The ADARB1 gene encodes the adenosine deaminase acting on RNA 2 (ADAR2) RNA editing enzyme, which edits the GRIA2 transcript Q/R editing site with almost 100% efficiency in the nervous system. The edited GRIA2 R transcript encodes the GLUA2 R subunit isoform of tetrameric α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which is essential to prevent seizures associated with aberrantly elevated AMPA receptor cation permeability. Rare biallelic variants in ADARB1 cause severe infant and childhood seizures and developmental delays in seven cases we previously described. Here, we report two new homozygous ADARB1 variants and study ADAR2 variant editing activities at the GRIA2 Q/R site and other editing sites in cell cultures. One new variant in the second double-stranded RNA binding domain (dsRBD II) retains up to 60% editing activity, whereas another, in the deaminase domain, eliminates RNA editing activity. Reduced GRIA2 Q/R site editing increases AMPA receptor permeability by upregulating the expression of the GLUA2 Q isoform and reducing overall GLUA2 subunit levels, resulting in AMPA receptors that lack GLUA2 and are calcium-permeable. Because failure to edit the GRIA2 Q/R site leads to failure of intron 11 splicing, we also examined the effects of ADAR2 variants on the splicing of a mouse Gria2-based reporter and concluded that ADAR2 variants affect splicing only through their effects on RNA editing activity. To expand the number of variants in ADARB1, some variants reported in ClinVar have also been analyzed by in silico methods to predict which are likely to be most deleterious and associated with seizures in patients.

Central European Institute for Technology Masaryk University Brno 62500 Czechia

Department of Genetics Necker Enfants Malades University Hospital AP HP Université Paris Cité 75015 Paris France

Department of Pediatric Neurology Reference Centre for Rare Epilepsies Necker Enfants Malades University Hospital AP HP Université Paris Cité 75015 Paris France

Greenwood Genetic Center Greenwood South Carolina 29646 USA

Laboratory of Translational Research for Neurological Disorders UMR 1163 Institut National de la Santé et de la Recherche Médicale Imagine Institute 75015 Paris France

National Centre for Biomolecular Research Faculty of Science Masaryk University Brno 62500 Czechia

See more in PubMed

Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR. 2010. A method and server for predicting damaging missense mutations. Nat Methods 7: 248–249. 10.1038/nmeth0410-248 PubMed DOI PMC

Cappannini A, Ray A, Purta E, Mukherjee S, Boccaletto P, Moafinejad SN, Lechner A, Barchet C, Klaholz BP, Stefaniak F, et al. 2024. MODOMICS: a database of RNA modifications and related information. 2023 update. Nucleic Acids Res 52: D239–D244. 10.1093/nar/gkad1083 PubMed DOI PMC

Cull-Candy SG, Farrant M. 2021. Ca PubMed DOI PMC

Desterro JM, Keegan LP, Lafarga M, Berciano MT, O'Connell M, Carmo-Fonseca M. 2003. Dynamic association of RNA-editing enzymes with the nucleolus. J Cell Sci 116: 1805–1818. 10.1242/jcs.00371 PubMed DOI

Feldmeyer D, Kask K, Brusa R, Kornau HC, Kolhekar R, Rozov A, Burnashev N, Jensen V, Hvalby O, Sprengel R, et al. 1999. Neurological dysfunctions in mice expressing different levels of the Q/R site-unedited AMPAR subunit GluR-B. Nat Neurosci 2: 57–64. 10.1038/4561 PubMed DOI

Greger IH, Khatri L, Ziff EB. 2002. RNA editing at arg607 controls AMPA receptor exit from the endoplasmic reticulum. Neuron 34: 759–772. 10.1016/S0896-6273(02)00693-1 PubMed DOI

Greger IH, Khatri L, Kong X, Ziff EB. 2003. AMPA receptor tetramerization is mediated by Q/R editing. Neuron 40: 763–774. 10.1016/S0896-6273(03)00668-8 PubMed DOI

Heale BS, Keegan LP, McGurk L, Michlewski G, Brindle J, Stanton CM, Caceres JF, O'Connell MA. 2009. Editing independent effects of ADARs on the miRNA/siRNA pathways. EMBO J 28: 3145–3156. 10.1038/emboj.2009.244 PubMed DOI PMC

Higuchi M, Single FN, Kohler M, Sommer B, Sprengel R, Seeburg PH. 1993. RNA editing of AMPA receptor subunit GluR-B: a base-paired intron–exon structure determines position and efficiency. Cell 75: 1361–1370. 10.1016/0092-8674(93)90622-W PubMed DOI

Higuchi M, Maas S, Single F, Hartner J, Rozov A, Burnashev N, Feldmeyer D, Sprengel R, Seeburg P. 2000. Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2. Nature 406: 78–81. 10.1038/35017558 PubMed DOI

Isaac JT, Ashby MC, McBain CJ. 2007. The role of the GluR2 subunit in AMPA receptor function and synaptic plasticity. Neuron 54: 859–871. 10.1016/j.neuron.2007.06.001 PubMed DOI

Konen LM, Wright AL, Royle GA, Morris GP, Lau BK, Seow PW, Zinn R, Milham LT, Vaughan CW, Vissel B. 2020. A new mouse line with reduced GluA2 Q/R site RNA editing exhibits loss of dendritic spines, hippocampal CA1-neuron loss, learning and memory impairments and NMDA receptor-independent seizure vulnerability. Mol Brain 13: 27. 10.1186/s13041-020-0545-1 PubMed DOI PMC

Livingston JH, Lin JP, Dale RC, Gill D, Brogan P, Munnich A, Kurian MA, Gonzalez-Martinez V, De Goede CG, Falconer A, et al. 2014. A type I interferon signature identifies bilateral striatal necrosis due to mutations in ADAR1. J Med Genet 51: 76–82. 10.1136/jmedgenet-2013-102038 PubMed DOI

Lorenz R, Bernhart SH, Honer Zu Siederdissen C, Tafer H, Flamm C, Stadler PF, Hofacker IL. 2011. ViennaRNA Package 2.0. Algorithms Mol Biol 6: 26. 10.1186/1748-7188-6-26 PubMed DOI PMC

Maroofian R, Sedmik J, Mazaheri N, Scala M, Zaki MS, Keegan LP, Azizimalamiri R, Issa M, Shariati G, Sedaghat A, et al. 2021. Biallelic variants in ADARB1, encoding a dsRNA-specific adenosine deaminase, cause a severe developmental and epileptic encephalopathy. J Med Genet 58: 495–504. 10.1136/jmedgenet-2020-107048 PubMed DOI PMC

Minoche AE, Lundie B, Peters GB, Ohnesorg T, Pinese M, Thomas DM, Zankl A, Roscioli T, Schonrock N, Kummerfeld S, et al. 2021. ClinSV: clinical grade structural and copy number variant detection from whole genome sequencing data. Genome Med 13: 32. 10.1186/s13073-021-00841-x PubMed DOI PMC

Ng PC, Henikoff S. 2003. SIFT: predicting amino acid changes that affect protein function. Nucleic Acids Res 31: 3812–3814. 10.1093/nar/gkg509 PubMed DOI PMC

Pejaver V, Urresti J, Lugo-Martinez J, Pagel KA, Lin GN, Nam HJ, Mort M, Cooper DN, Sebat J, Iakoucheva LM, et al. 2020. Inferring the molecular and phenotypic impact of amino acid variants with MutPred2. Nat Commun 11: 5918. 10.1038/s41467-020-19669-x PubMed DOI PMC

Pettersen EF, Goddard TD, Huang CC, Meng EC, Couch GS, Croll TI, Morris JH, Ferrin TE. 2021. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci 30: 70–82. 10.1002/pro.3943 PubMed DOI PMC

Quin J, Sedmík J, Vukić D, Khan A, Keegan LP, O'Connell MA. 2021. ADAR RNA modifications, the epitranscriptome and innate immunity. Trends Biochem Sci 46: 758–771. 10.1016/j.tibs.2021.02.002 PubMed DOI

Rice GI, Kasher PR, Forte GM, Mannion NM, Greenwood SM, Szynkiewicz M, Dickerson JE, Bhaskar SS, Zampini M, Briggs TA, et al. 2012. Mutations in ADAR1 cause Aicardi–Goutières syndrome associated with a type I interferon signature. Nat Genet 44: 1243–1248. 10.1038/ng.2414 PubMed DOI PMC

Salpietro V, Dixon CL, Guo H, Bello OD, Vandrovcova J, Efthymiou S, Maroofian R, Heimer G, Burglen L, Valence S, et al. 2019. AMPA receptor GluA2 subunit defects are a cause of neurodevelopmental disorders. Nat Commun 10: 3094. 10.1038/s41467-019-10910-w PubMed DOI PMC

Schneider CA, Rasband WS, Eliceiri KW. 2012. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9: 671–675. 10.1038/nmeth.2089 PubMed DOI PMC

Schoft VK, Schopoff S, Jantsch MF. 2007. Regulation of glutamate receptor B pre-mRNA splicing by RNA editing. Nucleic Acids Res 35: 3723–3732. 10.1093/nar/gkm314 PubMed DOI PMC

Shihab HA, Gough J, Cooper DN, Stenson PD, Barker GL, Edwards KJ, Day IN, Gaunt TR. 2013. Predicting the functional, molecular, and phenotypic consequences of amino acid substitutions using hidden Markov models. Hum Mutat 34: 57–65. 10.1002/humu.22225 PubMed DOI PMC

Sinigaglia K, Wiatrek D, Khan A, Michalik D, Sambrani N, Sedmik J, Vukic D, O'Connell MA, Keegan LP. 2019. ADAR RNA editing in innate immune response phasing, in circadian clocks and in sleep. Biochim Biophys Acta Gene Regul Mech 1862: 356–369. 10.1016/j.bbagrm.2018.10.011 PubMed DOI

Sinigaglia K, Cherian A, Du Q, Lacovich V, Vukic D, Melicherova J, Linhartova P, Zerad L, Stejskal S, Malik R, et al. 2024. An ADAR1 dsRBD3–PKR kinase domain interaction on dsRNA inhibits PKR activation. Cell Rep 43: 114618. 10.1016/j.celrep.2024.114618 PubMed DOI

Sommer B, Kohler M, Sprengel R, Seeburg PH. 1991. RNA editing in brain controls a determinant of ion flow in glutamate-gated channels. Cell 67: 11–19. 10.1016/0092-8674(91)90568-J PubMed DOI

Tan MH, Li Q, Shanmugam R, Piskol R, Kohler J, Young AN, Liu KI, Zhang R, Ramaswami G, Ariyoshi K, et al. 2017. Dynamic landscape and regulation of RNA editing in mammals. Nature 550: 249–254. 10.1038/nature24041 PubMed DOI PMC

Tan TY, Sedmik J, Fitzgerald MP, Halevy RS, Keegan LP, Helbig I, Basel-Salmon L, Cohen L, Straussberg R, Chung WK, et al. 2020. Bi-allelic ADARB1 variants associated with microcephaly, intellectual disability, and seizures. Am J Hum Genet 106: 467–483. 10.1016/j.ajhg.2020.02.015 PubMed DOI PMC

Thuy-Boun AS, Thomas JM, Grajo HL, Palumbo CM, Park S, Nguyen LT, Fisher AJ, Beal PA. 2020. Asymmetric dimerization of adenosine deaminase acting on RNA facilitates substrate recognition. Nucleic Acids Res 48: 7958–7972. 10.1093/nar/gkaa532 PubMed DOI PMC