The common G60G variant (c.180C > T) of CTRC is associated with increased risk of chronic pancreatitis in children

Abstract

s / Pancreatology 16 (2016) S1eS130 S6 1 KG Jebsen Center for Diabetes Research, Department of Clinical Science, University of Bergen, Norway Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, Children's Hospital of Pittsburgh of UPMC, Pit, United States Introduction: We have previously identified disease-associated mutations in the last exon of the CEL gene, localized in a variable number of tandem repeats (VNTR) domain. Two single base deletions (DEL1 and DEL4) lead to frameshifts, predicting a new C-terminus of the CEL protein. Patients with these mutations suffer from exocrine pancreatic dysfunction and diabetes. Aims: To understand the role of the C-terminal domain of CEL in human pancreatic disease by investigating expression, secretion and intracellular localization of different protein-variants in cellular models. Materials & methods: cDNAs of CEL-wild-type, CEL-DEL1, CEL-DEL4 and CEL-TRUNC (lacking the VNTR) were cloned into pcDNA3.1 vectors, with or without epitope tags (V5/His), and transfected into HEK293 cells. The expressed CEL-proteins were studied by Western-blotting, immunostaining and confocal imaging. Results: Forty-eight hours post-transfection all CEL-variants were detectable in the lysate, pellet and medium fractions of HEK293 cells. The amount of CEL protein in each fraction varied between variants and also depended upon the presence of epitope tags. Comparing with V5/Histagged protein variants, we observed a higher amount of untagged proteins in the pellet and less in the medium. Significantly more of the disease-causing CEL-variants were detected in the pellet as compared with the wild-type protein. When co-expressing FLAGand V5/His-tagged CELvariants, we observed that CEL-WT could be detected in the pellet fraction together with CEL-DEL1. Conclusion: The cellular fate of CEL protein variants is highly influenced by the C-terminus of the protein. Epitope tags may cause increased solubility of disease-causing CEL variants, leading to underestimation of their propensity to aggregate in cellular model systems. Abstract ID: 1346, Oral-20.ID: 1346, Oral-20. The common G60G variant (c.180C > T) of CTRC is associated with increased risk of chronic pancreatitis in children Agnieszka Magdalena Rygiel , Alicja Monika Grabarczyk , Grzegorz Oracz , Aleksandra Kujko , Katarzyna Wertheim-Tysarowska , Elwira Kolodziejczyk , Dorota Koziel , Artur Kowalik , Stanislaw Gluszek , Jerzy Bal 1 Department of Medical Genetics, Institute of Mother and Child Warsaw, Poland Department of Gastroenterology, Hepatology and Feeding Disorders, The Children's Memorial Health Institute, Warsaw, Poland Department of Surgery and Surgical Nursing, Jan Kochanowski University of Kielce, Poland Department of Molecular Diagnostics, Oncology Center of the Holy Cross, Kielce, Poland Introduction: Loss-of-function variants in chymotripsin C (CTRC) increase the risk for chronic pancreatitis (CP). The most commonly found pathogenicmutations of CTRC include K247_R254del, and R254W but their frequencies are low. Studies with adults indicated the association of G60G (c.180C>T) common variant of CTRC with CP but the available data are limited and obscure. Aims: To evaluate the risk of CP associated with CTRC mutations and G60G variant in case (CP children)-control study. Patients & methods: We sequenced exones 2-7 of the CTRC in 136 children including idiopathic CP (ICP, n1⁄461) and CP with various etiological factors (n1⁄475).Children (median age at onset 8 years) with CP verified by imagingmethods were included. Control group (n1⁄4401, median age 45) of healthy volunteers was screened by HRM-PCR and detected mutations were confirmed by direct sequencing. Results: In CP, we identified R254W(4,5%) and K247_R254del (4,5%) mutations, and G60G variant (c.180 CT or TT, 49%). Compared with controls, CTRC mutations are associated with 7 times increased risk of CP (9% vs.1.2%, OR1⁄47.95% CL:2.5-19.7, P1⁄48,6*10-6). The c.180 CT and TT genotype is associated with 1,8 (OR1⁄4 1.8; 95% CL:1.3-3.2) and 15 (OR1⁄414.7;95% CL:5.1-42.3) times increased risk of CP with frequencies of 35% vs.18% (P1⁄43,5*10-4) and 15% vs.0.9% (P1⁄41,5*10-9), respectively. This association was also significant when ICP and control group were compared (c.180 CT: OR1⁄41,9; 95% CL (1.28-2.8), P1⁄40,0039 and c.180 TT: OR1⁄414.8;95% CL: 4.6-46.5, P1⁄43,2*10-6). Conclusion: We showed, for the first time that G60G variant of CTRC, especially c.180 TT genotype is strongly associated with idiopathic CP in children.