PRSS1 c.455-33C>T突变在遗传性胰腺炎中的致病性分析  

Pathogenicity of PRSS1 c.455-33C>T mutation in hereditary pancreatitis

作　　者：张雨 李晓宇[2] 王佳[1] 张文清 赵向忠 雷珂[3] ZHANG Yu;LI Xiaoyu;WANG Jia;ZHANG Wenqing;ZHAO Xiangzhong;LEI Ke(Qingdao University Medical College,Qingdao 266071,China)

机构地区：[1]青岛大学医学部,山东青岛266071 [2]青岛大学附属医院消化内科,山东青岛266071 [3]青岛大学医学研究中心,山东青岛266071

出　　处：《青岛大学学报（医学版）》2024年第1期23-27,共5页Journal of Qingdao University(Medical Sciences)

基　　金：国家自然科学基金资助项目(82270676);2021山东省研究生教学改革研究项目(SDYJG21110);青岛市中医药科技项目(2021-zyym26);青岛市医药卫生科研计划项目(2021-WJZD-191)。

摘　　要：目的探讨人阳离子胰蛋白酶原(PRSS1)c.455-33C>T内含子杂合突变在遗传性胰腺炎(HP)中的致病性。方法报告1例反复发作的急性胰腺炎病人,病人携带PRSS1 c.455-33C>T杂合突变,检索文献及网站初步分析其突变。提取健康对照者外周血基因组DNA,通过PCR扩增将PRSS1基因3号内含子序列、4号外显子序列和4号内含子序列与pSPL3载体连接构建质粒,命名为SWT;在SWT质粒的基础上通过定点突变的方法构建PRSS1 c.455-33C>T突变质粒,命名为S33;转染人胚肾细胞(Hek293T)后提取总RNA并进行RT-PCR及凝胶电泳,分析PRSS1 c.455-33C>T内含子突变是否引起PRSS1 mRNA剪切的改变进而引起HP。PCR扩增健康对照者外周血基因组DNA,将PRSS1基因3号内含子序列与增强子分析质粒pGL4.23连接,命名为EWT质粒;并在EWT质粒的基础上构建PRSS1 c.455-33C>T定点突变的质粒,命名为E33;将EWT、E33质粒转染Hek293T后利用双荧光素酶报告基因分析系统进行荧光素酶活性测定,分析PRSS1 c.455-33C>T内含子突变是否通过具有增强子功能引起PRSS1基因功能发生改变进而导致HP。结果检索文献及网站确定PRSS1 c.455-33C>T突变为未报道的功能不明新突变。剪切分析实验显示,SWT和S33两组RT-PCR产物相同;双荧光素酶实验结果显示,E33组的荧光值低于EWT组,差异有统计学意义(t=12.23,P<0.001),E33组与EWT组相比没有增强子功能。结论PRSS1 c.455-33C>T内含子突变不引起PRSS1 mRNA剪切的改变,也不具有PRSS1基因增强子功能,与PRSS1基因直接导致的HP无关。Objective To investigate the pathogenicity of human cationic trypsinogen(PRSS1)c.455-33C>T intron hete-rozygous mutation in hereditary pancreatitis(HP).Methods This article reported a patient with recurrent acute pancreatitis carrying PRSS1 c.455-33C>T heterozygous mutation,which was analyzed based on related literature and websites.Peripheral blood genomic DNA was collected from healthy controls,and PCR amplification was used to connect the intron 3,exon 4,and intron 4 sequences of the PRSS1 gene with pSPL3 vector to construct SWT plasmid.The PRSS1 c.455-33C>T mutant plasmid,named the S33 plasmid,was constructed by site-directed mutagenesis on the basis of SWT plasmid.After human embryonic kidney Hek293T cells were transfected with the S33 plasmid,total RNA was extracted for RT-PCR and gel electrophoresis to investigate whether PRSS1 c.455-33C>T intron mutation caused the change of PRSS1 mRNA cleavage and led to HP.PCR amplification was performed for peripheral blood genomic DNA of healthy controls to connect the intron 3 sequence of the PRSS1 gene with pGL4.23 vector,namely the EWT plasmid,and the E33 plasmid of PRSS1 c.455-33C>T mutation was constructed based on the EWT plasmid.After Hek293T cells were transfected with EWT and E33 plasmids,dual-luciferase reporter assay was used to measure luci-ferase activity,and the results were analyzed to determine whether PRSS1 c.455-33C>T intron mutation had enhancer function to cause the change of PRSS1 gene function and lead to HP.Results The search of relatedliterature and websites identified PRSS1 c.455-33C>T mutationas an unreported new mutation of unknown function.The shear analysis experiment showed that the SWT and S33 groups had identical products.The dual-luciferase reporter assay showed that the E33 group had a significantly lower fluorescence value than the EWT group(t=12.23,P<0.001).The E33 group showed no enhancer function compared with the EWT group.Conclusion PRSS1 c.455-33C>T intron mutation does not cause changes in PRSS1 mRNA cleavage and does not have

关 键 词：胰腺炎 人阳离子胰蛋白酶原 基因 突变 

分 类 号：R576[医药卫生—消化系统]