Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy
Michaela Yuen, et al.
Michaela Yuen, et al.
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Research Article

Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy

Abstract

Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.

Authors

Michaela Yuen, Sarah A. Sandaradura, James J. Dowling, Alla S. Kostyukova, Natalia Moroz, Kate G. Quinlan, Vilma-Lotta Lehtokari, Gianina Ravenscroft, Emily J. Todd, Ozge Ceyhan-Birsoy, David S. Gokhin, Jérome Maluenda, Monkol Lek, Flora Nolent, Christopher T. Pappas, Stefanie M. Novak, Adele D’Amico, Edoardo Malfatti, Brett P. Thomas, Stacey B. Gabriel, Namrata Gupta, Mark J. Daly, Biljana Ilkovski, Peter J. Houweling, Ann E. Davidson, Lindsay C. Swanson, Catherine A. Brownstein, Vandana A. Gupta, Livija Medne, Patrick Shannon, Nicole Martin, David P. Bick, Anders Flisberg, Eva Holmberg, Peter Van den Bergh, Pablo Lapunzina, Leigh B. Waddell, Darcée D. Sloboda, Enrico Bertini, David Chitayat, William R. Telfer, Annie Laquerrière, Carol C. Gregorio, Coen A.C. Ottenheijm, Carsten G. Bönnemann, Katarina Pelin, Alan H. Beggs, Yukiko K. Hayashi, Norma B. Romero, Nigel G. Laing, Ichizo Nishino, Carina Wallgren-Pettersson, Judith Melki, Velia M. Fowler, Daniel G. MacArthur, Kathryn N. North, Nigel F. Clarke

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Figure 2

Most LMOD3 mutations abolish LMOD3 protein expression.

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Most LMOD3 mutations abolish LMOD3 protein expression. Western blots ass...
Western blots assessing LMOD3 expression in available (A) patient muscle tissue and (B) patient primary muscle cells. (A) In most patients with nonsense or frame-shift mutations, truncated protein of the predicted size were not detected. In some biopsies, replacement of muscle tissue by connective tissue may limit our ability to detect a muscle-specific truncated product (e.g., lanes 4, 10, 13, and 14; expression of sarcomeric actin and α-actinin-2 is low relative to that of GAPDH). Muscle from patient 14a shows expression of truncated protein from both mutant LMOD3 alleles (red arrow, mutations in red in Figure 1). The approximately 50-kDa band (lanes 4, 10, and 14 and weakly in controls) does not correlate with the predicted molecular weight of mutant LMOD3 in these patients (12–43 kDa). This band likely arises from Ab cross-reactivity with a nonmuscle LMOD isoform or an unrelated nonmuscle antigen. (B) Primary myoblasts were differentiated into myotubes, and primary fibroblasts were MyoD-converted into differentiated myotubes in culture. Primary cells from controls express LMOD3 (~80-kDa band) and other thin filament markers (sarcomeric actin and α-actinin-2) after 5 days (d5) of differentiation (lanes 6 and 8). Myogenic conversion of fibroblasts from patient 14a induces expression of mutant LMOD3 from both alleles (lane 10; R401*, red arrow). Primary myotubes and MyoD-converted fibroblasts from patient 12c (homozygous p.N367Qfs*11) express no mutant protein (lanes 12, 13, and 15). These results replicate LMOD3 expression obtained in patient and control muscle biopsies. L, leg muscle; P, paraspinalis muscle; Q, quadriceps; T, triceps.