Lack of significant association of an insertion/deletion polymorphism in the angiotensin converting enzyme (ACE) gene with tropical calcific pancreatitis
© Bhaskar et al; licensee BioMed Central Ltd. 2006
Received: 05 August 2006
Accepted: 12 December 2006
Published: 12 December 2006
The genetic basis of tropical calcific pancreatitis (TCP) is different and is explained by mutations in the pancreatic secretory trypsin inhibitor (SPINK1) gene. However, mutated SPINK1 does not account for the disease in all the patients, neither does it explain the phenotypic heterogeneity between TCP and fibro-calculous pancreatic diabetes (FCPD). Recent studies suggest a crucial role for pancreatic renin-angiotensin system during chronic hypoxia in acute pancreatitis and for angiotensin converting enzyme (ACE) inhibitors in reducing pancreatic fibrosis in experimental models. We investigated the association of ACE gene insertion/deletion (I/D) polymorphism in TCP patients using a case-control approach. Since SPINK1 mutations are proposed a modifier role, we also investigated its interaction with the ACE gene variant.
We analyzed the I/D polymorphism in the ACE gene (g.11417_11704del287) in 171 subjects comprising 91 TCP and 80 FCPD patients and compared the allelic and genotypic frequency in them with 99 healthy ethnically matched control subjects.
We found 46% and 21% of TCP patients, 56% and 19.6% of FCPD patients and 54.5% and 19.2% of the healthy controls carrying the I/D and D/D genotypes respectively (P>0.05). No significant difference in the clinical picture was observed between patients with and without the del allele at the ACE in/del polymorphism in both categories. No association was observed with the presence or absence of N34S SPINK1 mutation in these patients.
We conclude that the ACE insertion/deletion variant does not show any significant association with the pathogenesis, fibrosis and progression of tropical calcific pancreatitis and the fibro-calculous pancreatic diabetes.
Chronic Pancreatitis (CP) is a major health care problem worldwide and is associated with varied etiologies. CP is identified clinically by loss of endocrine and/or exocrine functions and pathologically by the presence of chronic inflammation and fibrosis of the pancreas [1, 2]. Tropical calcific pancreatitis (TCP) is a form of chronic pancreatitis of unknown etiology, more prevalent in the tropical regions of developing countries such as India whereas fibrocalculous pancreatic diabetes (FCPD) is a form of diabetes secondary to TCP . Both TCP and FCPD are associated with extensive fibrosis involving both intra and inter-lobular regions and not limited to one zone of the pancreas, although the extent of fibrosis varies and is usually more in FCPD . We and several others previously demonstrated an absence of mutations in the cationic trypsinogen gene (PRSS1) and a higher frequency of N34S mutation in the pancreatic secretory trypsin inhibitor (encoded by SPINK1 gene) in both FCPD and TCP patients without diabetes mellitus suggesting a common genetic basis for them [5, 6]. However, other genetic and/or environmental factors may explain the phenotypic variability in FCPD and TCP patients without diabetes .
The renin-angiotensin system (RAS) plays a central role in health and disease by maintaining the electrolyte and fluid balance and in turn, the blood pressure . Apart from circulating RAS, several studies suggest the existence of local RAS components in tissues such as brain, heart, kidney, pancreas, adrenals and gonads [8, 9]. Tissue specific RAS can act locally as paracrine and/or autocrine factors in meeting specific needs of individual tissues  and any functional alterations may be associated with pathophysiology of respective tissue/organ functions [11–13]. An intrinsic renin-angiotensin system (RAS) is present in the pancreas. Angiotensin converting enzyme (ACE), a zinc metallopeptidase is a key enzyme of this system . ACE and RAS activity is known to be enhanced during acute pancreatitis and chronic hypoxia in experimental animals [15, 16]. ACE cleaves angiotensin (AT)-I to AT-II, which induces proliferation of hepatic stellate cells (HSCs) and synthesis of extracellular matrix proteins by increasing the expression of transforming growth factor-beta [17, 18]. Isolated pancreatic stellate cells are similar to HSCs  and known to be involved in pathogenesis of pancreatic fibrosis in both experimental animals and humans . Increased accumulation of extra-cellular matrix is a histological characteristic of chronic pancreatitis that results in pancreatic fibrosis . In addition, ACE inhibitors have recently been demonstrated to attenuate pancreatic inflammation and fibrosis in spontaneously occurring chronic pancreatitis in male Wistar Bonn/Kobori rats .
A polymorphism within intron 16 (g.11417_11704del287) of the ACE gene is strongly related to the circulating enzyme levels in a dose dependent manner . The DD genotype that is associated with higher levels of circulating ACE than the II and ID genotypes, is known to be significantly more frequent in patients with myocardial infarction or patients with diabetic proteinuria than in controls [24, 25]. Thus, ACE gene polymorphisms may play a role in the pathogenesis of chronic pancreatitis and several other pancreatic diseases including acute pancreatitis, pancreatic cancer, diabetes mellitus and cystic fibrosis. Although a recent study failed to find any association of ACE I/D polymorphism in familial and sporadic chronic pancreatitis, we hypothesized a role for this polymorphism in view of the extensive fibrosis observed in the TCP patients . In this study, we investigated the ACE gene I/D polymorphism in TCP patients (without diabetes mellitus), FCPD patients and healthy controls to understand its association with the disease. Since, SPINK1 mutations are proposed to play a modifier role, we also investigated whether any association exists with the I/D polymorphism in the ACE gene.
Patients and controls
A total of 171 unrelated patients (120 M+51 F) comprising of 91 TCP and 80 FCPD patients were recruited in the study based on the WHO criteria . 99 unrelated ethnically matched individuals with complaints of dyspepsia were recruited as controls after a similar set of investigations excluded the disease in them. These individuals filled the same questionnaire and underwent similar investigations as the patients especially the imaging procedures which included computed tomography, endoscopic ultrasonography, magnetic resonance cholangio-pancreatography and endoscopic retrograde cholangio-pancreatography, wherever indicated . All the patients and healthy controls were explained the purpose of the study and the complications of investigative procedures and peripheral blood samples were collected after they signed the written informed consent. The study was approved by the institutional ethics committee of respective institutes following the Indian Council of Medical Research guidelines for handling human samples.
Allele frequency was calculated by the allele counting method, which was then utilized to determine the genotype frequency. The patients were divided into TCP and FCPD based on the presence or absence of diabetes mellitus and further based on the status of N34S SPINK1 mutation and ACE in/del polymorphism. Statistical comparisons were carried out by using Pearson's Chi-squared test, unpaired t-test and odds ratios (OR) along with the 95% confidence interval (CI). Two-tailed P values less than 0.05 were considered statistically significant. Post-hoc power analysis showed the study being adequately powered (84.6%) to accept the null hypothesis that the I/D polymorphism in ACE gene is not associated with TCP (α = 0.05, β = -1.426) .
Results and discussion
Tropical calcific pancreatitis is a form of chronic pancreatitis associated with fibrosis and pancreatic calcification. FCPD, a form of diabetes secondary to TCP, is thought to occur due to pancreatic destruction as a result of fibrosis and calcification . We investigated the association of I/D polymorphism in the ACE gene with TCP and the phenotype of diabetes in FCPD.
Characteristics of the study population
61 M/30 F
59 M/21 F
120 M/51 F
Age at onset (yrs)
Age at presentation (yrs)
Duration of symptoms (yrs)
Age at onset of diabetes (yrs)
Duration of diabetes (yrs)
Allele frequency and genotype distribution of I/D polymorphism at ACE locus in TCP patients & control subjects
TCP vs. FCPD
TCP vs. controls
FCPD vs. controls
Genotype frequency based on the presence or absence of the mutant allele
ID + DD
Clinical features of TCP and FCPD patients grouped according to their genotype at in/del polymorphism at ACE gene
Age at onset (yrs)
25.7 ± 11.1
24.8 ± 11.8
34.4 ± 15.1
31.2 ± 12.9
29.0 ± 13.3
28.0 ± 12.7
Age at presentation (yrs)
36.2 ± 11.8
32.7 ± 10.9
40.3 ± 17.0
38.9 ± 13.9
37.8 ± 14.0
35.8 ± 12.9
Age at onset of diabetes (yrs)
34.2 ± 13.9
33.7 ± 11.44
34.2 ± 13.9
33.7 ± 11.4
Allele frequency and genotype distribution of I/D polymorphism at ACE locus in TCP patients and controls based on their N34S SPINK1 status
N34S SPINK1 mutation status
Genotype frequency based on the presence or absence of the mutant allele
ID + DD
Angiotensin converting enzyme catalyzes the conversion of angiotensin I into the vasoactive and aldosterone-stimulating peptide angiotensin II , which carries out its biological functions by binding to two receptors, AT1R and AT2R . Several studies have suggested the presence of a pancreatic RAS, having physiological effects via a paracrine/autocrine pattern in the exocrine and endocrine pancreas, probably in the regulation of pancreatic microcirculation, ductal anion secretion and islet hormonal secretion . Both circulating and intrinsic pancreatic ACE activity is markedly elevated during acute and chronic inflammation of the pancreas [15, 16]. Recent studies have shown that ACE enzyme directs pancreatic fibrogenesis in experimental animals  and inhibition of the RAS system with an ACE inhibitor attenuates pancreatic inflammation and fibrosis in an animal model of chronic pancreatitis . Thus, angiotensinogen-II converted endogenously from angiotensinogen-I by circulating ACE may be directly involved in the induction of pancreatic inflammation and development of pancreatic fibrosis in the spontaneously occurring chronic pancreatitis in male Wistar Bonn/Kobori (WBN/Kob) rats . However, our study failed to observe any association between I/D variant and TCP or FCPD patients irrespective of N34S SPINK1 mutation status.
Our results are in agreement with the recent study on familial and sporadic chronic pancreatitis patients, which showed that the insertion/deletion polymorphism in the ACE gene does not make a significant contribution to the pathogenesis and progression of the disease . Although, recent evidence suggests that TCP is a specific genetic entity , there may be certain overlapping features with idiopathic chronic pancreatitis (ICP). In fact, the nomenclature of idiopathic chronic pancreatitis is itself a matter of debate since genetic etiology appears to play an important role in both ICP and TCP in India . Thus, ACE I/D polymorphism may not contribute to the pathogenesis of TCP despite the fact that the RAS system specifically affects the pancreas, an indication that local effects are more important than the systemic ones in pancreatic diseases. In the absence of intra-pancreatic ACE levels in these patients, there may exist a possibility that in contrast to other tissues such as heart and renal tissue, DD genotype does not influence the ACE gene expression and consequently its levels in the pancreas.
We conclude that the insertion/deletion polymorphism at the ACE locus does not play any role in the causation and progression of TCP. Further studies focusing on other environmental and/or genetic factors such as mutations in the CFTR gene may explain the phenotypic variability between TCP and FCPD patients.
- ACE :
Angiotensin converting enzyme
Fibrocalculous pancreatic diabetes
Hepatic stellate cells
Idiopathic chronic pancreatitis
- PRSS1 Protease:
serine, 1 (trypsin 1)
Tropical calcific pancreatitis
- SPINK1 :
Serine protease inhibitor Kazal type I
World Health Organization
The authors are grateful to all the patients and the normal individuals for agreeing to participate in the study and particularly for giving consent for genetic studies. Thanks are also due to Dr Ramakrishna, Asian Institute of Gastroenterology for his help in recruitment of patients and collection of blood samples. The financial support of Council of Scientific and Industrial Research, Ministry of Science and Technology, Government of India is gratefully acknowledged.
- Ammann RW: Natural history of chronic pancreatitis. Dig Surg. 1994, 11: 267-274.View ArticleGoogle Scholar
- Steer MI, Waxman I, Freedman S: Chronic pancreatitis. N Engl J Med. 1995, 332: 1482-90. 10.1056/NEJM199506013322206.View ArticlePubMedGoogle Scholar
- Mohan V, Chari ST, Hitman GA, Suresh S, Madanagopalan N, Ramachandran A, Viswanathan M: Familial aggregation in tropical fibrocalculous pancreatic diabetes. Pancreas. 1989, 4: 690-693. 10.1097/00006676-198912000-00006.View ArticlePubMedGoogle Scholar
- Nagalotimath SJ: Pancreatic pathology in pancreatic calcification with diabetes. Secondary diabetes: The spectrum of the diabetic syndromes. Edited by: Podolsky S, Viswanathan M. 1980, New York: Raven Press, 117-145.Google Scholar
- Chandak GR, Idris MM, Reddy DN, Bhaskar S, Sriram PVJ, Singh L: 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: 347-351. 10.1136/jmg.39.5.347.View ArticlePubMedPubMed CentralGoogle Scholar
- Hassan Z, Mohan V, Ali L, Allotey R, Barakat K, Faruque MO, Deepa R, McDermott MF, Jackson AE, Cassell P, Curtis D, Gelding SV, Vijayaravaghan S, Gyr N, Whitcomb DC, Khan AK, Hitman GA: SPINK1 is a susceptibility gene for fibrocalculous pancreatic diabetes in subjects from Indian subcontinent. Am J Hum Genet. 2002, 71: 964-968. 10.1086/342731.View ArticlePubMedPubMed CentralGoogle Scholar
- Stroth U, Unger T: The renin-angiotensin system and its receptors. J Cardiovasc Pharmacol. 1999, 33: S21-28. 10.1097/00005344-199900001-00005.View ArticlePubMedGoogle Scholar
- Campbell DJ: Circulating and tissue angiotensin systems. J Clin Invest. 1987, 79: 1-6.View ArticlePubMedPubMed CentralGoogle Scholar
- Leung PS, Carlsson PO: Tissue renin-angiotensin system: its expression, localization, regulation and potential role in the pancreas. J Mol Endocrinol. 2001, 26: 155-164. 10.1677/jme.0.0260155.View ArticlePubMedGoogle Scholar
- Phillips MI, Speakman EA, Kimura B: Levels of angiotensin and molecular biology of the tissue renin angiotensin systems. Regul Pept. 1993, 43: 1-20. 10.1016/0167-0115(93)90403-U.View ArticlePubMedGoogle Scholar
- Ruiz-Ortega M, Ruperez M, Esteban V, Egido J: Molecular mechanisms of angiotensin II-induced vascular injury. Curr Hypertens Rep. 2003, 5: 73-79.View ArticlePubMedGoogle Scholar
- Bataller R, Gines P, Nicolas JM, Gorbig MN, Garcia Ramealo E, Gasull X, Bosch J, Arroyo V, Rodes J: Angiotensin II induces contraction and proliferation of human hepatic stellate cells. Gastroenterology. 2000, 118: 1149-1156. 10.1016/S0016-5085(00)70368-4.View ArticlePubMedGoogle Scholar
- Marshall RP, McAnulty RJ, Laurent GJ: Angiotensin II is mitogenic for human lung fibroblasts via activation of the type 1 receptor. Am J Respir Crit Care Med. 2000, 161: 1999-2004.View ArticlePubMedGoogle Scholar
- Erdos E, Skidgel RA: The angiotensin I-converting enzyme. Lab Invest. 1987, 56: 345-348.PubMedGoogle Scholar
- Ip SP, Kwan PC, Williams CH, Pang S, Hooper NM, Leung PS: Changes of angiotensin-converting enzyme activity in the pancreas of chronic hypoxia and acute pancreatitis. Int J Biochem Cell Biol. 2003, 35: 944-954. 10.1016/S1357-2725(02)00181-4.View ArticlePubMedGoogle Scholar
- Leung PS, Chan HC, Nobiling R: Regulated expression of pancreatic renin angiotensin system in experimental pancreatitis. Mol Cell Endocrinol. 2000, 166: 121-128. 10.1016/S0303-7207(00)00275-6.View ArticlePubMedGoogle Scholar
- Friedman SL: Molecular regulation of hepatic fibrosis, an integrated response to injury. J Biol Chem. 2000, 275: 2247-2250. 10.1074/jbc.275.4.2247.View ArticlePubMedGoogle Scholar
- Okuno M, Akita K, Moriwaki H, Kawada N, Ikeda K, Kaneda K, Suzuki Y, Kojima S: Prevention of rat hepatic fibrosis by the protease inhibitor, camostat mesilate, via reduced generation of active TGF-β. Gastroenterology. 2001, 20: 1784-1800. 10.1053/gast.2001.24832.View ArticleGoogle Scholar
- Schneider E, Schmid-Kotsas A, Zhao J, Weidenbach H, Schmid RM, Menke A, Adler G, Waltenberger J, Grunert A, Bachem MG: Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells. Am J Physiol. 2001, 281: C532-C543.Google Scholar
- Apte MV, Haber PS, Darby SJ, Rodgers SC, McCaughan GW, Korsten MA, Pirola RC, Wilson JS: Pancreatic stellate cells are activated by proinflammatory cytokines: implications for pancreatic fibrogenesis. Gut. 1999, 44: 534-541.View ArticlePubMedPubMed CentralGoogle Scholar
- Haber PS, Keogh QW, Apte MV, Moran CS, Stewart NL, Crawford DHG, Pirola RC, McCaughan GW, Ramm GA, Wilson JS: Activation of pancreatic stellate cells in human and experimental pancreatic fibrosis. Am J Pathol. 1999, 155: 1087-1095.View ArticlePubMedPubMed CentralGoogle Scholar
- Kuno A, Yamada T, Masunda K, Ogawa K, Sogawa M, Nakamura S, Nakazawa T, Ohara H, Nomura T, Joh T, Shirai T, Itoh M: Angiotensin- Converting Enzyme attenuates pancreatic inflammation and fibrosis in male Bonn/Kabori rats. Gastroenterology. 2003, 124: 1010-1019. 10.1053/gast.2003.50147.View ArticlePubMedGoogle Scholar
- Rigat B, Hubert C, Alhenc-Gelas F, Corvol P, Soubrier F: An insertion/deletion polymorphism in angiotensin converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest. 1990, 86: 1343-1346.View ArticlePubMedPubMed CentralGoogle Scholar
- Cambien F, Poirier O, Lecerf L, Evans A, Cambou JP, Arveiler D, Luc G, Bard JM, Bara L, Richard S: Deletion polymorphism in the gene for angiotensin converting enzyme is a potent risk factor for myocardial infarction. Nature. 1992, 359: 641-644. 10.1038/359641a0.View ArticlePubMedGoogle Scholar
- Vishwanathan V, Zhu Y, Bala K, Dunn S, Snehalatha C, Ramachandran A, Jayaraman M, Sharma K: Association between ACE gene polymorphism and diabetic nephropathy in South Indian patients. JOP. 2001, 2: 83-87.Google Scholar
- Oruc N, Lamb J, Kutlu OC, Barmada MM, Money ME, Slivka A, Whitcomb DC: The functional angiotensin converting enzyme gene I/D polymorphism does not alter susceptibility to chronic pancreatitis. JOP. 2004, 5: 457-463.PubMedGoogle Scholar
- WHO study group Report on Diabetes Mellitus: WHO technical report series 727. Geneva. 1985Google Scholar
- Miller SA, Dykes DD, Polesky HF: Simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988, 16: 1215-View ArticlePubMedPubMed CentralGoogle Scholar
- 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 type1 are associated with chronic pancreatitis. Nat Genet. 2000, 25: 213-216. 10.1038/76088.View ArticlePubMedGoogle Scholar
- Lalouel JM, Rohrwasser A: Power and Replication in Case-Control Studies. Am J Hypertens. 2002, 15 (2 Pt 1): 201-205. 10.1016/S0895-7061(01)02285-3.View ArticlePubMedGoogle Scholar
- Mohan V, Barman KK, Rajan VS, Chari ST, Deepa R: Natural history of endocrine failure in tropical chronic pancreatitis: a longitudinal follow-up study. J Gastroenterol Hepatol. 2005, 20: 1927-1934. 10.1111/j.1440-1746.2005.04068.x.View ArticlePubMedGoogle Scholar
- Pueyo ME, N'Diaye N, Michael JB: Angiotensin II elicited signal transduction via AT-1 receptors in endothelial cells. Br J Pharmacol. 1996, 118: 79-84.View ArticlePubMedPubMed CentralGoogle Scholar
- Nagashio Y, Asaumi H, Watanabe S, Nomiyama Y, Taguchi M, Tashiro M, Sugaya T, Otsuki M: Angiotensin II type 1 receptor interaction is an important regulator for the development of pancreatic fibrosis in mice. Am J Physiol Gastrointest Liver Physiol. 2004, 287: G170-177. 10.1152/ajpgi.00005.2004.View ArticlePubMedGoogle Scholar
- Chandak GR, Idris MM, Reddy DN, Mani KR, Bhaskar S, Rao GV, Singh L: Absence of PRSS1 mutations and association of SPINK1 trypsin inhibitor mutations in hereditary and non-hereditary chronic pancreatitis. Gut. 2004, 53: 723-728. 10.1136/gut.2003.026526.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/6/42/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.