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

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

doi:10.1016/j.ajhg.2024.04.014

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

Am J Hum Genet. 2024 May 10:S0002-9297(24)00129-0. doi: 10.1016/j.ajhg.2024.04.014. Online ahead of print.

Authors

Fang Yang¹, Anais Begemann², Nadine Reichhart¹, Akvile Haeckel³, Katharina Steindl², Eyk Schellenberger³, Ronja Fini Sturm¹, Magalie Barth⁴, Sissy Bassani², Paranchai Boonsawat², Thomas Courtin⁵, Bruno Delobel⁶; EuroEPINOMICS-RES Dravet working group; Boudewijn Gunning⁷, Katia Hardies⁸, Mélanie Jennesson⁹, Louis Legoff⁴, Tarja Linnankivi¹⁰, Clément Prouteau⁴, Noor Smal⁸, Marta Spodenkiewicz¹¹, Sandra P Toelle¹², Koen Van Gassen¹³, Wim Van Paesschen¹⁴, Nienke Verbeek¹³, Alban Ziegler⁴, Markus Zweier², Anselm H C Horn¹⁵, Heinrich Sticht¹⁶, Holger Lerche¹⁷, Sarah Weckhuysen¹⁸, Olaf Strauß¹, Anita Rauch¹⁹

Affiliations

¹ Experimental Ophthalmology, Department of Ophthalmology, Charité - Universitätsmedizin Berlin, a Corporate Member of Freie Universität, Humboldt-University, the Berlin Institute of Health, Berlin, Germany.
² Institute of Medical Genetics, University of Zurich, Schlieren-Zurich, Switzerland.
³ 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.
⁴ University Hospital of Angers, Department of Genetics, Angers, France.
⁵ 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.
⁶ Service de Cytogénétique, GH de l'Institut Catholique de Lille, Hopital Saint Vincent de Paul, Lille, France.
⁷ Stichting Epilepsie Instellingen Nederland, Zwolle, the Netherlands.
⁸ Applied & Translational Neurogenomics Group, VIB Center for Molecular Neurology, VIB, University of Antwerp, 2610 Antwerp, Belgium.
⁹ Department of Pediatrics, CHU, Reims, France.
¹⁰ 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.
¹¹ Department of Genetics, La Réunion University Hospital, Saint-Pierre, France.
¹² Department of Pediatric Neurology, Children's University Hospital Zurich, Zurich, Switzerland.
¹³ University Medical Center Utrecht, Department of Genetics, Utrecht, the Netherlands.
¹⁴ Laboratory for Epilepsy Research, KU Leuven, and Neurology Department, University Hospitals Leuven, 3000 Leuven, Belgium.
¹⁵ Institute of Medical Genetics, University of Zurich, Schlieren-Zurich, Switzerland; Division of Bioinformatics, Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
¹⁶ Division of Bioinformatics, Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
¹⁷ Department of Neurology and Epileptology, Hertie-Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany.
¹⁸ 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.
¹⁹ Institute of Medical Genetics, University of Zurich, Schlieren-Zurich, Switzerland; Children's University Hospital Zurich, Zurich, Switzerland. Electronic address: anita.rauch@medgen.uzh.ch.

PMID: 38744284
DOI: 10.1016/j.ajhg.2024.04.014

Abstract

Anoctamins are a family of Ca²⁺-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 Ca²⁺ 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 Ca²⁺-independent annexin A5 at the cell surface suggested an increased apoptosis rate in DEE-mutant expressing cells, but no changes in Ca²⁺-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.

Keywords: ANO4; Ca(2+)-dependent ion channel; GEFS+; TMEM16D; anoctamin; developmental and epileptic encephalopathy; phospholipid scramblase; temporal lobe epilepsy.