- Email: [email protected]
G Polymorphism Is Not a Susceptibility Factor for Chronic Pancreatitis
Original Paper Pancreatology 2006;6:297–300 DOI: 10.1159/000092692
Received: August 30, 2005 Accepted after revision: December 24, 2005 Published online: April 19, 2006
The MCP-1 –2518 A/G Polymorphism Is Not a Susceptibility Factor for Chronic Pancreatitis David A. Sass a Georgios I. Papachristou a Janette Lamb a M. Michael Barmada c Randall E. Brand e Mary E. Money f Robert H. Hawes g Peter B. Cotton g Adam Slivka a David C. Whitcomb a–d Departments of a Medicine, b Cell Biology and Physiology, and c Human Genetics, University of Pittsburgh School of Medicine; d University of Pittsburgh Cancer Institute, Pittsburgh, Pa.; e Department of Medicine, Evanston Northwestern Healthcare, Evanston, Ill.; f Washington County Hospital, Hagarstown, Md., and g Digestive Disorder Center, Medical University of South Carolina, Charlestown, S.C., USA
Key Words Chronic pancreatitis Genetics Monocyte chemotactic protein-1 Fibrosis Inflammation
Abstract Background and Aims: Chronic pancreatitis (CP) is an inflammatory process initiated by recurrent acute pancreatitis and characterized by progressive parenchyma destruction and fibrosis. Genetic factors influence susceptibility and modify progression. The monocyte chemotactic protein-1 (MCP-1) –2518 G allele, which modifies the severity of acute pancreatitis, was investigated as a susceptibility factor for CP. Methods: A genetic association study was performed on 177 CP patients and 116 healthy controls from the NAPS2 Study. The MCP-1 A/G genotype was determined by RFLP and confirmed by DNA sequencing. Results: Compared to the control group the MCP-1 –2518 genotypes were similar: A/A (57% vs. 50%), A/G (34.5% vs. 40%) and G/G (8.5% vs. 10%). These allele frequencies were not statistically different (p = 0.267). Conclusions: Although the pro-inflammatory chemokine MCP-1 –2518 G allele is a severity factor for AP, it does not significantly alter susceptibility to CP.
Introduction
Chronic pancreatitis (CP) remains a major source of morbidity in the United States and presents a challenging and frustrating clinical problem [1]. Pathologically, CP is a progressive, destructive inflammatory process that often leads to pancreatic parenchymal fibrosis with loss of exocrine and endocrine function and development of unrelenting pain [1]. The clinical observation is that some patients with major risk factors such as alcoholism and heavy tobacco smoking develop CP while others do not, and the rate of progression from normal pancreas to end stage organ destruction and fibrosis is highly variable and unpredictable [2, 3]. The discovery that mutations in the cationic trypsinogen gene (PRSS1), the pancreatic secretory trypsin inhibitor gene (SPINK1) and the cystic fibrosis transmembrane conductance regulation gene (CFTR) were associated with recurrent acute and chronic pancreatitis suggests that the process leading to chronic pancreatitis begins with recurrent acute pancreatitis and that risk is increased by genetic factors (see recent reviews [4, 5]). We recently observed that a common promoter polymorphism in the monocyte chemotactic protein-1 gene (MCP-1 –2518 A1G [6]) significantly increase the risk of severe acute pancreatitis regardless of etiology [7].
Copyright © 2006 S. Karger AG, Basel and IAP
© 2006 S. Karger AG, Basel and IAP Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com
Accessible online at: www.karger.com/pan
David C. Whitcomb, MD, PhD UPMC Presbyterian, Mezzanine 2, C-Wing 200 Lothrop Street Pittsburgh, PA 15213 (USA) Tel. +1 412 648 9604, Fax +1 412 383 7236, E-Mail [email protected]
MCP-1 is an important early pro-inflammatory ‘CC’ chemokine [8] primarily released from monocyte-macrophages and other inflammatory cells. The presence of the –2518 G allele (GG or GA) results in a significantly greater MCP-1 experession in response to the same inflammatory stimulus than the normal A/A homozygous genotype [6]. In addition to modifying the severity of acute pancreatitis [7] the MCP-1 –2518 G allele modifies susceptibility or severity of a variety of other inflammatory disorders (e.g. hepatitis C [9], bronchial asthma [10], cutaneous lupus vasculitis [11], Crohn’s disease [12] and coronary artery disease [13]). While MCP-1 is an important marker of inflammation in experimental and clinical acute pancreatitis [14–17] it is also expressed in CP in experimental models and human pancreas specimens [16, 18, 19]. The MCP-1 gene is not expressed in normal pancreas, but in chronic pancreatitis MCP-1 mRNA localizes to centroacinar cells, endothelia, fibroblasts, macrophages, T cells and occasionally in nerves [19]. However, the role of MCP-1 in the development of chronic pancreatitis has not been fully defined. We tested the hypothesis that the MCP-1 –2518 A/G polymorphism increase susceptibility to chronic pancreatitis by comparing the frequency of the two alleles in prospectively ascertained and well-characterized patients with chronic pancreatitis and prospectively collected controls. The findings help clarify the role of MCP-1 and related inflammatory processes in the development of chronic pancreatitis.
Materials and Methods Patients and Controls One hundred and seventy-seven patients and 116 controls from the initial phases of the North American Pancreatitis 2 (NAPS2) Study were studied. The NAPS2 study is a multicenter molecular epidemiologic study designed to evaluating the genetic and environmental factors predisposing to recurrent acute and chronic pancreatitis. Study eligibility criteria include: [1] a diagnosis of recurrent (62 episodes) acute pancreatitis as determined by amylase elevation 13! upper limit of normal with abdominal pain and/or nausea or [2] diagnosis of CP, as determined by ERCP (with a classification of mild or higher using the Cambridge criteria) or [3] diagnosis of CP by computed tomography criteria or [4] both recurrent acute pancreatitis and CP. The current study included patients and controls from the University of Pittsburgh Medical Center, Evanston Northwestern Healthcare system, Medical University of South Carolina, and additional phenotyped controls (over age 50 years) from the Washington County Hospital. IRB approval was obtained from all centers prior to patient recruitment and all patients signed an informed written consent prior to study enroll-
298
Pancreatology 2006;6:297–300
ment. Blood samples were collected in EDTA-containing tubes for DNA extraction. DNA Preparation Genomic DNA was extracted using the Gentra Systems Puregene system (Minneapolis, Minn., USA) according to the manufacturer instructions as described by Pfützer et al. [20]. PCR Amplification Polymerase chain reaction (PCR) was performed in a total reaction volume of 25 l containing 50 ng of genomic DNA using primers 5´-CCGAGATGTTCC-CAGCACAG-3´ and 5´-CTGCTTTGCTTGTGCCTCTT-3´ as described by Mühlbauer et al. [9] with modifications. The cycling conditions were modified by increasing the annealing temperature to 57 ° C and extending the amplification to 35 cycles which was necessary to optimize the PCR product for RFLP and sequencing. RFLP Analysis The genotype of the distal MCP-1 promoter was determined by restriction fragment length polymorphism (RFLP) analysis of the 930 bp amplicon [10]. PvuII (Invitrogen Life Technologies, Carlsbad, Calif., USA) cleaves the G allele of the –2518G1A polymorphism to yield fragments of 708 and 222 bp. Restriction fragments were separated by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. DNA Sequencing Because of variability and inconsistency in initial RFLP analysis, samples were verified by DNA sequencing of the PCR products. Following PCR, samples were purified with the exonuclease I, shrimp alkaline phosphatase enzymatic purification system (ExoSAP-it; USB Corp. Cleveland, Ohio, USA) according to the manufacturer recommendations. Cycle sequencing was performed using the ABI Prism Big Dye Terminator Sequencing Kit v3.1 (ABI, Foster City, Calif., USA) diluted 1:4. Reaction products were purified by ethanol EDTA precipitation following the ABI sequencing kit protocol. Sequence products were run on an ABI Prism 3730 Genetic Analyzer and sequence data was analyzed using Sequencher 4.1.4 (Gene Codes Corporation, Ann Arbor, Mich., USA). Statistical Analysis Genotype frequencies are reported with their group percentages. Comparisons between genotype frequencies were made using Armitage trend tests. The MCP-1 –2518 A/G polymorphism is known to be functional and associated with acute pancreatitis [7], so the prior probability of a statistically significant test result being true was high [21]. Furthermore, a single SNP was tested, so no corrections were needed for multiple comparisons. Thus, p ! 0.05 was considered statistically significant with a low false-positive report probability [21].
Results
Patient Characteristics Table 1 contains the demographic data of our study population and table 2 the underlying etiologic risk factors according to the TIGAR-O classification of CP. The
Sass et al.
MCP-1 Genotypes The frequencies of the MCP-1 A/A homozygotes, A/G heterozygotes and G/G homozygotes in CP patients and controls are summarized in figure 1. In the control group, the genotype frequencies were: A/A (66/116, 57%), A/G (40/116, 34.5%) and G/G (10/116, 8.5%). Hardy-Weinberg estimation of allele frequencies were met, with the control samples matching the frequencies seen in other studies [9–11, 13]. In the chronic pancreatitis group, the genotype frequencies were: A/A (89/177, 50%), A/G (70/177, 40%) and G/G (18/177, 10%). There was no statistically significant difference between the genotypes of CP subjects and controls (p = 0.267).
60
Genotype frequency (%)
TIGAR-O classification is an etiological classification system developed by the Midwest Multicenter Pancreatic Study Group (renamed the North American Pancreatic Study Group) to categorize risk factors for CP according to various mechanisms [1].
A/A A/G G/G
50 40 30 20 10 0 Chronic pancreatitis
Healthy controls
Fig. 1. Frequencies of the –2518 MCP-1 genotypes in patients with CP and in healthy controls.
Table 1. Demographic data on patients and controls
Discussion
Acute and chronic pancreatitis are complex disorders with a strong genetic basis. The interaction between environmental and genetic factors determines susceptibility to acute pancreatitis, frequency of acute pancreatitis and progression to chronic pancreatitis [4, 5]. The effect of the MCP-1 –2518 polymorphism in acute pancreatitis is to alter the immunological response to pancreatic injury [7]. The possibility that the MCP-1 –2518 G allele also increases the risk of chronic pancreatitis has not been previously reported. Our results suggest that the association between this gene and susceptibility to chronic pancreatitis is minimal and not clinically relevant. We tested the hypothesis that the MCP-1 –2518 G allele was associated with a susceptibility to chronic pancreatitis. This hypothesis is relevant to questions about the mechanism linking acute pancreatitis to chronic pancreatitis. For example, is chronic pancreatitis more dependent on the severity of acute pancreatitis (as implied by the necrosis-fibrosis sequence model of chronic pancreatitis), or primarily an increased frequency of acute pancreatitis attacks (as seen in subjects with hereditary pancreatitis due to cationic trypsinogen mutations [4, 22, 23])? Based on our acute pancreatitis study, the MCP-1 –2518 polymorphism affects the severity of acute pancreatitis and therefore the likelihood of developing chronic pancreatitis based on the necrosis-fibrosis hypothesis. This did not appear to be the case in our population, sug-
MCP-1 and Chronic Pancreatitis
Gender Age range, years Mean age, years
Patients (n = 177)
Controls (n = 116)
86 male; 91 female 7–91 48.4
49 male; 67 female 25–84 62.7
Table 2. Etiologic risk factors associated with CP: TIGAR-O clas-
sification system Toxic-metabolic
alcohol hypercalcemia hyperlipidemia
Idiopathic Genetic
49 3 9 73
cationic trypsinogen (PRSS1) and SPINK1 (PSTI) CFTR
4 3
Autoimmune
5
Recurrent and severe acute pancreatitis
1
Obstructive
pancreas divisum sphincter of Oddi dysfunction duct obstruction (tumor) other
15 8 4 3
PRSS1 = UniGene name: protease, serine, 1; PSTI = pancreatic secretory trypsin inhibitor, UniGene name; SPINK 1 = serine protease inhibitor, Kazal type 1; CFTR = cystic fibrosis transmembrane conductance regulator.
Pancreatology 2006;6:297–300
299
gesting that other mechanisms are more important in determining susceptibility to chronic pancreatitis. Our results also show some important distinction between inflammatory processes in the pancreas compared to the liver. In hepatitis C the liver inflammation is chronic and anti-viral in nature. Under these circumstances the MCP-1 –2518 G allele may drive fibrosis by amplifying the intensity of the overall immune response [9]. In chronic pancreatitis, the nature of the chronic inflammation is not fully characterized, but the MCP-1 –2518 G allele does not seem to affect the threshold for developing fibrosis. Although the MCP-1 –2518 G allele does not appear to increase susceptibility to chronic pancreatitis, the study did not test whether the MCP-1 –2518 G allele altered the rate of progression of chronic pancreatitis from early to end-stage disease.
Determining the exact role of genetic susceptibility factors, disease modifier genes and environmental factors in complex diseases is highly dependent on the quality of phenotyping, the population being investigated, environmental exposures and epigenetic factors phenomena [24, 25]. The current study of 177 subjects and 116 well-characterized controls is sufficiently large to exclude a clinically relevant effect of the MCP-1 –2518 polymorphism as a susceptibility factor for chronic pancreatitis, but does not exclude other effects that are highly relevant to other aspects of pancreatic diseases.
Acknowledgement This study was supported by NIH DK 061451-01 (D.C.W.).
References 1 Etemad B, Whitcomb DC: Chronic pancreatitis: diagnosis, classification, and new genetic developments. Gastroenterology 2001; 120: 682–707. 2 Ammann RW, Heitz PU, Kloppel G: Course of alcoholic chronic pancreatitis: a prospective clinicomorphological long-term study. Gastroenterology 1996;111:224–231. 3 Ammann RW, Muellhaupt B: Progression of alcoholic acute to chronic pancreatitis. Gut 1994;35:552–556. 4 Whitcomb DC: Value of genetic testing in management of pancreatitis. Gut 2004; 53: 1710–1717. 5 Whitcomb DC: Advances in understanding the mechanisms leading to chronic pancreatitis. Nat Clin Practice Gastro-Hepatol 2004; 1: 46–52. 6 Rovin BH, Lu L, Saxena R: A novel polymorphism in the MCP-1 gene regulatory region that influences MCP-1 expression. Biochem Biophys Res Commun 1999;259:344–348. 7 Papachristou GI, Sass DA, Avula H, Lamb J, Lokshin A, Barmada MM, et al: Is the monocyte chemotactic protein-1 –2518 G allele a risk factor for severe acute pancreatitis? Clin Gastroenterol Hepatol 2005;3:475–481. 8 Adams DH, Lloyd AR: Chemokines: leukocyte recruitment and activation cytokines. Lancet 1997;349:490–495. 9 Muhlbauer M, Bosserhoff AK, Hartmann A, Thasler WE, Weiss TS, Herfarth H, et al: A novel MCP-1 gene polymorphism is associated with hepatic MCP-1 expression and severity of HCV-related liver disease. Gastroenterology 2003;125:1085–1093. 10 Szalai C, Kozma GT, Nagy A, Bojszko A, Krikovszky D, Szabo T, et al: Polymorphism in the gene regulatory region of MCP-1 is associated with asthma susceptibility and sever-
300
11
12
13
14
15
16
17
ity. J Allergy Clin Immunol 2001; 108: 375– 381. Aguilar F, Gonzalez-Escribano MF, SanchezRoman J, Nunez-Roldan A: MCP-1 promoter polymorphism in Spanish patients with systemic lupus erythematosis. Tissue Antigens 2001;58:335–338. Herfrath H, Goke M, Hellerbrand C, Muhlbauer M, Vogl D, Scholmerich J, et al: Polymorphism of monocyte chemoattractant protein-1 in Crohn’s disease. Int J Colorectal Dis 2003;18:401–405. Szalai C, Duba J, Prohaszka Z, Kalina A, Szabo T, Nagy B, et al: Involvement of polymorphisms in the chemokine system in susceptibility for coronary artery disease (CAD). Coincidence of Lp(a) and MCP-1 –2518G/G genotype in CAD patients. Atherosclerosis 2001;158:233–239. Grady T, Liang P, Ernst SA, Logsdon CD: Chemokine gene expression in rat pancreatic acinar cells is an early event associated with acute pancreatitis. Gastroenterology 1997; 113: 1966–1975. Yang BM, Demaine AG, Klingsnorth A: Chemokines MCP-1 and RANTES in isolated rat pancreatic acinar cells treated with CCK and ethanol in vitro. Pancreas 2000;21:22–31. Deng X, Wang L, Elm MS, Gabazadeh D, Diorio GJ, Eagon PK, et al: Chronic alcohol consumption accelerates fibrosis in response to cerulein-induced pancreatitis in rats. Am J Pathol 2005;166:93–106. Rau B, Baumgart K, Kruger CM, Schilling M, Beger HG: CC-chemokine activation in acute pancreatitis: enhanced release of monocyte chemoattractant protein-1 in patients with local and systemic complications. Intens Care Med 2003;29:622–629.
Pancreatology 2006;6:297–300
18 Inoue M, Ino Y, Gibo J, Ito T, Hisano T, Arita Y, et al: The role of monocyte chemoattractant protein-1 in experimental chronic pancreatitis model induced by dibutyltin dichloride in rats. Pancreas 2002;25:e64–e70. 19 Saurer L, Reber P, Schaffner T, Buchler MW, Buri C, Kappeler A, et al: Differential expression of chemokines in normal pancreas and in chronic pancreatitis. Gastroenterology 2000; 118:356–367. 20 Pfützer RH, Barmada MM, Brunskil APJ, Finch R, Hart PS, Neoptolemos J, et al: SPINK1/PSTI polymorphisms act as disease modifiers in familial and idiopathic chronic pancreatitis. Gastroenterology 2000;119:615– 623. 21 Wacholder S, Chanock S, Garcia-Closas M, El Ghormli L, Rothman N: Assessing the probability that a positive report is false: an approach for molecular epidemiology studies. J Natl Cancer Inst 2004;96:434–442. 22 Whitcomb DC, Gorry MC, Preston RA, Furey W, Sossenheimer MJ, Ulrich CD, et al: Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nature Genetics 1996;14:141–145. 23 Gorry MC, Gabbaizedeh D, Furey W, Gates LK Jr, Preston RA, Aston CE, et al: Mutations in the cationic trypsinogen gene are associated with recurrent acute and chronic pancreatitis. Gastroenterology 1997;113:1063–1068. 24 Cardon LR, Bell JI: Association study designs for complex diseases. Nat Rev Genet 2001; 2: 91–99. 25 Ioannidis JP, Trikalinos TA, Ntzani EE, Contopoulos-Ioannidis DG: Genetic associations in large versus small studies: an empirical assessment. Lancet 2003;361:567–571.
Sass et al.
Received: August 30, 2005 Accepted after revision: December 24, 2005 Published online: April 19, 2006
The MCP-1 –2518 A/G Polymorphism Is Not a Susceptibility Factor for Chronic Pancreatitis David A. Sass a Georgios I. Papachristou a Janette Lamb a M. Michael Barmada c Randall E. Brand e Mary E. Money f Robert H. Hawes g Peter B. Cotton g Adam Slivka a David C. Whitcomb a–d Departments of a Medicine, b Cell Biology and Physiology, and c Human Genetics, University of Pittsburgh School of Medicine; d University of Pittsburgh Cancer Institute, Pittsburgh, Pa.; e Department of Medicine, Evanston Northwestern Healthcare, Evanston, Ill.; f Washington County Hospital, Hagarstown, Md., and g Digestive Disorder Center, Medical University of South Carolina, Charlestown, S.C., USA
Key Words Chronic pancreatitis Genetics Monocyte chemotactic protein-1 Fibrosis Inflammation
Abstract Background and Aims: Chronic pancreatitis (CP) is an inflammatory process initiated by recurrent acute pancreatitis and characterized by progressive parenchyma destruction and fibrosis. Genetic factors influence susceptibility and modify progression. The monocyte chemotactic protein-1 (MCP-1) –2518 G allele, which modifies the severity of acute pancreatitis, was investigated as a susceptibility factor for CP. Methods: A genetic association study was performed on 177 CP patients and 116 healthy controls from the NAPS2 Study. The MCP-1 A/G genotype was determined by RFLP and confirmed by DNA sequencing. Results: Compared to the control group the MCP-1 –2518 genotypes were similar: A/A (57% vs. 50%), A/G (34.5% vs. 40%) and G/G (8.5% vs. 10%). These allele frequencies were not statistically different (p = 0.267). Conclusions: Although the pro-inflammatory chemokine MCP-1 –2518 G allele is a severity factor for AP, it does not significantly alter susceptibility to CP.
Introduction
Chronic pancreatitis (CP) remains a major source of morbidity in the United States and presents a challenging and frustrating clinical problem [1]. Pathologically, CP is a progressive, destructive inflammatory process that often leads to pancreatic parenchymal fibrosis with loss of exocrine and endocrine function and development of unrelenting pain [1]. The clinical observation is that some patients with major risk factors such as alcoholism and heavy tobacco smoking develop CP while others do not, and the rate of progression from normal pancreas to end stage organ destruction and fibrosis is highly variable and unpredictable [2, 3]. The discovery that mutations in the cationic trypsinogen gene (PRSS1), the pancreatic secretory trypsin inhibitor gene (SPINK1) and the cystic fibrosis transmembrane conductance regulation gene (CFTR) were associated with recurrent acute and chronic pancreatitis suggests that the process leading to chronic pancreatitis begins with recurrent acute pancreatitis and that risk is increased by genetic factors (see recent reviews [4, 5]). We recently observed that a common promoter polymorphism in the monocyte chemotactic protein-1 gene (MCP-1 –2518 A1G [6]) significantly increase the risk of severe acute pancreatitis regardless of etiology [7].
Copyright © 2006 S. Karger AG, Basel and IAP
© 2006 S. Karger AG, Basel and IAP Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com
Accessible online at: www.karger.com/pan
David C. Whitcomb, MD, PhD UPMC Presbyterian, Mezzanine 2, C-Wing 200 Lothrop Street Pittsburgh, PA 15213 (USA) Tel. +1 412 648 9604, Fax +1 412 383 7236, E-Mail [email protected]
MCP-1 is an important early pro-inflammatory ‘CC’ chemokine [8] primarily released from monocyte-macrophages and other inflammatory cells. The presence of the –2518 G allele (GG or GA) results in a significantly greater MCP-1 experession in response to the same inflammatory stimulus than the normal A/A homozygous genotype [6]. In addition to modifying the severity of acute pancreatitis [7] the MCP-1 –2518 G allele modifies susceptibility or severity of a variety of other inflammatory disorders (e.g. hepatitis C [9], bronchial asthma [10], cutaneous lupus vasculitis [11], Crohn’s disease [12] and coronary artery disease [13]). While MCP-1 is an important marker of inflammation in experimental and clinical acute pancreatitis [14–17] it is also expressed in CP in experimental models and human pancreas specimens [16, 18, 19]. The MCP-1 gene is not expressed in normal pancreas, but in chronic pancreatitis MCP-1 mRNA localizes to centroacinar cells, endothelia, fibroblasts, macrophages, T cells and occasionally in nerves [19]. However, the role of MCP-1 in the development of chronic pancreatitis has not been fully defined. We tested the hypothesis that the MCP-1 –2518 A/G polymorphism increase susceptibility to chronic pancreatitis by comparing the frequency of the two alleles in prospectively ascertained and well-characterized patients with chronic pancreatitis and prospectively collected controls. The findings help clarify the role of MCP-1 and related inflammatory processes in the development of chronic pancreatitis.
Materials and Methods Patients and Controls One hundred and seventy-seven patients and 116 controls from the initial phases of the North American Pancreatitis 2 (NAPS2) Study were studied. The NAPS2 study is a multicenter molecular epidemiologic study designed to evaluating the genetic and environmental factors predisposing to recurrent acute and chronic pancreatitis. Study eligibility criteria include: [1] a diagnosis of recurrent (62 episodes) acute pancreatitis as determined by amylase elevation 13! upper limit of normal with abdominal pain and/or nausea or [2] diagnosis of CP, as determined by ERCP (with a classification of mild or higher using the Cambridge criteria) or [3] diagnosis of CP by computed tomography criteria or [4] both recurrent acute pancreatitis and CP. The current study included patients and controls from the University of Pittsburgh Medical Center, Evanston Northwestern Healthcare system, Medical University of South Carolina, and additional phenotyped controls (over age 50 years) from the Washington County Hospital. IRB approval was obtained from all centers prior to patient recruitment and all patients signed an informed written consent prior to study enroll-
298
Pancreatology 2006;6:297–300
ment. Blood samples were collected in EDTA-containing tubes for DNA extraction. DNA Preparation Genomic DNA was extracted using the Gentra Systems Puregene system (Minneapolis, Minn., USA) according to the manufacturer instructions as described by Pfützer et al. [20]. PCR Amplification Polymerase chain reaction (PCR) was performed in a total reaction volume of 25 l containing 50 ng of genomic DNA using primers 5´-CCGAGATGTTCC-CAGCACAG-3´ and 5´-CTGCTTTGCTTGTGCCTCTT-3´ as described by Mühlbauer et al. [9] with modifications. The cycling conditions were modified by increasing the annealing temperature to 57 ° C and extending the amplification to 35 cycles which was necessary to optimize the PCR product for RFLP and sequencing. RFLP Analysis The genotype of the distal MCP-1 promoter was determined by restriction fragment length polymorphism (RFLP) analysis of the 930 bp amplicon [10]. PvuII (Invitrogen Life Technologies, Carlsbad, Calif., USA) cleaves the G allele of the –2518G1A polymorphism to yield fragments of 708 and 222 bp. Restriction fragments were separated by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. DNA Sequencing Because of variability and inconsistency in initial RFLP analysis, samples were verified by DNA sequencing of the PCR products. Following PCR, samples were purified with the exonuclease I, shrimp alkaline phosphatase enzymatic purification system (ExoSAP-it; USB Corp. Cleveland, Ohio, USA) according to the manufacturer recommendations. Cycle sequencing was performed using the ABI Prism Big Dye Terminator Sequencing Kit v3.1 (ABI, Foster City, Calif., USA) diluted 1:4. Reaction products were purified by ethanol EDTA precipitation following the ABI sequencing kit protocol. Sequence products were run on an ABI Prism 3730 Genetic Analyzer and sequence data was analyzed using Sequencher 4.1.4 (Gene Codes Corporation, Ann Arbor, Mich., USA). Statistical Analysis Genotype frequencies are reported with their group percentages. Comparisons between genotype frequencies were made using Armitage trend tests. The MCP-1 –2518 A/G polymorphism is known to be functional and associated with acute pancreatitis [7], so the prior probability of a statistically significant test result being true was high [21]. Furthermore, a single SNP was tested, so no corrections were needed for multiple comparisons. Thus, p ! 0.05 was considered statistically significant with a low false-positive report probability [21].
Results
Patient Characteristics Table 1 contains the demographic data of our study population and table 2 the underlying etiologic risk factors according to the TIGAR-O classification of CP. The
Sass et al.
MCP-1 Genotypes The frequencies of the MCP-1 A/A homozygotes, A/G heterozygotes and G/G homozygotes in CP patients and controls are summarized in figure 1. In the control group, the genotype frequencies were: A/A (66/116, 57%), A/G (40/116, 34.5%) and G/G (10/116, 8.5%). Hardy-Weinberg estimation of allele frequencies were met, with the control samples matching the frequencies seen in other studies [9–11, 13]. In the chronic pancreatitis group, the genotype frequencies were: A/A (89/177, 50%), A/G (70/177, 40%) and G/G (18/177, 10%). There was no statistically significant difference between the genotypes of CP subjects and controls (p = 0.267).
60
Genotype frequency (%)
TIGAR-O classification is an etiological classification system developed by the Midwest Multicenter Pancreatic Study Group (renamed the North American Pancreatic Study Group) to categorize risk factors for CP according to various mechanisms [1].
A/A A/G G/G
50 40 30 20 10 0 Chronic pancreatitis
Healthy controls
Fig. 1. Frequencies of the –2518 MCP-1 genotypes in patients with CP and in healthy controls.
Table 1. Demographic data on patients and controls
Discussion
Acute and chronic pancreatitis are complex disorders with a strong genetic basis. The interaction between environmental and genetic factors determines susceptibility to acute pancreatitis, frequency of acute pancreatitis and progression to chronic pancreatitis [4, 5]. The effect of the MCP-1 –2518 polymorphism in acute pancreatitis is to alter the immunological response to pancreatic injury [7]. The possibility that the MCP-1 –2518 G allele also increases the risk of chronic pancreatitis has not been previously reported. Our results suggest that the association between this gene and susceptibility to chronic pancreatitis is minimal and not clinically relevant. We tested the hypothesis that the MCP-1 –2518 G allele was associated with a susceptibility to chronic pancreatitis. This hypothesis is relevant to questions about the mechanism linking acute pancreatitis to chronic pancreatitis. For example, is chronic pancreatitis more dependent on the severity of acute pancreatitis (as implied by the necrosis-fibrosis sequence model of chronic pancreatitis), or primarily an increased frequency of acute pancreatitis attacks (as seen in subjects with hereditary pancreatitis due to cationic trypsinogen mutations [4, 22, 23])? Based on our acute pancreatitis study, the MCP-1 –2518 polymorphism affects the severity of acute pancreatitis and therefore the likelihood of developing chronic pancreatitis based on the necrosis-fibrosis hypothesis. This did not appear to be the case in our population, sug-
MCP-1 and Chronic Pancreatitis
Gender Age range, years Mean age, years
Patients (n = 177)
Controls (n = 116)
86 male; 91 female 7–91 48.4
49 male; 67 female 25–84 62.7
Table 2. Etiologic risk factors associated with CP: TIGAR-O clas-
sification system Toxic-metabolic
alcohol hypercalcemia hyperlipidemia
Idiopathic Genetic
49 3 9 73
cationic trypsinogen (PRSS1) and SPINK1 (PSTI) CFTR
4 3
Autoimmune
5
Recurrent and severe acute pancreatitis
1
Obstructive
pancreas divisum sphincter of Oddi dysfunction duct obstruction (tumor) other
15 8 4 3
PRSS1 = UniGene name: protease, serine, 1; PSTI = pancreatic secretory trypsin inhibitor, UniGene name; SPINK 1 = serine protease inhibitor, Kazal type 1; CFTR = cystic fibrosis transmembrane conductance regulator.
Pancreatology 2006;6:297–300
299
gesting that other mechanisms are more important in determining susceptibility to chronic pancreatitis. Our results also show some important distinction between inflammatory processes in the pancreas compared to the liver. In hepatitis C the liver inflammation is chronic and anti-viral in nature. Under these circumstances the MCP-1 –2518 G allele may drive fibrosis by amplifying the intensity of the overall immune response [9]. In chronic pancreatitis, the nature of the chronic inflammation is not fully characterized, but the MCP-1 –2518 G allele does not seem to affect the threshold for developing fibrosis. Although the MCP-1 –2518 G allele does not appear to increase susceptibility to chronic pancreatitis, the study did not test whether the MCP-1 –2518 G allele altered the rate of progression of chronic pancreatitis from early to end-stage disease.
Determining the exact role of genetic susceptibility factors, disease modifier genes and environmental factors in complex diseases is highly dependent on the quality of phenotyping, the population being investigated, environmental exposures and epigenetic factors phenomena [24, 25]. The current study of 177 subjects and 116 well-characterized controls is sufficiently large to exclude a clinically relevant effect of the MCP-1 –2518 polymorphism as a susceptibility factor for chronic pancreatitis, but does not exclude other effects that are highly relevant to other aspects of pancreatic diseases.
Acknowledgement This study was supported by NIH DK 061451-01 (D.C.W.).
References 1 Etemad B, Whitcomb DC: Chronic pancreatitis: diagnosis, classification, and new genetic developments. Gastroenterology 2001; 120: 682–707. 2 Ammann RW, Heitz PU, Kloppel G: Course of alcoholic chronic pancreatitis: a prospective clinicomorphological long-term study. Gastroenterology 1996;111:224–231. 3 Ammann RW, Muellhaupt B: Progression of alcoholic acute to chronic pancreatitis. Gut 1994;35:552–556. 4 Whitcomb DC: Value of genetic testing in management of pancreatitis. Gut 2004; 53: 1710–1717. 5 Whitcomb DC: Advances in understanding the mechanisms leading to chronic pancreatitis. Nat Clin Practice Gastro-Hepatol 2004; 1: 46–52. 6 Rovin BH, Lu L, Saxena R: A novel polymorphism in the MCP-1 gene regulatory region that influences MCP-1 expression. Biochem Biophys Res Commun 1999;259:344–348. 7 Papachristou GI, Sass DA, Avula H, Lamb J, Lokshin A, Barmada MM, et al: Is the monocyte chemotactic protein-1 –2518 G allele a risk factor for severe acute pancreatitis? Clin Gastroenterol Hepatol 2005;3:475–481. 8 Adams DH, Lloyd AR: Chemokines: leukocyte recruitment and activation cytokines. Lancet 1997;349:490–495. 9 Muhlbauer M, Bosserhoff AK, Hartmann A, Thasler WE, Weiss TS, Herfarth H, et al: A novel MCP-1 gene polymorphism is associated with hepatic MCP-1 expression and severity of HCV-related liver disease. Gastroenterology 2003;125:1085–1093. 10 Szalai C, Kozma GT, Nagy A, Bojszko A, Krikovszky D, Szabo T, et al: Polymorphism in the gene regulatory region of MCP-1 is associated with asthma susceptibility and sever-
300
11
12
13
14
15
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