SERPINA3基因突变致肌间神经丛兴奋性/抑制性神经失衡在贲门失弛缓症发病中的作用及其机制

Idiopathic achalasia is a rare disease with unclear etiology. Previous studies demonstrated that genetic susceptibility is associated with achalasia potentially via imbalance of activities between inhibitory and excitatory enteric neurons. However, the key susceptibility genes for achalasia are still largely unknown due to lack of systematic studies for genome-wide searching. As one of the major clinics for achlasia in China, our plenty of clinical samples make the genome-wide searching possible. In the preliminary study, we performed whole-exome sequencing of 100 Chinese Han achalasia patients, and identified that SERPINA3 gene may be a new susceptibility gene for achalasia. In this study, we aim to: ①identify the mutation frequency of SERPINA3 gene in large-scale samples using Sanger sequencing method; ②demonstrate the expression and histologic distribution of SERPINA3 in achalasia, and evaluate its predictive value for the prognosis in patients after peroral endoscopic myotomy (POEM); ③clarify the underlying molecular mechanisms of SERPINA3 gene mutation on the pathogeny (especially on imbalance of activities between inhibitory and excitatory enteric neuronses) using several molecular biological methods in vitro, in SERPINA3-deficient rat, as well as in achalasia patients. Accordingly, these findings will give new evidence for the etiology, and provide a potential target for etiological treatment of this rare disease.

贲门失弛缓症是一种病因不明的罕见病。遗传易感性导致的肠肌间神经丛兴奋性/抑制性神经失衡可能与其发病相关，但对致病的关键基因缺乏全面搜寻和系统研究。课题组是国内最大的贲门失弛缓症诊治中心之一，利用已建立起的大规模病例库，前期对100例患者采用全外显子组测序发现SERPINA3为贲门失弛缓症新的潜在发病易感基因。在此基础上，我们拟：①扩大样本量，采用Sanger测序法明确SERPINA3基因在贲门失弛缓症患者中的突变频率；②明确SERPINA3在贲门失弛缓症患者中的表达水平及组织学分布，评价其表达水平对经口内镜下肌切开（POEM）治疗疗效的预测价值；③在细胞层面、突变体动物模型和临床层面，结合系列分子生物学实验技术对SERPINA3基因突变致兴奋性/抑制性神经失衡进行后续验证，并阐明其致病的分子机制。由此为揭示此罕见病的病因提供新的依据，并为将治疗措施由目前的对症转向对因提供潜在靶点。

贲门失弛缓症是一种病因不明的罕见病。遗传易感性导致的肠肌间神经丛兴奋性/抑制性 神经失衡可能与其发病相关，但对致病的关键基因缺乏全面搜寻和系统研究。课题组是国内最 大的贲门失弛缓症诊治中心之一，利用已建立起的大规模病例库，前期对100例患者采用全外 显子组测序发现SERPINA3为贲门失弛缓症新的潜在发病易感基因。然而我们构建了Serpina3 基因敲除的纯合小鼠及同窝野生型小鼠，定期进行体重测定及上消化道钡剂造影，截至6月龄处死时，未出现贲门失弛缓症表型，且两组体重无明显差异。后续食管下段免疫荧光染色及western blot测定兴奋性神经元及抑制性神经元分布均无明显差异，提示Serpina3敲除对食管下段括约肌的神经元分布无影响，Serpina3基因敲除无法出现贲门失弛缓症。课题组继续通过家系、散发病例的测序及验证确定了ADAR、ADCY5和SECISBP2三个关键候选致病基因。SECISBP2（Sec Insertion Sequence-Binding Protein 2）基因。Secisbp2是胚胎发育和增强硒蛋白表达所必需的。人类SECISBP2杂合突变会导致硒蛋白表达降低和功能丧失。而硒蛋白与神经退行性疾病密切相关。鉴于贲门失弛缓症和神经退行性疾病在发病机制上的共通性，我们进一步探讨了SECISBP2基因可能在贲门失弛缓症发病中发挥的作用。基因敲除动物模型研究发现Secisbp2 基因缺失可能会引起贲门失弛缓症表型，并猜想可能机制是通过导致Sepp1合成障碍影响ROS清除引起神经元损伤导致贲门失弛缓症发生，机制验证正在进行，研究结果将为阐明贲门失弛缓的发病机制提供新的线索和理论依据。