- Email: [email protected]
Hereditary pancreatitis: A model for understanding the genetic basis of acute and chronic pancreatitis
IAP/APA Meeting Pancreatology 2001;1:565–570
Hereditary Pancreatitis: A Model for Understanding the Genetic Basis of Acute and Chronic Pancreatitis David C. Whitcomb Departments of Medicine, Cell Biology and Physiology, and Human Genetics, University of Pittsburgh, and the VA Pittsburgh Health Care System, Pittsburgh, Pa., USA
Key Words Pancreatitis W Genetics W Hereditary
Abstract Progress in understanding pancreatic diseases has been limited by a number of factors. Primary problems include the absence of good animal models, and difficulty in understanding the origin of pancreatic disease since the disease is usually manifest by the progressive destruction of the gland itself. Beginning in 1995, our laboratory, with the support of the Midwest Multicenter Pancreatic Study Group, began investigating the genetic basis of hereditary pancreatitis. Utilization of information becoming available through the human genome project allowed us to map and identify the hereditary pancreatitis gene as cationic trypsinogen (PRSS1). Molecular modeling, and subsequent experimental evidence, has solved key elements of the mysteries surrounding the origin of acute pancreatitis and the progression of acute pancreatitis to chronic pancreatitis. The availability of new genetic information and genomic tools should produce a revolution in our understanding of pancreatic diseases. Copyright © 2001 S. Karger AG, Basel and IAP
Based on a lecture at the combined meeting of the International Association of Pancreatology and the American Pancreatic Association, Chicago, 2000.
ABC Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com
© 2001 S. Karger AG, Basel and IAP
Accessible online at: www.karger.com/journals/pan
Introduction
Acute and chronic pancreatitis continues to be among the most difficult diseases faced by physicians and surgeons. It has been especially difficult to understand the etiology and progression of acute pancreatitis because the symptoms usually become evident only after the disease is fully developed, and when subtle biochemical changes that may predispose to acute pancreatitis are destroyed by the disease process [1]. Furthermore, uncertainty surrounds most animal models thereby failing to provide clear insights into human disease [2, 3]. Likewise, chronic pancreatitis presents as a progressive inflammatory and fibrotic process of unclear etiology and mechanism of disease progression [4]. Indeed these two forms of pancreatic disease have frustrated physicians because the medical and surgical interventions have been primarily relegated to provision of symptomatic relief, attempts to replace lost organ function, and controlled pain. Thus, early diagnosis and effective intervention has been totally lacking. Even up until the last five years our basic understanding of chronic pancreatitis was limited. It was conceded that ‘chronic pancreatitis remains an enigmatic process of uncertain pathogenesis, unpredictable clinical course and unclear treatment’ [4]. However, a new frontier and resource opened, and tremendous possibilities for gaining insights into diseases such as acute and chronic pancreatitis became available.
David Whitcomb, MD, PhD Division of Gastroenterology, Hepatology and Nutrition, Center for Genomic Sciences University of Pittsburgh Medical Center, Mezzanine Level, C Wing, PUH 200 Lothrop Street, Pittsburgh, PA 15213 (USA) Tel. +1 412 648 9604, Fax +1 412 383 7236, E-Mail [email protected]
Fig. 1. The major steps to accomplish a genetic linkage study are demonstrated. First, a family must be identified with a well characterized disease. Second, a careful survey of the 23 human chromosomes in each family member is made to identify the one segment of DNA that is always inherited with the disease. This identifies a single region of a chromosome that contains the affective gene. Third, the genes in the segment of DNA identified in step 2 are sequenced in order to identify the diseasecausing mutation. Fourth, once the mutation is identified, the genetic code is solved to reveal the normal protein and abnormal protein caused by the mutation.
This frontier was human genome project. The human genome project was initiated on October 1, 1990 as a 15year project to map and sequence the complete set of human chromosomes and those of several other model organisms. The major goals of the project were to construct detailed genetic and physical maps of the human genome to determine the complete 3,200,000,000 nucleotide sequence of human DNA, to localize the 50,000 to 100,000 genes within the human genome and to perform analysis on several other organisms used extensively in research laboratories as model systems [5]. The development of closely spaced chromosome markers and detailed maps of the human chromosome [6] provided the opportunity for smaller labs to perform genetic linkage studies and pinpointing the very one mutation out of 3.2 billion possibilities that causes disease. The development of first long-range DNA sequencing techniques [7] proved invaluable in determining precisely the mutations associated with hereditary pancreatitis.
Hereditary Pancreatitis
Hereditary pancreatitis is an autosomal dominant genetic disorder characterized by recurrent attacks of acute pancreatitis in approximately 80% of individuals with the susceptibility gene [8, 9]. About half of the individuals who develop acute pancreatitis progressed to chronic pancreatitis and many of these individuals eventually develop pancreatic cancer [10, 11]. Of great importance was the observation that the clinical and pathologic appearance of
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both acute and chronic pancreatitis in these patients was indistinguishable from sporadic forms of acute and chronic pancreatitis [11]. This suggested that the mutated gene product in hereditary pancreatitis represented a central and critical component of the mechanisms normally protecting others from bouts of acute and chronic pancreatitis [12]. The cause of hereditary pancreatitis has been discovered. The basic steps are outlined in figure 1. Genetic linkage studies performed by our group with the help of a large kindred from Kentucky that was recruited by the Midwest Multicenter Pancreatic Study Group identified a loci for the hereditary pancreatitis gene on chromosome 7q35 [12] (fig. 2). The identical locus was simultaneously being mapped by a French group [13] and confirmed by a group from Virginia [14]. We identified the disease gene, cationic trypsinogen, using a candidate gene approach [15]. The information from human genome project was critical in this discovery because the long-range sequencing efforts revealed 9 highly homologous trypsin-like genes and pseudogenes within the human genome. We uncovered this finding before it was published in Science through a GenBank search using the cDNA of trypsin. To our surprise these genes were within a 685,000 base pair sequence that had just been deposited by Rowen et al. [7]. This information was critical because it revealed that all trypsinogen-like genes were divided into five exons with over 90% homology. Having the exact DNA sequence allowed utilization of a precise nested PCR strategy to guarantee that only the target exons in the cationic trypsinogen gene would be amplified and sequenced without co-amplification of portions of
Whitcomb
Fig. 2. Linkage of the hereditary pancreatitis gene to chromosome 7q35. To find the chromosome region containing the affective gene, microsatellite markers from a number of chromosomes were sequentially tested for linkage with the disease gene. This led to the identification of the hereditary pancreatitis locus on chromosome 7. This figure illustrates the LOD score plot of microsatellite markers along chromosome 7. This is a statistic plot that proved that this region of DNA was inherited through the family tree with the hereditary pancreatitis trait. The hereditary pancreatitis gene was on the same chromosome as the cystic fibrosis gene (requiring mutations on both alleles), and at the exact location of the T-cell receptor beta chain gene (bold arrow).
other trypsinogen genes. A single G to A transversion mutation was identified within exon 3 of the cationic trypsinogen gene. This mutation was present in every individual tested within 5 families who had classic autosomal dominant form of hereditary pancreatitis, but it was absent in control populations [15]. This verified that this mutation was associated with hereditary pancreatitis. Our conclusion, therefore, was that cationic trypsinogen was a key molecule in the pathogenesis of both acute and chronic pancreatitis. Why is trypsin such an important molecule? A review of biochemistry and pancreatic physiology reminds us that trypsin plays a central role in digestion [16]. The pancreas has three basic functions which includes secretion of sodium bicarbonate to neutralize hydrochloric acid from the stomach, secretion of digestive enzymes, and secretion of hormones to regulate intermediate metabolism following a meal. There are many different digestive enzymes which work together to breaking down most carbohydrates, proteins, lipids, and nucleic acids within a meal into forms that can either be absorbed directly or easily digested by brush border enzymes during the absorptive process. All pancreatic enzymes, with the exception of amylase and lipase, are synthesized and secreted in an inactive form to protect the pancreas from autodigestion. Once the proenzymes enter the duodenum, they are activated beginning with the conversion of trypsinogen to trypsin by the action of enterokinase. Once trypsin is activated it will activate trypsinogen and all of the other digestive proenzymes so that suddenly every major enzyme is available for the digestion of the meal. Indeed, the
digestive capacity of these enzymes is so great that most of the meal is digestion in the proximal small intestine. What protects the pancreas from autodigestion? A number of protective mechanisms appear to work synergistically to prevent premature activation of trypsinogen and the other prodigestive enzymes [16]. Specifically, the pancreas synthesizes pancreatic secretory trypsin inhibitor (PSTI, or serine protease inhibitor, Kazal type 1; SPINK1) along with trypsin and the other digestive enzymes. PSTI/SPINK1 is a trypsin inhibitor with a capacity of neutralizing approximately 20% of pancreatic trypsin activity [16]. Therefore, in the event of premature, activation, trypsinogen, PSTI/SPINK1 inhibits trypsin thereby preventing it from activating the digestive enzymes causing pancreatitis. Another important protective mechanism is low intracellular calcium concentrations [16]. Biochemical studies have repeatedly demonstrated that trypsin is affected by the calcium concentration and high calcium protects active trypsin from autodigestion [17–19]. Other mechanisms thought to be important are the sorting of the digestive enzymes into zymogen granules which are condensed and stored apart from other cellular elements until pancreatic acinar cells are stimulated to secrete. In addition, the pancreas is protected by a secretory duct system that sweeps the digestive enzymes into the duodenum with copious amounts of bicarbonaterich fluids. By solving the genetic code for trypsinogen to determine the consequence of the mutation, it becomes apparent that the amino acid at codon 122 (amino acid #117 using the chymotrypsinogen numbering system [10]) is
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Fig. 3. Fail-safe trypsin inactivation. The trypsin molecule is illustrated on the right side of the two structures in the figure and is shaped like a ‘pac-man’ with the jaws opening to the left. If a protein chain with an arginine or lysine enters the jaw it is rapidly cut into two pieces. Trypsinogen is made by the pancreas in an inactive form. It is one of the most important enzymes because once it is activated to trypsin in the intestine, it is responsible for activating nearly all of the other digestive enzymes to their active form, in the intestine. If trypsinogen becomes active in the pancreas it could activate all of the other digestive enzymes and cause the pancreas to digest itself. Forturnately, the pancreas also makes a trypsin inhibitor (SPINK1/PSTI
– on the left side of the two structures in the figure) that plugs the active site and prevents trypsin from activating other trypinogen molecules and all the other enzymes. However, the amount of SPINK1/PSTI is limited, so another protective mechanism is necessary. If too much trypsin is active, trypsin begins attacking itself at arginine #122 (R122). Cutting the connecting chain splits the trypsinogen molecule in half, permanently inactivating it, and saving the pancreas from auto-digestion. Solving the genetic code for hereditary pancreatitis proved that the mutation was at R122, changing the arginine to histidine (R122H mutation).
changed from the normal ariginine to a histidine (R122H). This mutation is located on the peptide side chain connecting the two globular domains of trypsin. Since arginine is a recognition site for trypsin hydrolysis and histidine is not, the mutation would eliminate the site for attack by a second trypsin molecule [15]. Therefore, we developed a mechanistic model suggesting that, under conditions of excessive trypsinogen activation, exceeding the inhibitory capacity of PSTI/SPINK1, the R122 would function as a ‘fail safe’ autolysis site initiating trypsin’s self destruction. Hydrolysis of the side chain at arginine 122 would promote separation of the two globular domains of trypsin, splitting the molecule through the active site and allowing the remaining components to be rapidly degraded. In hereditary pancreatitis, the arginine to histamine substitution eliminates this autolysis site so that trypsin continues to digest pancreatic proteins and activate other digestive proenzymes without being inhibited (fig. 3). The R122H mutation is not the only mutation associated with hereditary pancreatitis. Although the R122H mutation is common, a number of families were identi-
fied without the R122H mutation. We studied two such families with clinical characteristics nearly identical to the R122H families and identified an N29I substitution (chymotrypsinogen system #N21I) [20]. Additional mutations were identified in the trypsinogen activation peptide region which also increases susceptibility to pancreatitis possibly by facilitating early activation. These include A16V [21], D22G [22], K23R [23], N29T and R122C. In addition to trypsinogen, great interest developed with discovery of the mutations in the trypsinogen inhibitor PSTI/SPINK1. Mutations in the PSTI/SPINK1, especially N34S, which was initially reported by Chen et al. [24] and found to be associated with idiopathic chronic pancreatitis in children [25] and familial pancreatitis [26], further established the importance of excessive amounts of active trypsin inside the pancreas. Surprisingly, both homozygous and heterozygous PSTI/SPINK1 produce acute and chronic pancreatitis, and this only occurs in a minority of patients who harbor these mutations [24–26]. We therefore suggest that the PSTI/SPINK1 genes are modifier genes that increase the risk of pancreatitis without necessarily causing disease by themselves [26].
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Whitcomb
Taking together this work in molecular genetics is providing a clearer picture of factors that are important for initiating acute pancreatitis, and have clearly established a progressive link between acute pancreatitis and chronic pancreatitis [15]. Central to acute pancreatitis is the activation of trypsin. Enhanced activation of trypsin can occur through specific mutations such as the A16V, D22G, or K23R mutations and possibly N29I. Increased amounts of active trypsin can also be generated with the diminished inhibitory capacity of PSTI/SPINK1 through a variety of mutations [25, 26]. Finally, trypsin activity can be enhanced with reduced autolysis through codon 122 mutations or in the presence of elevated calcium which also prevents autolysis [19]. Thus, through a variety of scenarios, individuals with increased amounts of intracellular active trypsin are at risk of acute pancreatitis, and eventually chronic pancreatitis. Pancreatitis is also associated with failure of the duct system to secrete adequate amounts of bicarbonate-rich fluid and to transport digestive enzymes, including trypsin, to the duodenum promptly. A number of groups have demonstrated that mutations in the CFTR gene, which is central to this process, also predispose individuals to recurrent acute and chronic pancreatitis [27, 28]. Careful investigation of hereditary pancreatitis families has raised a number of unanswered and potentially important questions. The first question is why approximately 20% of individuals with the most severe genetic mutations of trypsinogen gene never develop pancreatitis. Indeed, there must be other genetic, epigenetic or environmental factors that are critical in protecting the pancreas of these individuals. Studies on identical twins and specific investigations of obligate carriers have emphasized this fact without bringing any clear answers [29]. Additional questions surround the families that have been thoroughly investigated and all known mutations excluded [10]. Therefore, although the genetics of hereditary pancreatitis have answered a number of extremely important questions, there is still much to be learned. What are some of the practical applications of these discoveries? One of the most important is in risk assessment [1]. The discovery of specific genetic mutations that run in families provide the opportunity for individuals with a history of pancreatitis or with early signs of pancreatitis to have a molecular diagnostic test done and determine whether or not they are, in fact, at risk for developing pancreatitis through these mechanisms. Identification of these patients and knowledge of the pathophysiologic process may allow for the development of semi-specific therapeutic and preventative trials to determine whether
Genomics of Hereditary Pancreatitis
or not we can prevent a disease for which we have no cure. Secondly, these discoveries aid in the categorization of patients according to risk and etiology of pancreatitis [1]. Understanding acute and chronic pancreatitis from the perspective of risk and etiology may uncover different clinical courses and responses to different therapies. The most obvious example at the present time is autoimmune pancreatitis in which immunosuppressive treatment appears to rapidly reverse many of the gross changes in the pancreas thought to be associated with pancreatitis [30– 32]. Progress in cardiovascular heart disease provides another example of how early risk assessment and intervention prevents premature deaths and untold sufferings. On the other hand the huge investment in heart transplantation and artificial heart development to replace an endstage organ has provided little overall benefit to date. Likewise, we believe that the risk assessment and early intervention in pancreatic diseases may be far more effective than the attempts to treat end-stage pancreatic disease with support measures and surgical or endoscopic intervention for pain, and screening for cancer. In conclusion, hereditary pancreatitis has indeed proven to be an extremely important clinical and basic research tool for understanding a variety of pancreatic diseases. In the future, we may have the opportunity to shift our goal from treating patients with end stage diseases to treating patients with high risk of developing pancreatic diseases so that our patients may enjoy a pain free and uncomplicated life. As pancreatologists, we should work to preserve the function of an organ that is close to our heart.
Acknowlegments Supported by NIH DK54709, AA20885, a VA Merit Review and the Center for Genomic Sciences, University of Pittsburgh, Pa., USA.
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References 1 Etemad B, Whitcomb DC: Chronic Pancreatitis: Diagnosis, Classification, and New Genetic Developments. Gastroenterology 2001;120: 682–707. 2 Steinberg W, Schlesselman S: Treatment of acute pancreatitis: Comparison of animal and human studies. Gastroenterology 1987;93: 1420–1427. 3 Whitcomb DC, Ulrich II CD: Hereditary Pancreatitis: New Insights, New Directions. Baillière’s Clinical Gastroenterol 1999;13:253– 263. 4 Steer ML, Waxman I, Freedman S: Chronic pancreatitis. N Engl J Med 1995;332:1482– 1490. 5 Collins FS, Patrinos A, Jordan E, Chakravarti A, Gesteland R, Walters L: New goals for the US Human Genome Project: 1998–2003. Science 1998;282:682–689. 6 Gyapay G, Morissette J, Vignal A, Dib C, Fizames C, Phillippe M, Marc S, Bernardi G, Lathrop M, Weissenbach J: The 1993–94 Genethon human genetic linkage map. Nat Genet 1994;7:247–339. 7 Rowen L, Koop BF, Hood L: The complete 685-kilobase DNA sequence of the human beta T cell receptor locus. Science 1996;272:1755– 1762. 8 Perrault J: Hereditary pancreatitis. Gastroenterol Clin N Am 1994;23:743–752. 9 Perrault J: Hereditry pancreatitis: Historical perspectives. Med Clin N Am 2000;84:519– 529. 10 Whitcomb DC: Genetic Predispositions to Acute and Chronic Pancreatitis. Med Clin N Am 2000;84:531–547. 11 Lowenfels A, Maisonneuve P, DiMagno E, Elitsur Y, Gates L, Perrault J, Whitcomb D: Hereditary pancreatitis and the risk of pancreatic cancer. J Nat Canc Inst 1997;89:442– 446. 12 Whitcomb DC, Gorry MC, Preston RA, Furey W, Sossenheimer MJ, Ulrich CD, Martin SP, Gates LK, Amann ST, Toskes PP, Liddle R, McGrath K, Uomo G, Post JC, Ehrlich GD: A gene for hereditary pancreatitis maps to chromosome 7q35. Gastroenterology 1996; 110:1975–1980. 13 Le Bodic L, Bignon JD, Raguenes O, Mercier B, Georgelin T, Schnee M, Soulard F, Gagne K, Bonneville F, Muller JY, Bachner L, Ferec C: The hereditary pancreatitis gene maps to long arm of chromosome 7. Hum Mol Genet 1996; 5:549–554.
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14 Pandya A, Blanton SH, Landa B, Javaheri R, Melvin E, Nance WE, Markello T: Linkage studies in a large kindred with hereditary pancreatitis confirms mapping of the gene to a 16cm region on 7q. Genomics 1996;38:227–230. 15 Whitcomb DC, Gorry MC, Preston RA, Furey W, Sossenheimer MJ, Ulrich CD, Martin SP, Gates Jr LK, 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;14:141–145. 16 Rinderknecht H: Pancreatic Secretory Enzymes; in Go VLW, DiMagno EP, Gardner JD, Lebenthal E, Reber HA, Scheele GA (eds): The Pancreas: Biology, Pathobiology, and Disease. 2nd ed. New York, Raven Press, 1993, pp 219– 251. 17 Colomb E, Guy O, Deprez P, Michel R, Figarella C: The two human trypsingens: Catalytic properties of the corresponding trypsins. Biochem Biophys Acta 1978;525:186–193. 18 Colomb E, Figarella C: Comparative studies on the mechanism of activation of the two human trypsinogens. Biochem Biophys Acta 1979;571: 343–351. 19 Sahin-Toth M: Human cationic trypsinogen: Role of Asn-21 in zymogen activation and implications in hereditary pancreatitis. J Biol Chem 2000;275:22750–22755. 20 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;113:1063–1068. 21 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. 22 Teich N, Ockenga J, Hoffmeister A, Manns M, Mössner J, Keim V: Chronic pancreatitis associated with an activation peptide mutation that facilitates trypsin activation. Gastroenterology 2000;119:461–465.
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23 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;36:228–232. 24 Chen J-M, Mercier B, Audrezet M-P, Ferec C: Mutational analysis of the human pancreatic secretory trypsin inhibitor (PSTI) gene in hereditary and sporadic chronic pancreatitis. J Med Genet 2000;37:67–69. 25 Witt H, Luck W, Hennies HC, Classen M, Kage A, Lass U, Landt O, Becker M: Mutations in the gene encoding the serine protease inhibitor, kazal type 1 are associated with chronic pancreatitis. Nat Genet 2000;25:213–216. 26 Pfützer RH, Barmada MM, Brunskil APJ, Finch R, Hart PS, Neoptolemos J, Furey WF, Whitcomb DC: SPINK1/PSTI polymorphisms act as disease modifiers in familial and idiopathic chronic pancreatitis. Gastroenterology 2000;119:615–623. 27 Cohn JA, Friedman KJ, Noone PG, Knowles MR, Silverman LM, Jowell PS: Relation between mutations of the cystic fibrosis gene and idiopathic pancreatitis. N Engl J Med 1998; 339:653–658. 28 Sharer N, Schwarz M, Malone G, Howarth A, Painter J, Super M, Braganza J: Mutations of the cystic fibrosis gene in patients with chronic pancreatitis. N Engl J Med 1998;339:645–652. 29 Amann ST, Gates LK Jr, Aston CE, Pandya A, Bartness M, Whitcomb DC: Expression and penetrance of the hereditary pancreatitis phenotype in monozygotic twins. Gut 2001;48: 542–547. 30 Okazaki K, Uchida K, Ohana M, Nakase H, Uose S, Inai M, Matsushima Y, Katamura K, Ohmori K, Chiba T: Autoimmune-related pancreatitis is associated with autoantibodies and a Th1/Th2-type cellular immune response. Gastroenterology 2000;118:573–581. 31 Furukawa N, Muranaka T, Yasumori K, Matsubayashi R, Hayashida K, Arita Y: Autoimmune pancreatitis: Radiologic findings in three histologically proven cases. J Comput Ass Tomogr 1998;22:880–883. 32 Ito T, Nakano I, Koyanagi S, Miyahara T, Migita Y, Ogoshi K, Sakai H, Matsunaga S, Yasuda O, Sumii T, Nawata H: Autoimmune pancreatitis as a new clinical entity. Three cases of autoimmune pancreatitis with effective steroid therapy. Dig Dis Sci 1997;42: 1458–1468.
Whitcomb
Hereditary Pancreatitis: A Model for Understanding the Genetic Basis of Acute and Chronic Pancreatitis David C. Whitcomb Departments of Medicine, Cell Biology and Physiology, and Human Genetics, University of Pittsburgh, and the VA Pittsburgh Health Care System, Pittsburgh, Pa., USA
Key Words Pancreatitis W Genetics W Hereditary
Abstract Progress in understanding pancreatic diseases has been limited by a number of factors. Primary problems include the absence of good animal models, and difficulty in understanding the origin of pancreatic disease since the disease is usually manifest by the progressive destruction of the gland itself. Beginning in 1995, our laboratory, with the support of the Midwest Multicenter Pancreatic Study Group, began investigating the genetic basis of hereditary pancreatitis. Utilization of information becoming available through the human genome project allowed us to map and identify the hereditary pancreatitis gene as cationic trypsinogen (PRSS1). Molecular modeling, and subsequent experimental evidence, has solved key elements of the mysteries surrounding the origin of acute pancreatitis and the progression of acute pancreatitis to chronic pancreatitis. The availability of new genetic information and genomic tools should produce a revolution in our understanding of pancreatic diseases. Copyright © 2001 S. Karger AG, Basel and IAP
Based on a lecture at the combined meeting of the International Association of Pancreatology and the American Pancreatic Association, Chicago, 2000.
ABC Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com
© 2001 S. Karger AG, Basel and IAP
Accessible online at: www.karger.com/journals/pan
Introduction
Acute and chronic pancreatitis continues to be among the most difficult diseases faced by physicians and surgeons. It has been especially difficult to understand the etiology and progression of acute pancreatitis because the symptoms usually become evident only after the disease is fully developed, and when subtle biochemical changes that may predispose to acute pancreatitis are destroyed by the disease process [1]. Furthermore, uncertainty surrounds most animal models thereby failing to provide clear insights into human disease [2, 3]. Likewise, chronic pancreatitis presents as a progressive inflammatory and fibrotic process of unclear etiology and mechanism of disease progression [4]. Indeed these two forms of pancreatic disease have frustrated physicians because the medical and surgical interventions have been primarily relegated to provision of symptomatic relief, attempts to replace lost organ function, and controlled pain. Thus, early diagnosis and effective intervention has been totally lacking. Even up until the last five years our basic understanding of chronic pancreatitis was limited. It was conceded that ‘chronic pancreatitis remains an enigmatic process of uncertain pathogenesis, unpredictable clinical course and unclear treatment’ [4]. However, a new frontier and resource opened, and tremendous possibilities for gaining insights into diseases such as acute and chronic pancreatitis became available.
David Whitcomb, MD, PhD Division of Gastroenterology, Hepatology and Nutrition, Center for Genomic Sciences University of Pittsburgh Medical Center, Mezzanine Level, C Wing, PUH 200 Lothrop Street, Pittsburgh, PA 15213 (USA) Tel. +1 412 648 9604, Fax +1 412 383 7236, E-Mail [email protected]
Fig. 1. The major steps to accomplish a genetic linkage study are demonstrated. First, a family must be identified with a well characterized disease. Second, a careful survey of the 23 human chromosomes in each family member is made to identify the one segment of DNA that is always inherited with the disease. This identifies a single region of a chromosome that contains the affective gene. Third, the genes in the segment of DNA identified in step 2 are sequenced in order to identify the diseasecausing mutation. Fourth, once the mutation is identified, the genetic code is solved to reveal the normal protein and abnormal protein caused by the mutation.
This frontier was human genome project. The human genome project was initiated on October 1, 1990 as a 15year project to map and sequence the complete set of human chromosomes and those of several other model organisms. The major goals of the project were to construct detailed genetic and physical maps of the human genome to determine the complete 3,200,000,000 nucleotide sequence of human DNA, to localize the 50,000 to 100,000 genes within the human genome and to perform analysis on several other organisms used extensively in research laboratories as model systems [5]. The development of closely spaced chromosome markers and detailed maps of the human chromosome [6] provided the opportunity for smaller labs to perform genetic linkage studies and pinpointing the very one mutation out of 3.2 billion possibilities that causes disease. The development of first long-range DNA sequencing techniques [7] proved invaluable in determining precisely the mutations associated with hereditary pancreatitis.
Hereditary Pancreatitis
Hereditary pancreatitis is an autosomal dominant genetic disorder characterized by recurrent attacks of acute pancreatitis in approximately 80% of individuals with the susceptibility gene [8, 9]. About half of the individuals who develop acute pancreatitis progressed to chronic pancreatitis and many of these individuals eventually develop pancreatic cancer [10, 11]. Of great importance was the observation that the clinical and pathologic appearance of
566
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both acute and chronic pancreatitis in these patients was indistinguishable from sporadic forms of acute and chronic pancreatitis [11]. This suggested that the mutated gene product in hereditary pancreatitis represented a central and critical component of the mechanisms normally protecting others from bouts of acute and chronic pancreatitis [12]. The cause of hereditary pancreatitis has been discovered. The basic steps are outlined in figure 1. Genetic linkage studies performed by our group with the help of a large kindred from Kentucky that was recruited by the Midwest Multicenter Pancreatic Study Group identified a loci for the hereditary pancreatitis gene on chromosome 7q35 [12] (fig. 2). The identical locus was simultaneously being mapped by a French group [13] and confirmed by a group from Virginia [14]. We identified the disease gene, cationic trypsinogen, using a candidate gene approach [15]. The information from human genome project was critical in this discovery because the long-range sequencing efforts revealed 9 highly homologous trypsin-like genes and pseudogenes within the human genome. We uncovered this finding before it was published in Science through a GenBank search using the cDNA of trypsin. To our surprise these genes were within a 685,000 base pair sequence that had just been deposited by Rowen et al. [7]. This information was critical because it revealed that all trypsinogen-like genes were divided into five exons with over 90% homology. Having the exact DNA sequence allowed utilization of a precise nested PCR strategy to guarantee that only the target exons in the cationic trypsinogen gene would be amplified and sequenced without co-amplification of portions of
Whitcomb
Fig. 2. Linkage of the hereditary pancreatitis gene to chromosome 7q35. To find the chromosome region containing the affective gene, microsatellite markers from a number of chromosomes were sequentially tested for linkage with the disease gene. This led to the identification of the hereditary pancreatitis locus on chromosome 7. This figure illustrates the LOD score plot of microsatellite markers along chromosome 7. This is a statistic plot that proved that this region of DNA was inherited through the family tree with the hereditary pancreatitis trait. The hereditary pancreatitis gene was on the same chromosome as the cystic fibrosis gene (requiring mutations on both alleles), and at the exact location of the T-cell receptor beta chain gene (bold arrow).
other trypsinogen genes. A single G to A transversion mutation was identified within exon 3 of the cationic trypsinogen gene. This mutation was present in every individual tested within 5 families who had classic autosomal dominant form of hereditary pancreatitis, but it was absent in control populations [15]. This verified that this mutation was associated with hereditary pancreatitis. Our conclusion, therefore, was that cationic trypsinogen was a key molecule in the pathogenesis of both acute and chronic pancreatitis. Why is trypsin such an important molecule? A review of biochemistry and pancreatic physiology reminds us that trypsin plays a central role in digestion [16]. The pancreas has three basic functions which includes secretion of sodium bicarbonate to neutralize hydrochloric acid from the stomach, secretion of digestive enzymes, and secretion of hormones to regulate intermediate metabolism following a meal. There are many different digestive enzymes which work together to breaking down most carbohydrates, proteins, lipids, and nucleic acids within a meal into forms that can either be absorbed directly or easily digested by brush border enzymes during the absorptive process. All pancreatic enzymes, with the exception of amylase and lipase, are synthesized and secreted in an inactive form to protect the pancreas from autodigestion. Once the proenzymes enter the duodenum, they are activated beginning with the conversion of trypsinogen to trypsin by the action of enterokinase. Once trypsin is activated it will activate trypsinogen and all of the other digestive proenzymes so that suddenly every major enzyme is available for the digestion of the meal. Indeed, the
digestive capacity of these enzymes is so great that most of the meal is digestion in the proximal small intestine. What protects the pancreas from autodigestion? A number of protective mechanisms appear to work synergistically to prevent premature activation of trypsinogen and the other prodigestive enzymes [16]. Specifically, the pancreas synthesizes pancreatic secretory trypsin inhibitor (PSTI, or serine protease inhibitor, Kazal type 1; SPINK1) along with trypsin and the other digestive enzymes. PSTI/SPINK1 is a trypsin inhibitor with a capacity of neutralizing approximately 20% of pancreatic trypsin activity [16]. Therefore, in the event of premature, activation, trypsinogen, PSTI/SPINK1 inhibits trypsin thereby preventing it from activating the digestive enzymes causing pancreatitis. Another important protective mechanism is low intracellular calcium concentrations [16]. Biochemical studies have repeatedly demonstrated that trypsin is affected by the calcium concentration and high calcium protects active trypsin from autodigestion [17–19]. Other mechanisms thought to be important are the sorting of the digestive enzymes into zymogen granules which are condensed and stored apart from other cellular elements until pancreatic acinar cells are stimulated to secrete. In addition, the pancreas is protected by a secretory duct system that sweeps the digestive enzymes into the duodenum with copious amounts of bicarbonaterich fluids. By solving the genetic code for trypsinogen to determine the consequence of the mutation, it becomes apparent that the amino acid at codon 122 (amino acid #117 using the chymotrypsinogen numbering system [10]) is
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Fig. 3. Fail-safe trypsin inactivation. The trypsin molecule is illustrated on the right side of the two structures in the figure and is shaped like a ‘pac-man’ with the jaws opening to the left. If a protein chain with an arginine or lysine enters the jaw it is rapidly cut into two pieces. Trypsinogen is made by the pancreas in an inactive form. It is one of the most important enzymes because once it is activated to trypsin in the intestine, it is responsible for activating nearly all of the other digestive enzymes to their active form, in the intestine. If trypsinogen becomes active in the pancreas it could activate all of the other digestive enzymes and cause the pancreas to digest itself. Forturnately, the pancreas also makes a trypsin inhibitor (SPINK1/PSTI
– on the left side of the two structures in the figure) that plugs the active site and prevents trypsin from activating other trypinogen molecules and all the other enzymes. However, the amount of SPINK1/PSTI is limited, so another protective mechanism is necessary. If too much trypsin is active, trypsin begins attacking itself at arginine #122 (R122). Cutting the connecting chain splits the trypsinogen molecule in half, permanently inactivating it, and saving the pancreas from auto-digestion. Solving the genetic code for hereditary pancreatitis proved that the mutation was at R122, changing the arginine to histidine (R122H mutation).
changed from the normal ariginine to a histidine (R122H). This mutation is located on the peptide side chain connecting the two globular domains of trypsin. Since arginine is a recognition site for trypsin hydrolysis and histidine is not, the mutation would eliminate the site for attack by a second trypsin molecule [15]. Therefore, we developed a mechanistic model suggesting that, under conditions of excessive trypsinogen activation, exceeding the inhibitory capacity of PSTI/SPINK1, the R122 would function as a ‘fail safe’ autolysis site initiating trypsin’s self destruction. Hydrolysis of the side chain at arginine 122 would promote separation of the two globular domains of trypsin, splitting the molecule through the active site and allowing the remaining components to be rapidly degraded. In hereditary pancreatitis, the arginine to histamine substitution eliminates this autolysis site so that trypsin continues to digest pancreatic proteins and activate other digestive proenzymes without being inhibited (fig. 3). The R122H mutation is not the only mutation associated with hereditary pancreatitis. Although the R122H mutation is common, a number of families were identi-
fied without the R122H mutation. We studied two such families with clinical characteristics nearly identical to the R122H families and identified an N29I substitution (chymotrypsinogen system #N21I) [20]. Additional mutations were identified in the trypsinogen activation peptide region which also increases susceptibility to pancreatitis possibly by facilitating early activation. These include A16V [21], D22G [22], K23R [23], N29T and R122C. In addition to trypsinogen, great interest developed with discovery of the mutations in the trypsinogen inhibitor PSTI/SPINK1. Mutations in the PSTI/SPINK1, especially N34S, which was initially reported by Chen et al. [24] and found to be associated with idiopathic chronic pancreatitis in children [25] and familial pancreatitis [26], further established the importance of excessive amounts of active trypsin inside the pancreas. Surprisingly, both homozygous and heterozygous PSTI/SPINK1 produce acute and chronic pancreatitis, and this only occurs in a minority of patients who harbor these mutations [24–26]. We therefore suggest that the PSTI/SPINK1 genes are modifier genes that increase the risk of pancreatitis without necessarily causing disease by themselves [26].
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Taking together this work in molecular genetics is providing a clearer picture of factors that are important for initiating acute pancreatitis, and have clearly established a progressive link between acute pancreatitis and chronic pancreatitis [15]. Central to acute pancreatitis is the activation of trypsin. Enhanced activation of trypsin can occur through specific mutations such as the A16V, D22G, or K23R mutations and possibly N29I. Increased amounts of active trypsin can also be generated with the diminished inhibitory capacity of PSTI/SPINK1 through a variety of mutations [25, 26]. Finally, trypsin activity can be enhanced with reduced autolysis through codon 122 mutations or in the presence of elevated calcium which also prevents autolysis [19]. Thus, through a variety of scenarios, individuals with increased amounts of intracellular active trypsin are at risk of acute pancreatitis, and eventually chronic pancreatitis. Pancreatitis is also associated with failure of the duct system to secrete adequate amounts of bicarbonate-rich fluid and to transport digestive enzymes, including trypsin, to the duodenum promptly. A number of groups have demonstrated that mutations in the CFTR gene, which is central to this process, also predispose individuals to recurrent acute and chronic pancreatitis [27, 28]. Careful investigation of hereditary pancreatitis families has raised a number of unanswered and potentially important questions. The first question is why approximately 20% of individuals with the most severe genetic mutations of trypsinogen gene never develop pancreatitis. Indeed, there must be other genetic, epigenetic or environmental factors that are critical in protecting the pancreas of these individuals. Studies on identical twins and specific investigations of obligate carriers have emphasized this fact without bringing any clear answers [29]. Additional questions surround the families that have been thoroughly investigated and all known mutations excluded [10]. Therefore, although the genetics of hereditary pancreatitis have answered a number of extremely important questions, there is still much to be learned. What are some of the practical applications of these discoveries? One of the most important is in risk assessment [1]. The discovery of specific genetic mutations that run in families provide the opportunity for individuals with a history of pancreatitis or with early signs of pancreatitis to have a molecular diagnostic test done and determine whether or not they are, in fact, at risk for developing pancreatitis through these mechanisms. Identification of these patients and knowledge of the pathophysiologic process may allow for the development of semi-specific therapeutic and preventative trials to determine whether
Genomics of Hereditary Pancreatitis
or not we can prevent a disease for which we have no cure. Secondly, these discoveries aid in the categorization of patients according to risk and etiology of pancreatitis [1]. Understanding acute and chronic pancreatitis from the perspective of risk and etiology may uncover different clinical courses and responses to different therapies. The most obvious example at the present time is autoimmune pancreatitis in which immunosuppressive treatment appears to rapidly reverse many of the gross changes in the pancreas thought to be associated with pancreatitis [30– 32]. Progress in cardiovascular heart disease provides another example of how early risk assessment and intervention prevents premature deaths and untold sufferings. On the other hand the huge investment in heart transplantation and artificial heart development to replace an endstage organ has provided little overall benefit to date. Likewise, we believe that the risk assessment and early intervention in pancreatic diseases may be far more effective than the attempts to treat end-stage pancreatic disease with support measures and surgical or endoscopic intervention for pain, and screening for cancer. In conclusion, hereditary pancreatitis has indeed proven to be an extremely important clinical and basic research tool for understanding a variety of pancreatic diseases. In the future, we may have the opportunity to shift our goal from treating patients with end stage diseases to treating patients with high risk of developing pancreatic diseases so that our patients may enjoy a pain free and uncomplicated life. As pancreatologists, we should work to preserve the function of an organ that is close to our heart.
Acknowlegments Supported by NIH DK54709, AA20885, a VA Merit Review and the Center for Genomic Sciences, University of Pittsburgh, Pa., USA.
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