Abstract
Hundreds of LMNA variants have been associated with several distinct disease phenotypes. However, genotype–phenotype relationships remain largely undefined and the impact for most variants remains unknown. We performed a functional analysis for 178 variants across five structural domains using two different overexpression models. We found that lamin A aggregation is a major determinant for skeletal and cardiac laminopathies. An in vitro solubility assay shows that aggregation-prone variants in the immunoglobulin-like domain correlate with domain destabilization. Finally, we demonstrate that myopathic-associated LMNA variants show aggregation patterns in induced pluripotent stem cell derived-cardiomyocytes (iPSC-CMs) in contrast to non-myopathic LMNA variants. Our data-driven approach (1) reveals that striated muscle laminopathies are predominantly protein misfolding diseases, (2) demonstrates an iPSC-CM experimental platform for characterizing laminopathic variants in human cardiomyocytes, and (3) supports a functional assay to aid in assessing pathogenicity for myopathic variants of uncertain significance.
Highlights
The nuclear lamina is composed of the intermediate filament protein lamins A/C, B1 and B2 encoded by LMNA, LMNB1, and LMNB2, respectively[1]
Lamins have a short N-terminal domain, long rod domain consisting of four coiled-coil domains (CCDs) (1A, 1B, 2A, and 2B)[8], and a C-terminal protein interaction rich immunoglobulin-like domain (IgD)[9], which polymerize to form intermediate filaments in the nucleus
We studied aggregation because (1) misfolding and aggregation are dominant mechanisms underlying inherited diseases and may help reveal genotype–phenotype relationships[17], (2) aggregation of several LMNA variants has been reported in a variety of model systems[10,14,15,18,19,20], and (3) it could serve as a relatively simple functional test to support pathogenic classification as defined by the American College of Medical Genetics (ACMG) guidelines for variant classification[21]
Summary
The nuclear lamina is composed of the intermediate filament protein lamins A/C, B1 and B2 encoded by LMNA, LMNB1, and LMNB2, respectively[1]. Nuclear membrane abnormalities including “blebs”, “honeycombs”, and lamin A/C foci are a hallmark of all classes of laminopathies[14,15,16] Despite these mechanisms, ascribing LMNA variants to tissuespecific phenotypes is a challenge. Validating aggregation as a major determinant of striated muscle Strikingly, 53/70 (76%) of skeletal and 27/60 (45%) of cardiac laminopathies These results (1) are a valuable resource for further disease-associated variants formed aggregates in either HEK 293 studies investigating laminopathies, (2) demonstrate an in vitro cells or C2C12 myoblasts. 1/7 (14%) of lipodystrophy functional assay to assist in classifying LMNA VUS associated with and 2/13 (15%) of progeria variants aggregated but all had striated muscle disease, (3) establish a human cardiac cell model crossover cardiac or skeletal muscle disease (e.g., R60G, A57P, for studying cardiac laminopathies, and (4) point to lamin A L59R).
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