原发性甲状旁腺功能亢进症新致病基因的鉴定及GGT1基因突变发病机制研究

The pathogenic genes and molecular pathogenesis of primary hyperparathyroidism (pHPT) is still largely unknown by now. It’s demonstrated that most parathyroid adenomas are monoclonal lesions, which suggest the existence of somatic mutations. To identify novel somatic mutations of pHPT, we have established the specimen bank of pHPT with 50 samples, and whole-exome sequencing (WES) was carried out in 16 blood-adenoma pairs. We identified 7 potentially functional mutated genes, among them, a hotspot somatic C530T/p.A177V mutation in GGT1 was discovered in 3 of 16 sequenced samples. Experimental data suggest that the expression of GGT may promote tumor progression by protecting cells from oxidative stress-associated apoptosis through the Ras signal transduction pathway. Thus, we conjecture that the GGT1 mutation may affect risk of pHPT through the Ras signal transduction pathway. Therefore, we intend to identify novel pathogenic somatic mutations of pHPT using WES strategy and use in vitro experiments and knock-in mouse model to clarify the exact molecular mechanism of the novel pathogenic genes(including the GGT1 gene) in the pathogenesis of pHPT. This study will help reveal the mechanism of pHPT and provide an important basis for research and development of new therapy for pHPT.

迄今散发性原发性甲旁亢的致病基因及分子机制尚未阐明，散发性甲状旁腺瘤多为单克隆性，提示存在体细胞突变。我们已建立原发性甲旁亢样本库共50例，前期对16对外周血和腺瘤组织DNA行全外显子组测序，筛选出7个功能性体细胞突变基因，其中3例样本出现GGT1基因相同位点（A177V）突变。GGT1基因编码γ-谷氨酰转肽酶，保护细胞抵抗氧化应激，抑制细胞凋亡，GGT1过度表达促进肿瘤发生。而GGT表达上调可能与Ras信号通路有关。我们推测GGT1基因突变可能经Ras通路参与了甲状旁腺瘤的发生。为此，本课题拟利用二代测序对50例原发性甲旁亢患者腺瘤组织行体细胞突变基因鉴定，并通过体外细胞实验和突变靶向敲入小鼠整体实验，阐明新致病基因（包括GGT1基因）在调控甲状旁腺细胞增殖及功能中的确切分子机制。本研究有望发现原发性甲旁亢新的致病基因，将为阐明甲旁亢分子机制及后续药物研发等提供重要依据。

原发性甲旁亢(PHPT)是一种相对常见的内分泌疾病，表现为高钙血症、高钙尿症、低磷血症、高磷尿症、肾结石和骨吸收增加等一系列临床症候群，严重者可出现高钙危象，危及患者生命。然而, 国内尚缺乏关于原发性甲旁亢发病率或患病率的确切数据，分子机制也尚未阐明，缺乏特异性的分子标志物，以致早期定性与定位诊断均较困难。为了确定原发性甲旁亢的发病机制,我们对PHPT患者进行血液及肿瘤标本二代测序，鉴定新型致病基因ASXL3，基因CDC73及基因EZH2新型突变位点。为进一步阐述原发性甲旁亢的发病机制,我们构建CDC73基因突变位点质粒，发现该突变位点影响了其编码蛋白表达并影响其在核仁中的定位，并进一步增加了细胞增殖能力及调控细胞周期。此外通过对2005年至2016年间甲旁亢患者进行回顾性分析，明确了甲旁亢近12年内的发病率，及明确无症状及有症状甲旁亢患者临床特征差异。本文首次揭示了原发性甲旁亢新型突变基因及临床特征，为原发性甲旁亢的发病机制提供了新的认识。