Department of Biochemistry, Cell and Systems Biology, ISMIB, University of Liverpool, Liverpool, UK.
Epilepsia Open. 2024 Aug;9(4):1458-1466. doi: 10.1002/epi4.12982. Epub 2024 May 30.
GABA receptor subunit mutations pose a significant risk for genetic generalized epilepsy; however, there are over 150 identified variants, many with unknown or unvalidated pathogenicity. We aimed to develop in vivo models for testing GABA receptor variants using the model organism, Caenorhabditis elegans.
CRISPR-Cas9 gene editing was used to create a complete deletion of unc-49, a C. elegans GABA receptor, and to create homozygous epilepsy-associated mutations in the endogenous unc-49 gene. The unc-49 deletion strain was rescued with transgenes for either the C. elegans unc-49B subunit or the α1, β3, and γ2 subunits for the human GABA receptor. All newly created strains were analyzed for phenotype and compared against existing unc-49 mutations.
Nematodes with a full genetic deletion of the entire unc-49 locus were compared with existing unc-49 mutations in three separate phenotypic assays-coordinated locomotion, shrinker frequency and seizure-like convulsions. The full unc-49 deletion exhibited reduced locomotion and increased shrinker frequency and PTZ-induced convulsions, but were not found to be phenotypically stronger than existing unc-49 mutations. Rescue with the unc-49B subunit or creation of humanized worms for the GABA receptor both showed partial phenotypic rescue for all three phenotypes investigated. Finally, two epilepsy-associated variants were analyzed and deemed to be loss of function, thus validating their pathogenicity.
These findings establish C. elegans as a genetic model to investigate GABA receptor mutations and delineate a platform for validating associated variants in any epilepsy-associated gene.
Epilepsy is a complex human disease that can be caused by mutations in specific genes. Many possible mutations have been identified, but it is unknown for most of them whether they cause the disease. We tested the role of mutations in one specific gene using a small microscopic worm as an animal model. Our results establish this worm as a model for epilepsy and confirm that the two unknown mutations are likely to cause the disease.
GABA 受体亚基突变是遗传性全身性癫痫的重大风险因素；然而，已经鉴定出超过 150 种变体，其中许多具有未知或未经证实的致病性。我们旨在使用模式生物秀丽隐杆线虫开发用于测试 GABA 受体变体的体内模型。
使用 CRISPR-Cas9 基因编辑创建完整缺失 unc-49（秀丽隐杆线虫的 GABA 受体），并在内源性 unc-49 基因中创建纯合性与癫痫相关的突变。unc-49 缺失株通过转基因进行拯救，转基因为线虫 unc-49B 亚基或人类 GABA 受体的 α1、β3 和 γ2 亚基。所有新创建的菌株均进行表型分析，并与现有的 unc-49 突变进行比较。
与三种不同表型测定中的现有 unc-49 突变相比，整个 unc-49 基因座的完全遗传缺失的线虫进行了比较-协调运动、缩小者频率和癫痫样抽搐。完整的 unc-49 缺失表现出运动减少和缩小者频率增加以及 PTZ 诱导的抽搐，但未发现比现有 unc-49 突变表型更强。用 unc-49B 亚基进行拯救或创建人类化的 GABA 受体线虫均显示出所有三种表型调查的部分表型挽救。最后，分析了两种癫痫相关变体，认为它们是功能丧失，从而验证了它们的致病性。
这些发现确立了秀丽隐杆线虫作为研究 GABA 受体突变的遗传模型，并为任何癫痫相关基因中相关变体的验证奠定了平台。
癫痫是一种复杂的人类疾病，可能由特定基因的突变引起。已经鉴定出许多可能的突变，但对于大多数突变，尚不清楚它们是否会引起疾病。我们使用小型微观蠕虫作为动物模型来测试一个特定基因的突变作用。我们的结果确立了这种蠕虫作为癫痫模型，并证实了这两个未知突变很可能导致疾病。
