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. Author manuscript; available in PMC: 2012 May 1.
Published in final edited form as: Pancreas. 2011 May;40(4):540–546. doi: 10.1097/MPA.0b013e3182152fdf

Intragenic duplication: a novel mutational mechanism in hereditary pancreatitis

Maiken T Joergensen 1,#,*, Andrea Geisz 2,*, Klaus Brusgaard 3, Ove B Schaffalitzky de Muckadell 1, Péter Hegyi 4, Anne-Marie Gerdes 3, Miklós Sahin-Tóth 2
PMCID: PMC3088488  NIHMSID: NIHMS280563  PMID: 21499207

Abstract

Objectives

In a hereditary pancreatitis family from Denmark we identified a novel intragenic duplication of 9 nucleotides in exon-2 of the human cationic trypsinogen (PRSS1) gene (c.63_71dup) which at the amino-acid level resulted in the insertion of three amino acids within the activation peptide of cationic trypsinogen (p.K23_I24insIDK). The aim of the present study was to characterize the effect of this unique genetic alteration on the function of human cationic trypsinogen.

Methods

Wild-type and mutant cationic trypsinogens were produced recombinantly and purified to homogeneity. Trypsinogen activation was followed by enzymatic assays and SDS-PAGE. Trypsinogen secretion was measured from transfected HEK 293T cells.

Results

Recombinant cationic trypsinogen carrying the p.K23_I24insIDK mutation exhibited >10-fold increased autoactivation. Activation by human cathepsin B was also accelerated by 10-fold. Secretion of the p.K23_I24insIDK mutant from transfected cells was diminished, consistent with intracellular autoactivation.

Conclusions

This is the first report of an intragenic duplication within the PRSS1 gene causing hereditary pancreatitis. The accelerated activation of p.K23_I24insIDK by cathepsin B is a unique biochemical property not found in any other pancreatitis-associated trypsinogen mutant. In contrast, the robust autoactivation of the novel mutant confirms the notion that increased autoactivation is a disease-relevant mechanism in hereditary pancreatitis.

Keywords: Hereditary pancreatitis, human cationic trypsinogen, PRSS1, intragenic duplication, autoactivation, cathepsin B, enteropeptidase

Introduction

Hereditary chronic pancreatitis is an autosomal dominant genetic disorder characterized by incomplete penetrance and variable expressivity [1-6]. Heterozygous mutations in the serine protease 1 (PRSS1) gene have been identified as causative genetic changes in 25-80% of cases in different studies. The PRSS1 gene encodes human cationic trypsinogen, the most abundant digestive proenzyme in human pancreatic secretions. Approximately 70% of the mutation positive hereditary pancreatitis families carry the p.R122H mutation, and about 20% the p.N29I mutation. In the remaining 10% of families at least 10 different, relatively rare mutations have been identified, including a subset of mutations affecting the trypsinogen activation peptide (p.A16V, p.D19A, p.D22G, and p.K23R) [reviewed in 7]. The literature also reports 23 additional rare PRSS1 variants, which have been found in patients with chronic pancreatitis, however, their pathogenic significance, if any, remains unknown [reviewed in 7]. Functional characterization of pancreatitis-associated PRSS1 mutants revealed that increased trypsinogen autoactivation (trypsin-mediated trypsinogen activation) is a common phenotypic alteration at the protein level [see 1-8 and references therein]. The increased propensity for autoactivaton was especially notable in the activation peptide mutants [9]. More recent studies demonstrated that increased autoactivation of activation peptide mutants can occur intracellularly and result in decreased trypsinogen secretion and apoptotic acinar cell death [10].

Although the vast majority of hereditary pancreatitis cases are caused by missense point mutations, other genetic mechanisms have been also recognized as potentially disease-relevant. Mutation p.R122H is rarely caused by a dinucleotide change, possibly through a gene-conversion mechanism [11, 12]. Gene conversion and duplication can also create functional hybrid trypsinogen genes carrying the p.N29I mutation [13, 14]. Finally, duplication and triplication of the trypsinogen locus was described to result in hereditary pancreatitis in all likelihood owing to a gene dosage effect [14-17]. In the present study we report a novel mutational mechanism in a hereditary pancreatitis family from Denmark. We found that intragenic duplication of a nine nucleotide sequence in exon-2 resulted in a three-amino-acid insertion in the trypsinogen activation peptide which dramatically altered the activation properties of human cationic trypsinogen.

Materials and methods

Materials

Human recombinant cathepsin B was a generous gift from Paul M. Steed (Research Department, Novartis Pharmaceuticals, Summit, New Jersey, USA). Before use, cathepsin B was activated with 90 mM dithiothreitol (final concentration) for 30 min on ice. The N-benzyloxycarbonyl-Gly-Pro-Arg-p-nitroanilide trypsin substrate was from Sigma-Aldrich (St. Louis, Missouri, USA). Recombinant human pro-enteropeptidase (holoenzyme) was from R & D Systems (Minneapolis, Minnesota, USA). Human pro-enteropeptidase (at 0.07 mg/mL, ~640 nM concentration) was activated with 50 nM human cationic trypsin in 0.1 M Tris-HCl (pH 8.0), 10 mM CaCl2 and 2 mg/mL bovine serum albumin (final concentrations) for 30 min at room temperature. Ecotin was expressed and purified as reported previously [18-20]. Cell culture media and reagents were obtained from Invitrogen (Carlsbad, California, USA).

Patients

This study was approved by the Scientific Ethics Committee and the Danish Data Protection Agency. The family received genetic counseling before they gave their informed consent to participate in the study. A questionnaire recording symptoms, clinical tests and medical history was completed. A blood sample was drawn from the index patient, his brother and his father into tubes with ethylene diamine tetraacetic acid (EDTA) and stored at −20 °C. After discovery of the mutation the grandparents and the siblings of the father were also tested.

Nomenclature

Nucleotide numbering reflects coding DNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence. Amino acid residues are numbered starting with Met1 of human cationic pre-trypsinogen. The first amino acid of cationic trypsinogen is Ala16.

Genetic analyses

Genomic DNA was extracted from full blood using the Maxwell® DNA purification robot (Promega, Ramcon Denmark). The samples were tested for small deletions, insertions and point mutations in all exons and the exon-intron boundaries of the PRSS1 (GenBank NM_002769.3) and SPINK1 (GenBank NM_003122.3) genes using DHPLC (WAVE 3500HT High Sensitivity System; Transgenomic Inc, Elancourt, France). Samples with deviating chromatographic profiles were sequenced in both directions using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and analyzed on an automated ABI PRISM® 3100 (Applied Biosystems). The presence of PRSS1 gene duplication or triplication was excluded using the rapid PCR-based method described by Chauvin et al., 2009 [17].

Genomic DNA was also tested for 33 CFTR (GenBank NM_000492.3) mutations: 394delTT, p.R553X, 621+1G>T, p.R1162X, 1717-1G>A, 3659delC, p.G542X, 2183A>G, p.W1282X, 1078delT, 711+1G>T, p.F508del, p.S549N, I507del, p.S549R, 2184delA, p.G551D, p.G85E, p.N1303K, p.R560T, p.R117H, p.R347H, p.R347P, p.R334W, 2789+5G>A, 3849+10kbC>T, p.A445E, 3120+1G>A, p.V520F, 1898+1G>A, 3876delA, 3905insT and IVS8-5T.

Plasmid construction and mutagenesis

The pTrapT7 PRSS1, pTrapT7 PRSS1 p.S200A and pcDNA3.1(−) PRSS1 expression plasmids were constructed previously [21-24]. The p.K23_I24insIDK mutation was generated by overlap extension PCR mutagenesis and cloned into the pTrapT7 and pcDNA3.1(−) expression plasmids. The p.K23_I24insIDK p.S200A and p.D22G p.S200A mutants were created in the pTrapT7 PRSS1 plasmid by cut-and-paste using the NcoI and XhoI restriction sites and the appropriate parent plasmids.

Expression and purification of cationic trypsinogen

Wild-type, p.K23_I24insIDK, p.S200A, p.K23_I24insIDK/p.S200A and p.D22G/p.S200A cationic trypsinogens were expressed in Escherichia coli BL21(DE3) as cytoplasmic inclusion bodies. Refolding and purification of trypsinogen on immobilized ecotin was carried out as reported previously [20-22] with the following modification. To stabilize the p.K23_I24insIDK mutant against autoactivation, 100 mM NaCl was included with the 50 mM HCl elution solution during ecotin affinity chromatography. Concentrations of trypsinogen solutions were determined from the UV absorbance at 280 nm using the extinction coefficient 36,160 M−1 cm−1 (http://ca.expasy.org/tools/protparam.html).

Trypsin activity assay

Trypsin activity was measured with the synthetic chromogenic substrate, N-benzyloxycarbonyl-Gly-Pro-Arg-p-nitroanilide at 0.14 mM final concentration. One-minute time courses of p-nitroaniline release were followed at 405 nm in 0.1 M Tris-HCl (pH 8.0) and 1 mM CaCl2 at room temperature using a Spectramax Plus 384 microplate reader (Molecular Devices, Sunnyvale, California, USA).

Cell culture and transfection

HEK 293T cells were cultured in six-well tissue culture plates (106 cells per well) in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 4 mM glutamine, and 1% penicillin/streptomycin at 37 °C in 5% CO2. Transfections were performed using 2 μg expression plasmid and 10 μL Lipofectamine 2000 in 2 mL DMEM. After overnight incubation at 37 °C, cells were washed and the transfection medium was replaced with 2 mL OptiMEM Reduced Serum Medium. Time courses of expression were measured starting from this medium change and were followed for 48 h.

Western blot analysis

Aliquots of conditioned media (20 μL per lane) were electrophoresed on Tris-glycine minigels and transferred onto an Immobilon-P membrane (Millipore, Billerica, MA). The membrane was blocked with 5% milk powder solution at 4 °C overnight; and incubated with sheep polyclonal antibody against human cationic trypsinogen (R&D Systems, #AF3848) at a dilution of 1:2000 for 1 h at room temperature; followed by incubation with horse-radish peroxidase (HRP)-conjugated donkey polyclonal anti-sheep IgG (R&D Systems, #HAF016); used at 1:2000 dilution, for 1 h at room temperature. HRP was detected using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, Illinois, USA).

Results

A novel intragenic duplication in the PRSS1 gene is associated with hereditary pancreatitis

The index patient is a 2 year-old boy from Denmark who presented with ascites and a history of recurrent attacks of abdominal pain, diarrhea and vomiting for 4 months. A preoperative MRCP revealed a pancreatic fistula as the cause for the ascites, and also showed pancreas divisum, duct irregularities and multiple cysts in the pancreatic head and tail. The cauda of the pancreas was resected and pancreatico-jejunostomy was performed. After resection, the ascites production ceased and the patient recovered. The father of the index patient was diagnosed with chronic pancreatitis at the age of 21, which was initially attributed to a bicycle accident, which had happened at age 13. The father underwent resection of the cauda of the pancreas at the age of 27 because of repeated attacks of upper abdominal pain. The index patient’s younger brother also developed abdominal pain, diarrhea and elevated blood amylase at the age of 2 but pancreatic edema was not detected by ultrasonography. The three affected members within this family satisfy the formal criteria of autosomal dominant hereditary pancreatitis (Figure 1A). Curiously, however, no other case of pancreatitis could be confirmed in the rest of the extended family, suggesting that the causative mutation may have occurred de novo in the father. This assumption was later confirmed by the genetic studies detailed below. Non-paternity was excluded by DNA microsatellite analysis (Identifiler kit, Applied Biosystems).

Figure 1.

Figure 1

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Association of the c.63_71dup intragenic duplication in the cationic trypsinogen (PRSS1) gene with hereditary pancreatitis in a family from Denmark. A. Pedigree of the study family. Heterozygous carriers of the c.63_71dup (p.K23_I24insIDK) mutation are indicated. Subjects affected with chronic pancreatitis are shown by solid black symbols. Crossed symbols designate deceased subjects. The arrow points to the index patient. B. Nucleotide and amino-acid sequence of the wild-type and c.63_71dup (p.K23_I24insIDK) mutant cationic trypsinogen activation peptides. The location of the previously described pancreatitis-associated PRSS1 mutations within the activation peptide is also indicated.

DNA sequence analysis of the PRSS1 gene in the index patient found no known mutations but showed a yet unreported duplication in exon-2. As demonstrated by the electropherograms (see Supplementary Figure 1, Supplemental Digital Content 1, http://links.lww.com/MPA/A38), the forward sequencing of exon-2 showed mixed signals starting at nucleotide position c.72 due to a heterozygous nine-nucleotide insertion. The inserted sequence is TGACAAGAT, which corresponds to PRSS1 sequence between c.63 and c.71. Thus, the insertion represents a short intragenic duplication (c.63_71dup), which has never been described in trypsinogen genes so far. Sequencing of exon-2 with a reverse primer confirmed the duplication (see Supplementary Figure 1, Supplemental Digital Content 1, http://links.lww.com/MPA/A38). No other mutations were identified in the PRSS1 gene. The previously reported large-scale trypsinogen duplication and triplication was excluded [14-17]. The c.63_71dup mutation was also present in the father and the brother but not in the grandparents or in the father′s sister and half-brother, indicating that the mutation was de novo created in the father (Figure 1A). All affected family members were negative for SPINK1 mutations and a select panel of CFTR mutations (see Materials and Methods). We did not find the c.63_71dup duplication in 200 healthy controls (400 chromosomes) from the same geographical region.

At the amino-acid level the c.63_71dup mutation creates an insertion of the Ile-Asp-Lys (IDK) sequence between amino acids Lys23 and Ile24 (p.K23_I24insIDK) (Figure 1B). This region of trypsinogen is the so called activation peptide, an 8 amino-acid long N-terminal extension which is cleaved off during activation at the Lys23-Ile24 peptide bond by the physiological activator enteropeptidase or by the pathological activators trypsin and cathepsin B.

The tetra-Asp motif (Asp19-Asp22) preceding Lys23 is an important suppressor of trypsin mediated activation (autoactivation) [23]. In the p.K23_I24insIDK mutant, the tetra-Asp motif before the activating peptide bond is replaced with an Asp-Lys-Ile-Asp sequence. Previously described mutations that alter the tetra-Asp motif (p.D19A, p.D22G) were shown to increase autoactivation of cationic trypsinogen, suggesting a similar phenotype for the p.K23_I24insIDK mutant as well [9, 10, 23, 25].

Activation characteristics of p.K23_I24insIDK mutant cationic trypsinogen

To study the effect of the p.K23_I24insIDK mutation on the activation of cationic trypsinogen, we have generated recombinant versions of wild-type and mutant trypsinogens and purified them to homogeneity. The mutant trypsinogen preparations were highly unstable and spontaneous conversion to trypsin occurred rapidly, suggesting that the p.K23_I24insIDK mutant exhibits markedly increased autoactivation. Indeed, when autoactivation of wild-type and mutant trypsinogens were compared in a quantitative manner (pH 8.0, 37 °C), the mutant autoactivated at rates that were >10-fold higher relative to wild-type trypsinogen (Figure 2A). In order to better characterize the autoactivation kinetics, we generated catalytically inactive versions of wild-type and mutant trypsinogens by mutation of Ser200 to Ala (p.S200A). The use of the p.S200A-trypsinogens allowed us to measure exact rates of trypsin-mediated trypsinogen activation by controlling the trypsin concentration in the reactions. Because activation of p.S200A-trypsinogen does not result in enzymatic activity, the activation reactions were followed by the mobility shift on SDS-PAGE. For comparison, in these experiments we included the p.D22G mutant as a previously well-characterized example of the activation peptide mutants [9, 10, 25]. As shown in Figure 2B, 100 nM human cationic trypsin converted the 2 μM p.S200A (wild-type) trypsinogen to trypsin at a very slow rate and appreciable trypsin levels were seen only after 60 min incubation (pH 8.0, 37 °C). In contrast, p.K23_I24insIDK/p.S200A trypsinogen was completely activated to trypsin within 30 min, whereas complete conversion of the p.D22G/p.S200A mutant took about 60 min. Thus, as judged from the half-lives of p.S200A trypsinogens, the rate of trypsin-mediated trypsinogen activation is >10-fold higher for both the p.K23_I24insIDK and p.D22G mutants, relative to wild-type trypsinogen. These data are in agreement with the activity based assay using catalytically competent trypsinogens, as shown in Figure 2A for the p.K23_I24insIDK mutant and published previously for the p.D22G mutant [9].

Figure 2.

Figure 2

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Effect of the p.K23_I24insIDK mutation on the activation of human cationic trypsinogen by trypsin (autoactivation). A. Trypsinogens at 2 μM concentration were incubated with 40 nM human cationic trypsin (initial concentration) at 37 °C in 0.1 M Tris-HCl (pH 8.0), 1 mM CaCl2, and 40 mM NaCl (final concentrations) in 100 μL final volume. Aliquots (2 μL) were withdrawn at indicated times and trypsin activity was determined. Trypsin activity was expressed as percentage of the maximal activity. The inset shows the first 10 min of the time course. B. Trypsinogens carrying the p.S200A mutation were incubated with 100 nM human cationic trypsin at 37 °C in 0.1 M Tris-HCl (pH 8.0) and 1 mM CaCl2 in 100 μL final volume. At the indicated times reactions were precipitated with 10% trichloroacetic acid (final concentration) and analyzed by 15% SDS-PAGE and Coomassie Blue staining. The lower molecular weight bands represent double-chain forms of trypsinogen and trypsin cleaved at the Arg122-Val123 peptide bond. p.insIDK = p.K23_I24insIDK.

Owing to the robust autoactivation of the p.K23_I24insIDK mutant, we were unable to measure enteropeptidase-mediated trypsinogen activation using the catalytically active proteins. Therefore, we monitored enteropeptidase-mediated activation of the p.S200A-trypsinogens on SDS-PAGE. Supplementary Figure 2 (Supplemental Digital Content 2, http://links.lww.com/MPA/A40) demonstrates that activation of the p.K23_I24insIDK and p.D22G mutants were comparable to that of wild-type (pH 8.0, 37 °C). The results are in accord with our previous studies showing that the trypsinogen activation peptide plays no significant role in the recognition of human cationic trypsinogen by human enteropeptidase [23]. This finding is somewhat surprising, as the tetra-Asp motif in the trypsinogen activation peptide has been known as a specific recognition motif for enteropeptidase, at least in the context of the bovine enzymes [26].

The lysosomal cysteine protease cathepsin B has been recognized as a pathological activator of trypsinogen in acute models of experimental pancreatitis [27-30]. We tested the effect of the p.K23_I24insIDK mutation on cathepsin B-mediated trypsinogen activation at pH 4.0, where autoactivation is minimal. Remarkably, the mutant was activated by cathepsin B at a markedly elevated rate, which seemed approximately 5-10-fold higher than that of wild-type (Figure 3A). Using p.S200A-trypsinogens, we measured the rates of conversion in a more precise manner and found that the p.K23_I24insIDK mutant was activated by cathepsin B 10-fold faster than wild-type cationic trypsinogen (Figure 3B). As described previously, mutant p.D22G was resistant to cathepsin B mediated activation [10, 31]. More recently, cathepsin L was shown to degrade trypsinogen and active trypsin accumulation during pancreatitis was attributed not only to cathepsin B-mediated activation but also to a defect in cathepsin L-mediated degradation [32, 33]. We found no change in the degradation of the p.K23_I24insIDK mutant by cathepsin L (pH 4.0, 37 °C) as compared to wild-type cationic trypsinogen (data not shown).

Figure 3.

Figure 3

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Effect of the p.K23_I24insIDK mutation on the activation of human cationic trypsinogen with cathepsin B. A. Trypsinogens at 2 μM concentration were activated with human cathepsin B (37 μg/mL, ~1.3 μM) at 37 °C in 0.1 M Na-acetate buffer (pH 4.0), 1 mM EDTA and 1 mM dithiothreitol (final concentrations) in 50 μL final volume. Aliquots (2 μL) were withdrawn at indicated times and trypsin activity was determined. Trypsin activity was expressed as percentage of the maximal activity. B. Trypsinogens carrying the p.S200A mutation were activated with human cathepsin B (37 μg/mL, ~1.3 μM; 74 μg/mL, ~2.6 μM for p.D22G) at 37 °C in 0.1 M Na-acetate buffer (pH 4.0) 1 mM K-EDTA and 1 mM dithiothreitol in 100 μL final volume. At the indicated times reactions were precipitated with 10% trichloroacetic acid (final concentration) and analyzed by 15% SDS-PAGE and Coomassie Blue staining. p.insIDK =p.K23_I24insIDK.

Cleavage of the activation peptide in the p.K23_I24insIDK mutant cationic trypsinogen

The mutant activation peptide sequence contains two Lys-Ile peptides bonds (Figure 1B). Although activation of trypsinogen to trypsin requires proteolysis of the second site, cleavage after the first Lys may modify the efficiency of the second cleavage. Therefore, we sought to clarify whether both sites were cleaved. For these experiments we used the p.S200A-trypsinogens which we activated with trypsin (pH 8.0), enteropeptidase (pH 8.0) and cathepsin B (pH 4.0). The activation reactions were separated on SDS-PAGE, transferred to PVDF membranes and trypsin bands were subjected to N-terminal sequence analysis by Edman degradation. To capture cleavage intermediates, we sequenced trypsin bands early in the reaction when less than half of the trypsinogen was converted to a trypsin band. We found that trypsin and cathepsin B cleaved the second (activating) Lys-Ile peptide bond only, whereas enteropeptidase cleaved both Lys-Ile peptide bonds with equal efficacy. Cleavage after the first Lys-Ile peptide bond resulted in an N-terminally truncated trypsinogen, which was eventually completely cleaved at the second Lys-Ile peptide bond by enteropeptidase (see Supplementary Figure 3, Supplemental Digital Content 3, http://links.lww.com/MPA/A41).

Secretion of the p.K23_I24insIDK mutant from transfected cells

Recently, we demonstrated that activation peptide mutants that undergo robust autoactivation in the test tube were also autoactivating inside living cells [10]. Intracellular autoactivation resulted in diminished trypsinogen secretion. To test whether or not secretion of the strongly autoactivating p.K23_I24insIDK mutant would be reduced, we transfected HEK 293T cells with wild-type and p.K23_I24insIDK mutant cationic trypsinogen and measured secretion of trypsinogens from the conditioned medium by activity assays and immunoblot. The transfected cells exhibited healthy morphology during the time course studied with no signs of cell death. As shown in Figure 4, mutant p.K23_I24insIDK was secreted to significantly lower levels than wild-type cationic trypsinogen, suggesting that the p.K23_I24insIDK mutant suffered intracellular autoactivation.

Figure 4.

Figure 4

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Secretion of the p.K23_I24insIDK cationic trypsinogen mutant from transiently transfected HEK 293T cells. At 8, 24, 32 and 48 hours after transfection conditioned media were collected and 20 μL medium was supplemented with 0.1 M Tris-HCl (pH 8.0) and 1 mM CaCl2 to 50 μL volume and trypsinogen was activated with 28 ng/mL human enteropeptidase for 1 h at 37 °C. Trypsin activity was then measured by adding 150 μL of the chromogenic substrate, N-benzyloxycarbonyl-Gly-Pro-Arg-p-nitroanilide to 0.14 mM final concentration. Trypsin activities were expressed as percentage of the 48 h wild-type activity. The average of 2 independent transfection experiments is shown. For clarity, the error bars have been omitted; the standard error of the mean was within 15%. Inset: Eight hours after transfection 20 μL aliquots of conditioned media were electrophoresed on 15% SDS-polyacrylamide gels and analyzed by western blotting as described in Materials and Methods.

Discussion

There are a number of important observations in this study which set it apart from a typical mutation report. This is the first account of an intragenic duplication within the PRSS1 gene in association with hereditary pancreatitis. The human trypsinogen genes are located on chromosome 7q35, intercalated between the beta T-cell receptor genes; at a locus highly active in recombination. This organization seems beneficial for the evolution of trypsinogens which tend to undergo extensive gene-duplication and gene-loss events during speciation resulting in distinctive trypsinogen gene families [9, 34]. On the other hand, unwanted genetic rearrangements -- such as gene-conversions, duplications or triplications -- can result in novel pathogenic alleles in hereditary pancreatitis [13-17, 34]. In contrast to the previously reported large-scale gene duplications, in our family the duplication was confined only to a nine nucleotide segment within exon-2 without any evidence of more extensive genetic changes.

Interestingly, the duplication was identified only in the father and his two sons (see Figure 1A), while it was absent in the grandparents or the father’s siblings, indicating that it was de novo generated in the father. Ours is the second report on capturing a mutational event leading to hereditary pancreatitis. Simon et al. (2002) found a de novo p.R122H mutation in their cohort and their subsequent studies indicated that PRSS1 mutations are characteristically not inherited from a common founder, even when local clustering of families is observed [35, 36].

Intragenic duplications are likely to result in a frame shift and truncated, non-functional protein. In this case, however, the reading frame was kept and at the amino-acid level the duplication generated an insertion within the trypsinogen activation peptide (see Figure 1B). As expected from this alteration, the activation properties of cationic trypsinogen have been affected in profound ways. Trypsin-mediated trypsinogen activation (autoactivation) and cathepsin B-mediated trypsinogen activation were both increased by an order of magnitude; an effect size never before seen with the known PRSS1 mutants. With respect to autoactivation, the p.K23_I24insIDK mutant’s phenotype is consistent with the reported properties of other PRSS1 mutations affecting the activation peptide (p.D19A, p.D22G, and p.K23R), which all result in markedly increased autoactivation [9, 10, 23, 25]. Mechanistically, the increased autoactivation of p.K23_I24insIDK is explained by the disruption of inhibitory interactions between the negatively charged tetra-Asp motif in the activation peptide and trypsin [23]. In the p.K23_I24insIDK mutant Asp21 is replaced with a hydrophobic Ile residue and Asp20 by a positively charged Lys (see Figure 1B). A recent study found that at the cellular level increased autoactivation results in diminished trypsinogen secretion and eventual apoptotic death of acinar cells [10]. We confirmed using HEK 293T cells that the p.K23_I24insIDK mutant was secreted at markedly reduced rates, indicating that it also undergoes autoactivation inside living cells.

Cathepsin B has long been known as a pathological activator of trypsinogen in experimental models of acute pancreatitis, cerulein-induced pancreatitis in particular [27-30]. The effect of hereditary pancreatitis-associated mutations on cathepsin B-mediated trypsinogen activation has been studied in detail previously. Mutants p.D19A, p.N29I, p.N29T, p.E79K and p.R122H exhibited unchanged activation characteristics; whereas mutant p.K23R was activated slowly, and mutant p.D22G was resistant to activation by cathepsin B [10, 29, 31, 37]. Mutant p.K23_I24insIDK is the first cationic trypsinogen variant that exhibits increased sensitivity to cathepsin B-mediated activation and thus stands in contrast with all other PRSS1 mutants studied to date. We believe this property is related to the longer activation peptide which allows extended contacts with the activating enzyme.

In summary, we identified a unique intragenic duplication within exon-2 of the PRSS1 gene (c.63_71dup) in a hereditary pancreatitis family from Denmark. The duplication results in the p.K23_I24insIDK insertional mutation within the activation peptide of cationic trypsinogen. Activation of the p.K23_I24insIDK mutant by trypsin (autoactivation) or by cathepsin B is markedly increased confirming the significance of the trypsin-dependent pathological pathway in hereditary pancreatitis.

Supplementary Material

1

Acknowledgments

We appreciate the participation of all family members. We are also grateful to Steen Gregersen for technical assistance and to Rasmus Gaardskær Nielsen, Marianne S. Jakobsen (Department of Pediatrics, Odense University Hospital, Denmark) and Claus Hovendal (Department of Upper Surgery, Odense University Hospital, Denmark) for referring the index patient for genetic testing. Protein sequencing was performed by David McCourt (Midwest Analytical, Inc., St. Louis, Missouri, USA).

Financial acknowledgments: This work was supported by grants from the Danish Cancer Society, the Dagmar Marshalls Foundation, the Fru Astrid Thaysens Legat, the Legat for Lægevidenskabelig grundforskning, the Ingemann O. Buch’s Foundation 2004; the Lægernes Forsikringsforening af 1991 (to M.T.J); the National Institute of Health (DK058088 to M.S.-T.) and a scholarship from the Rosztoczy Foundation (to A.G.).

Footnotes

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