A homozygous ATP2A2 variant alters sarcoendoplasmic reticulum Ca2+-ATPase 2 function in skeletal muscle and causes a novel vacuolar myopathy

Background Sarcoendoplasmic reticulum Ca2+-ATPase isoform 2 (SERCA2), encoded by ATP2A2 , is a key protein involved in intracellular Ca2+ homeostasis. The transcript SERCA2a is predominantly expressed in cardiac muscle and in type I myofibers, while SERCA2b is ubiquitously expressed including in skin cells. To date, variants in this gene were reported to be the cause of Darier disease, an autosomal dominant dermatologic disorder, but have never been linked to primary skeletal muscle disease. We describe four patients suffering from a novel hereditary myopathy caused by a homozygous missense variant in ATP2A2 . Methods We studied a family with four affected individuals suffering from an adult-onset progressive skeletal myopathy. We performed a comprehensive evaluation of the clinical phenotype, serum CK levels, muscle MRI, and muscle biopsy, with genetic workup by means of gene panel sequencing followed by whole genome sequencing and segregation analysis. Immunohistochemistry and western blot (WB) to evaluate SERCA2 and SERCA1 expression in skeletal muscle was performed. We evaluated kinetics of Ca2+handling following caffeine exposure or voltage-induced sarcolemma depolarization in patient myoblasts and myotubes, compared to healthy controls. Results Four siblings in their fifties developed in early adulthood symmetric proximal weakness in lower limbs, which was slowly progressive over time. They had no skin or cardiac involvement. Biopsy findings in two affected individuals showed small vacuoles restricted to type I myofibers. Ultrastructural analysis showed dilation and proliferation of T-tubules, swelling of sarcoplasmic reticulum and autophagic vacuoles. Genome sequencing revealed a homozygous variant in ATP2A2 (c.1117G>A, p.(Glu373Lys)) which segregated with the disease. Immunohistochemistry suggested SERCA2 mislocalization in patient myofibers compared to controls. WB did not show changes in the amount or molecular weight of the protein. In vitro functional studies revealed delayed sarcoendoplasmic reticulum Ca2+reuptake in patient myotubes, consistent with an altered pumping capacity of SERCA2 after cell stimulation with caffeine or depolarization. Conclusions We report a novel adult-onset vacuolar myopathy caused by a homozygous variant in ATP2A2 , resulting in a pure skeletal muscle phenotype with a limb-girdle distribution. Biopsy findings and functional studies demonstrating an impaired function of SERCA2 and consequent Ca2+ dysregulation in slow-twitch skeletal myofibers highly support the pathogenicity of the variant. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by the Instituto de Salud Carlos III co-funded by ERDF/FEDER (Una manera de hacer Europa) under grant FIS PI21/01621 awarded to MO, and Fundacion Isabel Gemio. LL is supported by a Grifols-funded grant (Beca de formacion en enfermedades neuromusculares Isabel Illa). Whole human genome sequencing for this study was provided by the Garvan Sequencing Platform at the Garvan Institute of Medical Research. GR is supported by an Australian National Health and Medical Research Council (NHMRC) EL2 Investigator Grant (APP2007769). This work was also supported by funding from the Australian NHMRC (APP2002640) and PID2020-116927RB-C21 to LH. LL, MC, AC, AV, RC, EG, and MO are members of the European Reference Network for Neuromuscular Diseases. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee of Hospital la Santa Creu i Sant Pau gave ethical approval for this work, included in the project FIS PI21/01621 awarded to MO. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the corresponding author. Data including exact ages, gender or place of origin has been removed following Medrxiv guidelines. This fact includes removal of Figure 1 (family pedigree) from this version to avoid possible identification of the patients. All this information can be available upon request.