The Pathogenic Role And Mechanism Of CEL-HYB Variants In Chronic Pancreatitis

Part Ⅰ.The distribution of CEL-HYB variants in Chinese subjects with early-onset chronic pancreatitis and preliminary investigation of its underlying pathogenic mechanismBackground & Objective: Chronic pancreatitis(CP)is a genetic-related inflammatory disease of the pancreas with complex etiology and unclear mechanism.The CEL-HYB1,a hybrid allele generated by homologous recombination between CEL(encoding carboxyl ester lipase)and its downstream pseudogene(CELP),was found to be associated with a significantly higher risk of CP in European populations.However,the distribution of this variant could have an ethnic specificity.An alternative hybrid allele(CEL-HYB2)appears to be dominant in Asian populations.Here,we explored the distribution of hybrid alleles of CEL in Chinese early-onset idiopathic CP(ICP)patients and investigated the potential impact of CEL-HYB1 and CEL-HYB2 on transfected HEK293 T cells.Methods: 225 genetically unexplained Chinese early-onset ICP patients were recruited from Shanghai Changhai Hospital.DNA was extracted from peripheral blood samples of the subjects.We used a long-range duplex PCR assay to detect the CEL-HYB allele.HEK293 T cell lines stably expressing CEL-WT,CEL-HYB1 or CEL-HYB2 were generated to investigate the secretion,intracellular accumulation and protein misfolding.Results: CEL-HYB variants were present in 10/225(4.4%)early-onset ICP patients and in 20/1028(1.9%)controls(odds ratio = 2.34;P = 0.049),while CEL-HYB variants were present in 12/679(1.8%)non-early-onset ICP patients(odds ratio = 0.91;P = 0.857).In vitro experiment,both of CEL-HYB1 and CEL-HYB2 had impaired secretion compared with CEL-WT in HEK293 T cells.CEL-HYB2 was barely detectable in the culture supernatants.After treatment of cycloheximide,CEL-HYB1 and CEL-HYB2 demonstrated increased intracellular accumulation to variable extents.The expression of CEL-HYB1 in HEK293 T cells resulted in elevated endoplasmic reticulum(ER)stress markers HSPA5 and s XBP1,while the m RNA levels of HSPA5 and s XBP1 in HEK293 T cells stably expressing CELHYB2 were comparable to the control group.Conclusions: Unlike the CEL-HYB1 variant in European populations,CEL-HYB2 is the major subtype of CEL hybrid allele in Chinese populations and is significantly associated with early-onset ICP.Expression of CEL-HYB1 and CEL-HYB2 in HEK293 T cells showed impaired secretion and intracellular accumulation.Compared with CEL-WT or CEL-HYB2,expression of CEL-HYB1 induce elevated levels of endoplasmic reticulum stress in cell culture.Part Ⅱ.Generation of the humanized CEL-HYB1 mouse model and detection of its phenotypeBackground & Objective: The hybrid allele of the carboxyl ester lipase gene(CELHYB)is a genetic variant suggested to increase the risk of CP,although the mechanism promoting disease development is largely unknown.Our aim was to develop a mouse model for CEL-HYB1 that enables studies of pancreatic disease mechanisms.Methods: CRISPR-Cas9 was used to generate humanized mice harboring a 3×Flagtagged CEL-HYB1 allele on a C57BL/6J background.Humanized CEL mice and C57BL/6J mice were used as controls.We investigated whether an additional 3×Flag tag had detectable effect on the property of CEL proteins in cell cultures.Standard tail genotyping procedures were used to genotype the mice.Pancreata were collected and analyzed by histology,immunohistochemistry,immunoblotting,and transcriptomics.Results: The Flag tag had no detectable effect on the the properties of CEL proteins,including protein secretion,aggregation and misfolding-induced ER stress.We successfully generated mouse models expressing humanized CEL-HYB1 or CEL,which was confirmed by Western blot and immunohistochemistry.The transcripts of CEL-HYB1 and CEL in mouse pancreas are present at only 46% of the level of the murine Cel transcripts.Humanized mice demonstrated no obvious physical or behavioral changes and bred normally.Pancreata from mice expressing CEL-HYB1 developed pathological features characteristic of focal pancreatitis that included acinar atrophy,vacuolization,inflammatory infiltrates,and fibrosis in a time-dependent manner.Positive staining for cleaved caspase-3and terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL)were observed in the pancreatic sections from h CEL-HYB1 mice.In addition,some aged h CEL-HYB1 mice developed eosinophilic inclusion bodies and increased vacuolization in the acinar cells;immunohistochemistry showed that these eosinophilic bodies were strongly positive for CEL-HYB1 protein staining.Conclusions: We have developed two novel humanized mouse strains expressing CELHYB1 or CEL.Transgene expression of CEL-HYB1 in mice induced focal pancreatic lesions in a time-dependent manner.These two humanized mouse models provides a useful platform to study the pathogenesis of CP in vivo.Part Ⅲ.Investigation of the role and mechanism of CEL‐HYB1-mediated protein misfolding and impaired autophagy in pancreatitisBackground & Objective: Genetic variants contribute to the risk of CP in adults and children.CEL‐HYB1 encodes a pathogenic variant of the pancreatic digestive enzyme carboxyl ester lipase.Although suggested to cause digestive enzyme misfolding,definitive in vivo evidence for this postulate has been lacking.Methods: Primary pancreatic acini were islotated from two humanized mice strains and cultured in vitro.To measure the secretion and aggregation of CEL-HYB1 protein,the CEL protein content was measured in intracellular fraction,medium,soluble fraction,and insoluble fraction by Western blot.The expression of ER stress markers in pancreatic tissues were measured by Western blot and q PCR.Autophagy-related immunofluorescence and proteins were then analysed in the pancreata from h CEL‐HYB1 and control mice at different ages.We also performed transmission electron microscopic analysis of the pancreatic tissues.h CEL-HYB1,h CEL and C57BL/6J mice were given caerulein injections to induce acute pancreatitis(AP)and CP.The severity of pancreatitis was evaluated by histochemistry,immunostaining,biochemical assays and q PCR.Results: The amount of CEL-HYB1 protein in the conditioned medium of pancreatic acini was significantly lower than that of the CEL protein;essentially all of the CEL protein was soluble whereas a considerable fraction of the intracellular CEL-HYB1 protein was present in an insoluble form.The expression levels of s Xbp1,Ddit3 and insoluble Hspa5 were significantly increased in h CEL-HYB1 mice as compared with controls.The m RNA levels of Ddit3,Hspa5,Hspa1 a,Hsp90aa1,and Hsp90ab1 were significantly upregulated in the pancreata of h CEL-HYB1 mice.LC3-II upregulation with decreased p62 levels was observed in the pancreata of h CEL-HYB1 mice aged 24 weeks.We observed LC3-positive autophagic vesicles in pancreatic acinar cells of aged h CEL-HYB1 mice with significant upregulation of LC3-II and p62.Colocalization of LC3 with Lamp1 further revealed that most of these large vesicles were autolysosomes.Transmission electron microscope demonstrated dilated endoplasmic reticula and vacuolization in acinar cells from h CELHYB1 mice.Administration of caerulein increased the severity of AP/CP in mice expressing CEL-HYB1 compared with control mice,accompanied by higher levels of endoplasmic reticulum stress.Conclusions: Expression of a humanized form of CEL-HYB1 protein in mice promotes endoplasmic reticulum stress and pancreatitis through a misfolding-dependent pathway.Impaired autophagy appears to be involved in the pancreatic injury in aged h CEL-HYB1 mice.Part Ⅳ.Association between CEL VNTR length and chronic pancreatitis in Chinese populationBackground & Objective: The CEL gene contains a variable number of tandem repeats(VNTR)region.It remains unclear whether the number of repeats in the CEL VNTR is related to the risk of pancreatic diseases.The aim of this study was to investigate whether CEL VNTR length is associated with ICP,alcoholic chronic pancreatitis(ACP),or pancreatic cancer in a cohort of Chinese patients.Methods: A total of 771 patients diagnosed with ICP,222 patients with ACP,and 263 patients with pancreatic cancer were enrolled at Changhai hospital.DNA was extracted from peripheral blood samples of the patients and healthy controls(n = 927).CEL VNTR lengths were determined using a screening method combining PCR and DNA fragment analysis.CEL VNTRs were genotyped in patients diagnosed with ICP,ACP,or pancreatic cancer,and in healthy controls.Results: Overall,the CEL VNTR lengths ranged from 5 to 22 repeats,with the 16-repeat allele(“normal” size,N)accounting for 73.82% of all observed alleles(73.85% in controls,72.31% in ACP,73.21% in ICP,and 76.78% in pancreatic cancer).The VNTR allele frequencies and genotype distributions were not significantly different between healthy controls and patients with ACP or pancreatic cancer.For the ICP group,allele frequencies did not differ significantly from the controls,while the frequency of the SS genotype(homozygosity for 5-15 repeats)was significantly higher in the patients(4.67%)than in the controls(1.94%)(P = 0.0014;odds ratio = 2.47;95% CI = 1.39-4.39).CEL duplication variants were found in 22 ICP patients(2.85%),11 ACP patients(4.95%),8pancreatic cancer patients(3.04%),and 35 controls(3.78%);there were no significant differences in the frequency of duplication alleles among the groups.The duplication genotype “SSS” was present only in ICP and ACP patients but not in pancreatic cancer patients or healthy controls.Conclusions: There were no associations between the CEL VNTR length and ACP or pancreatic cancer.However,homozygosity for short VNTR lengths may confer susceptibility to ICP.