SPINK1基因内含子突变的鉴定及其在慢性胰腺炎中的致病机制研究

Chronic pancreatitis (CP) is a kind of long-duration complex disease associated with genetic factors. Manifestations of CP include repeatedly abdominal pain, diabetes, and steatorrhea. The exact pathogenesis is still unknown, which may be associated with abnormal activation of pancreatic enzyme. SPINK1 is one of the most studied in susceptibility genes associated with CP, encoding the pancreatic enzyme inhibitory proteins. Our previous study found that the number of intronic mutations in SPINK1 was far more than expected, some of which might be associated with CP disease. Currently, discovery genetics studies tend to focus on exons and may thus miss many intronic mutations, partly due to the lack of functional analysis models. We have constructed wild-type plasmid containing the whole SPINK1 gene whose expression is similar to the condition in vivo. We firstly will perform sequencing the reported mutations of four susceptibility genes in teenager patients with idiopathic CP. Next we will deeply sequence the introns of SPINK1 gene in order to identify new intronic mutations. Then we will construct mutated expression vectors using the wild-type plasmid to explore the pathogenic mechanism of candidate mutations. Finally we expect to classify the clinical significance of the new mutations, thus providing potential early diagnostic markers and therapeutic targets for CP patients.

慢性胰腺炎（CP）是一种与遗传因素相关的复杂疾病，病程较长或伴随终身，临床表现为反复腹痛、糖尿病和脂肪泻等，其发病机制尚不明确，可能与胰酶异常激活有关。SPINK1是目前发现的主要CP易感基因之一，编码胰酶抑制蛋白，我们前期发现其内含子区突变数量较多，部分突变可能与CP致病相关。目前的遗传学研究对内含子突变关注较少，重要原因是缺少成熟的内含子功能分析模型，我们已成功构建与体内情况高度相近的SPINK1野生型质粒。为排除已知突变的影响，本研究首先拟对青少年特发性CP患者进行4个已知易感基因的外显子测序，筛选出不含已知致病突变的患者，然后对其SPINK1整个内含子区进行靶向测序，以期筛选出新的内含子突变；利用前期的野生型质粒构建突变表达载体，深入阐明突变发生mRNA异常剪接或被降解的具体机制，对突变的临床意义进行分类，为CP患者提供潜在的早期诊断标志物和治疗靶点。

慢性胰腺炎（CP）是一种与遗传因素相关的复杂疾病，临床表现为反复腹痛、糖尿病和脂肪泻等，其发病机制尚不明确，可能与胰酶异常激活有关。SPINK1是目前发现的主要CP易感基因之一，编码胰酶抑制蛋白。前期成功收集特发性CP患者（855例）、酒精性CP（206例）和健康人群（1196例）的临床资料和外周血标本，其中青少年特发性CP患者共227例，进行四个CP相关基因的靶向二代测序（SPINK1, PRSS1, CTRC 和CFTR），发现CP组中535例（50.42％）患者检测出罕见致病基因型，而对照组仅有71例（5.94％，OR值为 16.12；P <0.001）。与突变阴性的CP患者相比，突变阳性患者的疾病起病年龄以及胰腺结石、糖尿病和脂肪泻的诊断年龄显著更早。SPINK1致病基因型在ICP患者中携带率占61.5％，是最常见的致病基因。通过上述研究，227例青少年特发性CP患者中，共有52例患者无已知基因突变；对该52例患者进行SPINK1内含子区域的深度靶向测序，共发现16个深度内含子突变。采用> 5％的MAF人群频率过滤，有6个内含子突变被分类为非致病性。组合软件预测剩余10个深度内含子突变和5个数据库中未定义功能的内含子突变，显示c.194+5G>A可能具有功能意义。基于细胞功能实验分析c.194+5G>A以及7种预测的非功能性突变，从而验证了该套流程的准确性和实用性。本研究首次建立一套完整的CP易感基因内含子致病突变筛查流程，为后续进一步开展相关临床转化研究工作做铺垫。申请者以第一/通讯作者（含共同）发表相关SCI论文6篇，IF>10分1篇，获国家发明专利一项、软件著作权2项，入选上海市青年拔尖人才计划。