Table_1_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.xlsx

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.

携带α2亚基（GABRA2）的γ-氨基丁酸A型（GABA type-A, GABA-A）受体在多数脑区均有表达，对抑制性突触功能的调控发挥关键作用。GABRA2基因座的遗传变异与癫痫、情感障碍及精神疾病、酒精成瘾和药物滥用存在明确关联。Gabra2的表达水平随基因型而异，且在多种脑结构与种群中受序列变异调控，其中包括源自C57BL/6J（B6J）的F2杂交群体，以及由B6J与DBA/2J衍生的BXD重组近交系家族。本研究证实，相较于其他近交品系小鼠，B6J品系脑内GABRA2的蛋白质与mRNA水平呈全局性降低，并成功鉴定并验证了B6J中的致病性突变。该突变为单碱基缺失（single base pair deletion），位于靠近剪接受体位点（splice acceptor site）的内含子（intron）区域，且仅存在于B6J参考基因组中。该缺失于1976年至1991年间在B6J品系中固定，如今已广泛存在于多种工程化品系、1991年后构建的BXD重组近交系品系、协作杂交（Collaborative Cross）群体，以及多数同染色体替换系（consomic lines）中。在B6J遗传背景下，利用CRISPR-Cas9介导的基因编辑修复该缺失后，脑内GABRA2的蛋白质与mRNA水平完全恢复至正常水平。对B6J与修复后基因型小鼠的海马（hippocampus）、大脑皮层（cortex）及纹状体（striatum）中的转录本（transcript）表达进行比较后发现，GABA-A受体亚基的表达发生了显著改变，尤以纹状体最为突出。上述结果提示，B6J与其他亚系或近交品系之间天然存在的GABRA2水平差异，或许也可解释与纹状体功能相关的焦虑样行为、酒精及药物反应相关性状的品系间差异。对Gabra2基因中B6J品系特有的突变的表征，对神经生物学研究中的分子遗传学研究具有至关重要的意义：该品系不仅被广泛用于构建基因工程小鼠及小鼠遗传研究群体，同时也是评估焦虑样、抑郁样、疼痛、癫痫及药物反应相关性状的最常用实验品系——而这些性状可能部分受GABRA2功能调控。