- Email: [email protected]
Identification of a Novel Pancreatitis-Associated Missense Mutation, R116C, in the Human Cationic Trypsinogen Gene (PRSS1)
Molecular Genetics and Metabolism 74, 342–344 (2001) doi:10.1006/mgme.2001.3246, available online at http://www.idealibrary.com on
MUTATION REPORT Identification of a Novel Pancreatitis-Associated Missense Mutation, R116C, in the Human Cationic Trypsinogen Gene (PRSS1) trypsinogen can result in a gain of trypsin leading to the autodigestion of the pancreas itself (reviewed in 9). In this study, we report a new pancreatitis-associated missense mutation in the PRSS1 gene.
Over the past 5 years, several gain-of-function missense mutations in the human cationic trypsinogen gene (PRSS1, OMIM 276000) have been associated with hereditary and/or sporadic pancreatitis. This study reports a new pancreatitis-associated mutation—R116C (CGT > TGT: c.346C > T)—in the gene. © 2001 Academic Press Key Words: human cationic trypsinogen gene; denaturing high-performance liquid chromatography; gain of function; hereditary pancreatitis; idiopathic chronic pancreatitis; missense mutation; mutational analysis.
MATERIALS AND METHODS Patients. All patients with idiopathic chronic pancreatitis (ICP) were diagnosed by typical clinical manifestations, an increase in serum amylase or lipase, and pathological sonographic findings. Exclusion criteria included the presence of precipitating factors, such as alcohol, gallstones, trauma, medication, infection, metabolic disorders, and a positive family history, as described previously (8).
A classic perception in human genetics is that gainof-function is likely when only a specific mutation in a gene produces a given pathology (1). However, since the discovery of an apparent gain-of-function missense mutation—R122H (originally named R117H in chymotrypsin numbering; mutation nomenclature discussed in 2)—in the human cationic trypsinogen gene [PRSS1; Online Mendelian Inheritance in Man (OMIM) 276000, http://www.ncbi.nlm.nih.gov/omim] as a cause of hereditary pancreatitis (HP; OMIM 167800) (3), screening of this gene in both hereditary and sporadic pancreatitis has identified more diseaseassociated mutations than expected (4 – 8). This exceptional case appears to lie in the unique properties of trypsin(ogen): Trypsinogen has the ability to autoactivate itself; and the activated trypsin has the ability to autolyze itself. Indeed, the known PRSS1 mutations affect mainly these two processes; for example, the R122H mutation disrupts the primary R122 autolysis site of trypsin, and the K23R and D22G mutations facilitate trypsinogen autoactivation. Both increased stability of trypsin and enhanced autoactivation of
Mutation screening. All of the five exons of the PRSS1 gene were screened by denaturing high-performance liquid chromatography (D-HPLC). Isoform-specific primers were designed for each exon (primer sequences are available on request). Polymerase chain reaction (PCR) was performed in 50 l containing 0.5 M of each primer, 1.5 mM MgCl 2, 200 M of each dNTP, 1⫻ PCR buffer II (Applied Biosystems), 0.2 U AmpliTaq DNA polymerase (Applied Biosystems), and 50 ng DNA. D-HPLC was performed using the Transgenomic WAVE system, as described previously (10). A total of 400 patients with ICP were analyzed and 300 healthy blood donors were used as controls. Direct sequencing. Samples showing abnormal D-HPLC profiles were reamplified from genomic DNA. Direct DNA sequencing was performed using the ABI PRISM BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster City, CA) with the same PCR primers on an ABI 310 sequencer. 342
1096-7192/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
MUTATION REPORT
343
FIG. 1. Identification of a new heterozygous missense mutation in the PRSS1 gene. (A) Denaturing high-performance liquid chromatography (D-HPLC) profile of the mutant compared with the wild-type sequence in exon 3 of the PRSS1 gene. Oven temperature was 64°C. The initial and final concentrations of buffer B for the analytical gradient were 55 and 62%, respectively. (B) Direct DNA sequencing showing a C ⬎ T single nucleotide substitution at position c.346 of the PRSS1 gene (indicated by the arrow) that would result in a substitution of Arg (CGT) by Cys (TGT) at amino acid residue 116 (R116C) of the human cationic trypsinogen.
RESULTS AND DISCUSSION The first and second reported mutations—R122H (3) and N29I (4)—in the PRSS1 gene have been associated with most of the large, well-characterized HP families. In contrast, the more recently identified mutations, including K23R (5), A16V (6), and D22G (7), are associated mainly with relatively small HP families and/or sporadic pancreatitis subjects (9). This largely represents a transition from initial identification of high-penetrance mutations to subsequent identification of lower-penetrance mutations after a causative gene is identified. In screening for PRSS1 mutations in ICP subjects, we identified a novel heterozygous DNA variant—a
single nucleotide substitution at position 346 in the PRSS1 coding sequence (c.346C ⬎ T)—in two unrelated patients. This variant is predicted to result in an arginine-to-cysteine amino acid change at residue 116 (R116C) of the encoded protein (Fig. 1). That the R116C is a likely pancreatitis-predisposing factor is supported by the following considerations. First, in the two unrelated ICP subjects, R116C is the only variant we have detected in the three known pancreatitis-associated genes—the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the PRSS1 gene, and the pancreatic secretory trypsin inhibitor (PSTI) gene— by our screening practice (11,12). Second, R116C was not present in 600 control chromosomes evaluated by
344
MUTATION REPORT
D-HPLC. Third, the same variant was also detected in a Turkish family that did not fit the conventional definition of HP, published as an abstract (13). The identification of the R116C mutation led again to a challenging situation in which there is strong evidence of disease association but a lack of supporting biochemical data. However, it is interesting to note that R116C is not so far from the R122 autolysis site. Thus, it is possible that R116C may have an effect on the R122 primary autolysis site, resulting in an increased stability of trypsin. Alternatively, the R116C mutation may create a novel disulfide bridge stabilizing trypsin. The identification of a novel pancreatitis-associated mutation in the PRSS1 gene further suggests that our understanding of trypsin, one of the most familiar enzymes, is far from complete. It also strengthens the notion that the mutational spectrum in gain-of-function conditions is not always limited. Finally, further disease-associated mutations in the PRSS1 gene may even be identified if more ICP patients are screened, and functional analysis of disease-associated mutations may reveal new mechanisms that have evolved against premature trypsin activation within the pancreas. ACKNOWLEDGMENT
7.
8.
9.
10.
11.
12.
13.
This work was supported by PHRC.
REFERENCES 1.
Strachan T, Read AP. Human Molecular Genetics. Oxford: BIOS Scientific, 1999.
2.
Chen JM, Ferec C. Wanted: A consensus nomenclature for cationic trypsinogen mutations. Gastroenterology 119:277– 279, 2000.
3.
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 14:141–145, 1996.
4.
5.
6.
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 113:1063–1068, 1997. 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 36:228 –232, 1999. Witt H, Luck W, Becker M. A signal peptide cleavage site
mutation in the cationic trypsinogen gene is strongly associated with chronic pancreatitis. Gastroenterology 117:7–10, 1999. Teich N, Ockenga J, Hoffmeister A, Manns M, Mossner J, Keim V. Chronic pancreatitis associated with an activation peptide mutation that facilitates trypsin activation. Gastroenterology 119:461– 465, 2000. Chen JM, Piepoli Bis A, Le Bodic L, Ruszniewski P, Robaszkiewicz M, Deprez PH, Raguenes O, Quere I, Andriulli A, Ferec C. Mutational screening of the cationic trypsinogen gene in a large cohort of subjects with idiopathic chronic pancreatitis. Clin Genet 59:189 –193, 2001. Chen JM, Montier T, Ferec C. Molecular pathology and evolutionary and physiological implications of pancreatitisassociated cationic trypsinogen mutations. Hum Genet 109: 245–252, 2001. Kuklin A, Munson K, Gjerde D, Haefele R, Taylor P. Detection of single-nucleotide polymorphisms with the WAVE DNA fragment analysis system. Genet Test 1:201–206, 1997–98. Le Marechal C, Audrezet MP, Quere I, Raguenes O, Langonne S, Ferec C. Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid chromatography (D-HPLC): Major implications for genetic counselling. Hum Genet 108:290 –298, 2001. Chen JM, Mercier B, Audrezet MP, Raguenes O, Quere I, Ferec C. Mutations of the pancreatic secretory trypsin inhibitor (PSTI) gene in idiopathic chronic pancreatitis. Gastroenterology 120:1061–1064, 2001. Tautermann G, Ruebsamen H, Beck M, Dertinger S, Drexel H, Lohse P. R116C mutation in the cationic trypsinogen in a Turkish family illustrates genetic microheterogeneity of hereditary pancreatitis (Abstract). Gastroenterology 120 (Suppl. 1):A–340.
Cedric Le Mare´chal* Jean-Franc¸ois Bretagne† Odile Rague´ne`s* Isabelle Que´re´* Jian-Min Chen* Claude Ferec* ,1 *INSERM EMI-01 15 Ge´ne´tique Mole´culaire et Ge´ne´tique Epide´miologique Etablissement Franc¸ais du Sang-Bretagne Universite´ de Bretagne Occidentale Centre Hospitalier Universitaire 46 rue Fe´lix Le Dantec 29275 Brest, France †C H U Pontchaillou 35033 Rennes, France Received July 16, 2001; published online October 25, 2001
1
To whom correspondence should be addressed at EFSBretagne, UBO, CHU, 46 rue Felix Le Dantec, 29275 Brest Cedex, France. Fax: ⫹33 2 98 43 05 55. E-mail: Claude.Ferec@ univ-brest.fr.
MUTATION REPORT Identification of a Novel Pancreatitis-Associated Missense Mutation, R116C, in the Human Cationic Trypsinogen Gene (PRSS1) trypsinogen can result in a gain of trypsin leading to the autodigestion of the pancreas itself (reviewed in 9). In this study, we report a new pancreatitis-associated missense mutation in the PRSS1 gene.
Over the past 5 years, several gain-of-function missense mutations in the human cationic trypsinogen gene (PRSS1, OMIM 276000) have been associated with hereditary and/or sporadic pancreatitis. This study reports a new pancreatitis-associated mutation—R116C (CGT > TGT: c.346C > T)—in the gene. © 2001 Academic Press Key Words: human cationic trypsinogen gene; denaturing high-performance liquid chromatography; gain of function; hereditary pancreatitis; idiopathic chronic pancreatitis; missense mutation; mutational analysis.
MATERIALS AND METHODS Patients. All patients with idiopathic chronic pancreatitis (ICP) were diagnosed by typical clinical manifestations, an increase in serum amylase or lipase, and pathological sonographic findings. Exclusion criteria included the presence of precipitating factors, such as alcohol, gallstones, trauma, medication, infection, metabolic disorders, and a positive family history, as described previously (8).
A classic perception in human genetics is that gainof-function is likely when only a specific mutation in a gene produces a given pathology (1). However, since the discovery of an apparent gain-of-function missense mutation—R122H (originally named R117H in chymotrypsin numbering; mutation nomenclature discussed in 2)—in the human cationic trypsinogen gene [PRSS1; Online Mendelian Inheritance in Man (OMIM) 276000, http://www.ncbi.nlm.nih.gov/omim] as a cause of hereditary pancreatitis (HP; OMIM 167800) (3), screening of this gene in both hereditary and sporadic pancreatitis has identified more diseaseassociated mutations than expected (4 – 8). This exceptional case appears to lie in the unique properties of trypsin(ogen): Trypsinogen has the ability to autoactivate itself; and the activated trypsin has the ability to autolyze itself. Indeed, the known PRSS1 mutations affect mainly these two processes; for example, the R122H mutation disrupts the primary R122 autolysis site of trypsin, and the K23R and D22G mutations facilitate trypsinogen autoactivation. Both increased stability of trypsin and enhanced autoactivation of
Mutation screening. All of the five exons of the PRSS1 gene were screened by denaturing high-performance liquid chromatography (D-HPLC). Isoform-specific primers were designed for each exon (primer sequences are available on request). Polymerase chain reaction (PCR) was performed in 50 l containing 0.5 M of each primer, 1.5 mM MgCl 2, 200 M of each dNTP, 1⫻ PCR buffer II (Applied Biosystems), 0.2 U AmpliTaq DNA polymerase (Applied Biosystems), and 50 ng DNA. D-HPLC was performed using the Transgenomic WAVE system, as described previously (10). A total of 400 patients with ICP were analyzed and 300 healthy blood donors were used as controls. Direct sequencing. Samples showing abnormal D-HPLC profiles were reamplified from genomic DNA. Direct DNA sequencing was performed using the ABI PRISM BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster City, CA) with the same PCR primers on an ABI 310 sequencer. 342
1096-7192/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
MUTATION REPORT
343
FIG. 1. Identification of a new heterozygous missense mutation in the PRSS1 gene. (A) Denaturing high-performance liquid chromatography (D-HPLC) profile of the mutant compared with the wild-type sequence in exon 3 of the PRSS1 gene. Oven temperature was 64°C. The initial and final concentrations of buffer B for the analytical gradient were 55 and 62%, respectively. (B) Direct DNA sequencing showing a C ⬎ T single nucleotide substitution at position c.346 of the PRSS1 gene (indicated by the arrow) that would result in a substitution of Arg (CGT) by Cys (TGT) at amino acid residue 116 (R116C) of the human cationic trypsinogen.
RESULTS AND DISCUSSION The first and second reported mutations—R122H (3) and N29I (4)—in the PRSS1 gene have been associated with most of the large, well-characterized HP families. In contrast, the more recently identified mutations, including K23R (5), A16V (6), and D22G (7), are associated mainly with relatively small HP families and/or sporadic pancreatitis subjects (9). This largely represents a transition from initial identification of high-penetrance mutations to subsequent identification of lower-penetrance mutations after a causative gene is identified. In screening for PRSS1 mutations in ICP subjects, we identified a novel heterozygous DNA variant—a
single nucleotide substitution at position 346 in the PRSS1 coding sequence (c.346C ⬎ T)—in two unrelated patients. This variant is predicted to result in an arginine-to-cysteine amino acid change at residue 116 (R116C) of the encoded protein (Fig. 1). That the R116C is a likely pancreatitis-predisposing factor is supported by the following considerations. First, in the two unrelated ICP subjects, R116C is the only variant we have detected in the three known pancreatitis-associated genes—the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the PRSS1 gene, and the pancreatic secretory trypsin inhibitor (PSTI) gene— by our screening practice (11,12). Second, R116C was not present in 600 control chromosomes evaluated by
344
MUTATION REPORT
D-HPLC. Third, the same variant was also detected in a Turkish family that did not fit the conventional definition of HP, published as an abstract (13). The identification of the R116C mutation led again to a challenging situation in which there is strong evidence of disease association but a lack of supporting biochemical data. However, it is interesting to note that R116C is not so far from the R122 autolysis site. Thus, it is possible that R116C may have an effect on the R122 primary autolysis site, resulting in an increased stability of trypsin. Alternatively, the R116C mutation may create a novel disulfide bridge stabilizing trypsin. The identification of a novel pancreatitis-associated mutation in the PRSS1 gene further suggests that our understanding of trypsin, one of the most familiar enzymes, is far from complete. It also strengthens the notion that the mutational spectrum in gain-of-function conditions is not always limited. Finally, further disease-associated mutations in the PRSS1 gene may even be identified if more ICP patients are screened, and functional analysis of disease-associated mutations may reveal new mechanisms that have evolved against premature trypsin activation within the pancreas. ACKNOWLEDGMENT
7.
8.
9.
10.
11.
12.
13.
This work was supported by PHRC.
REFERENCES 1.
Strachan T, Read AP. Human Molecular Genetics. Oxford: BIOS Scientific, 1999.
2.
Chen JM, Ferec C. Wanted: A consensus nomenclature for cationic trypsinogen mutations. Gastroenterology 119:277– 279, 2000.
3.
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 14:141–145, 1996.
4.
5.
6.
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 113:1063–1068, 1997. 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 36:228 –232, 1999. Witt H, Luck W, Becker M. A signal peptide cleavage site
mutation in the cationic trypsinogen gene is strongly associated with chronic pancreatitis. Gastroenterology 117:7–10, 1999. Teich N, Ockenga J, Hoffmeister A, Manns M, Mossner J, Keim V. Chronic pancreatitis associated with an activation peptide mutation that facilitates trypsin activation. Gastroenterology 119:461– 465, 2000. Chen JM, Piepoli Bis A, Le Bodic L, Ruszniewski P, Robaszkiewicz M, Deprez PH, Raguenes O, Quere I, Andriulli A, Ferec C. Mutational screening of the cationic trypsinogen gene in a large cohort of subjects with idiopathic chronic pancreatitis. Clin Genet 59:189 –193, 2001. Chen JM, Montier T, Ferec C. Molecular pathology and evolutionary and physiological implications of pancreatitisassociated cationic trypsinogen mutations. Hum Genet 109: 245–252, 2001. Kuklin A, Munson K, Gjerde D, Haefele R, Taylor P. Detection of single-nucleotide polymorphisms with the WAVE DNA fragment analysis system. Genet Test 1:201–206, 1997–98. Le Marechal C, Audrezet MP, Quere I, Raguenes O, Langonne S, Ferec C. Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid chromatography (D-HPLC): Major implications for genetic counselling. Hum Genet 108:290 –298, 2001. Chen JM, Mercier B, Audrezet MP, Raguenes O, Quere I, Ferec C. Mutations of the pancreatic secretory trypsin inhibitor (PSTI) gene in idiopathic chronic pancreatitis. Gastroenterology 120:1061–1064, 2001. Tautermann G, Ruebsamen H, Beck M, Dertinger S, Drexel H, Lohse P. R116C mutation in the cationic trypsinogen in a Turkish family illustrates genetic microheterogeneity of hereditary pancreatitis (Abstract). Gastroenterology 120 (Suppl. 1):A–340.
Cedric Le Mare´chal* Jean-Franc¸ois Bretagne† Odile Rague´ne`s* Isabelle Que´re´* Jian-Min Chen* Claude Ferec* ,1 *INSERM EMI-01 15 Ge´ne´tique Mole´culaire et Ge´ne´tique Epide´miologique Etablissement Franc¸ais du Sang-Bretagne Universite´ de Bretagne Occidentale Centre Hospitalier Universitaire 46 rue Fe´lix Le Dantec 29275 Brest, France †C H U Pontchaillou 35033 Rennes, France Received July 16, 2001; published online October 25, 2001
1
To whom correspondence should be addressed at EFSBretagne, UBO, CHU, 46 rue Felix Le Dantec, 29275 Brest Cedex, France. Fax: ⫹33 2 98 43 05 55. E-mail: Claude.Ferec@ univ-brest.fr.