%0 Journal Article
%T Missense variants in ANO4 cause sporadic encephalopathic or familial epilepsy with evidence for a dominant-negative effect.
%A Yang F
%A Begemann A
%A Reichhart N
%A Haeckel A
%A Steindl K
%A Schellenberger E
%A Sturm RF
%A Barth M
%A Bassani S
%A Boonsawat P
%A Courtin T
%A Delobel B
%A  
%A Gunning B
%A Hardies K
%A Jennesson M
%A Legoff L
%A Linnankivi T
%A Prouteau C
%A Smal N
%A Spodenkiewicz M
%A Toelle SP
%A Van Gassen K
%A Van Paesschen W
%A Verbeek N
%A Ziegler A
%A Zweier M
%A Horn AHC
%A Sticht H
%A Lerche H
%A Weckhuysen S
%A Strauß O
%A Rauch A
%J Am J Hum Genet
%V 111
%N 6
%D 2024 Jun 6
%M 38744284
%F 11.043
%R 10.1016/j.ajhg.2024.04.014
%X 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.