GABRA1 uORFs和IRES多态影响卡马西平抗癫痫疗效的机制与临床研究

Carbamazepine (CBZ) is a broad-spectrum anti-epileptic drug, which was reported to potentiate the function of GABAA receptor and inhibit neurotransmission, indicating that GABAA receptor may be involved in carbamazepine treatment for epilepsy. The α1 subunit (GABAR α1, encoded by GABRA1) of GABA receptor plays a major role in its assembly and function. Mutations in GABRA1 are closely related with the incidence and development of epilepsy. But the mechanism, by which GABRA1 affects CBZ's antiepileptic efficacy, is unclear. Our previous studies showed that upstream open reading frames (uORFs) of the GABRA1 5'UTR regulated gene translation. We also found that the 5'UTR included IRES structure，another regulatory factor for translation. Based on these findings, we propose that the polymorphisms of uORFs and IRES in GABRA1 gene affect CBZ's anti-epileptic efficacy through regulating the GABRA1 expression. By conducting cellular, animal, and clinical research, the current project intends to verify the vital role of GABRA1 in the incidence and development of epilepsy, and to investigate how the polymorphisms of the uORFs and IRES influence the expression of the GABRA1 gene and CBZ's antiepileptic efficacy. These studies will clarify the mechanism and genetic basis of the effect of GABRA1 gene on individual differences in CBZ's antiepileptic efficacy.

卡马西平(CBZ)为广谱抗癫痫药，文献报道可强化GABAA受体的功能和抑制性神经传递的作用，表明GABAA受体可能参与CBZ对癫痫疗效的作用。该受体α1亚基在其组装及功能起主要作用，其编码基因GABRA1突变与癫痫发生发展密切相关，对CBZ抗癫痫疗效的影响机制尚不清楚。我们前期研究表明，GABRA1的5'UTR上游开放阅读框(uORFs)可调控基因的翻译，并发现该序列具有另一调控因子IRES结构。据此，我们提出GABRA1的uORFs和IRES多态性通过调控GABRA1的表达而影响CBZ抗癫痫疗效的个体化差异。本项目拟通过细胞、模型动物及临床研究，验证GABRA1在癫痫发生发展的重要作用，研究GABRA1的uORFs和IRES多态性对表达的影响，进而对CBZ抗癫痫疗效产生差异。提供GABRA1影响CBZ抗癫痫疗效个体化差异的机制，揭示其影响卡马西平抗癫痫疗效差异的遗传学基础。

本项目研究GABRA1基因5’-非翻译区（5’-untranslated region，5’-UTR）多态性对卡马西平（carbamazepine, CBZ）抗癫痫疗效的机制和临床的影响。研究发现：1. CBZ作用靶点SCN1A和GABAA受体亚型多态性与癫痫易感性相关。SCN1A基因的rs3812718多态性与部分发作显著相关；GABRA1受体亚型的单倍体AC（rs1265883-rs1129647）与混合类型癫痫发作显著相关。SCN1A单倍型GAGC（rs1813503-rs10497275-rs1020853-2298771）与部分发作癫痫和复杂发作癫痫易感性相关。单倍型CC和TG（rs3812718 -rs4667869）与混合类型癫痫发作相关。2. GABRA1基因5’-UTR 上游开放阅读框（uORFs）可影响该基因的表达，从而影响药物的临床疗效。GABRA1受体5’-UTR含有至少9个uORFs，其中rs11576001、rs12658835和rs4608967位点多态性理论上可影响uORFs编码的短肽并影响GABRA1表达水平，并进而影响CBZ的疗效。临床上我们发现rs11576001多态性可显著影响CBZ的疗效，体外研究证实了该位点可显著影响GABRA1的转录和翻译水平。3. 常规抗癫痫药物如苯妥因钠、丙戊酸钠等对卡马西平和奥卡西平代谢酶CYP3A4/5和ABCB1的活性有显著影响，对CBZ血药浓度的产生显著影响并影响临床疗效。4. 动物模型SD大鼠海马神经元细胞的膜受体GABRA1高表达和沉默时，卡马西平对海马神经元钠电流具有显著影响。