Hereditary pancreatitis is an autosomal dominant

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1 GASTROENTEROLOGY 2000;119: Chronic Pancreatitis Associated With an Activation Peptide Mutation That Facilitates Trypsin Activation NIELS TEICH,* JOHANN OCKENGA, ALBRECHT HOFFMEISTER,* MICHAEL MANNS, JOACHIM MÖSSNER,* and VOLKER KEIM* *Medizinische Klinik II, Universität Leipzig, Leipzig; and Gastroenterologie und Hepatologie, Medizinische Hochschule Hannover, Hannover, Germany Background & Aims: Mutations of the cationic trypsinogen have been described in hereditary pancreatitis. We report a new trypsinogen mutation in the activation peptide of the proenzyme in a family with chronic pancreatitis. Methods: The coding region of the cationic trypsinogen gene was sequenced after polymerase chain reaction amplification. The following peptides homologous to the N-terminal end of cationic trypsinogen were synthesized (one-letter code, mutated amino acid underlined): wild-type peptide, APFDDDDKIVGG; pd22g, APFDDDGKIVGG; pk23r, APFDDDDRIVGG. The sequences of pd22g and pk23r correspond to the recently identified mutation K23R and to the mutation described here (D22G). To mimic trypsinogen activation, these peptides were digested with trypsin for 30 minutes at ph , and the fragments were analyzed by high-performance liquid chromatography. Results: In a family with clinical evidence of hereditary chronic pancreatitis, a missense mutation of codon 22 (GAC3GGC) of the cationic trypsinogen was found. This mutation results in a substitution of aspartic acid by glycine; therefore, the mutation was called D22G. Chromatographic analysis of tryptic digests of the peptides pd22g and pk23r showed hydrolysis rates of 22% and 75%, respectively, whereas the wild-type peptide was hydrolyzed at only 6%. The cleavage rates were reduced at lower ph, and no hydrolysis occurred without trypsin. Conclusions: The activation peptides of the trypsinogen variants D22G and K23R could be released at a higher rate than in wild-type trypsinogen, resulting in increased amounts of trypsin in the pancreas, which could initiate pancreatitis. Hereditary pancreatitis is an autosomal dominant disorder with 80% penetrance. 1 Several hundred cases have been reported. The first identified point mutation in this disease was an A-to-T change in the exon 3 of the cationic trypsinogen. 2 Because of an arginineto-histidine change in the deduced amino acid sequence, the mutation was called R117H. Recently, additional hereditary pancreatitis associated mutations (N21I, K23R, A16V) have been reported in the same gene. 3 5 Although other genes also seem to be important, 6 the mutations N21I and R117H were found in most patients with hereditary pancreatitis. 2 4 There is considerable confusion concerning the nomenclature of the trypsinogen mutations because the chymotrypsin numbering was used in 2 publications, 2,3 whereas other authors 4,5 used a system based on the trypsin sequence. For clarity, and in accordance with a recent recommendation, 7 we use only the trypsin numbering system. In this system, the amino acids of the proenzyme are counted from the N-terminal end of the signal peptide, and the numbers of the codons and of the amino acids are identical. As a consequence, the frequent mutations N21I and R117H are called N29I and R122H, whereas the numbering of the mutations A16V and K23R requires no change. We describe a new trypsinogen mutation in a family with chronic pancreatitis and suggest a model that could explain its pathogenesis. Materials and Methods DNA Analysis After written informed consent was obtained from the subjects, leukocyte DNA was extracted from anticoagulated blood specimens using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). DNA was stored in Tris-EDTA buffer (ph 8.0) at 4 C. Exons of the cationic trypsinogen were amplified by polymerase chain reaction (PCR), as described recently. 8 Cycling conditions consisted of an initial 2-minute denaturation step at 94 C followed by 35 cycles at 94 C for 20 seconds, 61 C for 30 seconds, and 72 C for 45 seconds and a final 7-minute elongation step at 72 C. DNA sequencing of PCR products was performed using the sense primer, an ABI 377 DNA sequencer, and the ABI PRISM Dye Terminator Abbreviations used in this paper: HPLC, high-performance liquid chromatography; PCR, polymerase chain reaction by the American Gastroenterological Association /00/$10.00 doi: /gast

2 462 TEICH ET AL. GASTROENTEROLOGY Vol. 119, No. 2 Cycle Sequencing Reaction Kit (Perkin Elmer, Norwalk, CT) according to the manufacturer s instructions. Mutations were confirmed by repeated PCR and the antisense primer for sequencing. Oligopeptide Digestion Unless otherwise indicated, all reagents were obtained from Sigma (Deisenhofen, Germany). The oligopeptides shown in Table 1 were commercially synthesized (MWG Biotech, Ebersberg, Germany) and purified by high-performance liquid chromatography (HPLC), and their molecular masses were controlled by mass spectrometry. These dodecapeptides are homologous to the N-terminal part of cationic trypsinogen and correspond to the wild-type sequence (pwt), to the mutation K23R recently identified by Ferec et al., 4 and to the mutation D22G identified in our present study. They include the activation peptide cleavage site so that after tryptic hydrolysis, an octapeptide and a tetrapeptide will be released. The octapeptide pco, which is identical to the tryptic digestion product of the wild-type peptide pwt, served as a control for the HPLC separation. A solution of 2 mg dodecapeptide/ml of buffer (50 mmol HEPES, 20 mmol CaCl 2 ), with ph varying from 5.0 to 8.0, was used. Bovine trypsin at a final concentration of 2.5 g/ml was added, and the mixture was incubated for 30 minutes at 37 C. As control, the peptides were incubated without addition of trypsin. The samples were then centrifuged using the Microcon centrifugal filter device YM-10 (catalogue no ; Millipore, Bedford, MA) to remove trypsin from peptides or hydrolytic fragments. Next, 100 L of the eluates was then separated by HPLC. Rates of hydrolysis were determined by integration of the respective areas under each peak, and the digestion rates of the peptides were indicated as percentages of initial dodecapeptide. The HPLC analysis was controlled by separation of undigested peptides and the control peptide pco. Four separate experiments were performed with each peptide. Statistical Evaluation A nonparametric sign test was used for evaluation of the data; a significant difference was assumed at P Table 1. Sequences of Synthetic Peptides Homologous to the N-Terminal End of Human Cationic Trypsinogen pwt pd22g pk23r pco APFDDDDK-IVGG APFDDDGK-IVGG APFDDDDR-IVGG APFDDDDK NOTE. The sequences are shown in the 1-letter code; underlined letters represent the mutated amino acids. The tryptic cutting sites of the peptides are marked by dashes. pwt, wild-type peptide; pd22g, peptide D22G; pk23r, peptide K23R; pco, control peptide. Figure 1. Pedigree of a family with chronic pancreatitis and D22G mutation of the cationic trypsinogen. The patients with documented chronic pancreatitis are represented by closed symbols; individuals with suspected disease are depicted in grey. *D22G variant present. Results Family Characteristics A 22-year-old patient (subject III-2 in Figure 1) had experienced episodes of abdominal pain since his first year of life. When he was 8 years old, acute pancreatitis was diagnosed; during the following years, he was hospitalized several times with episodes of acute pancreatitis. In June 1998, computed tomographic scanning showed 2 pseudocysts in the pancreas tail and scatty calcifications. To evaluate the cause of the pancreatitis, endocrine and metabolic diseases (hypertriglyceridemia, hyperparathyroidism) were excluded. Serologic tests showed no evidence of a viral infection caused by adenoviruses; cytomegalovirus; Epstein Barr virus; herpes simplex virus; mumps; hepatitis A, B, and C viruses; or human immunodeficiency virus. Screening for the cystic fibrosis transmembrane regulator gene showed no mutation. The patient s mother (40 years old, subject II-3 in Figure 1) complained of recurrent abdominal discomfort that had been present since her youth. In 1996, cholecystolithiasis was diagnosed and treated with laparoscopic cholecystectomy, resulting in slight improvement of the symptoms, but she still had recurrent abdominal pain. An abdominal ultrasound scan showed typical signs of chronic pancreatitis. The patient s father (43 years old) and his relatives (parents, 2 brothers, 2 sisters, their 3 children; individuals not shown in Figure 1) had no evidence of pancreatic or abdominal disease. The 20- year-old sister of the patient had no symptoms of acute or chronic pancreatic disease. According to the anamnestic information, the uncle (II-2) and the grandmother (I-2) of the index patient complained of recurrent upper abdominal discomfort; the latter had developed diabetes after her second pregnancy. The family originates from Kiev/Ukraine, and individuals II-3, II-4, III-2, and III-3

3 August 2000 TRYPSIN ACTIVATION IN PANCREATITIS 463 migrated to Germany in No other relatives were available for genetic testing. DNA Sequence Data Sequencing of the cationic trypsinogen showed none of the known mutations but a heterozygosity (A3 G) at nucleotide (GenBank no. U66061 L36092). The mutation was identified in the index patient (III-2), his mother (II-3), and his sister (III-3), who was healthy up to now. The DNA sequence of the father (II-4) showed wild type. Because this mutation refers to the 22nd codon (GAC3 GGC) of the cationic trypsinogen and leads to a change of aspartic acid to glycine in the deduced amino acid sequence, it was called D22G. The altered residue is situated 2 amino acids upstream of the activation peptide cleavage site (Table 1). The D22G mutation was not found in 19 other families with hereditary pancreatitis and was also absent in more than 350 individuals with chronic pancreatitis of other origin. Oligopeptide Digestion To investigate the functional impact of the D22G mutation on the cationic trypsinogen, the corresponding N-terminal peptide pd22g was digested with trypsin. In addition, the peptide corresponding to the K23R mutation described recently by Ferec et al. 4 was used. After HPLC separation, the wild-type peptide pwt as Figure 2. HPLC separation of oligopeptide digests. (A) Wild-type peptide (pwt), no trypsin; (B) pwt, 30 minutes of trypsin; (C) pd22g, 30 minutes of trypsin; (D) pk23r, 30 minutes of trypsin. Arrows indicate the position of the octapeptide. Figure 3. ph-dependent hydrolysis of synthetic peptides by trypsin. Wild-type (pwt) and mutant (pd22g, pk23r) peptides were incubated with or without trypsin for 30 minutes. After chromatographic separation of the hydrolytic products, the percentage of peptide digestion was measured by integration of the areas under the respective peaks. Values are means SE; n 4; *P well as the mutated peptides pd22g and pk23r were eluted at a retention time of approximately 14 minutes. The control peptide pco was eluted after 11.6 minutes, and the residual tetrapeptide was seen as a very small peak after 8.6 minutes. After tryptic digestion, the peaks of the dodecapeptides pd22g, pk23r, and pwt decreased, whereas peaks at the retention time of the octapeptide pco and the tetrapeptide increased (Figure 2). These findings show that the synthetic peptides were cut at the predicted site. Quantitative evaluation of the hydrolysis rate was done by determination of the areas under the peaks. Without trypsin, less than 0.3% of the peptides used were hydrolyzed (Figure 3). After a 30-minute digestion with trypsin at ph 8.0, 6% of the wild-type peptide and 22% or 75% of the peptides pd22g and pk23r were hydrolyzed, respectively (Figure 3; P 0.05). At ph 7, the tryptic cleavage of all peptides was less than at ph 8, but the hydrolysis of the mutant peptides was still more than 4 11 times higher than the digestion rate of the wild-type peptide (Figure 3). At ph 6, only the cleavage rate of pk23r was significantly elevated, and at ph 5 the hydrolysis of all peptides was almost negligible (not shown). Discussion Mutations of the cationic trypsinogen in hereditary pancreatitis have been identified in North America, Europe, and Japan. 2 4,9 The point mutation D22G we describe is the first one detected in a family from Eastern Europe. Diagnosis of hereditary pancreatitis in this family may be questioned because only 2 members were

4 464 TEICH ET AL. GASTROENTEROLOGY Vol. 119, No. 2 affected with the disease and 1 individual with the mutation has been healthy up to now. However, we have clinical evidence of chronic disease in other relatives. In addition, because of a penetrance of 80%, unaffected individuals with mutations have been reported in most families with hereditary pancreatitis. 2 4 Similar to all other known point mutations in hereditary pancreatitis, the variant D22G identified affects the N-terminal part of the cationic trypsinogen. It substitutes the last of the 4 aspartic acids by a glycine residue. This (Asp) 4 group is a characteristic structural feature highly conserved in all mammalian trypsinogens. 10 Sequence alterations in this part of the molecule have not been described in humans, so it is unlikely that the D22G variant is associated with the disease only by chance. This conclusion is supported by our finding of only wild-type sequences in a significant number of controls. The mutations D22G and K23R alter the amino acid sequence adjacent to the cleavage site of the activation peptide. We speculated that this would influence the release of the activation peptide. To evaluate this hypothesis, we synthesized N-terminal peptides covering that region and studied their tryptic cleavage. One problem of this approach might be that the structural organization of the N-terminal part of native trypsinogen is different from that of the synthetic oligopeptide. However, analysis of the trypsinogen-to-trypsin conversion by radiographic studies indicates that the N-terminal end of trypsinogen can move freely in space. 11 In the past, the activation process of trypsinogen was analyzed in numerous studies using this technique. 12 We confirmed the validity of our analytical setup in further control experiments. The synthetic peptides separated by HPLC remained undigested without addition of trypsin. The tryptic fragments eluate at retention times anticipated from their peptide sequences. Trypsin activity is known to be highest at ph 8. Therefore, the highest rates of hydrolysis were found at this ph. Upon digestion, the wild-type peptide pwt was hydrolyzed at only 6%, whereas the rates for the mutant peptides pd22g and pk23r were 4 and 11 times higher, respectively. From these findings, we conclude that the activation peptides in the trypsinogen variants D22G and K23R may be released more easily than in the wild-type proenzyme. At first glance, facilitated activation of trypsinogen as a result of activation peptide mutation is a surprising finding. However, it was already noted 2 decades ago that the 4 aspartic acid residues not only act as a binding factor for enterokinase but also inhibit the activity of trypsin. 10 Higher hydrolysis rates of synthetic peptides were found with deletion of 1 or 2 asparagine residues. The (Asp) 4 group could therefore be an important protective mechanism that inhibits the premature activation of trypsinogen by trypsin in the pancreatic acinar cell. Thus, it is conceivable that the destruction of this inhibitory structure by the point mutation D22G could result in a facilitated activation of trypsinogen in the pancreas. In the K23R variant, the cutting site of the activation peptide is directly affected by a substitution of a lysine by an arginine residue. Both amino acids are known to be specific for trypsin cleavage. However, the Arg Ile bond in the activation peptide is a rather poor substrate for trypsin. 12 Important differences were also found when substrates with either lysine or arginine residues were compared: upon hydrolysis with trypsin, the lysine derivative of p-nitranilide exhibited a turnover rate of only 0.003/s, whereas the corresponding value for the arginine-p-nitranilide was 200 times higher. 13 This is consistent with our finding of a more than 10 times increased rate of hydrolysis after a lysine arginine exchange. The facilitated activation of D22G and K23R trypsinogens could result in increased levels of trypsin in the pancreas. Inhibitory principles such as the binding of trypsin to pancreatic secretory trypsin inhibitor or deactivation at the R122 site may still be effective. However, it is conceivable that a more than 10-fold increase in the rate of trypsin activation will exhaust these protective systems, which may finally lead to pancreatitis. The rate of tryptic digestion of pk23r was 3 4 times higher than with pd22g. Caused by the higher levels of free trypsin in the pancreas, the pancreatitis in K23R carriers might be more severe than that with D22G. However, information about the clinical course in these patients is difficult to evaluate because these mutations have been described in only a few individuals. 4 With respect to the mutations identified up to now, the pathogenesis of hereditary pancreatitis is clearly related to cationic trypsinogen. 14 At least 3 different mechanisms conceivably cause the increase in trypsin activity in the acinar cell (Figure 4). First, the mutations N29I and R122H could result in a reduced degradation of the active trypsin (either by reduced autolysis or by increased stability). Although experiments have been conducted with mutant rat trypsins, definite proof of this theory by investigation of recombinant human cationic trypsinogen is still lacking. Second, as shown by us, the variants D22G and K23R could result in a facilitated activation of trypsinogen. The pathogenesis of the A16V

5 August 2000 TRYPSIN ACTIVATION IN PANCREATITIS 465 Figure 4. Mutations and proposed pathogenetic models of hereditary pancreatitis. trypsinogen is purely speculative, but altered intracellular maturation may be the cause. The identification of trypsinogen mutations in hereditary pancreatitis has emphasized the pathogenetic importance of this enzyme in all types of pancreatitis. 14 In contrast to the R122H and N29I variants, wild-type trypsin is released in the D22G and K23R trypsinogens after cleavage of the activation peptides. Therefore, expression of the D22G and K23R trypsinogens in a cellular system could be a useful tool for studies of the pathogenesis of pancreatitis in general. References 1. Sibert, JR. Hereditary pancreatitis in England and Wales. J Med Genet 1978;15: Whitcomb DC, Gorry MC, Preston RA, Furey W, Sossenheimer MJ, Ulrich CD, Martin SP, Gates LK Jr, Amann ST, Toskes PP, Liddle R, McGrath K, Uomo G, Post JC, Ehrlich GD. Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nat Genet 1996;14: Gorry MC, Gabbaizedeh D, Furey W, Gates LK Jr, Preston RA, Aston CE, Zhang Y, Ulrich C, Ehrlich GD, Whitcomb DC. Mutations in the cationic trypsinogen gene are associated with recurrent acute and chronic pancreatitis. Gastroenterology 1997;113: Ferec C, Raguenes O, Salomon R, Roche C, Bernard JP, Guillot M, Quere I, Faure C, Mercier B, Audrezet MP, Guillausseau PJ, Dupont C, Munnich A, Bignon JD, Le Bodic L. Mutations in the cationic trypsinogen gene and evidence for genetic heterogeneity in hereditary pancreatitis. J Med Genet 1999;36: Witt H, Luck W, Becker M. A signal peptide cleavage site mutation in the cationic trypsinogen gene is strongly associated with chronic pancreatitis. Gastroenterology 1999;117: Dasouki MJ, Cogan J, Summar ML, Neblitt W III, Foroud T, Koller D, Phillips JA. Heterogeneity in hereditary pancreatitis. Am J Med Genet 1998;77: Antonarakis SE. Recommendations for a nomenclature system for human gene mutations. Nomenclature Working Group. Hum Mutat 1998;11: Teich N, Mössner J, Keim V. Screening for mutations in the cationic trypsinogen: are they of relevance in chronic alcoholic pancreatitis? Gut 1999;44: Nishimori I, Kamakura M, Fujikawa-Adachi K, Morita M, Onishi S, Yokoyama K, Makino I, Ishida H, Yamamoto M, Watanabe S, Ogawa M. Mutations in exons 2 and 3 of the cationic trypsinogen gene in Japanese families with hereditary pancreatitis. Gut 1999; 44: Brigteux-Grégoire S, Schyns R, Florkin M. Phylogeny of trypsinogen activation peptides. Comp Biochem Physiol 1972;42B: Bode W, Fehlhammer H, Huber R. Crystal structure of bovine trypsinogen at 1 8 Å resolution. I. Data collection, application of patterson search techniques and preliminary structural interpretation. J Mol Biol 1976;106: Abita JP, Delaage M, Lazdunski M. The mechanism of activation of trypsinogen. The role of the four N-terminal aspartyl-residues. Eur J Biochem 1969;8: Erlanger BF, Kokowski N, Cohen W. The separation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 1961;95: Whitcomb D. Early trypsinogen activation in acute pancreatitis. Gastroenterology 1999;116: Varallyay E, Pal G, Patthy A, Szilagyi L, Graf L. Two mutations in rat trypsin confer resistance against autolysis. Biochem Biophys Res Commun 1998;243: Sahin-Toth M, Graf L, Toth M. Trypsinogen stabilization by mutation Arg1173His: a unifying pathomechanism for hereditary pancreatitis? Biochem Biophys Res Commun 1999;264: Sahin-Toth M. Hereditary pancreatitis associated mutation asn (21)3ile stabilizes rat trypsinogen in vitro. J Biol Chem 1999; 274: Received July 14, Accepted March 23, Address requests for reprints to: Volker Keim, M.D., Medizinische Klinik und Poliklinik II, Universität Leipzig, Philipp-Rosenthal-Strasse 27, D Leipzig, Germany. keimv@medizin.uni-leipzig.de; fax: (49) Supported in part by grant Ke 347/8-1 from the Deutsche Forschungsgemeinschaft. The authors thank Susanne Kistner for expert technical assistance.