GCH1基因新发错义突变对多巴反应性肌张力障碍(DRD)的致病作用及机制研究

Dopa-responsive dystonia (DRD) is a group of rare monogenic hereditary disease. The affected children present with severe motor disorders. Mutations in GCH1 gene are the most common cause of DRD, which characters as incomplete dominance hereditary. Recently, we detected a novel mutation p.L117R of the GCH1 gene in a patient by next-generation sequencing and further confirmed by Sanger sequencing. Bio-information, sequence alignment, protein modeling and functional prediction, Q-PCR all showed that the GCH1 gene expression level and GTPCH function were defects, which indicated this novel mutation might be pathogenic. In present study we are generating a knock-in mouse model of DRD that recapitulates the human novel GCH1 mutation by CRISPR-Cas9 technology, and first to investigate motor performance, GCH1 gene transcription, translation and post-translational modification, enzymatic activity, neurotransmitter metabolites, the pathology of dopaminergic neurons and then to observe its response to dopa intervention in heterozygous, homozygous and wild-type mice. Our study will be the first one of generating the GCH1 mutation knock-in mouse model and revealing the pathogenesis of GCH1 p.L117R novel mutation in DRD from function phenotype, cell and molecular levels, which will bring a new way to prevention and treatment of DRD.

多巴反应性肌张力障碍（DRD）是一组罕见单基因遗传病，患儿运动能力严重丧失。具不完全外显性遗传特征的GCH1基因突变导致编码蛋白GTPCH异常是常见病因。我们近期通过二代测序首次在DRD病人中发现GCH1新错义突变p.L117R。前期序列比对、蛋白结构模拟、功能预测等生物信息学分析和Q-PCR实验，均表明该新突变可引起GCH1表达和GTPCH功能变化，提示其为新的致病突变位点。本项目中，我们正在采用最新的CRISPR-Cas9编辑技术，构建该新突变敲入小鼠模型，拟首先研究杂合、纯合和野生型小鼠的运动功能、脑GCH1转录、翻译及修饰、酶活性、神经递质、神经元病理等变化，然后给予多巴药物干预，再观察运动功能变化，从功能表型、细胞、分子机理及药理等不同层次深入阐明新突变在DRD发病进程中的作用，进一步揭示其分子致病机制。本研究有望在国际上率先获得GCH1突变敲入小鼠模型，为DRD防治提供新策略。

本项目组发现一个带GCH1基因新突变p.L117R家系，先证患儿及其母亲均为该新发突变杂合子，患儿表现为抬头无力、运动发育迟缓等多巴反应性肌张力障碍DRD的典型症状，患儿母亲无相关症状。 GCH1基因编码的GTP环化水解酶GTPCH是四氢生物喋呤BH4生成途径中的起始酶和限速酶，而BH4是多巴胺合成途径中不可缺少的辅酶。因此GCH1基因突变所致GTPCH缺陷症可引起DRD等症状。为明确GCH1基因新突变p.L117R致DRD的作用及机制，进行了实验探索：①利用CRISPR/Cas9技术构建模拟患者GCH1基因新突变p.L117R小鼠模型，分析各基因型幼鼠出生比例及生长情况，发现纯合突变小鼠出生比例下降50%，所有纯合突变在出生后19日内夭折；②进行翻正反射试验、后肢悬吊试验、超声实验、前肢爪抓力实验等行为学试验评估运动、发声功能，发现纯合突变幼鼠表现出与患者类似的婴儿期运动、发声障碍；③比较新突变小鼠肝脏、脑部Gch1基因mRNA、蛋白表达水平；对比GTPCH酶活、BH4水平、相关上游代谢产物水平含量，发现突变小鼠Gch1基因在mRNA、蛋白水平的表达均无明显差异，但纯合子中的GTPCH酶活性降低。纯合子小鼠的肝脏、脑部中均观察到BH4缺乏和苯丙氨酸累积的现象，纯合子脑部中多种神经递质明显降低，代谢紊乱较肝脏更严重；④对纯合子采用苄丝肼-左旋多巴联合治疗能延长纯合子的平均存活时间，说明该动物模型对多巴治疗具有反应性，符合DRD疾病特点。.本研究成功构建首个可存活的模拟人类GTPCH缺陷症临床表现的突变小鼠模型，明确了GCH1基因新突变p.L117R为致病突变，其遗传方式为隐性遗传。纯合突变小鼠脑部有严重的BH4缺乏及苯丙氨酸累积，提示脑部更容易受GCH1基因突变所致BH4缺乏症影响。通过多种药物治疗，发现美多芭与BH4联合治疗能有效提高突变纯合子的生存时间，降低死亡率。