PDF(Pubmed)
Abstract：
GABAB receptors (GABABRs) are G protein-coupled receptors for γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system. Pathogenic variants in the GABBR1 and GABBR2 genes, which encode the GB1 and GB2 subunits of GABABRs, are implicated in several neurological and developmental disorders, including epilepsy and autism. Here we present a 7-year-old boy with Level 3 Autism Spectrum Disorder who carries a de novo heterozygous missense GABBR2 p.Arg212Gln variant. This variant was identified through whole exome sequencing and classified as variant of unknown significance (VUS). Treatment with the GABABR agonist baclofen showed no clinical improvement, raising the question whether this VUS is responsible for the patient’s phenotype. We conducted a study to investigate the impact of the GABBR2 p.Arg212Gln and the previously reported GABBR2 p.Arg212Trp variants on protein structure and receptor activity. This study utilized a combination of molecular dynamics (MD) simulations, and in vitro experiments. Our simulations demonstrate that both amino acid substitutions locally alter amino acid interactions in the extracellular domain of GB2. Most importantly, the substitutions influence the positioning of transmembrane helices, shifting the conformation towards an active state with GABBR2 p.Arg212Gln and an inactive state with GABBR2 p.Arg212Trp. Functional assays confirmed the MD predictions, as evidenced by increased constitutive activity and enhanced potency of GABA for GABBR2 p.Arg212Gln, and a decreased constitutive activity with a loss of GABA potency for GABBR2 p.Arg212Trp. Our findings demonstrate the utility of MD simulations in predicting the functional consequences of VUS. Clarifying the pathogenic mechanisms associated with gene variants will aid in the identification of personalized treatment approaches.
摘要：
GABAB受体（GABABR）是γ-氨基丁酸（GABA）的G蛋白偶联受体，中枢神经系统中主要的抑制性神经递质。GABBR1和GABBR2基因的致病变异，编码GABABR的GB1和GB2亚基，与几种神经和发育障碍有关，包括癫痫和自闭症。在这里，我们介绍了一个患有3级自闭症谱系障碍的7岁男孩，他携带一个从头杂合错义GABBR2p.Arg212Gln变体。通过全外显子组测序鉴定该变体,并将其分类为意义未知的变体(VUS)。GABABR激动剂巴氯芬治疗未显示临床改善，提出了一个问题，即这种VUS是否与患者的表型有关。我们进行了一项研究，以调查GABBR2p.Arg212Gln和先前报道的GABBR2p.Arg212Trp变体对蛋白质结构和受体活性的影响。本研究结合了分子动力学(MD)模拟，和体外实验。我们的模拟表明，两个氨基酸取代都局部改变了GB2胞外域中的氨基酸相互作用。最重要的是,取代影响跨膜螺旋的定位，将构象向GABBR2p.Arg212Gln的活动状态和GABBR2p.Arg212Trp的非活动状态转移。功能测定证实了MD的预测，正如GABA对GABBR2p.Arg212Gln的组成活性增加和效力增强所证明的那样，GABBR2p.Arg212Trp.的组成活性降低，GABA效力丧失。我们的发现证明了MD模拟在预测VUS功能后果方面的实用性。阐明与基因变异相关的致病机制将有助于识别个性化治疗方法。
