학술논문
Novel chymotrypsin C (CTRC) variants from real-world genetic testing of pediatric chronic pancreatitis cases.
Document Type
Academic Journal
Author
Stefanovics R; Department of Surgery, University of California Los Angeles, Los Angeles, CA, USA; Hungarian Centre of Excellence for Molecular Medicine - University of Szeged, Translational Pancreatology Research Group, Szeged, Hungary; Center for Gastroenterology, Department of Medicine, Albert Szent-Györgyi Medical School, University of Szeged, Szeged, Hungary.; Sándor M; Department of Surgery, University of California Los Angeles, Los Angeles, CA, USA.; Demcsák A; Department of Surgery, University of California Los Angeles, Los Angeles, CA, USA.; Berke G; Department of Surgery, University of California Los Angeles, Los Angeles, CA, USA.; Németh BC; Hungarian Centre of Excellence for Molecular Medicine - University of Szeged, Translational Pancreatology Research Group, Szeged, Hungary; Center for Gastroenterology, Department of Medicine, Albert Szent-Györgyi Medical School, University of Szeged, Szeged, Hungary.; Zhang W; Division of Human Genetics, Cincinnati Children's Hospital Medical Center, University of Cincinnati, Cincinnati, OH, USA; Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, OH, USA.; Abu-El-Haija M; Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, OH, USA; Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, University of Cincinnati, Cincinnati, OH, USA.; Sahin-Tóth M; Department of Surgery, University of California Los Angeles, Los Angeles, CA, USA. Electronic address: msahintoth@mednet.ucla.edu.
Source
Publisher: Country of Publication: Switzerland NLM ID: 100966936 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1424-3911 (Electronic) Linking ISSN: 14243903 NLM ISO Abbreviation: Pancreatology Subsets: MEDLINE
Subject
Language
English
Abstract
Background: Chymotrypsin C (CTRC) protects the pancreas against unwanted intrapancreatic trypsin activity through degradation of trypsinogen. Loss-of-function CTRC variants increase the risk for chronic pancreatitis (CP). The aim of the present study was to characterize novel CTRC variants found during genetic testing of CP cases at a pediatric pancreatitis center.
Methods: We used next-generation sequencing to screen patients. We analyzed the functional effects of CTRC variants in HEK 293T cells and using purified enzymes.
Results: In 5 separate cases, we detected 5 novel heterozygous CTRC variants: c.407C>T (p.Thr136Ile), c.550G>A (p.Ala184Thr), c.627Cdup (p.Ser210Leufs∗?, where the naming indicates a frame shift with no stop codon), c.628T>C (p.Ser210Pro), and c.779A>G (p.Asp260Gly). Functional studies revealed that with the exception of p.Ser210Leufs∗?, the CTRC variants were secreted normally from transfected cells. Enzyme activity of purified variants p.Thr136Ile, p.Ala184Thr, and p.Asp260Gly was similar to that of wild-type CTRC, whereas variant p.Ser210Pro was inactive. The frame-shift variant p.Ser210Leufs∗? was not secreted but accumulated intracellularly, and induced endoplasmic reticulum stress, as judged by elevated mRNA levels of HSPA5 and DDIT3, and increased mRNA splicing of XBP1.
Conclusions: CTRC variants p.Ser210Pro and p.Ser210Leufs∗? abolish CTRC function and should be classified as pathogenic. Mechanistically, variant p.Ser210Pro directly affects the amino acid at the bottom of the substrate-binding pocket while the frame-shift variant promotes misfolding and thereby blocks enzyme secretion. Importantly, 3 of the 5 novel CTRC variants proved to be benign, indicating that functional analysis is indispensable for reliable determination of pathogenicity and the correct interpretation of genetic test results.
Competing Interests: Declaration of competing interest The authors have declared that no conflict of interest exists.
(Copyright © 2024. Published by Elsevier B.V.)
Methods: We used next-generation sequencing to screen patients. We analyzed the functional effects of CTRC variants in HEK 293T cells and using purified enzymes.
Results: In 5 separate cases, we detected 5 novel heterozygous CTRC variants: c.407C>T (p.Thr136Ile), c.550G>A (p.Ala184Thr), c.627Cdup (p.Ser210Leufs∗?, where the naming indicates a frame shift with no stop codon), c.628T>C (p.Ser210Pro), and c.779A>G (p.Asp260Gly). Functional studies revealed that with the exception of p.Ser210Leufs∗?, the CTRC variants were secreted normally from transfected cells. Enzyme activity of purified variants p.Thr136Ile, p.Ala184Thr, and p.Asp260Gly was similar to that of wild-type CTRC, whereas variant p.Ser210Pro was inactive. The frame-shift variant p.Ser210Leufs∗? was not secreted but accumulated intracellularly, and induced endoplasmic reticulum stress, as judged by elevated mRNA levels of HSPA5 and DDIT3, and increased mRNA splicing of XBP1.
Conclusions: CTRC variants p.Ser210Pro and p.Ser210Leufs∗? abolish CTRC function and should be classified as pathogenic. Mechanistically, variant p.Ser210Pro directly affects the amino acid at the bottom of the substrate-binding pocket while the frame-shift variant promotes misfolding and thereby blocks enzyme secretion. Importantly, 3 of the 5 novel CTRC variants proved to be benign, indicating that functional analysis is indispensable for reliable determination of pathogenicity and the correct interpretation of genetic test results.
Competing Interests: Declaration of competing interest The authors have declared that no conflict of interest exists.
(Copyright © 2024. Published by Elsevier B.V.)