SEC16A基因突变在慢性胰腺炎中的作用及其机制研究

Chronic pancreatitis (CP) is a persistent inflammatory disease of the pancreas, characterized by abdominal pain, pancreatic stones, steatorrhea, and diabetes. The pathogenesis of CP remains unclear currently. Most Chinese patients with CP are idiopathic, accounting for over 70% of the total. Genetic susceptibility has emerged during the last two decades as a strong determinant of disease risk. However, Chinese Han population has a distinct genetic background from the Caucasian. To identify Chinese-specific CP susceptibility genes, we performed whole exome sequencing in 16 trios of teenager CP and found one patient carrying a frameshift mutation in Sec16A gene. Further targeted sequencing were validated in idiopathic CP patients and controls, showing high frequency of non-synonymous mutations in cases (43.6%, OR=2.3). Based on the preliminary studies, we speculated that Sec16A gene mutations may indirectly regulate the function of CFTR and other molecular factors to affect the function of acinar cells, thus promoting the occurrence of CP. To evaluate the effects of Sec16A mutations on pancreatic functions and further explore the possible mechanisms of gene regulation, we prepared to construct in vitro cell lines carrying selected Sec16A mutations using CRISPR-Cas9, co-transfected with expression plasmids of pancreatic secretory proteins. Then we perform similar protein tests in fibroblasts and pancreatic tissues of mutation-carrying patients. Finally, we would perform the association analysis of genotype and clinical phenotype, to evaluate the effects of mutations on the disease course, thus providing novel targets for the early detection and treatment of CP.

慢性胰腺炎（CP）是一种胰腺持续性炎症性疾病，临床表现为腹痛、结石、脂肪泻、糖尿病等。我国大部分CP患者病因不明，发病机制不清，已知遗传因素起重要作用，东西方遗传背景差异大。为寻找我国CP特有的致病基因，我们前期对青少年三口之家进行全外显子测序，发现一例携带Sec16A基因移码突变，进一步在散发CP患者中进行验证，发现非同义突变在病例中高发（43.6%，OR=2.3）。基于前期研究基础，我们推测Sec16A基因突变可能间接调控CFTR等因子影响腺泡细胞功能，促进CP发生。本课题拟采用体外CRISPR-Cas9技术构建Sec16A突变位点细胞株，共转染腺泡分泌蛋白表达质粒，结合突变携带者的成纤维细胞和胰腺组织实验结果，评估突变对胰腺功能的影响，并进一步探讨该基因调控CP发生的可能机制。通过基因型与临床表型的关联分析，明确突变对疾病进程和临床疗效的影响，以期为CP早期检测和治疗提供新的靶点。

慢性胰腺炎（chronic pancreatitis，CP）是一种胰腺的进行性炎症性疾病，先前的研究大多聚焦于胰蛋白酶异常激活，很少有人关注蛋白酶激活途径上游调节基因的作用。我们对20个青少年trios进行了全外显子组测序，在 SEC16A基因中发现一个de novo移码突变，随后我们分析了1061名CP患者和1196名对照中SEC16A基因的完整编码序列，并确定了99名（9.33％）携带非同义突变的患者和70名（5.85％）携带了41种错义突变的对照（P = 0.0017，or值= 1.66（95％CI 1.20–2.26）。Kaplan-Meier模型分析发现SEC16A突变阳性患者的CP发病年龄中位数比变异阴性患者早5年。我们通过CRISPR/Cas9技术将病例中富集的前2个罕见错义突变（p.R1495Q，p.V1947L）和作为对照的一种常见的多态性（p.R1039C）导入工具细胞进行功能学研究。我们发现与对照和野生型相比，导入p.R1495Q和p.V1947L突变的细胞中的淀粉酶和胰蛋白酶原分泌完全丧失，并引起细胞内蛋白超载，并且p.R1495Q和p.V1947L突变导致COPⅡ囊泡生成失败并影响随后的分泌途径。进一步通过使用CRISPR/Cas9基因编辑技术构建出Sec16a敲除小鼠，发现Sec16a+/−小鼠中Sec16a蛋白的表达显著降低，急性胰腺炎模型结果显示，Sec16a+/−小鼠胰腺炎的相较于野生小鼠更为严重，TUNEL染色示Sec16a+/−小鼠存在更多腺泡细胞凋亡；慢性胰腺炎模型结果提示，Sec16a+/−小鼠的胰腺腺泡细胞显著萎缩、存在更高水平的胶原沉积、中等程度的星状细胞活化、巨噬细胞浸润及腺泡导管化生。最后，我们证明p.R1495Q和p.V1947L突变引起内质网（ER）应激，这是对ER中蛋白质超载的反应，而不是诱导蛋白质错误折叠的结果。上述结果表明，SEC16A变异与CP发生显著相关，可能通过诱导功能丧失后诱导ER-高尔基体转运阻滞和ER应激新机制，增加CP风险。