VSI: The anoctamins: Structure and function: "Intracellular" anoctamins.

Plasma membrane localized anoctamin 1, 2 and 6 (TMEM16A, B, F) have been examined in great detail with respect to structure and function, but much less is known about the other seven intracellular members of this exciting family of proteins. This is probably due to their limited accessibility in intracellular membranous compartments, such as the endoplasmic reticulum (ER) or endosomes. However, these so-called intracellular anoctamins are also found in the plasma membrane (PM) which adds to the confusion regarding their cellular role. Probably all intracellular anoctamins except of ANO8 operate as intracellular phospholipid (PL) scramblases, allowing for Ca2+-activated, passive transport of phospholipids like phosphatidylserine between both membrane leaflets. Probably all of them also conduct ions, which is probably part of their physiological function. In this brief overview, we summarize key findings on the biological functions of ANO3, 4, 5, 7, 8, 9 and 10 (TMEM16C, D, E, G, H, J, K) that are gradually coming to light. Compartmentalized regulation of intracellular Ca2+ signals, tethering of the ER to specific PM contact sites, and control of intracellular vesicular trafficking appear to be some of the functions of intracellular anoctamins, while loss of function and abnormal expression are the cause for various diseases.

Familial gigantiform cementoma with recurrent ANO5 p.Cys356Tyr mutations: Clinicopathological and genetic study with literature review.

BACKGROUND: Familial gigantiform cementoma (FGC) is a rare tumor characterized by the early onset of multi-quadrant fibro-osseous lesions in the jaws, causing severe maxillofacial deformities. Its clinicopathological features overlap with those of other benign fibro-osseous lesions. FGC eventually exhibits progressively rapid growth, but no suspected causative gene has been identified. METHODS: In this study, three patients with FGC were recruited, and genomic DNA from the tumor tissue and peripheral blood was extracted for whole-exome sequencing. RESULTS: Results showed that all three patients harbored the heterozygous mutation c.1067G > A (p.Cys356Tyr) in the ANO5 gene. Furthermore, autosomal dominant mutations in ANO5 at this locus have been identified in patients with gnathodiaphyseal dysplasia (GDD) and are considered a potential causative agent, suggesting a genetic association between FGC and GDD. In addition, multifocal fibrous bone lesions with similar clinical presentations were detected, including five cases of florid cemento-osseous dysplasia, five cases of polyostotic fibrous dysplasia, and eight cases of juvenile ossifying fibromas; however, none of them harbored mutations in the ANO5 gene. CONCLUSION: Our findings indicate that FGC may be an atypical variant of GDD, providing evidence for the feasibility of ANO5 gene testing as an auxiliary diagnostic method for complex cases with multiple quadrants.

Hypertrophic Cardiomyopathy Complicated by Post-COVID-19 Myopericarditis in Patient with ANO5-Related Distal Myopathy.

A 60-year-old male with hypertrophic cardiomyopathy, conduction disorders, post-COVID-19 myopericarditis and heart failure was admitted to the hospital's cardiology department. Blood tests revealed an increase in CPK activity, troponin T elevation and high titers of anticardiac antibodies. Whole exome sequencing showed the presence of the pathogenic variant NM_213599:c.2272C>T of the ANO5 gene. Results of the skeletal muscle biopsy excluded the diagnosis of systemic amyloidosis. Microscopy of the muscle fragment demonstrated sclerosis of the perimysium, moderate lymphoid infiltration, sclerosis of the microvessels, dystrophic changes and a lack of cross striations in the muscle fibers. Hypertrophy of the LV with a low contractile ability, atrial fibrillation, weakness of the distal skeletal muscles and increased plasma CPK activity and the results of the skeletal muscle biopsy suggested a diagnosis of a late form of distal myopathy (Miyoshi-like distal myopathy, MMD3). Post-COVID-19 myopericarditis, for which genetically modified myocardium could serve as a favorable background, caused heart failure decompensation.

Genetic disruption of Ano5 leads to impaired osteoclastogenesis for gnathodiaphyseal dysplasia.

OBJECTIVES: Gnathodiaphyseal dysplasia (GDD; OMIM#166260) is a rare skeletal genetic disorder characterized by sclerosis of tubular bones and cemento-osseous lesions in mandibles. TMEM16E/ANO5 gene mutations have been identified in patients with GDD. Here, Ano5 knockout (Ano5-/- ) mice with enhanced osteoblastogenesis were used to investigate whether Ano5 disruption affects osteoclastogenesis. SUBJECTS AND METHODS: The maturation of osteoclasts, formation of F-actin ring and bone resorption were detected by immunohistochemistry, TRAP, phalloidin staining and Coming Osteo assays. The expression of osteoclast-related factors was measured by qRT-PCR. Early signaling pathways were verified by western blot. RESULTS: Ano5-/- mice exhibited inhibitory formation of multinucleated osteoclasts with a reduction of TRAP activity. The expression of Nfatc1, c-Fos, Trap, Ctsk, Mmp9, Rank and Dc-stamp was significantly decreased in bone tissues and bone marrow-derived macrophages (BMMs) of Ano5-/- mice. Ano5-/- osteoclasts manifested disrupted actin ring and less mineral resorption. RANKL-induced early signaling pathways were suppressed in Ano5-/- osteoclasts and Ano5 knockdown RAW264.7 cells. Moreover, the inhibitory effects of NF-κB signalling pathway on osteoclastogenesis were partially attenuated with NF-κB signalling activator. CONCLUSIONS: Ano5 deficiency impairs osteoclastogenesis, which leads to enhanced osteogenic phenotypes mediated by bone homeostasis dysregulation.

Integration of metabolomics and transcriptomics provides insights into enhanced osteogenesis in Ano5Cys360Tyr knock-in mouse model.

Introduction: Gnathodiaphyseal dysplasia (GDD; OMIM#166260) is a rare autosomal dominant disorder characterized by diaphyseal sclerosis of tubular bones and cemento-osseous lesions in mandibles. GDD is caused by point mutations in the ANO5 gene. However, the mechanisms underlying GDD have not been disclosed. We previously generated the first knock-in mouse model for GDD expressing a human mutation (p.Cys360Tyr) in ANO5 and homozygous Ano5 knock-in (Ano5KI/KI ) mice exhibited representative traits of human GDD especially including enhanced osteogenesis. Methods: Metabolomics and transcriptomics analyses were conducted for wildtype (Ano5+/+ ) and Ano5KI/KI mature mouse calvarial osteoblasts (mCOBs) grown in osteogenic cultures for 14 days to identify differential intracellular metabolites and genes involved in GDD. Subsequently, related differential genes were validated by qRT-PCR. Cell proliferation was confirmed by CCK8 assay and calcium content in mineral nodules was detected using SEM-EDS. Results: Metabolomics identified 42 differential metabolites that are primarily involved in amino acid and pyrimidine metabolism, and endocrine and other factor-regulated calcium reabsorption. Concomitantly, transcriptomic analysis revealed 407 differentially expressed genes in Ano5KI/KI osteoblasts compared with wildtype. Gene ontology and pathway analysis indicated that Ano5Cys360Tyr mutation considerably promoted cell cycle progression and perturbed calcium signaling pathway, which were confirmed by validated experiments. qRT-PCR and CCK-8 assays manifested that proliferation of Ano5KI/KI mCOBs was enhanced and the expression of cell cycle regulating genes (Mki67, Ccnb1, and Ccna2) was increased. In addition, SEM-EDS demonstrated that Ano5KI/KI mCOBs developed higher calcium contents in mineral nodules than Ano5+/+ mCOBs, while some calcium-related genes (Cacna1, Slc8a1, and Cyp27b1) were significantly up-regulated. Furthermore, osteocalcin which has been proved to be an osteoblast-derived metabolic hormone was upregulated in Ano5KI/KI osteoblast cultures. Discussion: Our data demonstrated that the Ano5Cys360Tyr mutation could affect the metabolism of osteoblasts, leading to unwonted calcium homeostasis and cellular proliferation that can contribute to the underlying pathogenesis of GDD disorders.

Anoctamin-5 Muscular Dystrophy: Report of Two Cases with Different Phenotypes and Genotypes from the Indian Subcontinent.

Anoctaminopathies are a group of autosomal recessive skeletal muscle disorders with various clinical phenotypes, caused by anoctamin 5 (ANO5) gene mutations and the abnormal expression of ANO5 protein. Patients with recessive mutations in ANO5 present with variable symptoms ranging from asymptomatic hyperCKemia and exercise-induced myalgia to proximal and/or distal muscle weakness. Here, we describe the clinical, pathological, and molecular findings of two unrelated patients with ANO5-related muscular dystrophy (MD). Ninety-six histologically identified MD cases were subjected to next-generation sequencing using a customized panel of 54 genes (IIlumina Design Studio). Two patients were diagnosed with ANO5-related MD. One patient had a pathogenic homozygous mutation of c.1406G>A in exon 14, while the other patient had a novel heterozygous mutation of c.2141C>G in exon 19 of ANO5 gene. Both showed two different phenotypes (limb girdle MD and Miyoshi myopathy) and histomorphological patterns. Muscle biopsy of one patient in addition showed amyloid deposit in the walls of interstitial blood vessels. ANO5-related MD is a heterogeneous disease with different clinical phenotypes as well as genotypes. All muscle biopsies with unclassified muscular dystrophies should be subjected to Congo red stain. The results of this study suggest that screening for ANO5 gene should represent an early step in the diagnostic work-up of the patients with undiagnosed MD and persistent asymptomatic hyperCKemia, even when muscle biopsy histomorphology is normal.

Anoctamin 5 (ANO5) Muscle Disorders: A Narrative Review.

Anoctaminopathy-5 refers to a group of hereditary skeletal muscle or bone disorders due to mutations in the anoctamin 5 (ANO5)-encoding gene, ANO5. ANO5 is a 913-amino acid protein of the anoctamin family that functions predominantly in phospholipid scrambling and plays a key role in the sarcolemmal repairing process. Monoallelic mutations in ANO5 give rise to an autosomal dominant skeletal dysplastic syndrome (gnathodiaphyseal dysplasia or GDD), while its biallelic mutations underlie a continuum of four autosomal recessive muscle phenotypes: (1). limb-girdle muscular dystrophy type R12 (LGMDR12); (2). Miyoshi distal myopathy type 3 (MMD3); (3). metabolic myopathy-like (pseudometabolic) phenotype; (4). asymptomatic hyperCKemia. ANO5 muscle disorders are rare, but their prevalence is relatively high in northern European populations because of the founder mutation c.191dupA. Weakness is generally asymmetric and begins in proximal muscles in LGMDR12 and in distal muscles in MMD3. Patients with the pseudometabolic or asymptomatic hyperCKemia phenotype have no weakness, but conversion to the LGMDR12 or MMD3 phenotype may occur as the disease progresses. There is no clear genotype-phenotype correlation. Muscle biopsy displays a broad spectrum of pathology, ranging from normal to severe dystrophic changes. Intramuscular interstitial amyloid deposits are observed in approximately half of the patients. Symptomatic and supportive strategies remain the mainstay of treatment. The recent development of animal models of ANO5 muscle diseases could help achieve a better understanding of their underlying pathomechanisms and provide an invaluable resource for therapeutic discovery.

Gnathodiaphyseal dysplasia with a novel genetic variant in a large family from Iran.

BACKGROUND: Gnathodiaphyseal dysplasia (GDD) is an ultrarare autosomal dominant bone dysplasia characterized by cementoosseous lesions of the jawbones, bone fragility, frequent bone fractures at the young age, bowing of tubular bones, and diaphyseal sclerosis of long bones associated with generalized osteopenia. GDD is caused by point mutations in anoctamin-5 (ANO5) on chromosome 11p14.3. For the past few years, next generation sequencing (NGS) technology has facilitated the discovery of causative variants in genetically heterogeneous diseases. METHODS: In this study, exome sequencing (ES) was performed using the DNA sample of the proband. Family histories and clinical information were collected through comprehensive medical examination and genetic counseling. RESULTS: ES results identified a heterozygous variant, NM_213599.3:c.1078T>C(p.Cys360Arg) in the ANO5 gene. Sanger sequencing was performed to confirm the detected pathogenic variant in DNA samples of the entire family (except deceased individuals), which segregated with the disease within the family. Finally, in silico analysis was applied to test the pathogenicity of the variant using various online software. CONCLUSION: In summary, our investigation identified a novel pathogenic variant in the ANO5, responsible for gnathodiaphyseal dysplasia in a large Iranian family. Therefore, based on the present study, this variant can be helpful for diagnosis and effective management of GDD patients.

Unraveling the Molecular Basis of the Dystrophic Process in Limb-Girdle Muscular Dystrophy LGMD-R12 by Differential Gene Expression Profiles in Diseased and Healthy Muscles.

Limb-girdle muscular dystrophy R12 (LGMD-R12) is caused by two mutations in anoctamin-5 (ANO5). Our aim was to identify genes and pathways that underlie LGMD-R12 and explain differences in the molecular predisposition and susceptibility between three thigh muscles that are severely (semimembranosus), moderately (vastus lateralis) or mildly (rectus femoris) affected in this disease. We performed transcriptomics on these three muscles in 16 male LGMD-R12 patients and 15 age-matched male controls. Our results showed that LGMD-R12 dystrophic muscle is associated with the expression of genes indicative of fibroblast and adipocyte replacement, such as fibroadipogenic progenitors and immune cell infiltration, while muscle protein synthesis and metabolism were downregulated. Muscle degeneration was associated with an increase in genes involved in muscle injury and inflammation, and muscle repair/regeneration. Baseline differences between muscles in healthy individuals indicated that muscles that are the most affected by LGMD-R12 have the lowest expression of transcription factor networks involved in muscle (re)generation and satellite stem cell activation. Instead, they show relative high levels of fetal/embryonic myosins, all together indicating that muscles differ in their baseline regenerative potential. To conclude, we profiled the gene expression landscape in LGMD-R12, identified baseline differences in expression levels between differently affected muscles and characterized disease-associated changes.

Novel Variants of ANO5 in Two Patients With Limb Girdle Muscular Dystrophy: Case Report.

Here we report on two unrelated adult patients presenting with Limb girdle muscular dystrophy who were found to have novel variants in ANO5. Both patients had prominent weakness of their proximal lower limbs with mild weakness of elbow flexion and markedly elevated creatine kinase. Next generation sequencing using a custom-designed neuromuscular panel was performed in both patients. In one patient, 336 genes were targeted for casual variants and in the other patient (using a later panel design), 464 genes were targeted. One patient was homozygous for a novel splice variant [c.294+5G>A; p.(Ala98Ins4*)] in ANO5. Another patient was compound heterozygous for two variants in ANO5; a common frameshift variant [c.191dupA; p.(Asn64fs)] and a novel missense variant [c.952G>C; p.(Ala318Pro)]. These findings support the utility of next generation sequencing in the diagnosis of patients presenting with a Limb girdle muscular dystrophy phenotype and extends the genotypic spectrum of ANO5 disease.

Lidocaine Suppresses Gastric Cancer Development Through Circ_ANO5/miR-21-5p/LIFR Axis.

BACKGROUND: Lidocaine has been manifested to exert anti-tumor role in gastric cancer (GC) progression. However, the action mechanism by which Lidocaine functions in GC has not been fully elucidated. AIM: The study aimed to reveal the molecular mechanism of Lidocaine in GC progression. METHODS: Cell clonogenicity and viability were assessed by colony formation and methyl thiazolyl tetrazolium assays, respectively. Transwell assay was employed to detect cell migration and invasion. Flow cytometry was implemented to monitor cell apoptosis. Relative expression of circular RNA ANO5 (circ_ANO5), microRNA (miR)-21-5p and Leukemia inhibitory factor receptor (LIFR) was examined by quantitative reverse transcription-polymerase chain reaction. Western blot assay was performed to analyze the levels of LIFR and cell metastasis-related proteins. The target relationship between miR-21-5p and circ_ANO5 or LIFR was confirmed by dual-luciferase reporter assay. In addition, xenograft model was established to explore the role of Lidocaine in vivo. RESULTS: Lidocaine inhibited cell proliferation, migration and invasion, while promoted apoptosis of GC cells. Lidocaine upregulated circ_ANO5 and LIFR expression, but downregulated miR-21-5p expression in GC cells. Additionally, expression of circ_ANO5 and LIFR was decreased, while miR-21-5p expression was increased in GC cells. Circ_ANO5 depletion or miR-21-5p overexpression attenuated Lidocaine-induced anti-proliferative and anti-metastatic effects on GC cells. Circ_ANO5 could sponge miR-21-5p, and miR-21-5p targeted LIFR. Moreover, Lidocaine suppressed the tumor growth in vivo. CONCLUSION: Lidocaine might GC cell malignancy by modulating circ_ANO5/miR-21-5p/LIFR axis, highlighting a novel insight for GC treatment.

BVES is a novel interactor of ANO5 and regulates myoblast differentiation.

BACKGROUND: Anoctamin 5 (ANO5) is a membrane protein belonging to the TMEM16/Anoctamin family and its deficiency leads to the development of limb girdle muscular dystrophy R12 (LGMDR12). However, little has been known about the interactome of ANO5 and its cellular functions. RESULTS: In this study, we exploited a proximal labeling approach to identify the interacting proteins of ANO5 in C2C12 myoblasts stably expressing ANO5 tagged with BioID2. Mass spectrometry identified 41 unique proteins including BVES and POPDC3 specifically from ANO5-BioID2 samples, but not from BioID2 fused with ANO6 or MG53. The interaction between ANO5 and BVES was further confirmed by co-immunoprecipitation (Co-IP), and the N-terminus of ANO5 mediated the interaction with the C-terminus of BVES. ANO5 and BVES were co-localized in muscle cells and enriched at the endoplasmic reticulum (ER) membrane. Genome editing-mediated ANO5 or BVES disruption significantly suppressed C2C12 myoblast differentiation with little impact on proliferation. CONCLUSIONS: Taken together, these data suggest that BVES is a novel interacting protein of ANO5, involved in regulation of muscle differentiation.

Muscle biopsy and MRI findings in ANO5-related myopathy.

INTRODUCTION/AIMS: Mutations in the anoctamin 5 (ANO5) gene are a common cause of muscular dystrophy. We aimed to investigate whether inflammatory changes in muscle are present in patients with ANO5 myopathy when assessed by muscle biopsy and muscle magnetic resonance imaging (MRI). METHODS: Adults with pathogenic variations in ANO5 known to cause muscular dystrophy were included in our study. Muscle biopsies of pelvic and lower extremity muscles were reviewed retrospectively. Muscle MR short-tau inversion recovery (STIR) images of a subset of these patients were obtained prospectively. RESULTS: Muscle biopsies from 24 patients were reviewed. MR STIR images were performed in 17 of these patients. We found inflammatory changes in muscle biopsies of three patients and MRI revealed hyperintense signals on STIR images in 14 of 17 patients. DISCUSSION: In this study, we found that muscle edema is very common in patients with ANO5 myopathy and that some patients have inflammatory changes in muscle biopsies. Further studies are needed to determine whether the STIR+ lesions reflect inflammation.

Anoctamin 5 promotes osteosarcoma development by increasing degradation of Nel-like proteins 1 and 2.

Anoctamin 5 (ANO5) is a member of the Anoctamin (ANO) family of calcium-activated chloride channels. Although ANO5 expression is upregulated in various cancers, its role in osteosarcoma remains largely unknown. In this study, bioinformatics analysis, western blot, and immunohistochemical staining revealed that ANO5 was upregulated in osteosarcoma cell lines and osteosarcoma tissues, and ANO5 expression was positively associated with tumor size, tumor grade, and metastasis. Functional experiments demonstrated that inhibition of ANO5 decreased, while ANO5 overexpression increased, osteosarcoma cell proliferation and mobility in vitro. Immunoprecipitation, western blot, and confocal microscopy experiments showed that ANO5 bound to and promoted the degradation of Nel-like proteins 1 (NELL1) and 2 (NELL2). Moreover, a subcutaneous tumor transplantation model revealed that ANO5 knockdown reduced osteosarcoma cell proliferation and increased NELL1 and NELL2 expression in vivo. Finally, rescue experiments showed that knockdown of NELL1 or NELL2 reversed the inhibitory effects of ANO5 knockdown on osteosarcoma cell proliferation and migration. These results demonstrated that upregulation of ANO5 promoted osteosarcoma development by decreasing the stability of the NELL1 and NELL2 proteins and that ANO5 may be an effective target for the treatment of osteosarcoma.

Muscle involvement assessed by quantitative magnetic resonance imaging in patients with anoctamin 5 deficiency.

OBJECTIVE: Using magnetic resonance imaging (MRI) and stationary dynamometry, the aim was to investigate the muscle affection in paraspinal muscles and lower extremities and compare the muscle affection in men and women with anoctamin 5 (ANO5) deficiency. METHODS: Seventeen patients (seven women) with pathogenic ANO5-mutations were included. Quantitative muscle fat fraction of back and leg muscles were assessed by Dixon MRI. Muscle strength was assessed by stationary dynamometer. Results were compared with 11 matched, healthy controls. RESULTS: Muscle involvement pattern in men with ANO5-deficiency is characterized by a severe fat replacement of hamstrings, adductor and gastrocnemius muscles, while paraspinal muscles are only mildly affected, while preserved gracilis and sartorius muscles were hypertrophied. Women with ANO5-myopathy, of the same age as male patients, were very mildly affected, showing muscle affection and strength resembling that found in healthy persons, with the exception of the gluteus minimus and medius and gastrocnemii muscles that were significantly replaced by fat. Although individual muscles showed clear asymmetric involvement in a few muscle groups, the overall muscle involvement was symmetric. CONCLUSIONS: Patients with ANO5-deficiency have relatively preserved paraspinal muscles on imaging and only mild reduction of trunk extension strength in men only. Our study quantifies the large difference in muscle affection in lower extremity between women and men with ANO5-deficiency. The clinical notion is that affection may be very asymmetric in ANO5-deficiency, but the present study shows that while this may be true for a few muscles, the general impression is that muscle affection is very symmetric.

Novel ANO5 intronic Roma variant alters splicing causing muscular dystrophy.

The pathogenic role of intronic variants is generally difficult to assess, except for those near known splice sites for which aberrant splicing is suspected, although deeper intronic variants can also alter splicing. We have identified a novel (NM_213599.2:c.1180+6T>C) ANO5 variant that causes the exclusion of exon 12. The mutation, identified in a Roma individual, has an estimated carrier rate of 1.68% among the Iberian Roma population, this being the first ANO5 pathogenic variant communicated in this ethnic group. In this study, we have also characterized the ANO5 splice forms expressed in human muscle with the detection of an alternative transcript, in which exons 8 and 9 are spliced out.

Cardiac MRI identifies valvular and myocardial disease in a subset of ANO5-related muscular dystrophy patients.

Patients with bi-allelic loss-of-function mutations in the gene ANO5 most commonly present with muscular dystrophy. In some studies, patients with ANO5-related dystrophy (ANO5-RD) had evidence of mild cardiac abnormalities; however, cardiac magnetic resonance imaging (MRI) has not been used for myocardial characterization. Ten patients with genetically confirmed ANO5-RD were enrolled in a phenotyping study to better characterize cardiac involvement. Evaluations included medical history, neurological examination and cardiac evaluations (electrocardiogram, echocardiogram and cardiac MRI). All patients were clinically asymptomatic from a cardiac perspective. Muscle MRI was consistent with previous studies of ANO5-RD with increased T1 signal in the posterior and medial compartments of the upper leg and the posterior compartment of the lower leg. Cardiac studies using echocardiography and cardiac MRI revealed dilation of the aortic root and thickening of the aortic valve without significant stenosis in 3/10 patients. There was evidence of abnormal late gadolinium enhancement (LGE) on cardiac MRI in 2/10 patients. In ANO5-RD, the development of cardiac fibrosis, edema or inflammation as demonstrated by LGE has not yet been reported. Cardiac MRI can characterize cardiac tissue and may detect subtle changes before they appear on echocardiography, with potential prognostic implications.

Targeting of Intracellular TMEM16 Proteins to the Plasma Membrane and Activation by Purinergic Signaling.

Anoctamins such as TMEM16A and TMEM16B are Ca2+-dependent Cl- channels activated through purinergic receptor signaling. TMEM16A (ANO1), TMEM16B (ANO2) and TMEM16F (ANO6) are predominantly expressed at the plasma membrane and are therefore well accessible for functional studies. While TMEM16A and TMEM16B form halide-selective ion channels, TMEM16F and probably TMEM16E operate as phospholipid scramblases and nonselective ion channels. Other TMEM16 paralogs are expressed mainly in intracellular compartments and are therefore difficult to study at the functional level. Here, we report that TMEM16E (ANO5), -H (ANO8), -J (ANO9) and K (ANO10) are targeted to the plasma membrane when fused to a C-terminal CAAX (cysteine, two aliphatic amino acids plus methionin, serine, alanin, cystein or glutamin) motif. These paralogs produce Ca2+-dependent ion channels. Surprisingly, expression of the TMEM16 paralogs in the plasma membrane did not produce additional scramblase activity. In contrast, endogenous scrambling induced by stimulation of purinergic P2X7 receptors was attenuated, in parallel with reduced plasma membrane blebbing. This could suggest that intracellular TMEM16 paralogs operate differently when compared to plasma membrane-localized TMEM16F, and may even stabilize intracellular membranes. Alternatively, CAAX tagging, which leads to expression in non-raft compartments of the plasma membrane, may antagonize phosphatidylserine exposure by endogenous raft-located TMEM16F. CAAX-containing constructs may be useful to further investigate the molecular properties of intracellular TMEM16 proteins.

Persistent asymptomatic or mild symptomatic hyperCKemia due to mutations in ANO5: the mildest end of the anoctaminopathies spectrum.

BACKGROUND: The ANO5 gene encodes for anoctamin-5, a chloride channel involved in muscle cell membrane repair. Recessive mutations in ANO5 are associated with muscular diseases termed anoctaminopathies, which are characterized by proximal or distal weakness, or isolated hyperCKemia. We present the largest series of patients with asymptomatic/paucisymptomatic anoctaminopathy reported so far, highlighting their clinical and radiological characteristics. METHODS: Twenty subjects were recruited retrospectively from the Neuromuscular Disorders Units database of two national reference centers. All had a confirmed genetic diagnosis (mean age of diagnosis was 48 years) established between 2015 and 2019. Clinical and complementary data were evaluated through clinical records. RESULTS: None of the patients complained about weakness or showed abnormal muscular balance. Among paucisymptomatic patients, the main complaints or findings were generalized myalgia, exercise intolerance and calf hypertrophy, occasionally associated with calf pain. All patients showed persistent hyperCKemia, ranging from mild-moderate to severe. Muscle biopsy revealed inflammatory changes in three cases. Muscle magnetic resonance imaging revealed typical signs (preferential involvement of adductor and gastrocnemius muscles) in all but one patient. In two cases, abnormal findings were detectable only in STIR sequences (not in T1). Three patients showed radiological progression despite remaining asymptomatic. Twelve different mutations in ANO5 were detected, of which seven are novel. CONCLUSIONS: Recessive mutations in ANO5 are a frequent cause of undiagnosed asymptomatic/paucisymptomatic hyperCKemia. Patients with an apparent indolent phenotype may show muscle involvement in complementary tests (muscle biopsy and imaging), which may progress over time. Awareness of anoctaminopathy as the cause of nonspecific muscular complaints or of isolated hyperCKemia is essential to correctly diagnose affected patients.