Missense variants in ANO4 cause sporadic encephalopathic or familial epilepsy with evidence for a dominant-negative effect.

Holger Lerche, Markus Zweier, Alban Ziegler, Koen Van Gassen, Sandra P Toelle, Noor Smal, Clément Prouteau, Louis Legoff, Thomas Courtin, Paranchai Boonsawat, Sissy Bassani, Ronja Fini Sturm, Akvile Haeckel, Anais Begemann, Fang Yang, Magalie Barth, Boudewijn Gunning, Katharina Steindl, Anita Rauch, Sarah Weckhuysen, Katia Hardies, Nienke Verbeek, Marta Spodenkiewicz, Heinrich Sticht, Tarja Linnankivi, Anselm H C Horn, Wim Van Paesschen, Eyk Schellenberger, Olaf Strauß, Nadine Reichhart, Mélanie Jennesson, Bruno Delobel

Journal: American journal of human genetics 2024;111(6):1184-1205

PMID: 38744284

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Abstract

Anoctamins are a family of Ca-activated proteins that may act as ion channels and/or phospholipid scramblases with limited understanding of function and disease association. Here, we identified five de novo and two inherited missense variants in ANO4 (alias TMEM16D) as a cause of fever-sensitive developmental and epileptic or epileptic encephalopathy (DEE/EE) and generalized epilepsy with febrile seizures plus (GEFS+) or temporal lobe epilepsy. In silico modeling of the ANO4 structure predicted that all identified variants lead to destabilization of the ANO4 structure. Four variants are localized close to the Ca binding sites of ANO4, suggesting impaired protein function. Variant mapping to the protein topology suggests a preliminary genotype-phenotype correlation. Moreover, the observation of a heterozygous ANO4 deletion in a healthy individual suggests a dysfunctional protein as disease mechanism rather than haploinsufficiency. To test this hypothesis, we examined mutant ANO4 functional properties in a heterologous expression system by patch-clamp recordings, immunocytochemistry, and surface expression of annexin A5 as a measure of phosphatidylserine scramblase activity. All ANO4 variants showed severe loss of ion channel function and DEE/EE associated variants presented mild loss of surface expression due to impaired plasma membrane trafficking. Increased levels of Ca-independent annexin A5 at the cell surface suggested an increased apoptosis rate in DEE-mutant expressing cells, but no changes in Ca-dependent scramblase activity were observed. Co-transfection with ANO4 wild-type suggested a dominant-negative effect. In summary, we expand the genetic base for both encephalopathic sporadic and inherited fever-sensitive epilepsies and link germline variants in ANO4 to a hereditary disease.

Address: Experimental Ophthalmology, Department of Ophthalmology, Charité - Universitätsmedizin Berlin, a Corporate Member of Freie Universität, Humboldt-University, the Berlin Institute of Health, Berlin, Germany.; Institute of Medical Genetics, University of Zurich, Schlieren-Zurich, Switzerland.; Institute for Radiology and Children's Radiology, Charité-Universitätsmedizin Berlin, a Corporate Member of Freie Universität, Humboldt-University, the Berlin Institute of Health, Berlin, Germany.; University Hospital of Angers, Department of Genetics, Angers, France.; Sorbonne Université, INSERM, CNRS, Institut du Cerveau - Paris Brain Institute - ICM, 75013 Paris, France; Hôpital Pitié-Salpêtrière, DMU BioGe'M, AP-HP, 75013 Paris, France.; Service de Cytogénétique, GH de l'Institut Catholique de Lille, Hopital Saint Vincent de Paul, Lille, France.; Stichting Epilepsie Instellingen Nederland, Zwolle, the Netherlands.; Applied & Translational Neurogenomics Group, VIB Center for Molecular Neurology, VIB, University of Antwerp, 2610 Antwerp, Belgium.; Department of Pediatrics, CHU, Reims, France.; Epilepsia Helsinki, University of Helsinki and Helsinki University Hospital, 00029 HUS Helsinki, Finland; Department of Pediatric Neurology and Pediatric Research Center, New Children's Hospital, Helsinki University Hospital and University of Helsinki, 00029 HUS Helsinki, Finland.; Department of Genetics, La Réunion University Hospital, Saint-Pierre, France.; Department of Pediatric Neurology, Children's University Hospital Zurich, Zurich, Switzerland.; University Medical Center Utrecht, Department of Genetics, Utrecht, the Netherlands.; Laboratory for Epilepsy Research, KU Leuven, and Neurology Department, University Hospitals Leuven, 3000 Leuven, Belgium.; Institute of Medical Genetics, University of Zurich, Schlieren-Zurich, Switzerland; Division of Bioinformatics, Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.; Division of Bioinformatics, Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.; Department of Neurology and Epileptology, Hertie-Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany.; Applied & Translational Neurogenomics Group, VIB Center for Molecular Neurology, VIB, University of Antwerp, 2610 Antwerp, Belgium; Department of Neurology, Antwerp University Hospital, Antwerp, Belgium; Translational Neurosciences, Faculty of Medicine and Health Science, University of Antwerp, 2610 Antwerp, Belgium.; Institute of Medical Genetics, University of Zurich, Schlieren-Zurich, Switzerland; Children's University Hospital Zurich, Zurich, Switzerland. Electronic address: [email protected].

DOI: 10.1016/j.ajhg.2024.04.014

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MeSH Terms: Adolescent, Child, Child, Preschool, Female, Humans, Male, Anoctamins, Calcium, Epilepsy, Genes, Dominant, Genetic Association Studies, HEK293 Cells, Mutation, Missense, Pedigree, Phospholipid Transfer Proteins