A novel frameshift ACTN2 variant causes a rare adult‐onset distal myopathy with multi‐minicores

Abstract Introduction Distal myopathies are a group of rare muscle disorders characterized by selective or predominant weakness in the feet and/or hands. In 2019, ACTN2 gene was firstly identified to be a cause of a new adult‐onset distal muscular dystrophy calling actininopathy and another distinctly different myopathy, named multiple structured core disease (MsCD). Thus, the various phenotypes and limited mutations in ACTN2‐related myopathy make the genotype‐phenotype correlation hard to understand. Aims To investigate the clinical features and histological findings in a Chinese family with distal myopathy. Whole exome sequencing and several functional studies were performed to explore the pathogenesis of the disease. Results We firstly identified a novel frameshift variant (c.2504delT, p.Phe835Serfs*66) within ACTN2 in a family including three patients. The patients exhibited adult‐onset distal myopathy with multi‐minicores, which, interestingly, was more like a combination of MsCD and actininopathy. Moreover, functional analysis using muscle samples revealed that the variant significantly increased the expression level of α‐actinin‐2 and resulted in abnormal Z‐line organization of muscle fiber. Vitro studies suggested aggregate formations might be involved in the pathogenesis of the disease. Conclusion Our results expanded the phenotypes of ACTN2‐related myopathy and provided helpful information to clarify the molecular mechanisms.

structured core disease (MsCD) in congenital myopathy and adultonset distal muscular dystrophy calling actininopathy were reported caused by ACTN2 mutations. 15,16 Although myofibrillar features are the most common characteristics in various distal myopathies caused by genes encode Z-disk or Z-disk relevant protein, it seems that no aggregates are found in muscle biopsy of ACTN2-related myopathy. Instead, in MsCD, multiple structured cores are the main histological findings. Core pathology (central cores and multiple minicores) is rare in adult-onset distal myopathy. It has been reported that multicore-like unevenness of stain could occasionally occur in patients with distal myopathy caused by RYR1 mutations. 17,18 Herein, we firstly identified a novel ACTN2 variant causing an adult-onset distal myopathy with a morphological feature of multiple well-defined minicores, which, interestingly, was more like a combination of MsCD and actininopathy. Further, we investigated the functional characterization of this mutation, together with the previously reported mutations in ACTN2-related myopathy, providing insightful information to clarify the molecular mechanisms underlying. Written informed consents were obtained from all the participants.

| Genetic analysis
Genomic DNA was extracted from peripheral EDTA-treated blood using a commercial blood genomic extraction kit (Qiagen, Hilden).

| Immunofluorescence analysis
Frozen muscle sections were fixed at room temperature in acetone for 10 min and washed in PBS. Nonspecific sites were blocked in PBS with 1% BSA and 0.01% Triton X-100 for 1 h and incubated overnight at 4°C with rabbit anti-ACTN2 (A8939; Abclonal). After washing, the sections were incubated 1h with the secondary antibody: Alexa Fluor 594 goat anti-rabbit antibodies at room temperature.
Cell nuclei were then stained with 40, 6-diamidino-2-phenylindole (DAPI; Life Technologies). For C2C12 cell lines, after 24 h transfection with eGFP-tagged ACTN2, cells were fixed at room temperature in 4% PFA for 10 min. DAPI was used for the staining of cell nuclei.
Fluorescence images were captured by Olympus FV3000 OSR confocal system. Cell Signaling Technology).

| Statistical analysis
GraphPad Prism 8.0 and Adobe Photoshop CC 2017 software were used for data analysis. Data were first tested by Shapiro-Wilk test for normality and lognormality. All the experiments were repeated at least three times independently, and data were expressed as the mean ±standard deviation. One-way ANOVA test was performed followed by Turkey's multiple comparisons test for multiple comparisons. p value <0.05 was considered statistically significant.

| Clinical features
The proband of the family ( Figure 1A) is a 46-year-old male who

| Muscle pathology
Q uadriceps muscle biopsy was performed in the proband. H&E staining revealed fiber size variability with remarkable internal nuclei.
Oxidative enzyme reactions uncovered several small well-delimited cores, predominantly in type I fibers. ATPase reaction showed both type I and type II fibers-grouping ( Figure 1G). No necrotic fibers were observed, and no aggregates or nemaline rods were found in the histopathologic staining.

| Genetic analysis and the spectrum of ACTN2 mutations
After To date, a lot of heterozygous pathogenic variants have been reported related to cardiomyopathies and/or other cardiac abnormalities, while, only 4 variants were associated with skeletal myopathies (HGMD https://my.qiage ndigi talin sights.com/bbp/view/hgmd/pro/ start.php). These variants were widely distributed, and most were found in the SRs domain, followed by the ABD domain ( Figure 2C).

| The analysis of α-actinin-2 expression and distribution
To assess the impacts on the α-actinin-2 expression by the identified variant, we performed Western blot analysis of the muscle extracts obtained from the proband and two controls with normal pathology. The results revealed significantly increased level of α-actinin-2 and α-actinin in the proband compared with the controls (Figure 3A-B). Moreover, immunofluorescence analysis on muscle frozen sections showed a structural turbulence of α-actinin-2 staining in the proband compared with the control who exhibited a highly ordered structure ( Figure 3C), indicating the abnormal Z-disk organization.

| Protein subcellular localization
To further elucidate the biological effects, the plasmids with N-

| DISCUSS ION
In this study, we firstly reported a novel heterozygous pathogenic variant within ACTN2 causing distal myopathy with multi-minicores in a Chinese family. The variant resulted in an increased expression level of α-actinin-2, abnormal Z-disk organization, and aggregate formations, implying the disease pathogenesis, might relate to aggregate formations.
The presence of minicores with the loss of oxidative enzyme activity on muscle biopsy in our patient suggested the diagnosis of minicore disease. 23 Core myopathies including central core disease (CCD) and multi-minicore disease (MmD) are often considered preeminently pediatric conditions characterized by marked extensive weakness, early spinal rigidity, and respiratory impairment. 24 The most common causative gene for core myopathies is RYR1 and less frequent one is SEPN1. An unusual adult-onset, mild calf-predominant myopathy in combination with well-defined cores and multicore-like unevenness of stain was reported due to RYR1 mutations (Table 1).
In addition, a sporadic case with a missense RYR1 mutation was reported to exhibit an adult-onset, anterior tibial muscle-prominent distal myopathy with multiple cores pathology. 17 Compared to our patients, they showed mild lower limb distal muscle weakness and no upper limbs weakness and contractures were observed.
Patients with ACTN2 mutations could present with a wide clinical spectrum including cardiomyopathies and/or other cardiac abnormalities, MsCD, and actininopathy. MsCD is characterized by progressive early-onset extensive myopathy clinically manifesting with severe muscle atrophy, facial weakness, contractures, and respiratory symptoms and a unique histopathology of multiple structured cores in the biopsy, and actininopathy is a late-onset symmetric/ asymmetric distal muscular dystrophy (Table 1). In this study, our patient presented with an adult-onset distal myopathy with multiminicores, progressive to proximal upper and lower limbs with contractures, which, interestingly, was more like a combination of MsCD and actininopathy. Besides, cardiomyopathies were not remarkable in all reported patients with ACTN2-related myopathy, but heart failure or cardiac arrhythmia could be occurred in some patients. 16 Alpha-actinin-2 is composed of an N-terminal actin-binding domain (ABD), a central rod domain of four spectrin-like repeats (SRs), and a C-terminal calmodulin-like domain (CAMD) with two EF-hand motifs (EF1/2 and EF3/4). 11 It functionally acts as a homodimer where SRs repeats are thought to be responsible for the dimerization. 25 The EF3/4 domain interacts with the "neck" region between the ABD and first SR of the opposing monomer, making actinin to be a "closed" conformation. 10,11 This closed conformation will be changed into an "opening" structure when phospholipid bind-  core-like areas, nemaline bodies et al., 27 and various of titinopathy due to TTN variants. 28 Thus, the multiple interactions of proteins in the Zdisk possibly account for the various phenotypes. Therefore, additional patients with ACTN2-related myopathy are important to investigate the clinical features and more functional studies are needed to clarify the molecular mechanisms. Since most core diseases are caused by RYR1 variants, it may also be another target in excitation-contraction coupling for the studies of ACTN2-related myopathy. 24,29 In conclusion, we identified a novel ACTN2 pathogenic variant as the cause of adult-onset distal myopathy with multi-minicores

ACK N OWLED G EM ENTS
We would like to thank the patients and their family members for participating in this study. This work was supported by the research foundation for distinguished scholar of Zhejiang University to Zhi-Ying Wu (188020-193810101/089, Hangzhou) and the Natural Science Foundation of Zhejiang Province to Gong-Lu Liu (LY19H090023).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.