PRSS1 c.455-33C＞T突变在遗传性胰腺炎中的致病性分析OACSTPCD

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 in-tron 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 plas-mid.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 the function of PRSS1 gene enhancer,and it is not associated with HP directly caused by the PRSS1 gene.