Recurring homozygous ACTN2 variant (p. Arg506Gly ) causes a recessive myopathy

Figures

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  Figure 1. Pedigrees and clinical presentation of patients with biallelic ACTN2 variants. (A) Pedigrees of the five families. Circles indicate female, squares indicate male, clinically affected relatives are filled black, and unaffected relatives are unfilled white. (+) indicates presence of, (−) indicates absence of the c.1516A>G; (p.Arg506Gly) ACTN2 variant. (B) Visualization of muscle weakness using MuscleViz (https://muscleviz.github.io), based on the Medical Research Council (MRC) scores. (C) Patient F1P1 showing atrophy of the lower extremity muscles, with distal muscles more affected.

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  Figure 2. Muscle MRI imaging in patients with biallelic ACTN2 variants. Lower extremity T1 axial images in patients F1P1 (age 44 years), F2P2 (age 53 years), and F3P5 (age 61 years) reveal severe atrophy and T1 hyperintensity in the thigh muscles with almost complete replacement of the posterior compartment (hamstring muscles) and medial compartment (adductors) with fibroadipose tissue. There is also notable asymmetric involvement of the anterior thigh (quadriceps) muscles, with patchy abnormal signal (right > left) in patient F1P1 and (left > right) in patient F3P5. In the lower leg muscles, selective involvement of the soleus muscle is seen in all three patients. There is evidence of strikingly asymmetric involvement of the medial gastrocnemius, with fibroadipose tissue replacement on the right and relative sparing on the left (in F1P1 and F3P5). Similarly, the tibialis anterior muscle demonstrates strikingly asymmetric involvement, with severe involvement on the right and relative sparing on the left (in F2P2 and F3P5). Also of note is evidence of STIR positivity in muscles with normal T1 signal, as demonstrated in left tibialis anterior muscle of F1P1 and the medial and lateral…

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  Figure 3. Histopathological findings in patients with biallelic ACTN2 variants. (A) Hematoxylin and eosin (H&E) staining of the left quadriceps muscle biopsy obtained at 41 years of age from patient F2P3 shows myofiber atrophy and fiber size variability. (B) Gömöri trichrome (GT) staining highlights similar findings with variation in fiber size and internalized nuclei (black arrow). (C) Nicotinamide adenine dinucleotide (NADH) staining shows disruption of intermyofibrillar architecture with lobulated fibers (black arrow) and ring fibers (red arrow). (D) Cytochrome oxidase (COX) staining shows a similar lobulated appearance of fibers (black arrow). (E) ATPase (pH 9.4) reveals increase in Type 1 (pale) fibers which are atrophic (F) ATPase (pH 4.35) highlighting similar findings with Type 1 fiber (dark) predominance and selective Type 1 fiber atrophy. (G,H) Electron microscopy of muscle for patient F4P6 showing subsarcolemmal accumulation of mitochondria, and (I) normal appearing Z‐discs.

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  Figure 4. Pathogenic ACTN2 (NM_001103.2) skeletal muscle disease variants. (A) Schematic representation of ACTN2 with corresponding domains. Both recessive (top) and dominant (bottom) pathogenic variants associated with skeletal muscle disease are listed. (B) Alignment of ACTN2 showing that R506 impacts a conserved residue.

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  Figure 5. In vitro splicing analysis of ACTN2 c.1516A>G. (A) A schematic illustration of the pET01‐ACTN2 c.1516A>G Exontrap assay. Wild‐type (WT) or mutant (Mut) ACTN2 c.1516A>G exon 14 (yellow) and flanking introns (blue) were cloned into the XhoI and BamHI sites located in the vector's multiple cloning site (orange) between two pET01 exons (blue). Minigenes were transfected into HEK293T cells and RNA was isolated 24 hours later. (B) Agarose gel electrophoresis of reverse transcription PCR (RT‐PCR) products amplified using pET01 primers (red arrows). Composition and size of the amplicons is exemplified on the right: Empty pET01 vector (113 pb) and wild‐type (WT) and ACTN2 c.1516A>G (Mut) variant (254 bp). (C) Sanger sequencing chromatograms of the minigene splicing assay products. Wild‐type (top); Mutant c.1516A>G (bottom).