13.05.2024Missense variants in ANO4 cause sporadic encephalopathic or familial epilepsy with evidence for a dominant-negative effect

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 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 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, TMEM16D, anoctamin, developmental and epileptic encephalopathy, temporal lobe epilepsy, GEFS+, Ca²⁺-dependent ion channel, phospholipid scramblase

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

PMID: 38744284, DOI: 10.1016/j.ajhg.2024.04.014, S2CID: 269770731, American Journal of Human Genetics, S0002-9297(24)00129-0. Epub ahead of print. 2024 May 13.

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, 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