Stefanovics Regina, Sándor Máté, Demcsák Alexandra, Berke Gergő, Németh Balázs Csaba, Zhang Wenying, Abu-El-Haija Maisam, Sahin-Tóth Miklós
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.
Department of Surgery, University of California Los Angeles, Los Angeles, CA, USA.
Pancreatology. 2024 Aug;24(5):690-697. doi: 10.1016/j.pan.2024.06.003. Epub 2024 Jun 10.
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.
We used next-generation sequencing to screen patients. We analyzed the functional effects of CTRC variants in HEK 293T cells and using purified enzymes.
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.
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.
糜蛋白酶C（CTRC）通过降解胰蛋白酶原保护胰腺免受不必要的胰腺内胰蛋白酶活性影响。功能丧失的CTRC变异体增加了慢性胰腺炎（CP）的风险。本研究的目的是对在一家儿科胰腺炎中心对CP病例进行基因检测期间发现的新型CTRC变异体进行特征描述。
我们使用下一代测序技术对患者进行筛查。我们在HEK 293T细胞中并使用纯化的酶分析了CTRC变异体的功能影响。
在5个不同病例中，我们检测到5种新型杂合CTRC变异体：c.407C>T（p.Thr136Ile）、c.550G>A（p.Ala184Thr）、c.627Cdup（p.Ser210Leufs∗?，其中命名表示移码且无终止密码子）、c.628T>C（p.Ser210Pro）和c.779A>G（p.Asp260Gly）。功能研究表明，除了p.Ser210Leufs∗? 外，CTRC变异体从转染细胞中正常分泌。纯化变异体p.Thr136Ile、p.Ala184Thr和p.Asp260Gly的酶活性与野生型CTRC相似，而变异体p.Ser210Pro无活性。移码变异体p.Ser¬210Leufs∗? 未分泌而是在细胞内积累，并诱导内质网应激，这可通过HSPA5和DDIT3的mRNA水平升高以及XBP1的mRNA剪接增加来判断。
CTRC变异体p.Ser210Pro和p.Ser210Leufs∗? 消除了CTRC功能，应归类为致病性变异。从机制上讲，变异体p.Ser210Pro直接影响底物结合口袋底部的氨基酸，而移码变异体促进错误折叠，从而阻断酶的分泌。重要的是，5种新型CTRC变异体中有3种被证明是良性的，这表明功能分析对于可靠确定致病性和正确解释基因检测结果是必不可少的。
