Identification of a Functional Non-coding Variant in the GABA A Receptor α2 Subunit of the C57BL/6J Mouse Reference Genome: Major Implications for Neuroscience Research.

Megan K Mulligan,Jesse Ingels,Robert W Williams,Xusheng Wang,Cory Parks,M Trevor Houseal,Thomas M Shapaker,Lu Lu,Christine E Watkins,Camron D Bryant,Junmin Peng,Catherine C Kaczorowski,Timothy Abreo,Michael Hook,Haiyan Tan,Sarah M Neuner,Gregg E Homanics

doi:10.3389/fgene.2019.00188

Abstract

GABA type-A (GABA-A) receptors containing the α2 subunit (GABRA2) are expressed in most brain regions and are critical in modulating inhibitory synaptic function. Genetic variation at the GABRA2 locus has been implicated in epilepsy, affective and psychiatric disorders, alcoholism and drug abuse. Gabra2 expression varies as a function of genotype and is modulated by sequence variants in several brain structures and populations, including F2 crosses originating from C57BL/6J (B6J) and the BXD recombinant inbred family derived from B6J and DBA/2J. Here we demonstrate a global reduction of GABRA2 brain protein and mRNA in the B6J strain relative to other inbred strains, and identify and validate the causal mutation in B6J. The mutation is a single base pair deletion located in an intron adjacent to a splice acceptor site that only occurs in the B6J reference genome. The deletion became fixed in B6J between 1976 and 1991 and is now pervasive in many engineered lines, BXD strains generated after 1991, the Collaborative Cross, and the majority of consomic lines. Repair of the deletion using CRISPR-Cas9-mediated gene editing on a B6J genetic background completely restored brain levels of GABRA2 protein and mRNA. Comparison of transcript expression in hippocampus, cortex, and striatum between B6J and repaired genotypes revealed alterations in GABA-A receptor subunit expression, especially in striatum. These results suggest that naturally occurring variation in GABRA2 levels between B6J and other substrains or inbred strains may also explain strain differences in anxiety-like or alcohol and drug response traits related to striatal function. Characterization of the B6J private mutation in the Gabra2 gene is of critical importance to molecular genetic studies in neurobiological research because this strain is widely used to generate genetically engineered mice and murine genetic populations, and is the most widely utilized strain for evaluation of anxiety-like, depression-like, pain, epilepsy, and drug response traits that may be partly modulated by GABRA2 function.

Highlights

GABA-A receptors are pentameric ligand gated chloride channels responsible for fast inhibitory neurotransmission
Using a combination of genetic, genomic, functional, and bioinformatics approaches we have identified a private and non-coding single nucleotide deletion in the Gabra2 gene in the B6J strain that results in substantial reduction of brain mRNA and protein levels (Figures 1, 2)
Unlike previous studies in which function of Gabra2 was probed through complete genetic deletion, the mutation associated with the B6J allele of Gabra2 is hypomorphic and not a complete null

Summary

Introduction

GABA-A receptors are pentameric ligand gated chloride channels responsible for fast inhibitory neurotransmission. Over 500 common human variants have been identified at the GABRA2 locus, including numerous intronic variants, a handful localized to the UTRs, and one synonymous variant in exon 4 Genetic variation at this locus has been linked to alcohol dependence (Covault et al, 2004; Edenberg et al, 2004; Lappalainen et al, 2005; Agrawal et al, 2006; Enoch et al, 2006; Fehr et al, 2006; Lind et al, 2008a,b; Soyka et al, 2008; Villafuerte et al, 2013; Li et al, 2014), the subjective effects of alcohol (Pierucci-Lagha et al, 2005; Roh et al, 2011; Uhart et al, 2013; Arias et al, 2014), excess EEG fast activity (Lydall et al, 2011), cocaine reward (Dixon et al, 2010), substance dependence (Enoch et al, 2010), epilepsy (International League Against Epilepsy Consortium on Complex Epilepsies, 2014), and impulsivity and insula cortex activity during reward or loss anticipation (Villafuerte et al, 2013). The impact of these variants on expression, isoform usage, or function of GABRA2 protein or mRNA is unclear (Myers et al, 2007; Heinzen et al, 2008; Gibbs et al, 2010; Liu et al, 2010; Kim et al, 2014; Lieberman et al, 2015)

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