Diagnostic yield of repeat genetic testing in idiopathic chronic pancreatitis.

Genetic testing is performed for unexplained pancreatitis. The aim of this study was to evaluate the diagnostic value of repeating genetic testing in idiopathic pancreatitis when new predisposing genes are identified. We investigated 330 patients who were initially screened for PRSS1, SPINK1 and CFTR genes. A new analysis was performed by Next-Generation Sequencing (NGS) for PRSS1, SPINK1, CFTR, CTRC, CASR, CPA1, TRPV6 genes and the CEL-HYB1 allele in clinical practice, and patients were included in our cohort study. Additional rare variants were identified in 7.3 % of the patients. Screening for new pancreatitis genes is recommended when initial screening is limited. Routine use of NGS is a useful diagnostic tool in these cases.

Combining full-length gene assay and SpliceAI to interpret the splicing impact of all possible SPINK1 coding variants.

BACKGROUND: Single-nucleotide variants (SNVs) within gene coding sequences can significantly impact pre-mRNA splicing, bearing profound implications for pathogenic mechanisms and precision medicine. In this study, we aim to harness the well-established full-length gene splicing assay (FLGSA) in conjunction with SpliceAI to prospectively interpret the splicing effects of all potential coding SNVs within the four-exon SPINK1 gene, a gene associated with chronic pancreatitis. RESULTS: Our study began with a retrospective analysis of 27 SPINK1 coding SNVs previously assessed using FLGSA, proceeded with a prospective analysis of 35 new FLGSA-tested SPINK1 coding SNVs, followed by data extrapolation, and ended with further validation. In total, we analyzed 67 SPINK1 coding SNVs, which account for 9.3% of the 720 possible coding SNVs. Among these 67 FLGSA-analyzed SNVs, 12 were found to impact splicing. Through detailed comparison of FLGSA results and SpliceAI predictions, we inferred that the remaining 653 untested coding SNVs in the SPINK1 gene are unlikely to significantly affect splicing. Of the 12 splice-altering events, nine produced both normally spliced and aberrantly spliced transcripts, while the remaining three only generated aberrantly spliced transcripts. These splice-impacting SNVs were found solely in exons 1 and 2, notably at the first and/or last coding nucleotides of these exons. Among the 12 splice-altering events, 11 were missense variants (2.17% of 506 potential missense variants), and one was synonymous (0.61% of 164 potential synonymous variants). Notably, adjusting the SpliceAI cut-off to 0.30 instead of the conventional 0.20 would improve specificity without reducing sensitivity. CONCLUSIONS: By integrating FLGSA with SpliceAI, we have determined that less than 2% (1.67%) of all possible coding SNVs in SPINK1 significantly influence splicing outcomes. Our findings emphasize the critical importance of conducting splicing analysis within the broader genomic sequence context of the study gene and highlight the inherent uncertainties associated with intermediate SpliceAI scores (0.20 to 0.80). This study contributes to the field by being the first to prospectively interpret all potential coding SNVs in a disease-associated gene with a high degree of accuracy, representing a meaningful attempt at shifting from retrospective to prospective variant analysis in the era of exome and genome sequencing.

Expanding ACMG variant classification guidelines into a general framework.

BACKGROUND: The American College of Medical Genetics and Genomics (ACMG)-recommended five variant classification categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign) have been widely used in medical genetics. However, these guidelines are fundamentally constrained in practice owing to their focus upon Mendelian disease genes and their dichotomous classification of variants as being either causal or not. Herein, we attempt to expand the ACMG guidelines into a general variant classification framework that takes into account not only the continuum of clinical phenotypes, but also the continuum of the variants' genetic effects, and the different pathological roles of the implicated genes. MAIN BODY: As a disease model, we employed chronic pancreatitis (CP), which manifests clinically as a spectrum from monogenic to multifactorial. Bearing in mind that any general conceptual proposal should be based upon sound data, we focused our analysis on the four most extensively studied CP genes, PRSS1, CFTR, SPINK1 and CTRC. Based upon several cross-gene and cross-variant comparisons, we first assigned the different genes to two distinct categories in terms of disease causation: CP-causing (PRSS1 and SPINK1) and CP-predisposing (CFTR and CTRC). We then employed two new classificatory categories, "predisposing" and "likely predisposing", to replace ACMG's "pathogenic" and "likely pathogenic" categories in the context of CP-predisposing genes, thereby classifying all pathologically relevant variants in these genes as "predisposing". In the case of CP-causing genes, the two new classificatory categories served to extend the five ACMG categories whilst two thresholds (allele frequency and functional) were introduced to discriminate "pathogenic" from "predisposing" variants. CONCLUSION: Employing CP as a disease model, we expand ACMG guidelines into a five-category classification system (predisposing, likely predisposing, uncertain significance, likely benign, and benign) and a seven-category classification system (pathogenic, likely pathogenic, predisposing, likely predisposing, uncertain significance, likely benign, and benign) in the context of disease-predisposing and disease-causing genes, respectively. Taken together, the two systems constitute a general variant classification framework that, in principle, should span the entire spectrum of variants in any disease-related gene. The maximal compliance of our five-category and seven-category classification systems with the ACMG guidelines ought to facilitate their practical application.

Functionally deficient TRPV6 variants contribute to hereditary and familial chronic pancreatitis.

The recent discovery of TRPV6 as a pancreatitis susceptibility gene served to identify a novel mechanism of chronic pancreatitis (CP) due to Ca2+ dysregulation. Herein, we analyzed TRPV6 in 81 probands with hereditary CP (HCP), 204 probands with familial CP (FCP), and 462 patients with idiopathic CP (ICP) by targeted next-generation sequencing. We identified 25 rare nonsynonymous TRPV6 variants, 18 of which had not been previously reported. All 18 variants were characterized by a Ca2+ imaging assay, with 8 being identified as functionally deficient. Evaluation of functionally deficient variants in the three CP cohorts revealed two novel findings: (i) functionally deficient TRPV6 variants appear to occur more frequently in HCP/FCP patients than in ICP patients (3.2% vs. 1.5%) and (ii) functionally deficient TRPV6 variants found in HCP and FCP probands appear to be more frequently coinherited with known risk variants in SPINK1, CTRC, and/or CFTR than those found in ICP patients (66.7% vs 28.6%). Additionally, genetic analysis of available HCP and FCP family members revealed complex patterns of inheritance in some families. Our findings confirm that functionally deficient TRPV6 variants represent an important contributor to CP. Importantly, functionally deficient TRPV6 variants account for a significant proportion of cases of HCP/FCP.

Chronic Pancreatitis: The True Pathogenic Culprit within the SPINK1 N34S-Containing Haplotype Is No Longer at Large.

A diverse range of loss-of-function variants in the SPINK1 gene (encoding pancreatic secretory trypsin inhibitor) has been identified in patients with chronic pancreatitis (CP). The haplotype harboring the SPINK1 c.101A>G (p.Asn34Ser or N34S) variant (rs17107315:T>C) is one of the most important heritable risk factors for CP as a consequence of its relatively high prevalence worldwide (population allele frequency ≈ 1%) and its considerable effect size (odds ratio ≈ 11). The causal variant responsible for this haplotype has been intensively investigated over the past two decades. The different hypotheses tested addressed whether the N34S missense variant has a direct impact on enzyme structure and function, whether c.101A>G could affect pre-mRNA splicing or mRNA stability, and whether another variant in linkage disequilibrium with c.101A>G might be responsible for the observed association with CP. Having reviewed the currently available genetic and experimental data, we conclude that c.-4141G>T (rs142703147:C>A), which disrupts a PTF1L-binding site within an evolutionarily conserved HNF1A-PTF1L cis-regulatory module located ∼4 kb upstream of the SPINK1 promoter, can be designated as the causal variant beyond reasonable doubt. This case illustrates the difficulties inherent in determining the identity of the causal variant underlying an initially identified disease association.

The p.E152K-STIM1 mutation deregulates Ca2+ signaling contributing to chronic pancreatitis.

Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation, and stromal interaction molecule-1 (STIM1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) conformational change and enhanced SERCA pump activity leading to increased store-operated Ca2+ entry (SOCE). In pancreatic AR42J cells expressing the p.E152K variant, Ca2+ signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation.This article has an associated First Person interview with the first author of the paper.

The Experimentally Obtained Functional Impact Assessments of 5' Splice Site GT'GC Variants Differ Markedly from Those Predicted.

INTRODUCTION: 5' splice site GT>GC or +2T>C variants have been frequently reported to cause human genetic disease and are routinely scored as pathogenic splicing mutations. However, we have recently demonstrated that such variants in human disease genes may not invariably be pathogenic. Moreover, we found that no splicing prediction tools appear to be capable of reliably distinguishing those +2T>C variants that generate wild-type transcripts from those that do not. METHODOLOGY: Herein, we evaluated the performance of a novel deep learning-based tool, SpliceAI, in the context of three datasets of +2T>C variants, all of which had been characterized functionally in terms of their impact on pre-mRNA splicing. The first two datasets refer to our recently described "in vivo" dataset of 45 known disease-causing +2T>C variants and the "in vitro" dataset of 103 +2T>C substitutions subjected to full-length gene splicing assay. The third dataset comprised 12 BRCA1 +2T>C variants that were recently analyzed by saturation genome editing. RESULTS: Comparison of the SpliceAI-predicted and experimentally obtained functional impact assessments of these variants (and smaller datasets of +2T>A and +2T>G variants) revealed that although SpliceAI performed rather better than other prediction tools, it was still far from perfect. A key issue was that the impact of those +2T>C (and +2T>A) variants that generated wild-type transcripts represents a quantitative change that can vary from barely detectable to an almost full expression of wild-type transcripts, with wild-type transcripts often co-existing with aberrantly spliced transcripts. CONCLUSION: Our findings highlight the challenges that we still face in attempting to accurately identify splice-altering variants.

Characterization of CEL-DUP2: Complete duplication of the carboxyl ester lipase gene is unlikely to influence risk of chronic pancreatitis.

BACKGROUND/OBJECTIVES: Carboxyl ester lipase is a pancreatic enzyme encoded by CEL, an extremely polymorphic human gene. Pathogenic variants of CEL either increases the risk for chronic pancreatitis (CP) or cause MODY8, a syndrome of pancreatic exocrine and endocrine dysfunction. Here, we aimed to characterize a novel duplication allele of CEL (CEL-DUP2) and to investigate whether it associates with CP or pancreatic cancer. METHODS: The structure of CEL-DUP2 was determined by a combination of Sanger sequencing, DNA fragment analysis, multiplex ligation-dependent probe amplification and whole-genome sequencing. We developed assays for screening of CEL-DUP2 and analyzed cohorts of idiopathic CP, alcoholic CP and pancreatic cancer. CEL protein expression was analyzed by immunohistochemistry. RESULTS: CEL-DUP2 consists of an extra copy of the complete CEL gene. The allele has probably arisen from non-allelic, homologous recombination involving the adjacent pseudogene of CEL. We found no association between CEL-DUP2 carrier frequency and CP in cohorts from France (cases/controls: 2.5%/2.4%; P = 1.0), China (10.3%/8.1%; P = 0.08) or Germany (1.6%/2.3%; P = 0.62). Similarly, no association with disease was observed in alcohol-induced pancreatitis (Germany: 3.2%/2.3%; P = 0.51) or pancreatic cancer (Norway; 2.5%/3.2%; P = 0.77). Notably, the carrier frequency of CEL-DUP2 was more than three-fold higher in Chinese compared with Europeans. CEL protein expression was similar in tissues from CEL-DUP2 carriers and controls. CONCLUSIONS: Our results support the contention that the number of CEL alleles does not influence the risk of pancreatic exocrine disease. Rather, the pathogenic CEL variants identified so far involve exon 11 sequence changes that substantially alter the protein's tail region.

Natural history of SPINK1 germline mutation related-pancreatitis.

BACKGROUND: The 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). METHODS: All 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. FINDINGS: 209 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). INTERPRETATION: SPINK1 related pancreatitis is associated with earlier onset and pancreatic insufficiencies. p.N34S SPINK1 may well be associated with cancer.

Toward a clinical diagnostic pipeline for SPINK1 intronic variants.

BACKGROUND: The clinical significance of SPINK1 intronic variants in chronic pancreatitis has been previously assessed by various approaches including a cell culture-based full-length gene assay. A close correlation between the results of this assay and in silico splicing prediction was apparent. However, until now, a clinical diagnostic pipeline specifically designed to classify SPINK1 intronic variants accurately and efficiently has been lacking. Herein, we present just such a pipeline and explore its efficacy and potential utility in potentiating the classification of newly described SPINK1 intronic variants. RESULTS: We confirm a close correlation between in silico splicing prediction and results from the cell culture-based full-length gene assay in the context of three recently reported pathogenic SPINK1 intronic variants. We then integrated in silico splicing prediction and the full-length gene assay into a stepwise approach and tested its utility in the classification of two novel datasets of SPINK1 intronic variants. The first dataset comprised 16 deep intronic variants identified in 52 genetically unexplained Chinese chronic pancreatitis patients by sequencing the entire intronic sequence of the SPINK1 gene. The second dataset comprised five novel rare proximal intronic variants identified through the routine analysis of the SPINK1 gene in French pancreatitis patients. Employing a minor allele frequency of > 5% as a population frequency filter, 6 of the 16 deep intronic variants were immediately classified as benign. In silico prediction of the remaining ten deep intronic variants and the five rare proximal intronic variants with respect to their likely impact on splice site selection suggested that only one proximal intronic variant, c.194 + 5G > A, was likely to be of functional significance. Employing the cell culture-based full-length gene assay, we functionally analyzed c.194 + 5G > A, together with seven predicted non-functional variants, thereby validating their predicted effects on splicing in all cases. CONCLUSIONS: We demonstrated the accuracy and efficiency of in silico prediction in combination with the cell culture-based full-length gene assay for the classification of SPINK1 intronic variants. Based upon these findings, we propose an operational pipeline for classifying SPINK1 intronic variants in the clinical diagnostic setting.

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

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.

Severe infantile isolated exocrine pancreatic insufficiency caused by the complete functional loss of the SPINK1 gene.

Exocrine pancreatic insufficiency (EPI) is rare in children, with most if not all cases occurring as part of syndromic conditions such as cystic fibrosis and Shwachman-Diamond syndrome. Here we report two cases, both presenting with severe EPI around 5 months of age. Characterized by diffuse pancreatic lipomatosis, they otherwise exhibited no remarkable deficiencies in other organs. Novel non-identical homozygous variants (a deletion removing the entire SPINK1 gene and an insertion of a full-length inverted Alu element into the 3'-untranslated region of the SPINK1 gene) resulting in the complete functional loss of the SPINK1 gene (encoding pancreatic secretory trypsin inhibitor) were identified in each patient. Having correlated our findings with current knowledge of SPINK1's role in exocrine pancreas pathophysiology, we propose that complete and partial functional losses of the SPINK1 gene are associated with quite distinct phenotypes, the former causing a new pediatric disease entity of severe infantile isolated EPI.

Identification of a functional enhancer variant within the chronic pancreatitis-associated SPINK1 c.101A>G (p.Asn34Ser)-containing haplotype.

The haplotype harboring the SPINK1 c.101A>G (p.Asn34Ser) variant (also known as rs17107315:T>C) represents the most important heritable risk factor for idiopathic chronic pancreatitis identified to date. The causal variant contained within this risk haplotype has however remained stubbornly elusive. Herein, we set out to resolve this enigma by employing a hypothesis-driven approach. First, we searched for variants in strong linkage disequilibrium (LD) with rs17107315:T>C using HaploReg v4.1. Second, we identified two candidate SNPs by visual inspection of sequences spanning all 25 SNPs found to be in LD with rs17107315:T>C, guided by prior knowledge of pancreas-specific transcription factors and their cognate binding sites. Third, employing a novel cis-regulatory module (CRM)-guided approach to further filter the two candidate SNPs yielded a solitary candidate causal variant. Finally, combining data from phylogenetic conservation and chromatin accessibility, cotransfection transactivation experiments, and population genetic studies, we suggest that rs142703147:C>A, which disrupts a PTF1L-binding site within an evolutionarily conserved HNF1A-PTF1L CRM located ∼4 kb upstream of the SPINK1 promoter, contributes to the aforementioned chronic pancreatitis risk haplotype. Further studies are required not only to improve the characterization of this functional SNP but also to identify other functional components that might contribute to this high-risk haplotype.

Genetic and electrophysiological characteristics of recurrent acute pancreatitis.

OBJECTIVES: The aim was to present the workup of patients with acute recurrent pancreatitis (ARP) for genetic analysis and electrophysiological testing. METHODS: Patients with ARP with unknown etiology were referred for genetic testing and evaluation of cystic fibrosis transmembrane conductor regulator (CFTR) function by nasal potential difference (NPD) testing. RESULTS: A total of 67 patients were evaluated. The mean age was 23 ±â€Š17 years (median 17.0 years, range 1.5-72 years); 90% were Jewish and 10% Arab. Ten (15%) patients carried PRSS1 gene mutation (K23R(7), R122H(2), and D21A(1)). One patient had K172E/- (chymotrypsin C [CTRC]) mutation, 1 had I42M (serine protease inhibitor Kazal type 1 [SPINK1])/V235I (CTRC) together with ΔF508/5T, 1 patient had R67H (SPINK1)/V235I (CTRC), and 1 patient had V235I (CTRC)/-. Ten of 67 (15%) patients submitted for CFTR gene testing carried mutations (ΔF508/L997F, ΔF508/5T(11TG), W1282/5T(12TG), W1282X/Y1014C, ΔF508/R31C, R117H/-, R117H/Y1014C, D1152H/-, 5T(11TG)/-, and L997F/-). Fifty-four (80%) patients underwent sweat testing. Of these, 5 had sweat chloride ≥60 mEq/L, and 22 patients had sweat chloride from 40 to 60 mEq/L. Of the 56 (83%) patients had nasal potential difference testing, 4 (6%) with abnormal results. CONCLUSIONS: One-third (34%) of patients with ARP carry mutations for hereditary pancreatitis including rare mutations (K23R), and 12.5% have evidence of cftr mutations and 10% had CFTR dysfunction underscoring the importance of genetic and functional workup of these patients.

Mutational analysis of ATP8B1 in patients with chronic pancreatitis.

BACKGROUND: Mutations in genes encoding cationic trypsinogen (PRSS1), pancreatic secretory trypsin inhibitor (SPINK1) and chymotrypsinogen C (CTRC) are associated with chronic pancreatitis. However, in many patients with a familial chronic pancreatitis pattern suggesting a genetic cause, no mutations in either of these genes can be found, indicating that other, still unknown, associated genes exist. In this respect ATP8B1 is an interesting candidate due to its strong expression in the pancreas, its supposed general function in membrane organization and the higher incidence of pancreatitis in patients with ATP8B1 deficiency. METHODS: We analyzed all 27 ATP8B1 coding exons and adjacent non-coding sequences of 507 chronic pancreatitis patients by direct sequencing. Exons that harbored possible relevant variations were subsequently sequenced in 1,027 healthy controls. RESULTS: In the exonic regions, 5 novel non-synonymous alterations were detected as well as 14 previously described alterations of which some were associated with ATP8B1 deficiency. However, allele frequencies for any of these variations did not significantly differ between patients and controls. Furthermore, several non-synonymous variants were exclusively detected in control subjects and multiple variants in the non-coding sequence were identified with similar frequencies in both groups. CONCLUSIONS: We did not find an association between heterozygous ATP8B1 variants and chronic pancreatitis in our cohort of patients with hereditary and idiopathic chronic pancreatitis.

A conservative assessment of the major genetic causes of idiopathic chronic pancreatitis: data from a comprehensive analysis of PRSS1, SPINK1, CTRC and CFTR genes in 253 young French patients.

Idiopathic chronic pancreatitis (ICP) has traditionally been defined as chronic pancreatitis in the absence of any obvious precipitating factors (e.g. alcohol abuse) and family history of the disease. Studies over the past 15 years have revealed that ICP has a highly complex genetic architecture involving multiple gene loci. Here, we have attempted to provide a conservative assessment of the major genetic causes of ICP in a sample of 253 young French ICP patients. For the first time, conventional types of mutation (comprising coding sequence variants and variants at intron/exon boundaries) and gross genomic rearrangements were screened for in all four major pancreatitis genes, PRSS1, SPINK1, CTRC and CFTR. For the purposes of the study, synonymous, intronic and 5'- or 3'-untranslated region variants were excluded from the analysis except where there was persuasive evidence of functional consequences. The remaining sequence variants/genotypes were classified into causative, contributory or neutral categories by consideration of (i) their allele frequencies in patient and normal control populations, (ii) their presumed or experimentally confirmed functional effects, (iii) the relative importance of their associated genes in the pathogenesis of chronic pancreatitis and (iv) gene-gene interactions wherever applicable. Adoption of this strategy allowed us to assess the pathogenic relevance of specific variants/genotypes to their respective carriers to an unprecedented degree. The genetic cause of ICP could be assigned in 23.7% of individuals in the study group. A strong genetic susceptibility factor was also present in an additional 24.5% of cases. Taken together, up to 48.2% of the studied ICP patients were found to display evidence of a genetic basis for their pancreatitis. Whereas these particular proportions may not be extrapolable to all ICP patients, the approach employed should serve as a useful framework for acquiring a better understanding of the role of genetic factors in causing this oligogenic disease.

Characterization of two deletions of the CTRC locus.

Novel variants associated with chronic pancreatitis are being increasingly reported. However, most studies have so far only analyzed point mutations and small insertions or deletions. Here we report the characterization of two distinct deletions of the CTRC locus. Variants in four chronic pancreatitis genes, PRSS1, SPINK1, CTRC and CFTR, were systematically analyzed in the studied cases. Copy number change of the CTRC gene was analyzed by quantitative fluorescent multiplex PCR (QFM-PCR). Walking QFM-PCR followed by long-range PCR and direct sequencing were employed to identify the deletion breakpoints at the nucleotide level. A heterozygous CTRC-deleting complex rearrangement, which was co-inherited with different trans variants in SPINK1, CFTR or PRSS1, is associated with variable phenotypes (chronic pancreatitis; pancreatic cancer and chronic pancreatitis; and type 1 diabetes). Moreover, a different homozygous deletion of the CTRC locus was found in an unrelated patient with asymptomatic chronic pancreatitis. Our findings revealed a hitherto unrecognized level of complexity of genotype-phenotype correlation in chronic pancreatitis. The CTRC-deleting complex rearrangement probably resulted from LINE-1-mediated Alu insertion, which represents a novel mutational mechanism causing chronic pancreatitis.