Protein-extending ACTN2 frameshift variants cause variable myopathy phenotypes by protein aggregation

Abstract

Objective: The objective of the study is to characterize the pathomechanisms underlying actininopathies. Distal myopathies are a group of rare, inherited muscular disorders characterized by progressive loss of muscle fibers that begin in the distal parts of arms and legs. Recently, variants in a new disease gene, ACTN2, have been shown to cause distal myopathy. ACTN2, a gene previously only associated with cardiomyopathies, encodes alpha-actinin-2, a protein expressed in both cardiac and skeletal sarcomeres. The primary function of alpha-actinin-2 is to link actin and titin to the sarcomere Z-disk. New ACTN2 variants are continuously discovered; however, the clinical significance of many variants remains unknown. Thus, lack of clear genotype-phenotype correlations in ACTN2-related diseases, actininopathies, persists.

Methods: Functional characterization in C2C12 cell model of several ACTN2 variants is conducted, including frameshift and missense variants associated with dominant and recessive actininopathies. We assess the genotype-phenotype correlations of actininopathies using clinical data from several patients carrying these variants.

Results: The results show that the missense variants associated with a recessive form of actininopathy do not cause detectable alpha-actinin-2 aggregates in the cell model. Conversely, dominant frameshift variants causing a protein extension do form alpha-actinin-2 aggregates.

Interpretation: The results suggest that alpha-actinin-2 aggregation is the disease mechanism underlying some dominant actininopathies, and thus, we recommend that protein-extending frameshift variants in ACTN2 should be classified as pathogenic. However, this mechanism is likely elicited by only a limited number of variants. Alternative functional characterization methods should be explored to further investigate other molecular mechanisms underlying actininopathies.

Figure 1

(A–C) Pedigrees of reported families. Red coloring denotes strictly cardiac phenotype without skeletal muscle involvement; black coloring denotes skeletal muscle phenotype with or without cardiac involvement. Genotyped individuals are indicated by an asterisk.

Figure 2

(A–D) Muscle MRI studies of family B. (A) Foreleg of patient V‐1 shows severe fatty atrophy of the anterior tibialis, peroneus, and medial head of the gastrocnemius; moderate fatty atrophy of the soleus; and mild fatty atrophy of the distal portion of the posterior tibialis. (B) Foreleg for patient V‐2 shows diffuse bilateral muscle atrophy, mild fatty atrophy of the vastus medialis, and mild subcutaneous edema along the anterolateral aspect of the lower legs without organized fluid collection or mass. (C) Thigh of patient V‐1 shows moderate‐to‐severe fatty atrophy of the vastus intermedius muscle; and mild fatty atrophy of the proximal vastus lateralis, tensor fascia lata, sartorius, biceps femoris, semimembranosus, and gracilis. (D) Thigh of patient V‐2 shows diffuse bilateral muscle atrophy. (E–L) Muscle histopathology of patient V‐1 of family B. (E) Increased endomysial fatty infiltration, variation in myofiber size, and internalized nuclei (H&E). (F) Rimmed vacuoles (arrow) (Gomori trichrome). (G) Cores (NADH‐TR). (H) Increased desmin‐reactivity. (I and J) Predominance and mild atrophy of type 1 myofibers (myosin slow and fast). (K and L) Focal filamentous disruption of sarcomeres (electron microscopy).

Figure 3

Alpha‐actinin‐2 localization in C2C12 myotubes stably expressing WT or mutant eGFP‐ACTN2 (green). Nuclei are stained with Hoechst (blue). Scale bar is 20 μm.

Figure 4

(A) Soluble and insoluble protein fraction per variant with western blot probing for eGFP‐alpha‐actinin‐2. (B) Quantification of eGFP‐alpha‐actinin‐2 in the soluble versus insoluble protein fraction, shown as soluble over insoluble ratio. °mild outlier, *extreme outlier, bars denote minimum and maximum non‐outlier values. n = 3 biological replicates.