1. Missense variants in ANO4 cause sporadic encephalopathic or familial epilepsy with evidence for a dominant-negative effect
- Author
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Yang, Fang, Begemann, Anaïs; https://orcid.org/0000-0001-6762-0819, Reichhart, Nadine, Haeckel, Akvile, Steindl, Katharina; https://orcid.org/0000-0002-4425-3072, Schellenberger, Eyk, Sturm, Ronja Fini, Barth, Magalie, Bassani, Sissy; https://orcid.org/0000-0001-6800-8584, Boonsawat, Paranchai; https://orcid.org/0000-0003-3059-1916, Courtin, Thomas, Delobel, Bruno, Gunning, Boudewijn, Hardies, Katia, Jennesson, Mélanie, Legoff, Louis, Linnankivi, Tarja, Prouteau, Clément, Smal, Noor, Spodenkiewicz, Marta, Toelle, Sandra P; https://orcid.org/0000-0001-7999-9854, Van Gassen, Koen, Van Paesschen, Wim, Verbeek, Nienke, Ziegler, Alban; https://orcid.org/0000-0003-0287-3561, Zweier, Markus; https://orcid.org/0000-0001-8290-0413, Horn, Anselm H C; https://orcid.org/0000-0003-3539-8103, Sticht, Heinrich; https://orcid.org/0000-0001-5644-045X, Lerche, Holger, Weckhuysen, Sarah, Strauss, Olaf, Rauch, Anita; https://orcid.org/0000-0003-2930-3163, Yang, Fang, Begemann, Anaïs; https://orcid.org/0000-0001-6762-0819, Reichhart, Nadine, Haeckel, Akvile, Steindl, Katharina; https://orcid.org/0000-0002-4425-3072, Schellenberger, Eyk, Sturm, Ronja Fini, Barth, Magalie, Bassani, Sissy; https://orcid.org/0000-0001-6800-8584, Boonsawat, Paranchai; https://orcid.org/0000-0003-3059-1916, Courtin, Thomas, Delobel, Bruno, Gunning, Boudewijn, Hardies, Katia, Jennesson, Mélanie, Legoff, Louis, Linnankivi, Tarja, Prouteau, Clément, Smal, Noor, Spodenkiewicz, Marta, Toelle, Sandra P; https://orcid.org/0000-0001-7999-9854, Van Gassen, Koen, Van Paesschen, Wim, Verbeek, Nienke, Ziegler, Alban; https://orcid.org/0000-0003-0287-3561, Zweier, Markus; https://orcid.org/0000-0001-8290-0413, Horn, Anselm H C; https://orcid.org/0000-0003-3539-8103, Sticht, Heinrich; https://orcid.org/0000-0001-5644-045X, Lerche, Holger, Weckhuysen, Sarah, Strauss, Olaf, and Rauch, Anita; https://orcid.org/0000-0003-2930-3163
- Abstract
Anoctamins are a family of Ca$^{2+}$-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$^{2+}$ 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 patchclamp 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$^{2+}$-independent annexin A5 at the cell surface suggested an increased apoptosis rate in DEE-mutant expressing cells, but no changes in Ca$^{2+}$-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.
- Published
- 2024