RESUMEN
The serine protease chymotrypsin protects the pancreas against pancreatitis by degrading trypsinogen, the precursor to the digestive protease trypsin. Taking advantage of previously generated mouse models with either the Ctrb1 gene (encoding chymotrypsin B1) or the Ctrl gene (encoding chymotrypsin-like protease) disrupted, here we generated the novel Ctrb1-del × Ctrl-KO strain in the C57BL/6N genetic background, which harbors a naturally inactivated Ctrc gene (encoding chymotrypsin C). The newly created mice are devoid of chymotrypsin, yet the animals develop normally, breed well, and show no spontaneous phenotype, indicating that chymotrypsin is dispensable under laboratory conditions. When given cerulein, the Ctrb1-del × Ctrl-KO strain exhibited markedly increased intrapancreatic trypsin activation and more severe acute pancreatitis, relative to wild-type C57BL/6N mice. After the acute episode, Ctrb1-del × Ctrl-KO mice spontaneously progressed to chronic pancreatitis, whereas C57BL/6N mice recovered rapidly. The cerulein-induced pancreas pathology in Ctrb1-del × Ctrl-KO mice was highly similar to that previously observed in Ctrb1-del mice; however, trypsin activation was more robust and pancreatitis severity was increased. Taken together, the results confirm and extend prior observations demonstrating that chymotrypsin safeguards the pancreas against pancreatitis by limiting pathologic trypsin activity. In mice, the CTRB1 isoform, which constitutes about 90% of the total chymotrypsin content, is responsible primarily for the anti-trypsin defenses and protection against pancreatitis; however, the minor isoform CTRL also contributes to an appreciable extent.NEW & NOTEWORTHY Chymotrypsins defend the pancreas against the inflammatory disorder pancreatitis by degrading harmful trypsinogen. This study demonstrates that mice devoid of pancreatic chymotrypsins are phenotypically normal but become sensitized to secretagogue hyperstimulation and exhibit increased intrapancreatic trypsin activation, more severe acute pancreatitis, and rapid progression to chronic pancreatitis. The observations confirm and extend the essential role of chymotrypsins in pancreas health.
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Ceruletida , Quimotripsina , Ratones Endogámicos C57BL , Ratones Noqueados , Pancreatitis , Tripsina , Animales , Quimotripsina/metabolismo , Quimotripsina/genética , Ceruletida/toxicidad , Pancreatitis/inducido químicamente , Pancreatitis/patología , Pancreatitis/metabolismo , Pancreatitis/genética , Ratones , Tripsina/metabolismo , Secretagogos/metabolismo , Páncreas/metabolismo , Páncreas/patología , Modelos Animales de Enfermedad , MasculinoRESUMEN
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.
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Quimotripsina , Chaperón BiP del Retículo Endoplásmico , Pruebas Genéticas , Pancreatitis Crónica , Humanos , Pancreatitis Crónica/genética , Quimotripsina/genética , Quimotripsina/metabolismo , Células HEK293 , Masculino , Niño , Femenino , Adolescente , Mutación , Factor de Transcripción CHOPRESUMEN
BACKGROUND & AIMS: Heterozygous SPINK1 mutations are strong risk factors for chronic pancreatitis in humans, yet heterozygous disruption of mouse Spink1 yielded no pancreatic phenotype. To resolve this contradiction, we used CRISPR/Cas9-mediated genome editing to generate heterozygous Spink1-deleted mice (Spink1-KOhet) in the C57BL/6N strain and studied the effect of this allele in trypsin-independent and trypsin-dependent pancreatitis models. METHODS: We investigated severity of acute pancreatitis and progression to chronic pancreatitis in Spink1-KOhet mice after transient (10 injections) and prolonged (2 × 8 injections) cerulein hyperstimulation. We crossed Spink1-KOhet mice with T7D23A and T7D22N,K24R mice that carry strongly autoactivating trypsinogen mutants and exhibit spontaneous chronic pancreatitis. RESULTS: Prolonged but not transient cerulein stimulation resulted in increased intrapancreatic trypsin activity and more severe acute pancreatitis in Spink1-KOhet mice relative to the C57BL/6N control strain. After the acute episode, Spink1-KOhet mice developed progressive disease with chronic pancreatitis-like features, whereas C57BL/6N mice recovered rapidly. Trypsinogen mutant mice carrying the Spink1-KOhet allele exhibited strikingly more severe chronic pancreatitis than the respective parent strains. CONCLUSIONS: Heterozygous Spink1 deficiency caused more severe acute pancreatitis after prolonged cerulein stimulation and promoted chronic pancreatitis after the cerulein-induced acute episode, and in two strains of trypsinogen mutant mice with spontaneous disease. In contrast, acute pancreatitis induced with limited cerulein hyperstimulation was unaffected by heterozygous Spink1 deletion, in agreement with recent observations that trypsin activity does not mediate pathologic responses in this model. Taken together, the findings strongly support the notion that loss-of-function SPINK1 mutations in humans increase chronic pancreatitis risk in a trypsin-dependent manner.
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Modelos Animales de Enfermedad , Heterocigoto , Pancreatitis Crónica , Inhibidor de Tripsina Pancreática de Kazal , Tripsina , Animales , Inhibidor de Tripsina Pancreática de Kazal/genética , Inhibidor de Tripsina Pancreática de Kazal/metabolismo , Pancreatitis Crónica/genética , Pancreatitis Crónica/patología , Ratones , Tripsina/genética , Tripsina/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Ceruletida/toxicidad , Ceruletida/efectos adversos , Masculino , Páncreas/patología , Páncreas/metabolismo , Predisposición Genética a la Enfermedad , Glicoproteínas , Proteínas de Secreción ProstáticaRESUMEN
Chymotrypsin-like protease (CTRL) is one of the four chymotrypsin isoforms expressed in the human exocrine pancreas. Human genetic and experimental evidence indicate that chymotrypsins B1, B2, and C (CTRB1, CTRB2 and CTRC) are important not only for protein digestion but also for protecting the pancreas against pancreatitis by degrading potentially harmful trypsinogen. CTRL has not been reported to play a similar role, possibly due to its low abundance and/or different substrate specificity. To address this problem, we investigated the specificity of the substrate-binding groove of CTRL by evolving the substrate-like canonical loop of the Schistocerca gregaria proteinase inhibitor 2 (SGPI-2), a small-protein reversible chymotrypsin inhibitor to bind CTRL. We found that phage-associated SGPI-2 variants with strong affinity to CTRL were similar to those evolved previously against CTRB1, CTRB2 or bovine chymotrypsin A (bCTRA), indicating comparable substrate specificity. When tested as recombinant proteins, SGPI-2 variants inhibited CTRL with similar or slightly weaker affinity than bCTRA, confirming that CTRL is a typical chymotrypsin. Interestingly, an SGPI-2 variant selected with a Thr29His mutation in its reactive loop was found to inhibit CTRL strongly, but it was digested rapidly by bCTRA. Finally, CTRL was shown to degrade human anionic trypsinogen, however, at a much slower rate than CTRB2, suggesting that CTRL may not have a significant role in the pancreatic defense mechanisms against inappropriate trypsinogen activation and pancreatitis.
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Quimasas , Quimotripsina , Inhibidores de Proteasas , Animales , Bovinos , Humanos , Quimasas/antagonistas & inhibidores , Quimasas/química , Quimotripsina/química , Pancreatitis/prevención & control , Inhibidores de Proteasas/química , Inhibidores de Proteasas/aislamiento & purificación , Inhibidores de Proteasas/farmacología , Especificidad por Sustrato , Tripsinógeno , Biblioteca de PéptidosRESUMEN
Chymotrypsin C (CTRC) is a digestive serine protease produced by the pancreas that regulates intrapancreatic trypsin activity and provides a defensive mechanism against chronic pancreatitis (CP). CTRC exerts its protective effect by promoting degradation of trypsinogen, the precursor to trypsin. Loss-of-function missense and microdeletion variants of CTRC are found in around 4% of CP cases and increase disease risk by approximately 3-7-fold. In addition, a commonly occurring synonymous CTRC variant c.180C>T (p.Gly60=) was reported to increase CP risk in various cohorts but a global analysis of its impact has been lacking. Here, we analyzed the frequency and effect size of variant c.180C>T in Hungarian and pan-European cohorts, and performed meta-analysis of the new and published genetic association data. When allele frequency was considered, meta-analysis revealed an overall frequency of 14.2% in patients and 8.7% in controls (allelic odds ratio (OR) 2.18, 95% confidence interval (CI) 1.72-2.75). When genotypes were examined, c.180TT homozygosity was observed in 3.9% of CP patients and in 1.2% of controls, and c.180CT heterozygosity was present in 22.9% of CP patients and in 15.5% of controls. Relative to the c.180CC genotype, the genotypic OR values were 5.29 (95% CI 2.63-10.64), and 1.94 (95% CI 1.57-2.38), respectively, indicating stronger CP risk in homozygous carriers. Finally, we obtained preliminary evidence that the variant is associated with reduced CTRC mRNA levels in the pancreas. Taken together, the results indicate that CTRC variant c.180C>T is a clinically relevant risk factor, and should be considered when genetic etiology of CP is investigated.
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Pancreatitis Crónica , Humanos , Tripsina/genética , Pancreatitis Crónica/genética , Quimotripsina/genética , Quimotripsina/metabolismo , Estudios de Casos y Controles , Predisposición Genética a la Enfermedad , MutaciónRESUMEN
Mutation p.R122H in human cationic trypsinogen (PRSS1) is the most frequently identified cause of hereditary pancreatitis. The mutation blocks protective degradation of trypsinogen by chymotrypsin C (CTRC), which involves an obligatory trypsin-mediated cleavage at Arg122. Previously, we found that C57BL/6N mice are naturally deficient in CTRC, and trypsinogen degradation is catalyzed by chymotrypsin B1 (CTRB1). Here, we used biochemical experiments to demonstrate that the cognate p.R123H mutation in mouse cationic trypsinogen (isoform T7) only partially prevented CTRB1-mediated degradation. We generated a novel C57BL/6N mouse strain harboring the p.R123H mutation in the native T7 trypsinogen locus. T7R123H mice developed no spontaneous pancreatitis, and severity parameters of cerulein-induced pancreatitis trended only slightly higher than those of C57BL/6N mice. However, when treated with cerulein for 2 days, more edema and higher trypsin activity was seen in the pancreas of T7R123H mice compared to C57BL/6N controls. Furthermore, about 40% of T7R123H mice progressed to atrophic pancreatitis in 3 days, whereas C57BL/6N animals showed full histological recovery. Taken together, the observations indicate that mutation p.R123H inefficiently blocks chymotrypsin-mediated degradation of mouse cationic trypsinogen, and modestly increases cerulein-induced intrapancreatic trypsin activity and pancreatitis severity. The findings support the notion that the pathogenic effect of the PRSS1 p.R122H mutation in hereditary pancreatitis is dependent on its ability to defuse chymotrypsin-dependent defenses.
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Quimotripsina , Pancreatitis , Ratones , Humanos , Animales , Quimotripsina/genética , Tripsina/genética , Tripsinógeno/genética , Ceruletida , Ratones Endogámicos C57BL , Pancreatitis/patología , MutaciónRESUMEN
Inborn mutations in the digestive protease carboxypeptidase A1 (CPA1) gene may be associated with hereditary and idiopathic chronic pancreatitis (CP). Pathogenic mutations, such as p.N256K, cause intracellular retention and reduced secretion of CPA1, accompanied by endoplasmic reticulum (ER) stress, suggesting that mutation-induced misfolding underlies the phenotype. Here, we report the novel p.G250A CPA1 mutation found in a young patient with CP. Functional properties of the p.G250A mutation were identical to those of the p.N256K mutation, confirming its pathogenic nature. We noted that both mutations are in a catalytically important loop of CPA1 that is stabilized by the Cys248-Cys271 disulfide bond. Mutation of either or both Cys residues to Ala resulted in misfolding, as judged by the loss of CPA1 secretion and intracellular retention. We re-analyzed seven previously reported CPA1 mutations that affect this loop and found that all exhibited reduced secretion and caused ER stress of varying degrees. The magnitude of ER stress was proportional to the secretion defect. Replacing the naturally occurring mutations with Ala (e.g., p.V251A for p.V251M) restored secretion, with the notable exception of p.N256A. We conclude that the disulfide-stabilized loop of CPA1 is prone to mutation-induced misfolding, in most cases due to the disruptive nature of the newly introduced side chain. We propose that disease-causing CPA1 mutations exhibit abolished or markedly reduced secretion with pronounced ER stress, whereas CPA1 mutations with milder misfolding phenotypes may be associated with lower disease risk or may not be pathogenic at all.
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Carboxipeptidasas A , Predisposición Genética a la Enfermedad , Pancreatitis Crónica , Humanos , Carboxipeptidasas A/genética , Mutación , Pancreatitis Crónica/genética , FenotipoRESUMEN
INTRODUCTION: Cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in pancreatic ductal fluid secretion by mediating Cl- and HCO3- ion transport across the apical membrane. Severe CFTR mutations that diminish chloride conductance cause cystic fibrosis (CF) if both alleles are affected, whereas heterozygous carrier status increases risk for chronic pancreatitis (CP). It has been proposed that a subset of CFTR variants characterized by a selective bicarbonate conductance defect (CFTRBD) may be associated with CP but not CF. However, a rigorous genetic analysis of the presumed association has been lacking. AIMS: To investigate the role of heterozygous CFTRBD variants in CP by meta-analysis of published case-control studies. MATERIALS AND METHODS: A systematic search was conducted in the MEDLINE, Embase, Scopus, and CENTRAL databases for published studies that reported the CFTRBD variants p.R74Q, p.R75Q, p.R117H, p.R170H, p.L967S, p.L997F, p.D1152H, p.S1235R, and p.D1270N in CP patients and controls. RESULTS: Twenty-two studies were eligible for quantitative synthesis. Combined analysis of the 9 CFTRBD variants indicated enrichment in CP patients versus controls (OR = 2.31, 95% CI = 1.17-4.56). Individual analysis of CFTRBD variants revealed no association of p.R75Q with CP (OR = 1.12, 95% CI = 0.89-1.40), whereas variants p.R117H and p.L967S were significantly overrepresented in cases relative to controls (OR = 3.16, 95% CI = 1.94-5.14, and OR = 3.88, 95% CI = 1.32-11.47, respectively). The remaining 6 low-frequency variants gave inconclusive results when analyzed individually, however, their pooled analysis indicated association with CP (OR = 2.08, 95% CI = 1.38-3.13). CONCLUSION: Heterozygous CFTRBD variants, with the exception of p.R75Q, increase CP risk about 2-4-fold.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Pancreatitis Crónica , Humanos , Bicarbonatos/metabolismo , Cloruros , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Mutación , Pancreatitis Crónica/genéticaRESUMEN
Pancreatitis, the inflammatory disorder of the pancreas, has no specific therapy. Genetic, biochemical, and animal model studies revealed that trypsin plays a central role in the onset and progression of pancreatitis. Here, we performed biochemical and preclinical mouse experiments to offer proof of concept that orally administered dabigatran etexilate can inhibit pancreatic trypsins and shows therapeutic efficacy in trypsin-dependent pancreatitis. We found that dabigatran competitively inhibited all human and mouse trypsin isoforms (Ki range 10-79 nM) and dabigatran plasma concentrations in mice given oral dabigatran etexilate well exceeded the Ki of trypsin inhibition. In the T7K24R trypsinogen mutant mouse model, a single oral gavage of dabigatran etexilate was effective against cerulein-induced progressive pancreatitis, with a high degree of histological normalization. In contrast, spontaneous pancreatitis in T7D23A mice, which carry a more aggressive trypsinogen mutation, was not ameliorated by dabigatran etexilate, given either as daily gavages or by mixing it with solid chow. Taken together, our observations showed that benzamidine derivatives such as dabigatran are potent trypsin inhibitors and show therapeutic activity against trypsin-dependent pancreatitis in T7K24R mice. Lack of efficacy in T7D23A mice is probably related to the more severe pathology and insufficient drug concentrations in the pancreas.
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Dabigatrán , Pancreatitis , Animales , Humanos , Ratones , Modelos Animales de Enfermedad , Páncreas , Pancreatitis/inducido químicamente , Pancreatitis/tratamiento farmacológico , Pancreatitis/genética , Tripsina/genética , Tripsinógeno/genéticaRESUMEN
Pancreatic chymotrypsins (CTRs) are digestive proteases that in humans include CTRB1, CTRB2, CTRC, and CTRL. The highly similar CTRB1 and CTRB2 are the products of gene duplication. A common inversion at the CTRB1-CTRB2 locus reverses the expression ratio of these isoforms in favor of CTRB2. Carriers of the inversion allele are protected against the inflammatory disorder pancreatitis presumably via their increased capacity for CTRB2-mediated degradation of harmful trypsinogen. To reveal the protective molecular determinants of CTRB2, we compared enzymatic properties of CTRB1, CTRB2, and bovine CTRA (bCTRA). By evolving substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) inhibitory loop variants against the chymotrypsins, we found that the substrate binding groove of the three enzymes had overlapping specificities. Based on the selected sequences, we produced eight SGPI-2 variants. Remarkably, CTRB2 and bCTRA bound these inhibitors with significantly higher affinity than CTRB1. Moreover, digestion of peptide substrates, beta casein, and human anionic trypsinogen unequivocally confirmed that CTRB2 is a generally better enzyme than CTRB1 while the potency of bCTRA lies between those of the human isoforms. Unexpectedly, mutation D236R alone converted CTRB1 to a CTRB2-like high activity protease. Modeling indicated that in CTRB1 Met210 partially obstructed the substrate binding groove, which was relieved by the D236R mutation. Taken together, we identify CTRB2 Arg236 as a key positive determinant, while CTRB1 Asp236 as a negative determinant for chymotrypsin activity. These findings strongly support the concept that in carriers of the CTRB1-CTRB2 inversion allele, the superior trypsinogen degradation capacity of CTRB2 protects against pancreatitis.
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Quimotripsina , Pancreatitis , Animales , Bovinos , Quimotripsina/genética , Humanos , Páncreas/metabolismo , Pancreatitis/genética , Péptidos/metabolismo , Tripsinógeno/genéticaRESUMEN
ABSTRACT: The identification of the genetic basis of hereditary pancreatitis in 1996 confirmed the critical role of trypsinogen in this disease and opened a new avenue of research on pancreatitis-associated genetic risk factors and their mechanism of action. Through the following 25 years, the ensuing discoveries fundamentally changed our understanding of pancreatitis pathogenesis, clarified the role of trypsinogen autoactivation in disease onset and progression, and set the stage for future therapeutic interventions. This Frank Brooks Memorial Lecture was delivered on November 4, 2021, at the 52nd Annual Meeting of the American Pancreatic Association, held in Miami Beach, Florida.
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Pancreatitis Crónica , Tripsinógeno , Humanos , Páncreas/patología , Pancreatitis Crónica/genética , Pancreatitis Crónica/patología , Tripsinógeno/genéticaAsunto(s)
Pancreatitis , Tripsinógeno , Animales , Quimotripsina/genética , Ratones , Pancreatitis/genética , Fenotipo , Tripsina/genética , Tripsinógeno/genéticaRESUMEN
BACKGROUND: Genetic alterations in digestive enzymes have been associated with chronic pancreatitis (CP). Recently, chymotrypsin like elastase 3B (CELA3B) emerged as a novel risk gene. Thus, we evaluated CELA3B in two European cohorts with CP. METHODS: We analyzed all 8 CELA3B exons in 550 German non-alcoholic CP (NACP) patients and in 241 German controls by targeted DNA sequencing. In addition, we analyzed exons 6 and 7 by Sanger sequencing and the c.129+1G>A variant by melting curve analysis in 1078 further German controls. As replication cohort, we investigated up to 243 non-German European NACP patients and up to 1665 controls originating from Poland, Hungary, and Sweden. We assessed the cellular secretion and the elastase activity of recombinant CELA3B variants. RESULTS: In the German discovery cohort, we detected a splice-site variant in intron 2, c.129+1G>A, in 9/550 (1.64%) CP patients and in 5/1319 (0.38%) controls (P=0.007, OR=4.4, 95% CI=1.5-13.0). In the European replication cohort, this variant was also enriched in patients (9/178 [5.06%]) versus controls (13/1247 [1.04%]) (P=0.001, OR=5.1, 95% CI=2.1-12.0). We did not find the two previously reported codon 90 variants, p.R90C and p.R90L. CONCLUSIONS: Our data indicate that CELA3B is a susceptibility gene for CP. In contrast to previous reports suggesting that increased CELA3B activity is associated with CP risk, the splice-site variant identified here is predicted to cause diminished CELA3B expression. How reduced CELA3B function predisposes to pancreatitis remains to be elucidated.
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Quimotripsina , Elastasa Pancreática/genética , Pancreatitis Crónica , Quimotripsina/genética , Predisposición Genética a la Enfermedad , Humanos , Mutación , Elastasa Pancreática/metabolismo , Pancreatitis Crónica/metabolismoRESUMEN
OBJECTIVE: Non-alcoholic chronic pancreatitis (NACP) frequently develops in the setting of genetic susceptibility associated with alterations in genes that are highly expressed in the pancreas. However, the genetic basis of NACP remains unresolved in a significant number of patients warranting a search for further risk genes. DESIGN: We analyzed CUZD1, which encodes the CUB and zona pellucida-like domains 1 protein that is found in high levels in pancreatic acinar cells. We sequenced the coding region in 1163 European patients and 2018 European controls. In addition, we analyzed 297 patients and 1070 controls from Japan. We analyzed secretion of wild-type and mutant CUZD1 from transfected cells using Western blotting. RESULTS: In the European cohort, we detected 30 non-synonymous variants. Using different prediction tools (SIFT, CADD, PROVEAN, PredictSNP) or the combination of these tools, we found accumulation of predicted deleterious variants in patients (p-value range 0.002-0.013; OR range 3.1-5.2). No association was found in the Japanese cohort, in which 13 non-synonymous variants were detected. Functional studies revealed >50% reduced secretion of 7 variants, however, these variants were not significantly enriched in European CP patients. CONCLUSION: Our data indicate that CUZD1 might be a novel susceptibility gene for NACP. How these variants predispose to pancreatitis remains to be elucidated.
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Proteínas de la Membrana , Pancreatitis Crónica , Zona Pelúcida , Células Acinares/metabolismo , Western Blotting , Predisposición Genética a la Enfermedad , Humanos , Proteínas de la Membrana/genética , Pancreatitis Crónica/genética , Pancreatitis Crónica/patología , Zona Pelúcida/metabolismo , Zona Pelúcida/patologíaRESUMEN
The digestive protease chymotrypsin C (CTRC) protects the pancreas against pancreatitis by degrading potentially harmful trypsinogen. Loss-of-function genetic variants in CTRC increase risk for chronic pancreatitis (CP) with variable effect size, as judged by the reported odds ratio (OR) values. Here, we performed a meta-analysis of published studies on four variants that alter the CTRC amino-acid sequence, are clinically relatively common (global carrier frequency in CP >1%), reproducibly showed association with CP and their loss of function phenotype was verified experimentally. We found strong enrichment of CTRC variants p.A73T, p.V235I, p.K247_R254del, and p.R245W in CP cases versus controls, yielding OR values of 6.5 (95% confidence interval (CI) 2.4-17.8), 4.5 (CI 2.2-9.1), 5.4 (CI 2.6-11.0), and 2.6 (CI 1.6-4.2), respectively. Subgroup analysis demonstrated disease association of variants p.K247_R254del and p.R245W in alcoholic CP with similar effect sizes as seen in the overall CP group. Homozygosity or compound heterozygosity were rare and seemed to be associated with higher risk. We also identified a so far unreported linkage disequilibrium between variant p.K247_R254del and the common c.180C>T (p.G60 =) haplotype. Taken together, the results indicate that heterozygous loss-of-function CTRC variants increase the risk for CP approximately 3-7-fold. This meta-analysis confirms the clinical significance of CTRC variants and provides further justification for the genetic screening of CP patients.