Received: 22 October 2019 | Revised: 29 November 2019 | Accepted: 10 December 2019 DOI: 10.1002/mgg3.1106 ORIGINAL ARTICLE Structural analysis of pathogenic missense mutations in GABRA2 and identification of a novel de novo variant in the desensitization gate Alba Sanchis-Juan1,2 | Marcia A. Hasenahuer3,4 | James A. Baker3 | Amy McTague5 | Katy Barwick5 | Manju A. Kurian5 | Sofia T. Duarte6 | NIHR BioResource | Keren J. Carss1,2 | Janet Thornton3 | F. Lucy Raymond2,4 1Department of Haematology, University of Cambridge, NHS Blood and Transplant Abstract Centre, Cambridge, UK Background: Cys-loop receptors control neuronal excitability in the brain and their 2NIHR BioResource, Cambridge dysfunction results in numerous neurological disorders. Recently, six missense vari- University Hospitals NHS Foundation ants in GABRA2, a member of this family, have been associated with early infantile Trust, Cambridge Biomedical Campus, Cambridge, UK epileptic encephalopathy (EIEE). We identified a novel de novo missense variant 3European Molecular Biology Laboratory, in GABRA2 in a patient with EIEE and performed protein structural analysis of the European Bioinformatics Institute, seven variants. Wellcome Genome Campus, Hinxton, . Cambridge, UK Methods: The novel variant was identified by trio whole-genome sequencing We 4Department of Medical Genetics, performed protein structural analysis of the seven variants, and compared them to Cambridge Institute for Medical Research, previously reported pathogenic mutations at equivalent positions in other Cys-loop University of Cambridge, Cambridge, UK receptors. Additionally, we studied the distribution of disease-associated variants in 5Developmental Neurosciences, Great the transmembrane helices of these proteins. Ormond Street Institute of Child Health, University College London, London, UK Results: The seven variants are in the transmembrane domain, either close to the de- 6Hospital Dona Estefânia, Centro Hospitalar sensitization gate, the activation gate, or in inter-subunit interfaces. Six of them have de Lisboa Central, Lisbon, Portugal pathogenic mutations at equivalent positions in other Cys-loop receptors, emphasiz- Correspondence ing the importance of these residues. Also, pathogenic mutations are more common F. Lucy Raymond, NIHR BioResource, in the pore-lining helix, consistent with this region being highly constrained for vari- Cambridge University Hospitals NHS ation in control populations. Foundation Trust, Cambridge Biomedical Campus, Cambridge CB2 0QQ, UK. Conclusion: Our study reports a novel pathogenic variant in GABRA2, characterizes Email: [email protected] the regions where pathogenic mutations are in the transmembrane helices, and under- Funding information scores the value of considering sequence, evolutionary, and structural information as This work was supported by the National a strategy for variant interpretation of novel missense mutations. Institute for Health Research England (NIHR) for the NIHR BioResource KEYWORDS project (grant number RG65966) and the Cys-loop receptor, epileptic encephalopathy, GABRA2, protein structural analysis, whole-genome Cambridge Biomedical Research Centre. sequencing Alba Sanchis-Juan and Marcia A Hasenahuer are contributed equally to this work. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc. Mol Genet Genomic Med. 2020;00:e1106. wileyonlinelibrary.com/journal/mgg3 | 1 of 15 https://doi.org/10.1002/mgg3.1106 2 of 15 | SANCHIS-JUAN ET AL. 1 | INTRODUCTION Anderson, & Kearney, 2016), and increased expression ob- served in Pumilio-2–deficient mice resulted in enhanced sei- The dynamic partnership between excitatory principal cells zure susceptibility and the manifestation of epilepsy in the and inhibitory interneurons is essential for proper brain func- hippocampus (Follwaczny et al., 2017). GABRA2 is highly tion and needs to be maintained to avoid pathological con- expressed in the hippocampus, especially in early develop- sequences. Cys-loop ligand-gated ion channels are receptors ment, and is localized to the cell soma to mediate synaptic activated by neurotransmitters and play an important role transmission (Prenosil et al., 2006; Tian, Chen, Cross, & in the development and activity of the central and periph- Edenberg, 2005). Recently, six missense variants in GABRA2 eral nervous systems (Thompson, Lester, & Lummis, 2010). have been reported to cause early infantile epileptic enceph- These receptors mediate excitatory and inhibitory synaptic alopathy (EIEE) (Butler et al., 2018; Maljevic et al., 2019; transmissions depending on the distribution of ions at either Orenstein et al., 2018): five were de novo and one was present side of the membrane and the membrane potential of the cell. in two affected siblings, inherited from a father who also pre- There are two broad classifications of Cys-loop receptors, sented the variant at low level in blood, indicating mosaicism. each with different electrophysiological properties: firstly, Here, we describe the seventh variant in GABRA2 to be cation-selective receptors, corresponding to nicotinic ace- reported as de novo in an individual with EIEE identified tylcholine (nACh), serotonin (5-HT3), and zinc-activated re- by whole-genome sequencing (WGS). We perform protein ceptors and secondly, anion-selective members, that include structural mapping and analysis of all the seven variants in glycine (Gly) receptor and γ-aminobutyric acid receptors the GABAAR α2 and investigate the effect of the novel vari- type A (GABAA) and C (GABAC) (Lester, Dibas, Dahan, ant in the protein. We analyze the presence of variants in Leite, & Dougherty, 2004; Thompson et al., 2010). equivalent positions in other members of the Cys-loop recep- Cys-loop receptors are pentameric channels formed by tor family and compare their reported effect. Furthermore, five chains, each comprising an extracellular domain (where because the seven variants cluster in the TM domain, we also the ligand binds) and a transmembrane (TM) domain consist- analyze the distribution of previously reported pathogenic ing of four TM helices (TMH), M1 to M4 (Thompson et al., variants of the Cys-loop receptors. Our results demonstrate 2010). The pentamers can be assembled from different gene the utility of performing variant interpretation by gathering products that encode different subunits, but all belong to the together sequence, evolutionary, and structural information same type of receptors. For example, for the Cl−-selective from homologous Cys-loop receptors to facilitate the charac- GABAA receptors (GABAAR), there are 19 genes encoding terization of novel candidate missense variants. different subunits (α1-α6, β1-β3, γ1-γ3, ρ1-ρ3, δ, ε, π, θ) (Gonzalez-Nunez, 2015; D. D. Wang & Kriegstein, 2009), and the most abundant complex in adult human receptors is 2 | MATERIALS AND METHODS a combination of two α, two β, and one γ subunit (Sigel & Steinmann, 2012). Cys-loop receptors are conserved in hu- 2.1 | Ethical compliance mans and across species (Nys, Kesters, & Ulens, 2013; Ortells & Lunt, 1995). Dysfunctions of these receptors demonstrate a The proband and both unaffected parents were recruited to critical role in neurological development. Pathogenic variants the NIHR BioResource research study. The study was ap- in genes encoding receptors of nACh (CHRNA2, CHRNA4, proved by the East of England Cambridge South national and CHRNB2) and GABAA (GABRA1, GABRB1, GABRB2, institutional review board (13/EE/0325). The research con- GABRB3, GABRG2, and GABBR2) have previously been as- forms with the principles of the Declaration of Helsinki. All sociated with human epilepsy (Shinichi Hirose, 2014), and participants provided written informed consent to participate mutations in Gly receptors (GLRA1 and GLRB) cause hyper- in the study. ekplexia (Al-Owain et al., 2012; Shiang et al., 1993). For many years, GABRA2 (MIM: 137,140), that encodes for the GABAAR subunit α2 (GABAAR α2), remained as 2.2 | Genomic analysis a candidate gene for epilepsy. Multiple genome-wide as- sociation studies identified noncoding single-nucleotide WGS was performed on DNA extracted from whole blood at polymorphisms in GABRA2 associated with increased 30x coverage using Illumina HiSeq X Ten system (Illumina, risk for epilepsy (International League Against Epilepsy Inc., San Diego, CA, USA) with 150bp paired-end reads. Consortium on Complex Epilepsies, 2014, 2018), as well Reads were aligned to the human genome of reference as alcohol dependence and brain oscillations (Edenberg et GRCh37 using Isaac Aligner, and single-nucleotide variants al., 2004; Strac et al., 2015). Also, decreased expression of and indels were called with both Isaac Variant Caller (Raczy GABRA2 in Scn1a ± mice served as a model of Dravet syn- et al., 2013) and Platypus (http://github.com/andyrimmer/ drome (Follwaczny et al., 2017; Hawkins, Zachwieja, Miller, Platypus). Variant annotation was performed with Variant SANCHIS-JUAN ET AL. | 3 of 15 Effect Predictor (McLaren et al., 2016), which included al- 2002). The electrostatic surface visualization was done using lelic population frequencies from gnomAD (release 2.1.1) its APBS plugin (Baker, Sept, Joseph, Holst, & McCammon, (Lek et al., 2016) and deleteriousness scores from CADD 2001; Dolinsky et al., 2007). Residue