Screening And Functional Analysis Of CTRC Gene Mutation In Patients With Chronic Pancreatitis

Chronic pancreatitis(CP)can cause severe damage to pancreatic function,and its histological changes are irreversible.It seriously affects the quality of life of patients with CP,and its incidence is increasing year by year.Studies have shown that the pathogenesis of CP is closely related to genetic,environmental and metabolic factors.In this study,we analyzed the variation of CP-related chymotrypsin C(CTRC)gene,and explored the basic functional changes of CTRC gene mutation and its relationship with the development of CP in Sichuan Province Han population.Peripheral blood was collected from 142 patients with CP and 200 controls.The DNA of leukocytes was extracted for CTRC gene exon sequencing.The sequencing results were compared online and by DNAMAN.Clinical baseline data such as smoking,drinking,and blood calcium were collected to statistically analyze the clinical relationship between the CTRC gene variants and CP.The found mutation sites were predicted by bioinformatics,and the effect of mutation on protein expression was detected by Western Blotting assay.The enzyme activity assay was used to detect the effect of mutation on protein activity.Three cases of CTRC gene change were detected in the case group.No database and domestic and foreign literature reported the above variants.Bioinformatics analysis suggested that c.611G>A and c.703G>T mutations may affect protein function,which may be pathogenic mutations;c.733A>T mutation may be non-pathogenic mutation;basic function studies have shown that CTRC protein variants(p.G204E?p.V235 F and p.T245S)caused by 3 cases of mutation affect the protein expression and enzyme activity levels of chymotrypsin C,which are considered to be potential pathogenic changes.The protein expression and catalytic activity of p.E225 K and p.R254 Q are close to normal levels,and are considered to be non-pathogenic changes.Another six single nucleotide polymorphism(SNP)locus changes were detected.The loci c.40+92T>A,c.40+133 G>A is the newly discovered SNP change.The domestic and foreign literatures have not been reported,and the corresponding rs number is not found in the database.Clinical analysis showed that smoking,drinking,biliary tract disease,hyperlipidemia,c.40+133G>A locus and c.493+49G>C locus distribution were significantly different between CP group and control group(P<0.05),which can be considered as risk factors for CP;c.40+92T>A site changes significantly more in the control group than CP group,which suggesting that c.40+92T>A site variation may be a protective factor for CP;Multivariate analysis showed that smoking,biliary tract disease and c.493+49G >C site variation was statistically significant between the CP group and the control group and could be considered as an independent risk factor for CP,while there was no significant difference in drinking alcohol,blood lipids,c.40+92T>A site change and c.40+133G>A site change between CP and control group(P>0.05).This study found three new mutation sites and six SNPs site changes(including two new SNPs site changes and four known SNPs).Among them,c.40+92T>A locus variation may have a protective effect on CP;c.493+49G>C and c.40+133G>A locus variations and smoking,drinking,biliary tract disease,hyperlipidemia may increase the risk of CP in individuals;smoking,biliary disease and c.493+49G>C locus variation may be considered as independent risk factors for CP in this region.Functional studies suggested that p.G204 E,p.V235 F and p.T245 S mutations were considered as potential pathogenic changes.We explored the susceptibility of CP at the molecular level of gene and protein through screening the local Han population for CTRC gene variation and analyzing the effects of related mutations on protein function.In addition to,we combined the environmental factors and metabolic factors to analyze the risk factors associated with CP in Sichuan Han population.This study enriched the mutational spectrum and functional database of CTRC gene in this region,and for the first time,the clinical multivariate analysis of CTRC gene variation and CP was conducted through logistic regression.The basic research and rigorous clinical statistical analysis were integrated to better explore independent risk factors associated with CP.