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

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.

Polymorphic SNPs, short tandem repeats and structural variants are responsible for differential gene expression across C57BL/6 and C57BL/10 substrains

“…There were 23 genomic features that matched the SDP and thus could explain the gene expression difference. The gene expression was consistent with previous findings that C57BL/6J mice have reduced Gabra2 expression levels (40); in that prior study, an intronic indel adjacent to a splice acceptor site was identified at Chr5:71,014,638. Repairing that SNP restored normal expression (40), demonstrating causality.…”

The expanded BXD family of mice: A cohort for experimental systems genetics and precision medicine

“…The same phenomenon is true of spontaneous mutations that occurred in the 1980s in the C57BL/6J maternal parent that led to the deletion of Nnt exons and a striking reduction in Gabra2 expression in brain 68,72,73 .…”

“…detected a surprisingly large number of new variants (n = 47 out of about 13,000 SNPs) in the set of strains generated by Taylor in the early 1990s (BXD33 though BXD42), and a small number (n = 5) of even more recent mutations in BXDs generated in the late 1990s at UTHSC. While the majority of mutations are neutral or non-functional, we already know that a handful produce interesting differences in gene expression and higher order phenotypes among different phases of the BXD family68,69 .Analysis of expression data from different epochs highlights ~50 genes modulated bystrong cis-acting expression QTLs only in newer strains. For example, introduction of a premature stop codon in glycoprotein nmb (Gpnmb) and deletion of the last coding exon in killer cell lectin like receptor D1 (Klrd1; CD94) occurred spontaneously in the DBA/2J…”

Collaborative Cross Mouse Populations as a Resource for the Study of Epilepsy

“…An indel, rs225241970 located at 71,031,384 bp, was recently identified as a de novo deletion in B6J that significantly reduced gene expression 38 . When repaired using CRISPR-Cas9 editing, it fully restored expression of Gabra2 in B6J mice 38 . This finding is consistent with our observation on Gabra2 expression and sequence variation.…”