Trypsinogen Copy Number Mutations in Patients With Idiopathic Chronic Pancreatitis

Background & Aims: We have recently reported that the triplication of a ∼605 kilobase segment containing the PRSS1 (encoding cationic trypsinogen) and PRSS2 (encoding anionic trypsinogen) genes causes hereditary pancreatitis. Here we went further to investigate whether this copy number mutation could account for some unidentified French white patients with idiopathic chronic pancreatitis (ICP) or familial chronic pancreatitis (FCP) as well as Indian patients with tropical calcific pancreatitis (TCP). Methods: Patients and controls were screened by means of previously described quantitative fluorescent multiplex polymerase chain reaction and/or genotyping of the microsatellite marker rs3222967. Results: The ∼605 kilobase triplication and a novel duplication (confirmed by fluorescence in situ hybridization) of the trypsinogen locus were detected in 10 and 2 of 202 ICP patients, respectively (age of disease onset, ≤20 years) but were absent in 282 French controls. In addition, the duplication mutation was found in 2 of 1044 ICP patients whose age of disease onset was >20 years. However, the 2 trypsinogen copy number mutations were observed in neither 103 FCP patients nor 268 Indian TCP patients. Conclusions: Our findings revealed the molecular basis of 6% of the young ICP patients and further demonstrated that chronic pancreatitis is a genomic disorder. Our findings also add to the mounting evidence showing that trypsinogen gene mutations do not appear to play an important role in the pathogenesis of TCP in the Indian population. Finally, a dividend of this study is that we have provided convincing evidence to show that all 5 previously described copy number variations involving PRSS1 or/and PRSS2 are artifacts. Background & Aims: We have recently reported that the triplication of a ∼605 kilobase segment containing the PRSS1 (encoding cationic trypsinogen) and PRSS2 (encoding anionic trypsinogen) genes causes hereditary pancreatitis. Here we went further to investigate whether this copy number mutation could account for some unidentified French white patients with idiopathic chronic pancreatitis (ICP) or familial chronic pancreatitis (FCP) as well as Indian patients with tropical calcific pancreatitis (TCP). Methods: Patients and controls were screened by means of previously described quantitative fluorescent multiplex polymerase chain reaction and/or genotyping of the microsatellite marker rs3222967. Results: The ∼605 kilobase triplication and a novel duplication (confirmed by fluorescence in situ hybridization) of the trypsinogen locus were detected in 10 and 2 of 202 ICP patients, respectively (age of disease onset, ≤20 years) but were absent in 282 French controls. In addition, the duplication mutation was found in 2 of 1044 ICP patients whose age of disease onset was >20 years. However, the 2 trypsinogen copy number mutations were observed in neither 103 FCP patients nor 268 Indian TCP patients. Conclusions: Our findings revealed the molecular basis of 6% of the young ICP patients and further demonstrated that chronic pancreatitis is a genomic disorder. Our findings also add to the mounting evidence showing that trypsinogen gene mutations do not appear to play an important role in the pathogenesis of TCP in the Indian population. Finally, a dividend of this study is that we have provided convincing evidence to show that all 5 previously described copy number variations involving PRSS1 or/and PRSS2 are artifacts. Chronic pancreatitis is a persistent inflammation of the pancreas characterized by attacks of abdominal pain, irreversible morphologic changes, and deficiency of pancreatic enzymes.1Etemad B. Whitcomb D.C. Chronic pancreatitis: diagnosis, classification, and new genetic developments.Gastroenterology. 2001; 120: 682-707Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar Hereditary pancreatitis (MIM #167800)—a rare form of chronic pancreatitis—is inherited as an autosomal dominant disease.2Comfort M.W. Steinberg A.G. Pedigree of a family with hereditary chronic relapsing pancreatitis.Gastroenterology. 1952; 21: 54-63Abstract Full Text PDF PubMed Scopus (337) Google Scholar Nevertheless, genetic studies of hereditary pancreatitis have provided the strongest support for the century-old theory that chronic pancreatitis is an autodigestive disease,3Chiara H. Ueber Selbstverdauung des menschlichen Pankreas.Ztschr Heilkunde. 1896; 17: 70-96Google Scholar which was thought to be initiated by prematurely activated trypsin within the pancreas. Since the mapping and cloning of one gene for hereditary pancreatitis in 1996,4Le Bodic L. Bignon J.D. Raguénès O. et al.The hereditary pancreatitis gene maps to long arm of chromosome 7.Hum Mol Genet. 1996; 5: 549-554Crossref PubMed Scopus (182) Google Scholar, 5Whitcomb D.C. Preston R.A. Aston C.E. et al.A gene for hereditary pancreatitis maps to chromosome 7q35.Gastroenterology. 1996; 110: 1975-1980Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 6Pandya A. Blanton S.H. Landa B. et al.Linkage studies in a large kindred with hereditary pancreatitis confirms mapping of the gene to a 16-cM region on 7q.Genomics. 1996; 38: 227-230Crossref PubMed Scopus (91) Google Scholar, 7Whitcomb D.C. Gorry M.C. Preston R.A. et al.Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene.Nat Genet. 1996; 14: 141-145Crossref PubMed Scopus (1337) Google Scholar complementary observations have pointed to the pivotal role of a gain of trypsin in the etiology of chronic pancreatitis. First, several gain of function missense mutations (eg, p.D19A,8Chen J.M. 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Keller J. et al.A mouse model of hereditary pancreatitis generated by transgenic expression of R122H trypsinogen.Gastroenterology. 2006; 131: 1844-1855Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) in the PRSS1 gene (encoding cationic trypsinogen, the most abundant isoform of trypsinogen13Chen J.M. Férec C. Human trypsins.in: Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of proteolytic enzymes. 2nd ed. Elsevier, London2004: 1489-1493Google Scholar; MIM #276000) have been reported to be disease-causing. Second, loss of function variations in PRSS1 or PRSS2 (encoding anionic trypsinogen, the second major isoform of trypsinogen13Chen J.M. Férec C. Human trypsins.in: Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of proteolytic enzymes. 2nd ed. Elsevier, London2004: 1489-1493Google Scholar; MIM #601564) have been reported to be disease-protecting.14Chen J.M. Le Maréchal C. Lucas D. et al."Loss of function" mutations in the cationic trypsinogen gene (PRSS1) may act as a protective factor against pancreatitis.Mol Genet Metab. 2003; 79: 67-70Crossref PubMed Scopus (45) Google Scholar, 15Witt H. Sahin-Tóth M. Landt O. et al.A degradation-sensitive anionic trypsinogen (PRSS2) variant protects against chronic pancreatitis.Nat Genet. 2006; 38: 668-673Crossref PubMed Scopus (191) Google Scholar Finally, a diverse range of variations including missense, splicing, frame-shifting, and nonsense mutations in the SPINK1 gene (encoding trypsin's physiologic inhibitor; MIM #167790) have also been reported to be associated with idiopathic chronic pancreatitis (ICP), familial chronic pancreatitis (FCP), tropical calcific pancreatitis (TCP; MIM #608189), or even to cause autosomal dominant hereditary pancreatitis.16Chen J.M. Mercier B. Audrézet M.P. et al.Mutational analysis of the human pancreatic secretory trypsin inhibitor (PSTI) gene in hereditary and sporadic chronic pancreatitis.J Med Genet. 2000; 37: 67-69Crossref PubMed Scopus (138) Google Scholar, 17Witt H. Luck W. Hennies H.C. et al.Mutations in the gene encoding the serine protease inhibitor, Kazal type 1 are associated with chronic pancreatitis.Nat Genet. 2000; 25: 213-216Crossref PubMed Scopus (851) Google Scholar, 18Pfutzer R.H. Barmada M.M. Brunskill A.P. et al.SPINK1/PSTI polymorphisms act as disease modifiers in familial and idiopathic chronic pancreatitis.Gastroenterology. 2000; 119: 615-623Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 19Gaia E. Salacone P. Gallo M. et al.Germline mutations in CFTR and PSTI genes in chronic pancreatitis patients.Dig Dis Sci. 2002; 47: 2416-2421Crossref PubMed Scopus (42) Google Scholar, 20Chandak G.R. Idris M.M. Reddy D.N. et al.Mutations in the pancreatic secretory trypsin inhibitor gene (PSTI/SPINK1) rather than the cationic trypsinogen gene (PRSS1) are significantly associated with tropical calcific pancreatitis.J Med Genet. 2002; 39: 347-351Crossref PubMed Google Scholar, 21Bhatia E. Choudhuri G. Sikora S.S. et al.Tropical calcific pancreatitis: strong association with SPINK1 trypsin inhibitor mutations.Gastroenterology. 2002; 123: 1020-1025Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 22Schneider A. Suman A. Rossi L. et al.SPINK1/PSTI mutations are associated with tropical pancreatitis and type II diabetes mellitus in Bangladesh.Gastroenterology. 2002; 123: 1026-1030Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 23Le Maréchal C. Chen J.M. Le Gall C. et al.Two novel severe mutations in the pancreatic secretory trypsin inhibitor gene (SPINK1) cause familial and/or hereditary pancreatitis.Hum Mutat. 2004; 23: 205Crossref PubMed Scopus (62) Google Scholar, 24Kiraly O. Boulling A. Witt H. et al.Signal peptide variants that impair secretion of pancreatic secretory trypsin inhibitor (SPINK1) cause autosomal dominant hereditary pancreatitis.Hum Mutat. 2007; 28: 469-476Crossref PubMed Scopus (67) Google Scholar, 25Boulling A. Le Maréchal C. Trouvé P. et al.Functional analysis of pancreatitis-associated missense mutations in the pancreatic secretory trypsin inhibitor (SPINK1) gene.Eur J Hum Genet. 2007; 15: 936-942Crossref PubMed Scopus (57) Google Scholar, 26Kiraly O. Wartmann T. Sahin-Toth M. Missense mutations in pancreatic secretory trypsin inhibitor (SPINK1) cause intracellular retention and degradation.Gut. 2007; 56: 1433-1438Crossref PubMed Scopus (66) Google Scholar The above findings were invariably made from studies of point mutations or microinsertions/deletions. Recently, copy number mutations (CNMs) including 2 large genomic deletions of the SPINK1 gene27Masson E. Le Maréchal C. Chen J.M. et al.Detection of a large genomic deletion in the pancreatic secretory trypsin inhibitor (SPINK1) gene.Eur J Hum Genet. 2006; 14: 1204-1208Crossref PubMed Scopus (29) Google Scholar, 28Masson E. Le Maréchal C. Levy P. et al.Co-inheritance of a novel deletion of the entire SPINK1 gene with a CFTR missense mutation (L997F) in a family with chronic pancreatitis.Mol Genet Metab. 2007; 92: 168-175Crossref PubMed Scopus (23) Google Scholar and a ∼605 kilobase (kb) triplication containing both PRSS1 and PRSS2 on chromosome 729Le Maréchal C. Masson E. Chen J.M. et al.Hereditary pancreatitis caused by triplication of the trypsinogen locus.Nat Genet. 2006; 38: 1372-1374Crossref PubMed Scopus (166) Google Scholar (Figure 1A) have also been identified in patients with chronic pancreatitis. (The term CNM is used here to distinguish from the neutral term copy number variation [CNV]. CNV refers to a DNA segment of ≥1 kb that is present in different copy numbers with respect to a reference genome sequence.30Scherer S.W. Lee C. Birney E. et al.Challenges and standards in integrating surveys of structural variation.Nat Genet. 2007; 39: S7-S15Crossref PubMed Scopus (298) Google Scholar) The 2 large deletions in SPINK1 are clearly disease-causing; whereas the first one results in gene disruption,27Masson E. Le Maréchal C. Chen J.M. et al.Detection of a large genomic deletion in the pancreatic secretory trypsin inhibitor (SPINK1) gene.Eur J Hum Genet. 2006; 14: 1204-1208Crossref PubMed Scopus (29) Google Scholar the second one removes the entire gene.28Masson E. Le Maréchal C. Levy P. et al.Co-inheritance of a novel deletion of the entire SPINK1 gene with a CFTR missense mutation (L997F) in a family with chronic pancreatitis.Mol Genet Metab. 2007; 92: 168-175Crossref PubMed Scopus (23) Google Scholar The ∼605 kb trypsinogen triplication was also considered to be disease-causing because (1) it segregated with the disease in 5 French white families with hereditary pancreatitis, (2) it is absent in 200 healthy controls from the same population, and (3) an increased expression of trypsinogen resulting from an increased gene dosage of PRSS1/PRSS2 is consistent with our current understanding of the role of trypsin in the etiology of chronic pancreatitis.29Le Maréchal C. Masson E. Chen J.M. et al.Hereditary pancreatitis caused by triplication of the trypsinogen locus.Nat Genet. 2006; 38: 1372-1374Crossref PubMed Scopus (166) Google Scholar The initial aim of this study was to investigate whether the ∼605 kb trypsinogen triplication could account for some unidentified French white patients with ICP or FCP. Because previously reported mutations in PRSS1 are absent in TCP patients, we also investigated whether increased copy number of the trypsinogen locus could be the primary mechanism responsible for TCP. During this process, we had become aware of 5 "gain" or "loss" CNVs involving PRSS1 and/or PRSS2 detailed in the Database of Genomic Variants (http://projects.tcag.ca/variation/; Figure 1A and Table 1). That all of them, particularly the trypsinogen gain CNVs, were identified in subjects without any known phenotypes31Tuzun E. Sharp A.J. Bailey J.A. et al.Fine-scale structural variation of the human genome.Nat Genet. 2005; 37: 727-732Crossref PubMed Scopus (807) Google Scholar, 32Redon R. Ishikawa S. Fitch K.R. et al.Global variation in copy number in the human genome.Nature. 2006; 444: 444-454Crossref PubMed Scopus (3341) Google Scholar, 33Wong K.K. deLeeuw R.J. Dosanjh N.S. et al.A comprehensive analysis of common copy-number variations in the human genome.Am J Hum Genet. 2007; 80: 91-104Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar poses a serious challenge to our previous finding.29Le Maréchal C. Masson E. Chen J.M. et al.Hereditary pancreatitis caused by triplication of the trypsinogen locus.Nat Genet. 2006; 38: 1372-1374Crossref PubMed Scopus (166) Google Scholar To obtain further insights into this issue, we decided to screen a further set of control samples from different ethnic origins and, most importantly, to investigate whether we can replicate the presence of these CNVs in the original samples by using our own method, quantitative fluorescent multiplex polymerase chain reaction (QFM-PCR).29Le Maréchal C. Masson E. Chen J.M. et al.Hereditary pancreatitis caused by triplication of the trypsinogen locus.Nat Genet. 2006; 38: 1372-1374Crossref PubMed Scopus (166) Google ScholarTable 1Five CNVs Involving PRSS1 and/or PRSS2 Reported in Three Genome-Wide StudiesaFrom the Database of Genomic Variants (http://projects.tcag.ca/variation/; as of July 31, 2007). Variations were arranged in the same order (from top to the bottom) as they appeared in Figure 1A.VariationGenomic positionKnown gene(s)Frequency of gain or lossTotal no. of control samples analyzedReference0623chr7:141,948,871–142,002,444PRSS2Loss: 146 lymphoblastoid cell lines (Coriell Cell Repository) of diverse ethnic origin31Tuzun E. Sharp A.J. Bailey J.A. et al.Fine-scale structural variation of the human genome.Nat Genet. 2005; 37: 727-732Crossref PubMed Scopus (807) Google Scholar3707chr7:141,610,138–142,240,984C7orf34, PRSS1, PRSS2, KEL, EPHB6, TRPV6, TRPV5, OR9A2Loss: 28270 HapMap individuals32Redon R. Ishikawa S. Fitch K.R. et al.Global variation in copy number in the human genome.Nature. 2006; 444: 444-454Crossref PubMed Scopus (3341) Google Scholar2715chr7:141,739,700–142,018,646PRSS2, PRSS1Loss: 26270 HapMap individuals32Redon R. Ishikawa S. Fitch K.R. et al.Global variation in copy number in the human genome.Nature. 2006; 444: 444-454Crossref PubMed Scopus (3341) Google Scholar4567chr7:141,887,558–142,030,287PRSS1, PRSS2Gain: 3, loss: 295 individuals33Wong K.K. deLeeuw R.J. Dosanjh N.S. et al.A comprehensive analysis of common copy-number variations in the human genome.Am J Hum Genet. 2007; 80: 91-104Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar4566chr7:141,813,914–141,973,680PRSS1Gain: 1, loss: 295 individuals33Wong K.K. deLeeuw R.J. Dosanjh N.S. et al.A comprehensive analysis of common copy-number variations in the human genome.Am J Hum Genet. 2007; 80: 91-104Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholara From the Database of Genomic Variants (http://projects.tcag.ca/variation/; as of July 31, 2007). Variations were arranged in the same order (from top to the bottom) as they appeared in Figure 1A. Open table in a new tab Clinical diagnosis of chronic pancreatitis was based on 2 or more of the following criteria: presence of a typical history of recurrent pancreatitis, radiologic findings such as pancreatic calcifications and/or pancreatic irregularities revealed by endoscopic retrograde pancreatography or by magnetic resonance imaging of the pancreas and/or pathologic sonographic findings, as previously described.15Witt H. Sahin-Tóth M. Landt O. et al.A degradation-sensitive anionic trypsinogen (PRSS2) variant protects against chronic pancreatitis.Nat Genet. 2006; 38: 668-673Crossref PubMed Scopus (191) Google Scholar Patients were classified as having ICP when both precipitating factors (eg, alcohol abuse, trauma, medication, infection, metabolic disorders) and a positive family history were not reported. The only difference between ICP and FCP is that a positive family history was reported in the latter, but it did not satisfy the criteria we previously used to define hereditary pancreatitis (ie, 3 or more affected members involving at least 2 generations29Le Maréchal C. Masson E. Chen J.M. et al.Hereditary pancreatitis caused by triplication of the trypsinogen locus.Nat Genet. 2006; 38: 1372-1374Crossref PubMed Scopus (166) Google Scholar). TCP was diagnosed in accordance with the established World Health Organization criteria as described earlier.20Chandak G.R. Idris M.M. Reddy D.N. et al.Mutations in the pancreatic secretory trypsin inhibitor gene (PSTI/SPINK1) rather than the cationic trypsinogen gene (PRSS1) are significantly associated with tropical calcific pancreatitis.J Med Genet. 2002; 39: 347-351Crossref PubMed Google Scholar Four groups of patients participated in this study: all patients had not been found to carry any known point mutations or microinsertions/deletions in the PRSS1 and SPINK1 genes after mutational screening by our previously established methods;20Chandak G.R. Idris M.M. Reddy D.N. et al.Mutations in the pancreatic secretory trypsin inhibitor gene (PSTI/SPINK1) rather than the cationic trypsinogen gene (PRSS1) are significantly associated with tropical calcific pancreatitis.J Med Genet. 2002; 39: 347-351Crossref PubMed Google Scholar, 21Bhatia E. Choudhuri G. Sikora S.S. et al.Tropical calcific pancreatitis: strong association with SPINK1 trypsin inhibitor mutations.Gastroenterology. 2002; 123: 1020-1025Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 22Schneider A. Suman A. Rossi L. et al.SPINK1/PSTI mutations are associated with tropical pancreatitis and type II diabetes mellitus in Bangladesh.Gastroenterology. 2002; 123: 1026-1030Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 23Le Maréchal C. Chen J.M. Le Gall C. et al.Two novel severe mutations in the pancreatic secretory trypsin inhibitor gene (SPINK1) cause familial and/or hereditary pancreatitis.Hum Mutat. 2004; 23: 205Crossref PubMed Scopus (62) Google Scholar, 34Le Maréchal C. Bretagne J.F. Raguénès O. et al.Identification of a novel pancreatitis-associated missense mutation, R116C, in the human cationic trypsinogen gene (PRSS1).Mol Genet Metab. 2001; 74: 342-344Crossref PubMed Scopus (33) Google Scholar some of them (ie, groups Ia and II) had also not been found to carry any known mutations in the CFTR gene, by means of denaturing high-performance liquid chromatography35Le Maréchal C. Audrézet M.P. Quéré I. et al.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. 2001; 108: 290-298Crossref PubMed Scopus (112) Google Scholar (Table 2). Eighty-two unrelated healthy white-French subjects (newly recruited after the completion of our previous study29Le Maréchal C. Masson E. Chen J.M. et al.Hereditary pancreatitis caused by triplication of the trypsinogen locus.Nat Genet. 2006; 38: 1372-1374Crossref PubMed Scopus (166) Google Scholar) and 63 healthy Dravidians (ethnically matched to the Indian TCP patients) served as controls. The ethical review committee of respective institutions approved this study, and all patients gave informed consent for genetic analysis.Table 2Classification of Participating PatientsGroupNo.EthnicityDiagnosisPrevious mutational screeningIa202French whiteICP (age of disease onset was either known to be ≤20 y or the diagnosis was made at the age of ≤20 y)None of them carried any of the known point mutations or microinsertions/deletions in the PRSS1, SPINK1 and CFTR genes after mutational screening by our previously established methods23Le Maréchal C. Chen J.M. Le Gall C. et al.Two novel severe mutations in the pancreatic secretory trypsin inhibitor gene (SPINK1) cause familial and/or hereditary pancreatitis.Hum Mutat. 2004; 23: 205Crossref PubMed Scopus (62) Google Scholar, 34Le Maréchal C. Bretagne J.F. Raguénès O. et al.Identification of a novel pancreatitis-associated missense mutation, R116C, in the human cationic trypsinogen gene (PRSS1).Mol Genet Metab. 2001; 74: 342-344Crossref PubMed Scopus (33) Google Scholar, 35Le Maréchal C. Audrézet M.P. Quéré I. et al.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. 2001; 108: 290-298Crossref PubMed Scopus (112) Google ScholarIb1044French whiteICP (the remaining subjects)Same as above except that CFTR gene was not screenedII103French whiteFCPSame as IaIII268Dravidian (India)TCPNone of them carried any of the known point mutations or microinsertions/deletions in the PRSS1 and SPINK1 genes after mutational analysis as described before20Chandak G.R. Idris M.M. Reddy D.N. et al.Mutations in the pancreatic secretory trypsin inhibitor gene (PSTI/SPINK1) rather than the cationic trypsinogen gene (PRSS1) are significantly associated with tropical calcific pancreatitis.J Med Genet. 2002; 39: 347-351Crossref PubMed Google Scholar Open table in a new tab In addition, DNA samples from 25 Moroccans, 25 Ivorians, and 25 Vietnamese were further recruited; and several DNA samples reported to carry trypsinogen CNVs were purchased from the Coriell Cell Repositories (Camden, NJ) (Table 3).Table 3Trypsinogen CNVs Containing DNA SamplesReference no.aCoriell Cell Repositories.DescriptionReported CNVbSee Table 1 for indicated CNV.Copy number of TRY6 and TRY7cDetermined in this study.NA06985CEPH/UTAH pedigree 1341 (father)Unknown1NA06991CEPH/UTAH pedigree 1341 (child)Loss: variation 37070NA06993CEPH/UTAH pedigree 1341 (mother)Unknown1NA10469Pygmy populationGain: variation 45670NA10843CEPH/UTAH pedigree 1423 (mother)Unknown2NA11919CEPH/UTAH pedigree 1423 (child)Gain: variation 45671NA17076Human variation panel: Puerto RicanGain: variations 4566 and 45672NA18524International HapMap project: Han Chinese in Beijing, ChinaLoss: variation 37070NA18537International HapMap project: Han Chinese in Beijing, ChinaLoss: variation 37070NA18855International HapMap project: Yoruba in Ibadan, Nigeria (mother)dFrom same family.Loss: variation 37070NA18857International HapMap project: Yoruba in Ibadan, Nigeria (child)dFrom same family.Unknown1a Coriell Cell Repositories.b See Table 1 for indicated CNV.c Determined in this study.d From same family. Open table in a new tab Except for group Ib, all the patients and controls were screened by means of QFM-PCR as previously described.29Le Maréchal C. Masson E. Chen J.M. et al.Hereditary pancreatitis caused by triplication of the trypsinogen locus.Nat Genet. 2006; 38: 1372-1374Crossref PubMed Scopus (166) Google Scholar It is important to point out that because of the presence of a common deletion polymorphism involving TRY6 and TRY7,36McCarroll S.A. Hadnott T.N. Perry G.H. et al.Common deletion polymorphisms in the human genome.Nat Genet. 2006; 38: 86-92Crossref PubMed Scopus (587) Google Scholar all the QFM-PCR analyzed samples in this study were normalized against a same control sample that was known to possess 2 non–TRY6/TRY7-deletion alleles. This served to ensure an accurate estimation of the TRY6/TRY7 copy number in the analyzed subjects. The group Ib patients were initially genotyped for the microsatellite marker rs3222967, which is located ∼55 kb telomeric to TRY1 (Figure 1A), as previously described.29Le Maréchal C. Masson E. Chen J.M. et al.Hereditary pancreatitis caused by triplication of the trypsinogen locus.Nat Genet. 2006; 38: 1372-1374Crossref PubMed Scopus (166) Google Scholar Samples found to carry at least one 163 allele were then subjected to the abovementioned QFM-PCR analysis; the frequency of the 163 allele in the French white control population was determined to be only 1.2%; and the trypsinogen triplication and duplication mutations identified by QFM-PCR were found to carry at least one 163 allele (Table 4). For cross-platform comparison, the genotype of CEPH-134702 is 169-169.Table 4ICP Patients With Triplication or Duplication of Trypsinogen LocusPatientSexCNMAge of disease onset (y)Age at diagnosis (y)Genotype of rs3222967Group Ia (n = 202; see Table 2)523MTriplication1111163-163-169-169637FTriplication1515163-163-169-169663MTriplication1818163-163-169-169802MTriplicationUnknown13163-163-169-1671016MTriplicationUnknown19163-163-169-1691521MTriplication1010163-163-169-1691926MTriplication1112163-163-169-1692129MTriplication1315163-163-169-1692246FTriplication2047163-163-169-1692326MTriplication1024163-163-169-1692193MDuplication615163-169-1691620FDuplication419163-169-169Group Ib (n = 1044; see Table 2)344FDuplicationUnknown39149-163-1691215MDuplication3840163-169-169 Open table in a new tab Previous studies have shown that pancreatitis-predisposing genetic factors are more easily found in children and adolescents with ICP than in adults.17Witt H. Luck W. Hennies H.C. et al.Mutations in the gene encoding the serine protease inhibitor, Kazal type 1 are associated with chronic pancreatitis.Nat Genet. 2000; 25: 213-216Crossref PubMed Scopus (851) Google Scholar, 37Chen J.M. Mercier B. Audrézet M.P. et al.Mutations of the pancreatic secretory trypsin inhibitor (PSTI) gene in idiopathic chronic pancreatitis.Gastroenterology. 2001; 120: 1061-1064Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 38Witt 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: 7-10Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 39Chen J.M. Raguenes O. Ferec C. et al.The A16V signal peptide cleavage site mutation in the cationic trypsinogen gene and chronic pancreatitis.Gastroenterology. 1999; 117: 1508-1509Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar Thus, we initially analyzed group Ia of ICP patients (age of disease onset, <20 years) for both theoretical and practical considerations. Triplication of the entire trypsinogen gene family (top panel, Figure 1B) was found in 10 of the 202 ICP individuals (5%). (Because both the nonfunctional TRY6 and TRY7 genes are flanked by other trypsinogen members (Figure 1A) and the common TRY6 and TRY7 deletion polymorphism36McCarroll S.A. Hadnott T.N. Perry G.H. et al.Common deletion polymorphisms in the human genome.Nat Genet. 2006; 38: 86-92Crossref PubMed Scopus (587) Google Scholar appears not to be associated with the disease [unpublished preliminary data], we consider the trypsinogen locus on chromosome 7q34 to be intact if CNMs or CNVs were not found in the other gene members.) More importantly, we identified a novel CNM, duplication of the trypsinogen locus (middle panel, Figure 1B), in 2 of these ICP subjects. Altogether, trypsinogen triplication and duplication account for 6% (12/202) of group Ia of ICP patients (Table 4). Encouraged by this finding, we further screened group Ib of ICP patients (age of disease onset, >20 years) but found only the duplication of the trypsinogen locus in 2 subjects (Table 4). Neither the triplication nor the duplication was found in the 103 FCP subjects as well as in a total of 282 healthy French controls (the previous 20029Le Maréchal C. Masson E. Chen J.M. et al.Hereditary pancreatitis caused by triplication of the trypsinogen locus.Nat Genet. 2006; 38: 1372-1374Crossref PubMed Scopus (166) Google Scholar plus the newly recruited 82). In addition, both the above CNMs at the trypsinogen locus were not detected in the 268 Indian TCP patients (as well as the 63 Indian controls); this adds to the growing evidence showing that trypsinogen gene mutations might not play a role in the pathogenesis of TCP as well as other types of chronic pancreatitis from India.40Chandak G.R. Idris M.M. Reddy D.N. et al.Absence of PRSS1 mutations and association of SPINK1 trypsin inhibitor mutations in hereditary and non-hereditary chronic pancreatitis.Gut. 2004; 53: 723-728Crossref PubMed Scopus (97) Google Scholar, 41Idris M.M. Bhaskar S. Reddy D.N. et al.Mutations in anionic trypsinogen gene are not associated with tropical calcific pancreatitis.Gut. 2005; 54: 728-729Crossref PubMed Scopus (22) Google Scholar, 42Mahurkar S. Idris M.M. Reddy D.N. et al.Association of cathepsin B gene polymorphism with tropical calcific pancreatitis.Gut. 2006; 55: 1270-1275Crossref PubMed Scopus (73) Google Scholar The size of the triplicated segment identified in the 10 ICP patients was found to be identical to that of our previously found ∼605 kb triplication, as analyzed by walking QFM-PCR.29Le Maréchal C. Masson E. Chen J.M. et al.Hereditary pancreatitis caused by triplication of the trypsinogen locus.Nat Genet. 2006; 38: 1372-1374Crossref PubMed Scopus (166) Google Scholar Moreover, genotyping of the microsatellite marker rs3222967 suggested that the triplication in the 10 ICP patients probably occurred on the same haplotype that harbored the known ∼605 kb triplication; the triplication in all informative hereditary pancreatitis patients from our previous study always had the genotype of 163-163-169,29Le Maréchal C. Masson E. Chen J.M. et al.Hereditary pancreatitis caused by triplication of the trypsinogen locus.Nat Genet. 2006; 38: 1372-1374Crossref PubMed Scopus (166) Google Scholar and all 10 ICP triplication carriers were found to have at least two 163 and one 169 alleles (Table 4). (Note that the frequency of the 163 allele in the French population is only 1.2%.) Finally, it is hard to envisage how this kind of triplication could occur recurrently from the standpoint of mutational mechanism. The above observations, when considered together, strongly suggest that the triplication identified in the 10 ICP subjects and that previously found in the 5 hereditary pancreatitis families most probably arose through a common founder. If this were the case, one might argue that the ICP subjects carrying the triplication might belong to families with hereditary pancreatitis that escaped clinical attention. Although the ICP patients were recruited through different clinics, this seems very unlikely because of the following reasons. First, our other patients, diagnosed with either hereditary pancreatitis29Le Maréchal C. Masson E. Chen J.M. et al.Hereditary pancreatitis caused by triplication of the trypsinogen locus.Nat Genet. 2006; 38: 1372-1374Crossref PubMed Scopus (166) Google Scholar or FCP, were also referred to us by these clinics. Second, we have revisited some of the ICP patients carrying the triplication mutation, and in all revisited cases, we found no evidence to contradict the original diagnosis. Third, if one assumes that the triplication mutation should always cause hereditary pancreatitis, this mutation should more likely be found in the subjects with FCP rather than in those with ICP. Contrary to this expectation, none of the FCP patients was found to carry the triplication mutation. Last, variable penetrance of some human genetic diseases caused by CNMs has also been previously demonstrated; for example, in both Charcot-Marie-Tooth disease type 1A43Lupski J.R. de Oca-Luna R.M. Slaugenhaupt S. et al.DNA duplication associated with Charcot-Marie-Tooth disease type 1A.Cell. 1991; 66: 219-232Abstract Full Text PDF PubMed Scopus (1162) Google Scholar and hereditary neuropathy with liability to pressure palsies,44Chance P.F. Alderson M.K. Leppig K.A. et al.DNA deletion associated with hereditary neuropathy with liability to pressure palsies.Cell. 1993; 72: 143-151Abstract Full Text PDF PubMed Scopus (718) Google Scholar the disease-causing CNMs in some probands were found to be inherited from healthy parents. Cascade mutation screening in the triplication carriers should help to clarify this issue. The size of the newly identified duplicated segment was also shown to be identical to that of the known ∼605 kb triplication by walking QFM-PCR, and genotyping of rs3222967 suggested that either the duplication arose from the loss of 1 of the 2 allele-163–containing copies from the triplication, or the triplication arose from gain of an additional allele-163–containing copy from the duplication (Table 4). We are currently working on the mechanisms underlying the triplication/duplication mutations. Irrespective of its exact origin, the duplication was independently confirmed by fluorescence in situ hybridization (FISH) performed on cultured peripheral blood lymphocytes from one of the patients carrying this mutation (Figure 2). Because the duplication was only detected in 4 ICP subjects, it was impossible to perform a genotype-phenotype correlation. However, on the basis of the observations that triplication of the SNCA gene and the PLP1 gene appeared to cause more severe disease than their respective duplications,45Wolf N.I. Sistermans E.A. Cundall M. et al.Three or more copies of the proteolipid protein gene PLP1 cause severe Pelizaeus-Merzbacher disease.Brain. 2005; 128: 743-751Crossref PubMed Scopus (86) Google Scholar, 46Singleton A.B. Farrer M. Johnson J. et al.alpha-Synuclein locus triplication causes Parkinson's disease.Science. 2003; 302: 841Crossref PubMed Scopus (3531) Google Scholar, 47Chartier-Harlin M.C. Kachergus J. Roumier C. et al.Alpha-synuclein locus duplication as a cause of familial Parkinson's disease.Lancet. 2004; 364: 1167-1169Abstract Full Text Full Text PDF PubMed Scopus (1607) Google Scholar, 48Ibanez P. Bonnet A.M. Debarges B. et al.Causal relation between alpha-synuclein gene duplication and familial Parkinson's disease.Lancet. 2004; 364: 1169-1171Abstract Full Text Full Text PDF PubMed Scopus (864) Google Scholar trypsinogen locus triplication should be expected to cause a more severe phenotype than trypsinogen locus duplication (provided that each of the duplicated/triplicated copies is equally expressed). In this regard, it is worth mentioning that (1) the duplication was not found in any of our families with hereditary pancreatitis,29Le Maréchal C. Masson E. Chen J.M. et al.Hereditary pancreatitis caused by triplication of the trypsinogen locus.Nat Genet. 2006; 38: 1372-1374Crossref PubMed Scopus (166) Google Scholar and (2) only the duplication was identified in group Ib of ICP patients (Table 4). Finally, although we previously suggested that early mortality of subjects with chromosome 7q duplication syndromes might preclude acute or chronic pancreatitis to get clinical attention, we cautioned that a threshold level of trypsinogen copy number might be important in causing the disease.29Le Maréchal C. Masson E. Chen J.M. et al.Hereditary pancreatitis caused by triplication of the trypsinogen locus.Nat Genet. 2006; 38: 1372-1374Crossref PubMed Scopus (166) Google Scholar The new finding from this study provides support to the first theory. In short, gain of only one copy of the trypsinogen locus (ie, duplication) appears to be capable of causing the disease, although it might be associated with a less severe phenotype as compared with the gain of 2 copies of the trypsinogen locus (ie, triplication). When considered together with our negation of the previously described CNVs involving PRSS1 and/or PRSS2, this further potentiated the importance of a tightly regulated trypsin activation and inhibition balance for pathophysiology. As shown in Table 1, the 5 trypsinogen CNVs involve both loss and gain of DNA sequence, and some of them appear to be quite frequent in the general population. As a matter of fact, these CNVs should have been detected by our QFM-PCR if they were really to exist in any of our analyzed French and Indian patients and controls. Nevertheless, on the basis of previous findings,14Chen J.M. Le Maréchal C. Lucas D. et al."Loss of function" mutations in the cationic trypsinogen gene (PRSS1) may act as a protective factor against pancreatitis.Mol Genet Metab. 2003; 79: 67-70Crossref PubMed Scopus (45) Google Scholar, 15Witt H. Sahin-Tóth M. Landt O. et al.A degradation-sensitive anionic trypsinogen (PRSS2) variant protects against chronic pancreatitis.Nat Genet. 2006; 38: 668-673Crossref PubMed Scopus (191) Google Scholar we surmised initially that trypsinogen CNV loss could potentially protect one from the disease in the respective carriers. But the detection of trypsinogen CNV gain in controls poses a serious challenge to our previous finding.29Le Maréchal C. Masson E. Chen J.M. et al.Hereditary pancreatitis caused by triplication of the trypsinogen locus.Nat Genet. 2006; 38: 1372-1374Crossref PubMed Scopus (166) Google Scholar To obtain further insights into this issue, we went on to screen additional 75 samples from different ethnic origins (25 Moroccans, 25 Ivorians, and 25 Vietnamese) by using our QFM-PCR. Again, neither loss nor gain of trypsinogen CNVs was identified in any of these samples. Given that both trypsinogen triplication and duplication have been confirmed independently by FISH and QFM-PCR has also been used successfully by us to identify large genomic deletions in the CFTR49Audrézet M.P. Chen J.M. Raguénès O. et al.Genomic rearrangements in the CFTR gene: extensive allelic heterogeneity and diverse mutational mechanisms.Hum Mutat. 2004; 23: 343-357Crossref PubMed Scopus (111) Google Scholar, 50Férec C. Casals T. Chuzhanova N. et al.Gross genomic rearrangements involving deletions in the CFTR gene: characterization of six new events from a large cohort of hitherto unidentified cystic fibrosis chromosomes and meta-analysis of the underlying mechanisms.Eur J Hum Genet. 2006; 14: 567-576Crossref PubMed Scopus (82) Google Scholar and SPINK128Masson E. Le Maréchal C. Levy P. et al.Co-inheritance of a novel deletion of the entire SPINK1 gene with a CFTR missense mutation (L997F) in a family with chronic pancreatitis.Mol Genet Metab. 2007; 92: 168-175Crossref PubMed Scopus (23) Google Scholar genes, it seems unlikely that the absence of trypsinogen CNVs in our control samples was due to any inherent problems with our analytical technique. Therefore, we speculated that all 5 previously reported trypsinogen CNVs might be artifacts. Then we decided to investigate whether we could replicate the presence of these CNVs in the original samples by using our QFM-PCR. To this end, we purchased several relevant DNA samples from the Coriell Cell Repositories (Table 3). Not surprisingly, none of them was found to carry their respective CNV. That the common TRY6 and TRY7 deletion polymorphism36McCarroll S.A. Hadnott T.N. Perry G.H. et al.Common deletion polymorphisms in the human genome.Nat Genet. 2006; 38: 86-92Crossref PubMed Scopus (587) Google Scholar was found in most of these samples (lower panel, Figure 1B; Table 3) serves as an important internal control confirming that our analytical technique is highly reliable. In summary, we have for the first time analyzed trypsinogen CNMs in a large cohort of chronic pancreatitis subjects in 2 contrasting populations. Although increased number of trypsinogen copies was found in 6% of French ICP patients whose age of disease onset was ≤20 years, they were not found in any of the 268 TCP patients from India, indicating an ethnic specificity of these mutations. In particular, we identified a novel trypsinogen duplication that was confirmed by FISH analysis. Our results have thus revealed the molecular basis of a subset (6%) of the so-called ICP and further demonstrated the view that chronic pancreatitis is a genomic disorder.29Le Maréchal C. Masson E. Chen J.M. et al.Hereditary pancreatitis caused by triplication of the trypsinogen locus.Nat Genet. 2006; 38: 1372-1374Crossref PubMed Scopus (166) Google Scholar It is pertinent to emphasize 2 issues. First, the trypsinogen triplication and duplication mutations actually represent the second most frequent genetic factor predisposing to ICP in the French white population (the first being the SPINK1 N34S polymorphism). Second, this kind of rare mutation, unlike the common disease-associated N34S polymorphism, is of significant importance; whenever such a mutation is detected in a patient, a genetic etiology can usually be assigned, and genetic counseling can be given to the patient and his/her relatives. The above notwithstanding, our finding raises more questions than answers. For example, why was the triplication mutation always found in patients with either hereditary pancreatitis or ICP but not in FCP? Is there any difference in terms of trypsinogen expression in vivo between the triplication found in ICP and that found in hereditary pancreatitis? Could trypsinogen CNMs also account for some unidentified patients with hereditary pancreatitis or ICP in other populations?