Anoctamin 5 (ANO5) is a protein that belongs to a family of calcium-activated chloride channels and phospholipid scramblases (1). The exact function of the ANO5 protein is not completely understood although it has been shown to play a role in sarcolemma repair (1). Recessive variants of ANO5 have been associated with a range of muscle diseases including limb girdle muscular dystrophy (LGMD R12), (2, 3) distal myopathy (Miyoshi-like muscular dystrophy type 3), (4) exercise induced myalgia, (5) recurrent rhabdomyolysis, (6) axial myopathy (7) and asymptomatic hyperCKemia (5).

Patients with LGMD R12 typically present in adulthood with slowly progressive proximal lower limb and bicep weakness that is often asymmetric. This is associated with markedly elevated creatine kinase (CK) levels (2, 3). For unknown reasons females homozygous for ANO5 variants tend to have a less severe phenotype than males (5). Dilated cardiomyopathy and cardiac conduction disease have been reported but are rare (8). Magnetic resonance imaging (MRI) of the lower limbs often shows selective atrophy and fatty infiltration of the posterior calf and thigh muscles (9).

Here we report the clinical, pathologic, and genetic findings of two unrelated patients presenting with LGMD who were found to have novel variants in ANO5.

We describe two patients presenting with LGMD who were found to have novel ANO5 variants after NGS using a custom-designed neuromuscular genetic panel. The use of multigene panels is especially helpful for the diagnosis of LGMD R12 as its phenotype can overlap with other types of LGMD (11) and muscle biopsies in these patients are often non-diagnostic and show non-specific myopathic or dystrophic changes (7, 12) although skeletal muscle amyloid deposits have been identified in some cases (13). The identification of disease causing variants with NGS also helps with genetic counseling in those with LGMD by allowing for targeted genetic testing in family members that could be carriers or at risk of inheriting the disease.

Patient 1 was a 35-year-old Iranian man who was homozygous for a novel splice variant [c.294+5G>A; p (Ala98Ins4^*)]. This variant leads to intron inclusion resulting in premature truncation of the protein, is absent from controls (14–17) and segregates with disease in the affected sibling (13, 18) (Figure 2 and Table 1). Patient 1 has Iranian ancestry. Most reported cases of LGMD R12 have been in European patients (3). However, LGMD R12 has been reported in patients from the Middle East, including Saudi Arabia (18), Jordan (19) and Afghanistan (20).

Patient 2 was a 55-year-old Caucasian man who was compound heterozygous for the frameshift variant [c.191dupA; p.(Asn64fs)] and a novel missense variant [c.952G>C; p.(Ala318Pro)]. Through a founder effect, the frameshift variant [c.191dupA; p.(Asn64fs)] is the most common pathogenic ANO5 variant identified in European patients with LGMD R12 (3). The missense variant [c.952G>C; p.(Ala318Pro)] in patient 2 is absent from controls, is strongly conserved (21) and predicted to be damaging in silico (22, 23) (Table 1). Currently considered a variant of uncertain significance as per the American College of Medical Genetics and Genomics—Association for Molecular Pathology (ACMG-AMP) guidelines (24) additional functional evidence could help elucidate its impact. Detailed, mutation-specific assessments across ANO5 are lacking in the literature but loss of ANO5 has been shown to cause defective membrane repair (1). The novel missense variant we report in patient 2 is located within the first transmembrane domain of the protein (25) and could influence the effectiveness of membrane transport for repair (ie. coordination of annexins), particularly in muscle where it is highly expressed (26). The clinical presentation of patient 2 with adult onset, slowly progressive proximal lower limb and biceps weakness and significantly elevated CK is typical of other cases of LGMD R12 reported in the literature (2, 3).

ANO5 variants are not uncommon in LGMD but occur with a spectrum of LGMD severity. Our findings extend the genotypic spectrum of ANO5 disease and supports the utility of next generation sequencing in the diagnostic process. Further work on understanding disease mechanisms underlying ANO5 associated LGMD is needed to address relevant therapeutic avenues.

The datasets presented in this article are not readily available because of ethical and privacy restrictions. Requests to access the datasets should be directed to the corresponding author/s.

Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

FG and MD assisted with the genetic data and review of paper. RH and PM assisted with overall review of the paper. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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