Phenotypic Spectrum of Mutations in the LMNA Gene

Primary organ involved	Disease	Phenotypic features	Cardiac involvement
Systemic
	Hutchinson-Gilford Progeria Syndrome (HGPS)	Premature aging, short stature, loss of hair, joint immobility, premature myocardial infarction, and heart failure	Coronary calcification, myocardial infarction, ischemic heart failure, conduction defects, and arrhythmias
	Progeroid syndromes	Premature aging, similar to HGPS but milder	Atherosclerosis, ischemic heart failure, conduction defects, and valvular disease
Cardiovascular	Dilated cardiomyopathy (DCM)	4-chamber dilatation Bi-ventricular dysfunction Conduction defect, including left bundle branch block and brady arrhythmia	Main phenotype
	Arrhythmogenic cardiomyopathy (ACM)	Fibro-fatty infiltration of the right ventricle	Isolated or in conjunction with Charcot-Marie Tooth disease
	Hypertrophic cardiomyopathy (HCM)	No specific feature	Isolated case, unclear role in HCM
	Cardiac conduction defect	Sinus pause, sinus bradycardia, and atrioventricular block	usually in conjunction with DCM
	Atrial fibrillation	Slow atrioventricular conduction	Usually in conjunction with bradyarrhythmia and DCM
	Cardiac progeria	Involvement of multiple components of the heart	Rare case reports. Premature MI, conduction defects, arrhythmias, and valvular degeneration
Skeletal muscle	Emery-Dreifuss muscular dystrophy	Autosomal dominant and recessive forms, muscle wasting, and contracture	DCM, bradyarrhythmia, conduction defect
	Congenital muscular dystrophy	Severe and early onset muscular dystrophy	DCM and conduction defect
	Limb-girdle muscular dystrophy 1B	Muscle wasting and weakness of proximal hip and shoulder muscles	DCM and bradyarrhythmias
Peripheral nerves	Charcot-Marie-Tooth disease, type 2B1	Motor and sensory neuropathies, muscle wasting	Isolated and rarely in conjunction with ACM
Bone	Mandibuloacral dysplasia	Typically presents with lipodystrophy, Growth retardation, Mandibular hypoplasia, and other skeletal abnormalities	Not a feature
Adipose tissue	Familial partial lipodystrophy, Dunnigan type		Cardiomyopathy and myocardial infarction
	Insulin resistance	In conjunction with lipodystrophy	Not reported
Skin	Restrictive dermopathy	Tight and rigid skin, developmental abnormalities	Not a feature (likely because of premature death)
Syndromic	Heart-hand syndrome	Cardiomyopathy, conduction defect, and brachydactyly	DCM, conduction defects, and arrhythmias
	Hypergonadotropic hypogonadism syndrome	Often in conjunction with dilated cardiomyopathy	DCM (Malouf syndrome)

Cardiac involvement is also common in the subset of laminopathies wherein the main phenotype is premature aging, such as Hutchinson Gilford Progeria Syndrome (HGPS) and progeroid syndromes. It manifests as premature myocardial infarction and ischemic heart failure, the former resulting from coronary calcification and atherosclerosis, and the latter, a consequence of multiple bouts of myocardial cell death. (3) In addition to involving the myocardium, conduction system, and vasculature, mutations causing progeroid syndrome also cause premature valvular disease. (4, 5) Given the above, one might denote cardiac involvement as the core phenotype in laminopathies, manifesting either as the primary feature or occurring later in life following the development of disease in other organs.

The molecular basis of the phenotypic diversity of the LMNA mutations is largely unknown and subject to various suppositions. Ab initio, it is important to recognize that LMNA is a diffusely expressed gene with a low tissue and cell type specificity, albeit its expression levels differ among various cell types. (6) Given the ubiquitous expression of the LMNA, the involvement of various organs by the LMNA mutations is not surprising. What is perplexing, however, is the predominance involvement of one organ vs. others in laminopathies, despite the pervasive expression of the LMNA gene, even though cardiac involvement, by and large, is a common feature in various laminopathies. To explain such phenotypic diversity, the focus from the outset has been on the topography of the mutations, through genotype-phenotype correlation studies, determining the association of each subset of laminopathies with the topography of the mutation. The approach was based on the hypothesis that mutations in different domains of the LMNA gene would lead to the preferential involvement of certain organs. However, the association between the topography of the mutation and the differential expression of the phenotypes in laminopathies is less than compelling. It is mainly restricted to a subset of laminopathies such as HGPS whereby the mutations impair the maturation of the pre-LMNA to LMNA, which occurs through post-translational modifications and subsequent cleavage of the pre-lamin A to mature LMNA. (7, 8) The hypothesis is not without merit and is based on the principle that the mutations primarily impact the structure and organization of the LMNA protein, and consequently its interactions with the binding partners, which affect the LMNA biological functions.

The biological functions of the LMNA protein are mainly dictated by its localization on the inner side of the nuclear membrane in proximity to chromatin, where it contributes to the spatial organization of the genome. LMNA interacts with several hundred proteins, including those involved in maintaining chromatin and genomic integrity, structure, and function (https://thebiogrid.org/110186/summary/homo-sapiens/lmna.html). Notable among the LMNA interactomes is retinoblastoma 1 (RB1), which is the classic binding partner of the LMNA protein. (9) RB1 is a suppressor of a subset of E2F transcription factors (E2F1, E2F2, E2F3a) that activate gene expression. (9) LMNA binds to and stabilizes RB1 and maintains its suppressive effects on the activator E2F2 transcription factors. In laminopathies, impaired binding of the LMNA to RB1 leads to hyperphosphorylation and degradation of RB1 by the proteasome leaving the activator E2F transcription factors unchecked. The E2F2s, freed from the suppressive effects of the LMNA in laminopathies, induce the expression of genes involved in cell cycle progression and cell proliferation. (10)

LMNA also regulates gene expression through interactions with several transcriptional regulators, such as AT hook containing transcription factor 1 (AHCTF1), which is a regulator of nuclear pore complex assembly and cytokinesis; sterol regulatory element binding transcription factor 1 (SREBF1), which regulates cholesterol homeostasis; and TEA domain family member 1 (TEAD1), which is a regulator of cellular differentiation and proliferation. (11–13) Thus the LMNA through interactions with the transcriptional regulators influences the expression of diverse arrays of genes involved in various biological processes. Given that the expression and transcriptional activities of these interactomes are differential among various cell types, mutations, by affecting these interactions, could induce cell type and organ dominant gene expression and hence, clinical phenotypes. Similarly, several transcriptional regulators of gene expression are also dysregulated in laminopathies, including the Forkhead box O (FOXO), bromodomain-containing protein 4 (BRD4), lysin demethylase 5 (KDM5) family, and mammalian target of rapamycin (mTORC1). (14–17) These transcriptional regulators are involved in a multitude of biological functions ranging from metabolism to autophagy. Consequently, their dysregulation, which occurs differentially in various cell types would impart preferential effects in certain cell types and tissues contributing to the phenotypic expression of laminopathies. Moreover, the mutations could affect post-translational modifications of the LMNA, such as phosphorylation, ubiquitination, and neddylation, and therefore, the LMNA biological functions and the phenotypic diversity of laminopathies. (18)

Altered protein-protein interactions in the presence of the LMNA mutations are also expected to affect interactions of the LMNA with chromatin through lamin-associated domains (LADs). The latter comprise approximately 20 to 30% of the genome in cardiac myocytes and include invariant and variant LADs. (19). The former is constitutive and to some extent cell type autonomous and the latter is facultative cell type-dependent that shifts in response to internal and external stressors, including mutations. (19–21) The LADs are mainly located at the heterochromatin and gene-poor regions and are associated with increased CpG methylation and suppressed gene expression. (21) Mutations by altering the LMNA interactions with the chromatin lead to genome-wide reorganization of LADs, resulting in the gain and loss- of LADs. (21) The gain of LADs suppresses gene expression whereas the loss of LADs has the opposite effect. (21) Because the facultative LADs are cell-type dependent, the mutations would affect the genome-wide reorganization of the facultative LADs and consequently gene expression in a cell-type-specific manner. Thus, it is plausible to postulate that the phenotypic diversity of laminopathies is in part because of the differential effects of the mutations on the interactions of the LMNA with chromatin, re-distribution of LADs, and consequently cell type-dependent gene expression. According to this postulate, the phenotype will predominate in the organ wherein the redistribution of LADs and consequently, gene expression are affected the most.

LMNA is a guardian of genomic stability, likely through suppression of transcription, either directly through LADs or through its interactions with suppressor proteins such as RB1 and tumor protein 53 (TP53). (9, 11) Through this mechanism, LMNA mitigates the generation of transcription-coupled DNA double-stranded breaks (DSBs), as indicated by an increased number of DSBs in the LMNA-deficient cardiac myocytes. (20) DSBs are the most dangerous forms of DNA lesions, as their repair occurs mainly through the cell cycle independent mechanism of non-homologous end joining (NHEJ), particularly in cells with low or minimal replicative capacity such as the post-natal cardiac myocytes. (22, 23) The process is mutagenic as the repair through NHEJ introduces insertions and deletions at the break sites. The DSBs activate the DNA damage response (DDR) pathways, including the ataxia-telangiectasia mutated (ATM)-TP53 and the cytosolic DNA-sensing protein (CDSP) pathways, the latter upon the release of the damaged nuclear DNA (or mitochondrial DNA) to the cytosol. (16, 24) Whereas the activation of the ATM-TP53 pathway shuts down the cell cycle progression, the activation of the CDSPs induces an innate immune response through sensing of the cytosolic DNA by Cyclic GMP-AMP synthase (CGAS) and other cytosolic DNA sensors. (25) Downstream to CGAS is the stimulator of interferon response cGAMP interactor 1 (STING1), which provides a platform for autophosphorylation and activation of TANK-binding kinase 1 (TBK1) proteins. The latter activates interferon regulatory factor 3 (IRF3) upon phosphorylation and inactivates the inhibitors of the nuclear factor kappa B (NFκB). The net effects are increased expression of the interferon-responsive genes and the pro-inflammatory cytokines. (23, 25) Because the LMNA also regulates mitochondrial function, the release of mitochondrial DNA from the damaged mitochondria, in addition to the nuclear DNA, also activates the CDSP pathway and contributes to the pro-inflammatory phenotype in the laminopathies. (26, 27)

In addition to its role in protection from DNA breaks, the LMNA is also involved in the repair of damaged DNA. LMNA interacts with several key proteins involved in the repair of DBs, including poly (ADP-ribose) polymerase 1 (PARP1), X-Ray Repair Cross Complementing 6 (XRCC6, aka Ku70), TP53 binding protein 1 (TP53BP1), replication protein A1 (RPA1), and histone H2A.X Variant. (11, 28) Thus, in laminopathies, the repair mechanisms are likely to be impaired. Given the role of the LMNA in genomic stability and the repair of the damaged DNA, it is likely that the activation of the DDR including the CDSP pathway, secondary to the release of nuclear and/or mitochondrial DNA into the cytosol, to be a common mechanism in the induction of the pro-inflammatory phenotype observed in laminopathies. However, whether different mutations differentially activate the DDR pathways or impede the repair mechanisms in different cell types and hence, contribute to the predominant involvement of certain cells and organs in laminopathies is an empiric question that remains to be studied.

LMNA, as a nuclear inner membrane protein has a major function in the stability of the nuclear membrane as indicated by the distortion and blebbing of the nuclear membrane, which is a classic finding, in laminopathies. (29) Impaired mechanical stability of the nuclear membrane not only contributes to the extrusion of the chromatin fragments containing nuclear DNA into the cytoplasm and consequent activation of the CDSP pathway, but also de-stabilizes microtubules and linker between the cytoskeleton and nucleoplasm complex (LINC) leading to the activation of mechano-sensing pathways, including the Hippo, canonical WNT, mitogen-activated protein kinase (MAPK), mTORC1, and transforming growth factor β (TGFβ) pathways, among others. (30) The activated signaling pathways have been targeted for therapeutic gains in laminopathies. However, given the multifarity of the involved signaling and other pathways in the pathogenesis of laminopathies, targeting a single pathway with an undefined effect size may not be effective. A recent phase 3 clinical trial using a selective oral p38α MAPK inhibitor in patients with symptomatic LMNA-cardiomyopathy was discontinued because of the lack of effectiveness. (31) The null effect of targeting a single pathway, even if it is genuinely dysregulated in laminopathy, emphasizes the presence of multiple pathogenic pathways, which renders the effect size of each dysregulated pathway relatively modest and difficult to discern or target for therapeutic gain.

In conclusion, the phenotypic diversity of the mutations in the LMNA gene is unique among the genetic disorders. At the core of the involved mechanisms, to explain the phenotypic diversity, are the differential effects of the mutations on the interactions of the LMNA protein with its binding partners. These interactions are expected to be partly cell-type dependent because of the differential expression of the interactome among various cell types. The multifarious differential interactome would activate various secondary mechanisms, such as chromatin remodeling, gain- and loss-of LADs, signaling pathways, and transcriptional regulation, differentially among various cell types leading to differential gene expression among various cell types. The latter would lead to the predominance of the clinical phenotype in a specific cell type and organ caused by a diverse array of LMNA mutations. A subset of mechanisms, however, such as the activation of the DDR pathways, are expected to be common in various forms of laminopathies and therefore, might be desirable targets of interventions. Other mechanisms would be expected to be cell-type and tissue-specific, requiring in-depth characterization of the pathogenic pathways in various cell types to harvest the knowledge gained for therapeutic targeting of laminopathies.