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

Abstract

32GABA type-A (GABA-A) receptors containing the α2 subunit (Gabra2) are expressed in most brain regions and are 33 critical in modulating inhibitory synaptic function. Genetic variation at the GABRA2 locus has been implicated in epilepsy, 34 affective and psychiatric disorders, alcoholism and drug abuse. Gabra2 expression varies as a function of genotype and is 35 modulated by sequence variants in several brain structures and populations, including F2 crosses originating from 36 C57BL/6J (B6J) and the BXD recombinant inbred family derived from B6J and DBA/2J. Here we demonstrate a global 37 reduction of Gabra2 brain mRNA and protein in the B6J strain relative to other inbred strains, and identify and validate 38 the causal mutation in B6J. The mutation is a single base pair intronic deletion located adjacent to a splice acceptor site 39 that only occurs in the B6J reference genome. The deletion became fixed in B6J between 1976 and 1991 and is now 40 pervasive in many engineered lines, BXD strains generated after 1991, the Collaborative Cross, and the majority of 41 consomic lines. Repair of the deletion using CRISPR-Cas9 mediated gene editing on a B6J genetic background completely 42 restored brain levels of Gabra2 mRNA and protein. Comparison of transcript expression in hippocampus, cortex, and 43 striatum between B6J and repaired genotypes revealed alterations in GABA-A receptor subunit expression, especially in 44 striatum. These results suggest that naturally occurring variation in GABRA2 levels between B6J and other substrains or 45 inbred strains may also explain strain differences in anxiety-like or alcohol and drug response traits related to striatal 46 function. Characterization of the B6J private mutation in the Gabra2 gene is of critical importance to molecular genetic 47 studies in neurobiological research as this strain is widely used to generate genetically engineered mice and murine genetic 48 populations, and is the most widely utilized strain for evaluation of anxiety-like, depression-like, pain, epilepsy, and drug 49

Expression of the Neuronal tRNA n-Tr20 Regulates Synaptic Transmission and Seizure Susceptibility

“…136 genes were differentially expressed between the B6N and B6J strains independent of n-Tr20 genotype and intriguingly, they were enriched for synaptic localization (Figure 4A; Table S3D and S3E). SNPs and indels have been found between B6J and B6N mice ll Article in some of these genes (Table S3F), including an intronic mutation in Gabra2 in B6J mice that disrupts its splicing, resulting in reduced expression (Mulligan et al, 2019). These data suggest that differential expression of synaptic genes between the B6J and B6N strains leads to different neurophysiological setpoints and likely underlies the different effects of n-Tr20 deletion on synaptic physiology on the two genetic backgrounds.…”