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The A16V signal peptide cleavage site mutation in the cationic trypsinogen gene and chronic pancreatitis
1508
CORRESPONDENCE
6. Hartley BS, Shotton DM. Pancreatic elastase. In: Boyer RD, ed. The enzymes. New York: Academic, 1971:323–373. 7. Whitcomb DC. Hereditary pancreatitis: new insights into acute and chronic pancreatitis. Gut 1999;45:317–322. 8. OMIM, Online Mendelian Inheritance in Man; http://www.ncbi.nlm. nih.gov/htbin-post/Omim/dispmim?167800. 9. Antonarakis SE, Nomenclature Working Group. Recommendations for a nomenclature system for human gene mutations. Hum Mutat 1998;11:1–3. 10. HGMD, Human Gene Mutation Database: http://www.uwcm. ac.uk/uwcm/mg/hgmd0.html. 11. Nyaruhucha CN, Kito M, Fukuoka SI. Identification and expression of the cDNA-encoding human mesotrypsin(ogen), an isoform of trypsin with inhibitor resistance. 1997;272:10573–10578. 12. Rowen L, Trask B, Boysen C, et al. Sequence of a large duplication from human chromosome 7 to chromosome 9 containing a portion of the T cell receptor beta locus and trypsinogen locus. GenBank 1997; Accession AF029308: (unpublished).
The A16V Signal Peptide Cleavage Site Mutation in the Cationic Trypsinogen Gene and Chronic Pancreatitis Dear Sir: We read with great interest the study by Witt et al.1 in the July 1999 issue of GASTROENTEROLOGY in which they describe a strong association of a novel DNA variant—a heterozygous g.131906C . T transition in the second exon of the cationic trypsinogen gene resulting in an amino acid substitution of Ala with Val at residue 16 of the cationic pretrypsinogen (A16V)—with chronic pancreatitis. This genetic finding confirms for the first time that although the molecular mechanism underlying the pancreatitis phenotype may delimit the variety or number of causal mutations in the cationic trypsinogen gene, still other mutations may be found as more comprehensive searches are done, as suggested by Elitsur et al.2 In support of the Witt report, we also identified this new DNA variant in 2 unrelated subjects of 312 patients with sporadic pancreatitis, but not in 200 control subjects, using a previously established denaturing gradient gel electrophoresis.3 The exceptionally high detection frequency—4 of 44 patients had the A16V mutation in the Witt study—may be explained by their carefully selected participants. Only children and adolescents with chronic pancreatitis were enrolled, an age group thought to be suitable for such a prevalence study of genetic defects. As in the case of the N29I mutation,4,5 the identification of an A16V substitution has again led to a challenging situation in which there is strong molecular genetic evidence for a disease-associated mutation but an inadequate understanding of the basic biochemistry of the corresponding protein and its function. Moreover, the penetrance of the A16V mutation is obviously low compared with the ,80% penetrance of the N29I and R122H6 mutations: only 1 of the 7 first-degree relatives carrying the A16V mutation was affected,1 and neither of the 2 A16V carriers in our study had a family history of pancreatitis. Interestingly, an Ile is present at residue 29 of the functional anionic pretrypsinogen and a Val is present at residue 16 of the functional premesotrypsinogen (Figure 1). However, whereas an N29I mutation results in a nonconservative amino acid substitution, which is considered to be important in causing the disease,5 an A16V mutation results in a conservative substitution. Perhaps it is this kind of conservative substitution that underlies the lower penetrance of the A16V mutation. Nevertheless, residue 16 constitutes the signal peptide cleavage site and provides an important clue as to how an A16V substitution could predispose someone to pancreatitis. Of the
GASTROENTEROLOGY Vol. 117, No. 6
Figure 1. Alignment of the N-terminal amino acids of the 3 pancreatic pretrypsinogens: cationic, anionic, and meso isoforms. Dashes indicate identity with the cationic amino acid sequence. Note the presence of Val at residue 16 of the premesotrypsinogen and the presence of Ile at residue 29 of the anionic pretrypsinogen.
possible mechanisms proposed by Witt et al., we favor the hypothesis that an A16V substitution may disrupt the intracellular transportation of the pretrypsinogen; in this vein, it may even be tempting to speculate that this substitution could lead to colocalization of pretrypsinogen with lysosomal hydrolases, a phenomenon well demonstrated in dissimilar models of pancreatitis in experimental animals.7 On the contrary, the facts that residue 16 is the first amino acid of the activation peptide of the cationic trypsinogen and that the first 13 N-terminal amino acids of the mutated cationic trypsinogen are identical to those of the wild-type mesotrypsinogen (Figure 1) may make an enhanced autoactivation hypothesis less likely. Also, based on the nature of the A16V mutation (located near the N terminus of the pretrypsinogen, a conservative amino acid substitution, and the presence of Val at residue 16 of the wild-type mesotrypsinogen) and its lower penetrance, it may be difficult to prove exactly how this mutation works by in vitro or in vivo experiments. Above all, there is an urgent need to correlate the different DNA variants (genotypes) with their clinical manifestations (phenotypes). Finally, the identification of the A16V mutation, together with another recent report in which 9 of 48 patients with a presumed diagnosis of idiopathic chronic pancreatitis were shown to have the R122H or N29I mutations,8 strongly suggest that genetic testing for cationic trypsinogen mutations even in the absence of a family history of pancreatitis is warranted. JIAN–MIN CHEN, Ph.D. ODILE RAGUENES CLAUDE FEREC, M.D., Ph.D. Centre de Biogenetique University, Hospital, ETSBO Brest, France PIERRE H. DEPREZ, M.D. Pediatric Unit Clinques Universitaires St-Luc Brussels, Belgium CHRISTINE VERELLEN–DUMOULIN, M.D. Centre for Human Genetics Universite Catholique de Louvain Brussels, Belgium ANGELO ANDRIULLI, M.D. Division of Gastroenterology Casa Sollievo della Sofferenza Hospital S. Giovanni Rotondo, Italy 1. 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;117:7–10. 2. Elitsur Y, Chertow BC, Jewell RD, et al. Identification of a hereditary
December 1999
3.
4.
5.
6.
7. 8.
pancreatitis mutation in four West Virginia families. Pediatr Res 1998;44:927–930. Ferec C, Raguenes O, Salomon R, et al. Mutations in the cationic trypsinogen gene and evidence for genetic heterogeneity in hereditary pancreatitis. J Med Genet 1999;36:228–232. Gorry MC, Gabbaizedeh D, Furey W, et al. Mutations in the cationic trypsinogen gene are associated with recurrent acute and chronic pancreatitis. Gastroenterology 1997;113:1063–1068. Chen JM, Mercier B, Ferec C. Strong evidence that the N21I substitution in the cationic trypsinogen gene causes disease in hereditary pancreatitis. Gut (in press). Whitcomb DC, Gorry MC, Preston RA, et al. Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nat Genet 1996;14:141–145. Whitcomb DC. Early trypsinogen activation in acute pancreatitis. Gastroenterology 1999;116:770–772. Creighton J, Lyall R, Wilson DI, et al. Mutations of the cationic trypsinogen gene in patients with chronic pancreatitis. Lancet 1999;354:42–43.
Reply. We appreciate that our recent results are confirmed by Drs. Pfu¨tzer and Whitcomb and by Chen et al. Both research groups detected the A16V mutation in the cationic trypsinogen gene in patients with chronic pancreatitis. However, the frequency of this mutation in their cohorts was less than 1%. These findings are in contrast to our results showing a frequency of nearly 10% (4 of 44 patients). This difference may be due to a different selection of participants. In our study, unrelated children and adolescents were enrolled: an age group in which alcohol abuse, the most common etiologic factor for chronic pancreatitis in adults, can be excluded. The difference may also be related to the fact that families with several affected members were analyzed. Both letters do not include a characterization of the cohorts, and it is not apparent how many of the patients were related. The penetrance of the A16V mutation seems to be lower than the R122H mutation, because only 5 of 11 patients (not 2 of 8 patients as stated by Pfu¨tzer and Whitcomb) were affected. It might be possible that a combination of the A16V mutation with a mutation in another gene is necessary for development of chronic pancreatitis. Pfu¨tzer and Whitcomb found in 1 patient with the A16V mutation an alteration in the CFTR gene. However, none of our patients with the A16V mutation showed a frequent CFTR mutation such as DF508 or R117H, although we did not analyze the entire coding sequence of the CFTR gene. As stated by Pfu¨tzer and Whitcomb, the chymotrypsinogen numbering is a simple and clear system for protein chemists, ‘‘but a confusing one for molecular biologists.’’ It was not our intention to contribute to confusion, and we fully agree that a standardized nomenclature should be reached. Therefore, we applied the generally accepted nomenclature system as recommended by the Nomenclature Working Group2 and used by the Human Gene Mutation Database (HGMD). As mentioned by Pfu¨tzer and Whitcomb and by Chen et al., the A16V mutation represents a change to an amino acid residue that is observed in mesotrypsinogen at this position. The activation of mesotrypsinogen is postulated to be a part of the feedback mechanism for inactivating trypsinogen and other zymogens by hydrolyzing these enzymes to inert products.3 Therefore, an activation of mesotrypsinogen is unlikely to cause pancreatitis. The confirmation of our findings that the A16V mutation in the cationic trypsinogen gene is associated with chronic pancreatitis underlines our claim for genetic testing of patients with so-called idiopathic disease. Functional studies on protein level will help to
CORRESPONDENCE
1509
clarify the pathophysiological mechanisms of mutant trypsinogens in chronic pancreatitis. HEIKO WITT WERNER LUCK MICHAEL BECKER Kinderklinik, Charite´, Campus Virchow-Klinikum Humboldt-Universita¨t Berlin, Germany 1. 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;117:7–10. 2. Antonarakis SE, Nomenclature Working Group. Recommendations for a nomenclature system for human gene mutations. Hum Mutat 1998;11:1–3. 3. Whitcomb DC, Gorry MC, Preston RA, et al. Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nat Genet 1996;14:141–145.
Importance of Adequate Acid Suppression in the Management of Barrett’s Esophagus Dear Sir: Ouatu-Lascar et al.1 recently reported on the effect of improved control of esophageal acid exposure on histological markers of differentiation and proliferation of biopsy specimens from the epithelium of patients with Barrett’s esophagus. This excellent report on 42 patients with Barrett’s esophagus in whom reflux symptoms were controlled with lansoprazole at doses of 15–60 mg daily confirmed the dynamic effect of acid exposure on this tissue previously reported by these investigators in ex vivo studies.2 The patients in whom intraesophageal pH was ‘‘normalized’’ on lansoprazole therapy showed a significant decrease in proliferating cell nuclear antigen (PCNA) and a significant increase in villin expression, i.e., evidence of decreased proliferation and increased differentiation in the tissue specimens. The positive correlation identified by the authors between the PCNA score (as an index of proliferation) and the degree of dysplasia in these biopsy specimens led them to suggest a strong hope that acid suppression could be used effectively to prevent dysplasia in patients with Barrett’s esophagus. Would that it were so! The fascinating series of experiments performed by investigators from this laboratory provide strong support for the concept that control of acid reflux might provide great promise for the long-term management of Barrett’s esophagus. As emphasized in this article, and previously reported from 2 laboratories,3,4 simple relief of reflux symptoms does not provide adequate evidence of control of esophageal acid exposure because of the well-known decreased sensitivity of the Barrett epithelium to acid.5 This point also emphasizes the importance of performing pH monitoring on these patients while undergoing therapy to ensure adequate acid control. It also brings up the question of just how much control is adequate. As in previous reports on acid suppression therapy in patients with Barrett’s esophagus,6 the authors use ‘‘normalization’’ of distal esophageal acid exposure as their endpoint. One should remember that these normal values were obtained in healthy controls who were not receiving acid suppression therapy. When one evaluates ‘‘normal’’ acid exposure while on therapy with a proton pump inhibitor (PPI), much different normal values are obtained. For example, in our laboratory, the upper limit of normal in healthy volunteers receiving 40 mg of omeprazole per day was only 0.9%.7 Because the prior studies by Dr. Fitzgerald and her colleagues have
CORRESPONDENCE
6. Hartley BS, Shotton DM. Pancreatic elastase. In: Boyer RD, ed. The enzymes. New York: Academic, 1971:323–373. 7. Whitcomb DC. Hereditary pancreatitis: new insights into acute and chronic pancreatitis. Gut 1999;45:317–322. 8. OMIM, Online Mendelian Inheritance in Man; http://www.ncbi.nlm. nih.gov/htbin-post/Omim/dispmim?167800. 9. Antonarakis SE, Nomenclature Working Group. Recommendations for a nomenclature system for human gene mutations. Hum Mutat 1998;11:1–3. 10. HGMD, Human Gene Mutation Database: http://www.uwcm. ac.uk/uwcm/mg/hgmd0.html. 11. Nyaruhucha CN, Kito M, Fukuoka SI. Identification and expression of the cDNA-encoding human mesotrypsin(ogen), an isoform of trypsin with inhibitor resistance. 1997;272:10573–10578. 12. Rowen L, Trask B, Boysen C, et al. Sequence of a large duplication from human chromosome 7 to chromosome 9 containing a portion of the T cell receptor beta locus and trypsinogen locus. GenBank 1997; Accession AF029308: (unpublished).
The A16V Signal Peptide Cleavage Site Mutation in the Cationic Trypsinogen Gene and Chronic Pancreatitis Dear Sir: We read with great interest the study by Witt et al.1 in the July 1999 issue of GASTROENTEROLOGY in which they describe a strong association of a novel DNA variant—a heterozygous g.131906C . T transition in the second exon of the cationic trypsinogen gene resulting in an amino acid substitution of Ala with Val at residue 16 of the cationic pretrypsinogen (A16V)—with chronic pancreatitis. This genetic finding confirms for the first time that although the molecular mechanism underlying the pancreatitis phenotype may delimit the variety or number of causal mutations in the cationic trypsinogen gene, still other mutations may be found as more comprehensive searches are done, as suggested by Elitsur et al.2 In support of the Witt report, we also identified this new DNA variant in 2 unrelated subjects of 312 patients with sporadic pancreatitis, but not in 200 control subjects, using a previously established denaturing gradient gel electrophoresis.3 The exceptionally high detection frequency—4 of 44 patients had the A16V mutation in the Witt study—may be explained by their carefully selected participants. Only children and adolescents with chronic pancreatitis were enrolled, an age group thought to be suitable for such a prevalence study of genetic defects. As in the case of the N29I mutation,4,5 the identification of an A16V substitution has again led to a challenging situation in which there is strong molecular genetic evidence for a disease-associated mutation but an inadequate understanding of the basic biochemistry of the corresponding protein and its function. Moreover, the penetrance of the A16V mutation is obviously low compared with the ,80% penetrance of the N29I and R122H6 mutations: only 1 of the 7 first-degree relatives carrying the A16V mutation was affected,1 and neither of the 2 A16V carriers in our study had a family history of pancreatitis. Interestingly, an Ile is present at residue 29 of the functional anionic pretrypsinogen and a Val is present at residue 16 of the functional premesotrypsinogen (Figure 1). However, whereas an N29I mutation results in a nonconservative amino acid substitution, which is considered to be important in causing the disease,5 an A16V mutation results in a conservative substitution. Perhaps it is this kind of conservative substitution that underlies the lower penetrance of the A16V mutation. Nevertheless, residue 16 constitutes the signal peptide cleavage site and provides an important clue as to how an A16V substitution could predispose someone to pancreatitis. Of the
GASTROENTEROLOGY Vol. 117, No. 6
Figure 1. Alignment of the N-terminal amino acids of the 3 pancreatic pretrypsinogens: cationic, anionic, and meso isoforms. Dashes indicate identity with the cationic amino acid sequence. Note the presence of Val at residue 16 of the premesotrypsinogen and the presence of Ile at residue 29 of the anionic pretrypsinogen.
possible mechanisms proposed by Witt et al., we favor the hypothesis that an A16V substitution may disrupt the intracellular transportation of the pretrypsinogen; in this vein, it may even be tempting to speculate that this substitution could lead to colocalization of pretrypsinogen with lysosomal hydrolases, a phenomenon well demonstrated in dissimilar models of pancreatitis in experimental animals.7 On the contrary, the facts that residue 16 is the first amino acid of the activation peptide of the cationic trypsinogen and that the first 13 N-terminal amino acids of the mutated cationic trypsinogen are identical to those of the wild-type mesotrypsinogen (Figure 1) may make an enhanced autoactivation hypothesis less likely. Also, based on the nature of the A16V mutation (located near the N terminus of the pretrypsinogen, a conservative amino acid substitution, and the presence of Val at residue 16 of the wild-type mesotrypsinogen) and its lower penetrance, it may be difficult to prove exactly how this mutation works by in vitro or in vivo experiments. Above all, there is an urgent need to correlate the different DNA variants (genotypes) with their clinical manifestations (phenotypes). Finally, the identification of the A16V mutation, together with another recent report in which 9 of 48 patients with a presumed diagnosis of idiopathic chronic pancreatitis were shown to have the R122H or N29I mutations,8 strongly suggest that genetic testing for cationic trypsinogen mutations even in the absence of a family history of pancreatitis is warranted. JIAN–MIN CHEN, Ph.D. ODILE RAGUENES CLAUDE FEREC, M.D., Ph.D. Centre de Biogenetique University, Hospital, ETSBO Brest, France PIERRE H. DEPREZ, M.D. Pediatric Unit Clinques Universitaires St-Luc Brussels, Belgium CHRISTINE VERELLEN–DUMOULIN, M.D. Centre for Human Genetics Universite Catholique de Louvain Brussels, Belgium ANGELO ANDRIULLI, M.D. Division of Gastroenterology Casa Sollievo della Sofferenza Hospital S. Giovanni Rotondo, Italy 1. 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;117:7–10. 2. Elitsur Y, Chertow BC, Jewell RD, et al. Identification of a hereditary
December 1999
3.
4.
5.
6.
7. 8.
pancreatitis mutation in four West Virginia families. Pediatr Res 1998;44:927–930. Ferec C, Raguenes O, Salomon R, et al. Mutations in the cationic trypsinogen gene and evidence for genetic heterogeneity in hereditary pancreatitis. J Med Genet 1999;36:228–232. Gorry MC, Gabbaizedeh D, Furey W, et al. Mutations in the cationic trypsinogen gene are associated with recurrent acute and chronic pancreatitis. Gastroenterology 1997;113:1063–1068. Chen JM, Mercier B, Ferec C. Strong evidence that the N21I substitution in the cationic trypsinogen gene causes disease in hereditary pancreatitis. Gut (in press). Whitcomb DC, Gorry MC, Preston RA, et al. Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nat Genet 1996;14:141–145. Whitcomb DC. Early trypsinogen activation in acute pancreatitis. Gastroenterology 1999;116:770–772. Creighton J, Lyall R, Wilson DI, et al. Mutations of the cationic trypsinogen gene in patients with chronic pancreatitis. Lancet 1999;354:42–43.
Reply. We appreciate that our recent results are confirmed by Drs. Pfu¨tzer and Whitcomb and by Chen et al. Both research groups detected the A16V mutation in the cationic trypsinogen gene in patients with chronic pancreatitis. However, the frequency of this mutation in their cohorts was less than 1%. These findings are in contrast to our results showing a frequency of nearly 10% (4 of 44 patients). This difference may be due to a different selection of participants. In our study, unrelated children and adolescents were enrolled: an age group in which alcohol abuse, the most common etiologic factor for chronic pancreatitis in adults, can be excluded. The difference may also be related to the fact that families with several affected members were analyzed. Both letters do not include a characterization of the cohorts, and it is not apparent how many of the patients were related. The penetrance of the A16V mutation seems to be lower than the R122H mutation, because only 5 of 11 patients (not 2 of 8 patients as stated by Pfu¨tzer and Whitcomb) were affected. It might be possible that a combination of the A16V mutation with a mutation in another gene is necessary for development of chronic pancreatitis. Pfu¨tzer and Whitcomb found in 1 patient with the A16V mutation an alteration in the CFTR gene. However, none of our patients with the A16V mutation showed a frequent CFTR mutation such as DF508 or R117H, although we did not analyze the entire coding sequence of the CFTR gene. As stated by Pfu¨tzer and Whitcomb, the chymotrypsinogen numbering is a simple and clear system for protein chemists, ‘‘but a confusing one for molecular biologists.’’ It was not our intention to contribute to confusion, and we fully agree that a standardized nomenclature should be reached. Therefore, we applied the generally accepted nomenclature system as recommended by the Nomenclature Working Group2 and used by the Human Gene Mutation Database (HGMD). As mentioned by Pfu¨tzer and Whitcomb and by Chen et al., the A16V mutation represents a change to an amino acid residue that is observed in mesotrypsinogen at this position. The activation of mesotrypsinogen is postulated to be a part of the feedback mechanism for inactivating trypsinogen and other zymogens by hydrolyzing these enzymes to inert products.3 Therefore, an activation of mesotrypsinogen is unlikely to cause pancreatitis. The confirmation of our findings that the A16V mutation in the cationic trypsinogen gene is associated with chronic pancreatitis underlines our claim for genetic testing of patients with so-called idiopathic disease. Functional studies on protein level will help to
CORRESPONDENCE
1509
clarify the pathophysiological mechanisms of mutant trypsinogens in chronic pancreatitis. HEIKO WITT WERNER LUCK MICHAEL BECKER Kinderklinik, Charite´, Campus Virchow-Klinikum Humboldt-Universita¨t Berlin, Germany 1. 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;117:7–10. 2. Antonarakis SE, Nomenclature Working Group. Recommendations for a nomenclature system for human gene mutations. Hum Mutat 1998;11:1–3. 3. Whitcomb DC, Gorry MC, Preston RA, et al. Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nat Genet 1996;14:141–145.
Importance of Adequate Acid Suppression in the Management of Barrett’s Esophagus Dear Sir: Ouatu-Lascar et al.1 recently reported on the effect of improved control of esophageal acid exposure on histological markers of differentiation and proliferation of biopsy specimens from the epithelium of patients with Barrett’s esophagus. This excellent report on 42 patients with Barrett’s esophagus in whom reflux symptoms were controlled with lansoprazole at doses of 15–60 mg daily confirmed the dynamic effect of acid exposure on this tissue previously reported by these investigators in ex vivo studies.2 The patients in whom intraesophageal pH was ‘‘normalized’’ on lansoprazole therapy showed a significant decrease in proliferating cell nuclear antigen (PCNA) and a significant increase in villin expression, i.e., evidence of decreased proliferation and increased differentiation in the tissue specimens. The positive correlation identified by the authors between the PCNA score (as an index of proliferation) and the degree of dysplasia in these biopsy specimens led them to suggest a strong hope that acid suppression could be used effectively to prevent dysplasia in patients with Barrett’s esophagus. Would that it were so! The fascinating series of experiments performed by investigators from this laboratory provide strong support for the concept that control of acid reflux might provide great promise for the long-term management of Barrett’s esophagus. As emphasized in this article, and previously reported from 2 laboratories,3,4 simple relief of reflux symptoms does not provide adequate evidence of control of esophageal acid exposure because of the well-known decreased sensitivity of the Barrett epithelium to acid.5 This point also emphasizes the importance of performing pH monitoring on these patients while undergoing therapy to ensure adequate acid control. It also brings up the question of just how much control is adequate. As in previous reports on acid suppression therapy in patients with Barrett’s esophagus,6 the authors use ‘‘normalization’’ of distal esophageal acid exposure as their endpoint. One should remember that these normal values were obtained in healthy controls who were not receiving acid suppression therapy. When one evaluates ‘‘normal’’ acid exposure while on therapy with a proton pump inhibitor (PPI), much different normal values are obtained. For example, in our laboratory, the upper limit of normal in healthy volunteers receiving 40 mg of omeprazole per day was only 0.9%.7 Because the prior studies by Dr. Fitzgerald and her colleagues have