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 and two inherited missense variants in (alias ) 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. 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 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 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 to a hereditary disease.

中文翻译：

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ANO4 的错义变异导致散发性脑病或家族性癫痫，有证据表明存在显性负效应


Anoctamin 是 Ca 激活蛋白家族，可充当离子通道和/或磷脂扰乱酶，但对功能和疾病关联的了解有限。在这里，我们确定了 (alias) 中的五种和两种遗传性错义变异，作为发热敏感型发育性癫痫或癫痫性脑病 (DEE/EE) 和全身性癫痫伴热性惊厥附加 (GEFS+) 或颞叶癫痫的原因。 ANO4 结构的建模预测所有已识别的变体都会导致 ANO4 结构的不稳定。四个变体位于 ANO4 的 Ca 结合位点附近，表明蛋白质功能受损。蛋白质拓扑结构的变体映射表明了初步的基因型-表型相关性。此外，在健康个体中观察到的杂合缺失表明功能失调的蛋白质是疾病机制，而不是单倍体不足。为了检验这一假设，我们通过膜片钳记录、免疫细胞化学和膜联蛋白 A5 的表面表达（作为磷脂酰丝氨酸乱序酶活性的衡量标准）检查了异源表达系统中突变 ANO4 的功能特性。所有变体均显示离子通道功能严重丧失，DEE/EE 相关变体由于质膜运输受损而出现表面表达轻度丧失。细胞表面 Ca 不依赖性膜联蛋白 A5 水平的增加表明 DEE 突变表达细胞的凋亡率增加，但没有观察到 Ca 依赖性扰乱酶活性的变化。与 ANO4 野生型共转染表明存在显性失活效应。总之，我们扩大了脑病散发性和遗传性发热敏感型癫痫的遗传基础，并将种系变异与遗传性疾病联系起来。