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


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.

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Children's Hospital Zurich > Medical Clinic
04 Faculty of Medicine > Institute of Medical Genetics
Dewey Decimal Classification:610 Medicine & health
570 Life sciences; biology
Uncontrolled Keywords:Genetics, Genetics (clinical), ANO4, TMEM16D, anoctamin, developmental and epileptic encephalopathy, temporal lobe epilepsy, GEFS+, Ca2+-dependent ion channel, phospholipid scramblase
Language:English
Date:1 June 2024
Deposited On:14 May 2024 10:18
Last Modified:07 Jun 2024 01:06
Publisher:Elsevier
ISSN:0002-9297
Additional Information:Supplemental information can be found online at https://doi.org/10.1016/j.ajhg.2024.04.014.
OA Status:Hybrid
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1016/j.ajhg.2024.04.014
Related URLs:https://api.semanticscholar.org/CorpusID:269770731
PubMed ID:38744284
Other Identification Number:Corpus ID: 269770731
Project Information:
  • : FunderSNSF
  • : Grant ID179547
  • : Project TitleGenetic causes and molecular mechanisms in severe intellectual disability
  • : FunderUniversity of Zurich (UZH)
  • : Grant IDURPP ITINERARE
  • : Project TitleUniversity Research Priority Program ITINERARE: Innovative Therapies in Rare Diseases, Zurich, Switzerland
  • : Project Websitehttps://www.itinerare.uzh.ch/en.html
  • : FunderUniversity of Zurich (UZH)
  • : Grant IDCRPP praeclare
  • : Project TitleClinical Research Priority Program praeclare: Clarification of genetic variants for personalized prenatal and reproductive medicine, Zurich, Switzerland
  • : Project Websitehttps://www.praeclare.uzh.ch/en.html
  • : FunderDeutsche Forschungsgemeinschaft
  • : Grant IDSTR480/14-1
  • : Project Title
  • : FunderFWO
  • : Grant ID1861424N and G056122N
  • : Project Title
  • : FunderFP7
  • : Grant ID288028
  • : Project TitleThe DECIPHER Project (Distributed European Community Individual Patient Healthcare Electronic Record)
  • Content: Published Version
  • Language: English
  • Licence: Creative Commons: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)