Nemaline myopathy with minicores caused by mutation of the CFL2 gene encoding the skeletal muscle actin-binding protein, cofilin-2

Abstract

Nemaline myopathy (NM) is a congenital myopathy characterized by muscle weakness and nemaline bodies in affected myofibers. Five NM genes, all encoding components of the sarcomeric thin filament, are known. We report identification of a sixth gene, CFL2, encoding the actin-binding protein muscle cofilin-2, which is mutated in two siblings with congenital myopathy. The proband's muscle contained characteristic nemaline bodies, as well as occasional fibers with minicores, concentric laminated bodies, and areas of F-actin accumulation. Her affected sister's muscle was reported to exhibit nonspecific myopathic changes. Cofilin-2 levels were significantly lower in the proband's muscle, and the mutant protein was less soluble when expressed in Escherichia coli, suggesting that deficiency of cofilin-2 may result in reduced depolymerization of actin filaments, causing their accumulation in nemaline bodies, minicores, and, possibly, concentric laminated bodies.

Figure  1.

Pathologic and genetic findings in a family with CFL2 mutation A35T. A, Partial pedigree of the family illustrates several consanguineous loops. The proband is indicated by an arrow. The two affected sisters (filled circles) are homozygous for A35T, whereas an unaffected sister, both parents, and several other members of the extended family (half-filled circles) are heterozygous for the change and for a shared haplotype spanning ∼4.6-Mb pairs around the CFL2 gene. Green symbols indicate tested individuals with WT sequence. Light microscopic findings in proband’s muscle include presence of nemaline bodies (B, arrow) on Gomori trichrome staining and occasional minicores (C, arrow) on nicotinamide adenine nucleotide dehydrogenase-tetrazolium reductase staining. Electron microscopy confirmed identity of nemaline bodies (D, arrow), unstructured minicores (E, arrow), and concentric laminated bodies (F, arrow). G, DNA sequence analysis of genomic PCR products illustrating three genotypes for CFL2 c.103G→A seen in the family. H, Schematic representation of cofilin-2. Residue 35 is located next to NLS (30–34 aa); ABD = actin-binding domain. I, Altered alanine residue (red) is evolutionarily conserved among AC proteins (i.e., cofilin-2, cofilin-1, and destrin) across all sequenced vertebrates.

Figure  2.

Fluorescence microscopic analysis revealing α-actinin-2–positive nemaline bodies and actin-filament accumulations. Unaffected control muscle (A–C) and the proband’s muscle (D–I) were immunostained with anti-α-actinin-2 (A and D), anti-skeletal actin (clone 5C5 anti-sarcomeric actin [Sigma A2172]) (G), and phalloidin Alexa Fluor 546 (Invitrogen) (B, E, and H). Merged images, including blue DAPI-stained nuclei, are shown in panels C, F, and I. Several α-actinin–positive nemaline bodies are indicated by arrows in panels D and F, whereas nonoverlapping F-actin accumulations are indicated by arrowheads in panels E–I. Scale bars equal 20 μm (C, F, and I).

Figure  3.

Effects of the A35T mutation on cofilin-2 structure and expression in vivo. A–F, Indirect immunofluorescence analysis of cofilins (green stain [A, C, D, and F]) and skeletal actin (clone 5C5 anti-sarcomeric actin [Sigma A2172]) (red stain [B, C, E, and F]) merged with DAPI for visualization of nuclei (blue stain [C and F]) in muscle from control (A–C) and proband (D–F). Scale bar equals 50 μm. The anti-cofilin-2 polyclonal rabbit antibody (US Biologicals C7506-50) recognizes both sarcomeric cofilin-2 and nonmuscle cofilin-1 (seen in actin-negative connective tissue). Cofilin-2 staining in myofibers is markedly less intense in the proband compared with that in the control, but cofilin-1 staining is preserved in connective tissue and capillaries. G, Two-dimensional gel and Western blot analysis of cofilins in control (c) and proband muscle (pt) performed as described elsewhere. On each gel, 200 μg total muscle lysate proteins were loaded. Equal protein concentrations of lysates were confirmed by immunoblotting parallel SDS-PAGE gels that were stained for glyceraldehyde-3-phosphate dehydrogenase. Isoelectric focusing was accomplished on a pH gradient of 3–10, and immunodetection used an antibody that detects both phosphorylated and unphosphorylated cofilin-1 and cofilin-2 (catalog number C8736 [Sigma]). Identities of the various spots were confirmed using additional isoform-specific and phosphorylation-specific antibodies (not shown). Although unphosphorylated cofilin-1 spots are similar in intensity, both spots for cofilin-2 are significantly smaller in the patient’s muscle. H, A35T mutation, modeled using and Molscript, illustrating side-chain clash of T35 with I55. Residue 35 is in the middle of a β-sheet, with its backbone amide and carbonyl making hydrogen bonds to the backbone carbonyl and amide of I55. In this model, the T35 side-chain hydroxyl forms part of a narrow canyon wall on the molecular surface, which is probably filled with solvent, allowing hydrogen bonds between water molecules and the T35 hydroxyl.

Figure  4.

Differential solubility of WT and mutant cofilin-2. A, SDS-PAGE analysis of cofilin-2 with His/V5 tag expressed in E. coli. The amount of mutant cofilin-2 (arrowhead) in the soluble and purified fractions (lanes 1 and 3) is markedly lower than in the WT (lanes 2 and 4), even though equal amounts of both proteins were present in whole bacterial cell lysates (not shown). In contrast, when 6 M urea is added to the lysis buffer, the amounts of mutant and WT cofilin-2 are similar in both supernatant and purified fractions (lanes 5–8). B, SDS-PAGE analysis of the native cofilin-2 proteins expressed in E. coli without epitope tags. Identity of cofilin-2 was confirmed by western blotting (not shown). As above, A35T protein failed to purify by standard methods (lanes 1 and 2). Analysis of whole bacterial-cell lysates showed roughly equal amounts of A35T and WT protein produced (lanes 3 and 4), but only WT protein was soluble and present in centrifuged supernatants (lanes 5 and 6). Treatment of the insoluble fractions with 6 M urea resulted in recovery and purification of the A35T proteins (lanes 7–10).