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Defective binding of SPINK1 variants is an uncommon mechanism for impaired trypsin inhibition in chronic pancreatitis.

ABSTRACT: The serine protease inhibitor Kazal type 1 (SPINK1) protects the pancreas from intrapancreatic trypsin activation that can lead to pancreatitis. Loss-of-function genetic variants of SPINK1 increase the risk for chronic pancreatitis, often by diminishing inhibitor expression or secretion. Variants that are secreted normally have been presumed to be pathogenic because of defective trypsin inhibition, but evidence has been lacking. Here, we report quantitative studies on the inhibition of human trypsins by wildtype SPINK1 and seven secreted missense variants. We found that tyrosine sulfation of human trypsins weakens binding of SPINK1 because of altered interactions with Tyr43 in the SPINK1 reactive loop. Using authentic sulfated human trypsins, we provide conclusive evidence that SPINK1 variants N34S, N37S, R65Q, and Q68R have unimpaired inhibitory activity, whereas variant P55S exhibits a small and clinically insignificant binding defect. In contrast, rare variants K41N and I42M that affect the reactive-site peptide bond of SPINK1 decrease inhibitor binding by 20,000- to 30,000-fold and three- to sevenfold, respectively. Taken together, the observations indicate that defective trypsin inhibition by SPINK1 variants is an uncommon mechanism in chronic pancreatitis. The results also strengthen the notion that a decline in inhibitor levels explains pancreatitis risk associated with the large majority of SPINK1 variants.

SUBMITTER: Szabo A

PROVIDER: S-EPMC7949130 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Functional significance of SPINK1 promoter variants in chronic pancreatitis.

Project description:Chronic pancreatitis is a progressive inflammatory disorder of the pancreas, which often develops as a result of genetic predisposition. Some of the most frequently identified risk factors affect the serine protease inhibitor Kazal type 1 (SPINK1) gene, which encodes a trypsin inhibitor responsible for protecting the pancreas from premature trypsinogen activation. Recent genetic and functional studies indicated that promoter variants in the SPINK1 gene might contribute to disease risk in carriers. Here, we investigated the functional effects of 17 SPINK1 promoter variants using luciferase reporter gene expression assay in four different cell lines, including three pancreatic acinar cell lines (rat AR42J with or without dexamethasone-induced differentiation and mouse 266-6) and human embryonic kidney 293T cells. We found that most variants caused relatively small changes in promoter activity. Surprisingly, however, we observed significant variations in the effects of the promoter variants in the different cell lines. Only four variants exhibited consistently reduced promoter activity in all acinar cell lines, confirming previous reports that variants c.-108G>T, c.-142T>C, and c.-147A>G are risk factors for chronic pancreatitis and identifying c.-52G>T as a novel risk variant. In contrast, variant c.-215G>A, which is linked with the disease-associated splice-site mutation c.194 + 2T>C, caused increased promoter activity, which may mitigate the overall effect of the pathogenic haplotype. Our study lends further support to the notion that sequence evaluation of the SPINK1 promoter region in patients with chronic pancreatitis is justified as part of the etiological investigation.

| S-EPMC4421017 | biostudies-literature

Signal peptide variants that impair secretion of pancreatic secretory trypsin inhibitor (SPINK1) cause autosomal dominant hereditary pancreatitis.

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| S-EPMC2765331 | biostudies-literature

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| S-EPMC2677899 | biostudies-literature

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| S-EPMC7704661 | biostudies-literature

PRSS1, SPINK1, CFTR, and CTRC Pathogenic Variants in Korean Patients With Idiopathic Pancreatitis.

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| S-EPMC5011109 | biostudies-literature

Association of rare SPINK1 gene mutation with another base substitution in chronic pancreatitis patients.

Project description:AIM:To verify and expand the known spectrum of serine protease inhibitor Kazal type 1 (SPINK1) gene mutations in chronic pancreatitis. METHODS:DNA extracted from 172 chronic pancreatitis patients was assayed for SPINK1 gene mutations by PCR and DNA sequencing. A control cohort of 90 unrelated healthy individuals was analysed by the same methods for presence of common populational polymorphisms, and frequency of five-loci haplotypes was calculated. Linkages of gene aberrations in single SPINK1 gene copies were analysed by long-distance PCR followed by allele-specific PCR and DNA sequencing. RESULTS:The most frequent SPINK1 gene mutation N34S was found at a frequency of 6%. Furthermore, we detected the heterozygous intervening sequence (IVS) 3 + 2 T > C mutated gene in 2 German patients and 1 Macedonian chronic pancreatitis patient. In all three SPINK1 gene copies an additional rare base substitution was found: 5'untranslated region (UTR)-215 G > A. Polymorphism analysis revealed that all three affected genes carried the same five-loci haplotype. DNA sequencing of another chronic pancreatitis-related gene PRSS1 (cationic trypsinogen) did not reveal any mutations in these 3 patients. CONCLUSION:We found in 3 (2%) of 172 chronic pancreatitis patients an IVS3 + 2 T > C SPINK1 gene mutation and a base substitution 5'UTR-215 G > A in the same gene copy. Most probably the 5'UTR-215 G > A represents a rare polymorphism and not a mutation as previously concluded. Haplotype analysis suggests a common origin of the IVS3 + 2 T > C mutation in these patients.

| S-EPMC4088203 | biostudies-literature

Genetic Risk in Chronic Pancreatitis: The Trypsin-Dependent Pathway.

Project description:Genetic investigations have provided unique insight into the mechanism of chronic pancreatitis in humans and firmly established that uncontrolled trypsin activity is a central pathogenic factor. Mutations in the PRSS1, SPINK1, and CTRC genes promote increased activation of trypsinogen to trypsin by stimulation of autoactivation or by impairing protective trypsinogen degradation and/or trypsin inhibition. Here we review key genetic and biochemical features of the trypsin-dependent pathological pathway in chronic pancreatitis.

| S-EPMC5487703 | biostudies-other

No significant enrichment of rare functionally defective CPA1 variants in a large Chinese idiopathic chronic pancreatitis cohort.

Project description:Rare functionally defective carboxypeptidase A1 (CPA1) variants have been reported to predispose to nonalcoholic chronic pancreatitis, mainly the idiopathic subtype. However, independent replication has so far been lacking, particularly in Asian cohorts where initial studies employed small sample sizes. Herein we performed targeted next-generation sequencing of the CPA1 gene in 1,112 Han Chinese idiopathic chronic pancreatitis (ICP) patients-the largest ICP cohort so far analyzed in a single population-and 1,580 controls. Sanger sequencing was used to validate called variants, and the CPA1 activity and secretion of all newly found variants were measured. A total of 18 rare CPA1 variants were characterized, 11 of which have not been previously described. However, no significant association was noted with ICP irrespective of whether all rare variants [20 out of 1,112 (1.8%) in patients vs. 24 out of 1,580 (1.52%) in controls; P = 0.57] or functionally impaired variants [three out of 1,112 (0.27%) in patients vs. two out of 1,580 (0.13%) in controls; P = 0.68] were considered.

| S-EPMC5542845 | biostudies-literature

SPINK1, PRSS1, CTRC, and CFTR Genotypes Influence Disease Onset and Clinical Outcomes in Chronic Pancreatitis.

Project description:OBJECTIVES:Rare pathogenic variants in the SPINK1, PRSS1, CTRC, and CFTR genes have been strongly associated with a risk of developing chronic pancreatitis (CP). However, their potential impact on the age of disease onset and clinical outcomes, as well as their potential interactions with environmental risk factors, remain unclear. These issues are addressed here in a large Chinese CP cohort. METHODS:We performed targeted next-generation sequencing of the four CP-associated genes in 1061 Han Chinese CP patients and 1196 controls. To evaluate gene-environment interactions, the patients were divided into three subgroups, idiopathic CP (ICP; n?=?715), alcoholic CP (ACP; n?=?206), and smoking-associated CP (SCP; n?=?140). The potential impact of rare pathogenic variants on the age of onset of CP and clinical outcomes was evaluated using the Kaplan-Meier model. RESULTS:We identified rare pathogenic genotypes involving the SPINK1, PRSS1, CTRC, and/or CFTR genes in 535 (50.42%) CP patients but in only 71 (5.94%) controls (odds ratio?=?16.12; P?<?0.001). Mutation-positive patients had significantly earlier median ages at disease onset and at diagnosis of pancreatic stones, diabetes mellitus and steatorrhea than mutation-negative ICP patients. Pathogenic genotypes were present in 57.1, 39.8, and 32.1% of the ICP, ACP, and SCP patients, respectively, and influenced age at disease onset and clinical outcomes in all subgroups. CONCLUSIONS:We provide evidence that rare pathogenic variants in the SPINK1, PRSS1, CTRC, and CFTR genes significantly influence the age of onset and clinical outcomes of CP. Extensive gene-environment interactions were also identified.

| S-EPMC6232107 | biostudies-literature

Natural history of SPINK1 germline mutation related-pancreatitis.

Project description:BackgroundThe aim was to describe genetic, clinical and morphological features in a large, multicentre European cohort of patients with SPINK1 related pancreatitis, in comparison with patients with idiopathic pancreatitis (IP).MethodsAll SPINK1 mutation carriers with pancreatic symptoms from two French and one English centers were included. Patients with IP were included in a control group. Genetic, clinical, radiological and biochemical data were collected.Findings209 and 302 patients were included in the SPINK1 and control groups (median follow-up: 8.3 years (3.7-17.4) vs 5.3 (2.5-8.8)). The median age at onset of symptoms was 20.1 years (17.5-22.8) in the SPINK1 group versus 41.2 (35.2-45.2). The age of exocrine pancreatic insufficiency (EPI) onset in the SPINK1 group was 49.5 (44.5-54.6) years vs. 65.2 (62.1-68.3), p < 0.001. SPINK1 patients with EPI were 5.3%, 14.7%, 28.3% and 52.4% at 20, 30, 40 and 50 years. Diabetes occurred 37.7 (33.3-42.1) years following the onset of symptoms in the SPINK1 group vs. 30.6 (17.3-43.8) (p = 0.002). SPINK1 patients with diabetes were 7.8%, 13.4%, 26.3% and 43.4% at 30, 40, 50 and 60 years. Seven patients (3.3%) developed pancreatic cancer in the SPINK1 group (versus 3 (0.99%), p = 0.1), at a median age of 60 vs 66 years. The cancer risk was 0.8% before 50 years, 11.9%, 27.7%, 51.8% at 60, 70 and 80 years and was 12 times higher than in controls (Cox HR 12.0 (3.0-47.8), p < 0.001).InterpretationSPINK1 related pancreatitis is associated with earlier onset and pancreatic insufficiencies. p.N34S SPINK1 may well be associated with cancer.

| S-EPMC6838417 | biostudies-literature

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