CGREF1基因调控MAPK信号通路在芳香族抗癫痫药诱发的严重皮肤不良反应中的作用及分子机制研究

Aromatic antiepileptic drugs (AEDs) induced severe cutaneous drug hypersensitivities mainly include SJS and TEN, with the mortality up to 50%. The pathogenesis is unclear, it is considered that genetic susceptibility and innate immune response together contributed to apoptosis and necrosis of keratinocytes, which lead to the occurrence of SJS/TEN. It is reported that AEDs induces innate immune response through mitogen-activated protein kinase (MAPK) signaling pathways in HL-60 cells, a human DC-like cell lines, and the novel secretory protein CGREF1 inhibits the phosphorylation of MAPK pathway. Preliminary study about 4 cases of AEDs-SJS/TEN, with negative HLA alleles, identified CGREF1 gene mutations through all exon sequencing and bioinformatics analysis. Our next study plans are to expand the samples and verify the CGREF1 gene mutations in patients with AEDs-SJS/TEN; to extract peripheral blood mononuclear cells (PBMC) and dendritic cells (DC) and culture in vitro with different concentration of AEDs. Then, we will detect level of MAPK by western blotting; the immune cells functions by flow cytometric methods, and cytokine levels by RT-PCR. Our aim is to identify the effect and molecular mechanisms of CGREF1 gene regulated MAPK signaling pathway in AEDs-SJS/TEN, to provide the basis and proof for individualized and accurate medical care and genetic biomarkers for screening and prevention of AEDs-SJS/TEN.

芳香族抗癫痫药(AEDs)引发严重皮肤药物超敏反应主要包括SJS和TEN，致死率高达50%。目前认为遗传因素和固有免疫共同导致角质形成细胞凋亡坏死是SJS/TEN发病原因。文献报道AEDs经MAPK信号通路活化树突状细胞(DC)而激活固有免疫反应可导致SJS/TEN，而新型分泌蛋白CGREF1可抑制MAPK通路。前期研究针对4例HLA特定等位基因型阴性SJS/TEN患者经全外显子测序及生物信息分析确定CGREF1基因突变。本课题拟扩大样本量行一代测序验证AEDs-SJS/TEN患儿CGREF1基因突变，并提取外周血单个核细胞和DC加入不同浓度AEDs体外培养，通过蛋白印迹法检测MAPK通路激酶水平，流式细胞术检测免疫细胞功能，RT-PCR方法检细胞因子水平，验证CGREF1基因调控MAPK信号通路在SJS/TEN发病机制中作用和具体分子机制，为精准医疗提供依据，为筛查和预防提供遗传标记物。

研究背景：芳香族抗癫痫药引发重症药疹（AEDs-SCARs）主要包括SJS/TEN和DRESS，致死率最高可达50%。目前认为遗传因素和固有免疫共同导致体内适应性免疫活化经Th/Tc细胞介导发病。有文献报道药物可通过MAPK信号通路诱导DC细胞活化参与SCARs发生，而新型分泌蛋白CGREF1可抑制MAPK信号通路，结合课题组前期研究发现特定HLA等位基因型阴性AEDs-SCARs患者中存在的CGREF1基因突变可能与AEDs-SCARs有关，因此，本次研究扩大样本量验证AEDs-SCARs患者CGREF1基因突变情况以及AEDs与CGREF1基因调控MAPK信号通路在AEDs-SCARs发病机制中的作用。.研究内容：①扩大样本量进行AEDs-SCARs相关HLA等位基因型和CGREF1基因突变检测；②选取人DC细胞系HL-60细胞体外培养后，进行CGREF1突变基因的功能检测及AEDs刺激后检测细胞功能和MAPK信号通路变化。.研究结果：①研究发现HLA-A*03:01:01:01,-A*30:01:01,-B*07:02:01, -B*13:01:01和-C*06:02:01共5个等位基因可能是AEDs-SCARs的易感基因或与易感基因相连锁；②24例患者均发现CGREF1基因上有低频突变，最多见的为c.759C＞T, p. P253P、c.745G＞A, p. E249K和c.375C＞G, p. Thr125Thr；不排除此三个变异可导致基因功能异常后可与发病相关；③四种AEDs中，卡马西平体外孵育HL-60细胞可激活ERK，抑制p38 MAPK，具有诱导DC细胞活化并促进后续免疫反应向Th2方向发展可能；苯妥英钠体外孵育HL-60细胞则激活JNK，抑制ERK，具有诱导DC细胞活化并促进后续免疫反应向Th1方向发展可能；奥卡西平和拉莫三嗪则对MAPK信号通路作用尚不确定。.研究意义：以上结果说明不同AEDs可通过MAPK信号通路的不同活化途径诱导DC细胞活化并促进后续免疫反应向不同的Th/Tc方向发展参与AEDs-SCARs发病，为进一步探索重症药疹免疫机制和治疗方法提供了新的理论依据；特定HLA等位基因型和CGREF1可能是中国汉族人群中AEDs-SCARs发病的易感基因，为进一步转化为临床用药前筛查和疾病预防提供了理论基础。