Genetic causes of pancreatitis have been continuously discovered. Molecular testing for at least 1 gene-PRSS1 (Protease, Serine, 1, OMIM 276000)-has been suggested (1). The PRSS1 encodes cationic trypsinogen, and its defects are the most common cause of hereditary pancreatitis (HP, OMIM 167800). It is assumed that these defects result in trypsin modification that is associated with increased resistance to autolysis and premature zymogen activation. Most PRSS1 defects are autosomal dominant inherited, displaying a high penetrance, and seem to be associated with an increased risk of pancreatic cancer (2).
Mutations in 3 other genes-SPINK1 (Serine Protease Inhibitor Kazal type 1, OMIM 167790), CFTR (Cystic Fibrosis Transmembrane Conductance Regulator, OMIM 602421) and AAT (OMIM 107400)-are thought to be factors predisposing to pancreatitis. SPINK1 is a serine protease inhibitor that regulates trypsin activity. As the most common SPINK1 mutation, N34S, is quite frequent and is present also in unaffected individuals, the SPINK1 defects seem to have a modifying rather than causative role in the pathogenesis of pancreatitis (3). Another member of the serpin superfamily, α1-antitrypsin (AAT), plays a very similar physiological role as SPINK1. AAT defects have been proposed to be associated with pancreatitis (4).
As cystic fibrosis (CF, OMIM 219700) patients can clinically present with exocrine pancreatic insufficiency and pancreatitis, a role of CFTR mutations in pathogenesis of pancreatitis was successfully inferred (5,6). According to current knowledge, not only the most common but also specific CFTR mutations can be associated with pancreatitis.
This work focused on the mutations screening of PRSS1, SPINK1, AAT and CFTR defects in pancreatitis. We present results of molecular analysis of defects in juvenile chronic and acute recurrent pancreatitis.
PATIENTS AND METHODS
Characterisation of Patients
Patients with chronic pancreatitis (CP) and acute recurrent pancreatitis (ARP) were admitted to the Department of Gastroenterology, Hepatology and Immunology of The Children's Memorial Health Institute between 2000 and 2004 from all of Poland (the Institute is a reference health service centre for children in Poland). They were enrolled for genetic and clinical investigation. A total of 92 unrelated Polish patients (age 5.2-24.3 years, mean age 14.6 years, 48 female and 44 male) and 55 family members were studied. The cohort of patients included 52 CP patients (age 5.2-23.9 years, mean age 15.1 years) and 40 with ARP (age 6.3-24.3 years, mean age 14.0 years). Chronic pancreatitis and ARP were defined according to the Cambridge classification (7,8). Criteria for ARP were defined as 3 or more episodes of acute pancreatitis without evidence of CP in imaging studies. Chronic pancreatitis was diagnosed as pancreatitis with evidence of CP in imaging studies according to the Cambridge classification system. For all patients, alcohol consumption was excluded during the taking of the history of the patient.
A positive family history of pancreatitis was found for 25 patients. For a group of patients, establishing of probable cause of pancreatitis was possible: hypertriglyceridemia (8 patients), anatomical anomalies as pancreas divisum or ansa pancreatica (14 patients), biliary disease (choledochocele, choledocholithiasis, cholelithiasis and primary sclerosing cholangitis) (10 patients), trauma (5 patients), autoimmune pancreatitis (2 patients), lambliosis, dermatomiositis, colitis ulcerosa and ascariasis (1 patient each) (Table 1). Twenty-seven patients were classified as idiopathic pancreatitis.
The patients were informed on the aims of the project, and they signified their written consent form for clinical and molecular procedures to be used. Fifty random DNA samples from the DNA bank of the Department of Medical Genetics, Institute of Mother and Child, were included in a control group. These control DNA samples were collected from patients from different parts of Poland. The local ethical committee approved this study.
The sweat chloride concentration was measured by pilocarpine iontophoresis and expressed in milliequivalents per liter (mEq/L). The faecal fat excretion (in grams per 24 hours) was evaluated by collecting stool samples pooled over a 3-day period (laboratory reference value: 0.8-4.0 g/d) (9). No special diet was prescribed by physician or used by patient. Faecal elastase 1 concentration was examined with a commercially available enzyme-linked immunosorbent assay kit (Schebo-Tech, Wettenberg, Germany; laboratory reference value: >200 μg/g). Chymotrypsin activity was determined by a colorimetric method (Monotest Chymotrypsin, Boeringher Mannheim Diagnostica, Mannheim, Germany; laboratory reference value: >6 U/g).
DNA was extracted from leukocytes of EDTA-anticoagulated blood according to Miller et al. (10). The PRSS1 variants were detected as described before with modifications (1,11-13). Additionally, all coding regions of the PRSS1 for a group of the first 30 patients have been sequenced. As only the most common R122H/C mutations were found, for the rest of the patients, standard analysis in the PRSS1 was used. Screening of the SPINK1 N34S mutation was performed as reported previously (14). Genotyping for E264V (PiS) and E342K (PiZ) variants of AAT was done in accordance with Henry et al. (15). For the first 50 patients enrolled in this study, the CFTR mutations F508del, G542X, G551D, R553X, N1303K, W1282X, 1717-1G/A, I507del, S1251N, R560T, 3905insT, Q552X (INNO-LiPA CFTR12, Innogenetics, Gent, Belgium), CFTRdele2,3 (16) and polyT variant in intron 8 (IVS8-T) (17) were analyzed. For the remaining patients (n = 42), only F508del (18), CFTRdele2,3 and polyT variant were analyzed, as they were shown to be present in the first group of patients and none of the rare mutations were found in that group.
Most of the statistical analysis was done using the 1-sided Fisher exact test. Significance was assumed at P < 0.05. Odds ratios (OR) were used for effect estimation with 95% confidence intervals (95% CI) (SSPS v8.0 for Windows, SSPS Inc, Chicago, IL). It was always stated when the 2-sided Fisher exact test had to be used.
In 31 of 92 pancreatitis patients (33.7% vs 24%, 12 of 50 in the control group, P = 0.157), we identified mutations in at least 1 of 4 genes examined (PRSS1, SPINK1, CFTR and AAT). Mutations were found more frequently in CP patients than in ARP patients, but the difference was not statistically significant (38.5% vs 27.5%; P = 0.379). In both groups, mean age at onset was similar (Table 2). A cohort of 16 patients from a group of 25 with positive family history for pancreatitis had identified mutations in tested genes. Molecular defects were found in 12 control samples: 5 cases of IVS8-5T/− (CFTR), 2 cases of N34S/− (SPINK1) and 5 cases of the E264V: 1x E264V/E264V, 4 x E264V/− (AAT).
Mutations in the SPINK1 and the PRSS1 were most frequent (8.7% vs 6.5% of total alleles, P = 0.020 and P = 0.005, respectively, Table 3). The PRSS1 (9.6% of CP alleles vs 2.5% of ARP alleles, P = 0.0942) and the CFTR mutations (7.7% of CP alleles vs 1.2% of ARP alleles, P = 0.0862) were detected mainly in CP patients, whereas the N34S SPINK1 mutation was found with similar frequency both in CP and ARP patients (7.7% vs 10.0%, P = 0.768).
Substitutions in R122 residue of the PRSS1 (R122H and R122C) were the most frequent defects. In 2 cases, we found N29I mutation; A16V mutation in the PRSS1 was not found in any of the patients examined (Table 4).
The frequency of identified mutations in the CFTR alleles was similar to the control group (4.9% vs 5%, P = 0.587, Table 3). The most often found mutation was the F508del. In 5 of 92 patients, we identified the 5T variant in IVS8-T locus (Table 4). The frequency of this variant in the Polish population is near 10% (5% for allele population). All patients with CFTR mutations had the normal value of the sweat test (<60 mEq/L) and had no other CF symptoms (Table 4). Only patient 1 (Table 4) had an increased level of fat excretion (laboratory reference value: 0.8-4.0 g/d).
In contrast, the overall frequency of mutations E264V and E342K in the AAT was lower than in the control group (Table 3).
The patients with the CFTR and/or the SPINK1 mutations had family history of pancreatitis not as often as PRSS1 patients. Moreover, not in all family members with PRSS1 mutations was the clinical history positive for CP, ARP or other pancreatic disease (eg, family members of patients 19, 22, 25 and 27; Table 4). Analysis of disease course in patients and their families with identified mutations in the examined genes showed that genes defects were of incomplete penetrance.
In the group of patients who did not have mutations in the examined genes, changes in pancreas tissue (1 + 2 + 3 degrees in Cambridge classification) appeared nearly one and a half times as rarely (OR = 3.147; 95% CI: 1.060, 9.344) as in patients carrying mutations (P = 0.027, Table 5). Furthermore, nearly twice as many patients with identified mutations had more severe changes (third degree) in pancreas tissue (OR = 2.900; 95% CI: 1.184, 7.108); and these results were statistically significant (P = 0.017).
A genetic predisposition to the familial form of pancreatitis was reported by Comfort and Steinberg in 1952 (19). More frequent detection of PRSS1, SPINK1 and CFTR defects in pancreatitis patients than in the general population opened a new chapter in diagnostics of idiopathic pancreatitis and understanding of molecular mechanisms of the disease. We analysed 92 patients with CP or ARP with the major objective of estimating the causative role of examined genes in disease pathology.
Several groups have reported an increased prevalence of the CFTR mutations in patients with CP (5,6,20). First reports suggested that idiopathic chronic pancreatitis was associated with a CFTR mutation in 1 allele, although other results suggested that CP patients may be compound heterozygotes. In our study, the frequency of the examined CFTR mutations and the IVS8-5T variant was similar to that observed in the control group and the Polish population (21). However, we tested only the most frequently known CFTR mutations. This analysis may underestimate the presence of the CFTR mutations because patients with CP or ARP are more likely to carry at least 1 mild mutation, whereas the test was designed to identify mainly severe CFTR defects. Therefore, it is possible that more comprehensive DNA testing (sequencing of the entire coding region) may have detected additional mutations. Similar to the French group study (20), our investigation did not show that the IVS8-5T allele is more common than expected among patients with pancreatitis (3.8% of CP and ARP alleles vs 5% of control alleles). Only ~2% of CF patients develop pancreatitis (22). Pancreatic insufficiency is a common symptom of CF because of the destruction of pancreatic exocrine tissue by an inflammatory process leading to fibrosis and fatty replacement. The absence of clinical pancreatitis in most CF patients is in agreement with the histopathological findings in those patients (23). Because CP or ARP progressing into CP during the course of the disease is not observed in most of the CFTR mutations carriers, in patients with pancreatic disease brought about only by CFTR mutations, some additional genetic or environmental factors may be required for disease development.
One of these genetic factors could be the SPINK1, which plays a major role in protecting the pancreas from premature trypsinogen activation. The N34S mutation was found 4 to 5 times more frequently (OR = 4.667; 95% CI: 1.051, 20.726) in patients with pancreatitis than in control group (P = 0.020). Several groups have confirmed that the most common N34S mutation is associated with chronic idiopathic pancreatitis in >20% of patients with pancreatitis (24,25). This mutation was also found in 6% of patients with chronic alcoholic pancreatitis (26). In this study, we excluded the N34S mutation as a potential dominant defect that causes the disease (Table 4). Many of our patients with pancreatitis are N34S carriers and developed the disease. However, many related N34S carriers do not present symptoms of pancreatitis. Patient 9 and her brother had the same genotype at SPINK1 locus (N34S/N34S), but the brother has not developed pancreatitis so far. He should be under special medical care because of the higher risk of pancreatitis. It is important to remember that patient 9, despite having N34S mutation in both alleles, also presents with anatomical anomalies (pancreas divisum), and this possibly was an additional factor for progression to pancreatitis. However, these data suggest that SPINK1 mutations alone are not capable of initiating pancreatitis, but they may be defects associated with the disease (3). The results of investigation to define a SPINK1 role in pancreatitis are still unclear.
Cationic trypsinogen (PRSS1) plays a central role in hydrolysing dietary proteins and is crucial for activating other digestive proenzymes (27). Premature activation of trypsinogen within the pancreas with activation of other enzymes leads to autodigestion of pancreas. This mechanism is believed to be a crucial one for development of acute pancreatitis. Recurrent episodes of acute pancreatitis cause chronic pancreatitis. The PRSS1 mutations N29I and R122H increase autoactivation. Furthermore, the R122H and R122C mutations stabilize cationic trypsin against autolysis ("super-trypsin" hypothesis) by eliminating the autolytic cleavage site R122 (13,28,29).
All mutations may affect the pancreatic protease-antiprotease equilibrium. This imbalance may initiate further activation of trypsinogen and other pancreatic zymogens, with subsequent autodigestion of the pancreas. In our studies, the analysis of family history of patients with PRSS1 mutations have confirmed hereditary pancreatitis in most of the cases (Table 4). However, some patients with PRSS1 mutations have negative family history of the disease. The penetrance of PRSS1 mutations was estimated to be ~80% 1(30). About 70% to 80% of affected patients from the EUROPAC study manifested symptoms by the age of 20 years (31). One should remember that patients can really have ancestors who were/are unaffected, but also in many of them, the etiology of abdominal pain in previous generations was not diagnosed.
Moreover, it was suggested that patients with PRSS1 mutation have a more than 50-fold increased risk of pancreatic ductal cancer as compared with expected pancreatic cancers in the general population (32). An international consensus recommended screening using the least invasive imaging to detect pancreatic cancer for PRSS1 patients older than 40 years (33). Unfortunately, there is no good screening test for early diagnosis of pancreatic cancer in high-risk groups.
AAT is human serum inhibitor of serine proteases such as neutrophil elastase, cathepsin G and trypsin. Inherited AAT deficiency is associated with pulmonary emphysema and liver disease. There is a hypothesis that increased levels of pancreatic proteinases or a decrease in pancreatic antiproteinases can lead to pancreatitis. In our study, the frequency of the PIS (E264V) and PIZ (E342K) alleles was lower than for control group (Table 3). The results did not confirm the association of AAT variants with CP or ARP. This is in agreement with other studies (34). The lack of the association of AAT deficiency with pancreatitis in this study is supported by the results of an analysis of 246 PIZ homozygous (E342K/E342K) individuals in which no case of pancreatitis symptoms was reported (35). Teich et al. (36) and Witt et al. (37) also found only moderating or no effect of AAT variants in the course of chronic pancreatitis.
Because of the low patient and control individual numbers presented, results of statistical analysis can be unstable. Every estimation could have been biased by this factor, especially when a frequency of analysed mutation is very low in the general population (eg, PRSS1 defects).
The PRSS1 defects seem to be strongly causative for CP or ARP, whereas defects in SPINK1 are suggested to be only defects associated with the disease. In the analyzed group of patients, the CFTR mutations are not associated with pancreatitis. The importance of AAT variants for CP or ARP pathogenesis remains speculative.
The authors thank Dr Niels Teich from Medizinische Klinik und Poliklinik II, Universitätsklinikum Leipzig, Leipzig, Germany, for reference DNA sample of N29I PRSS1 mutation. The technical assistance of Mrs Violetta Hryniewicz, Mrs Danuta Sielska and Mrs Malgorzata Rozwadowska is gratefully acknowledged.
1. Whitcomb DC, Gorry MC, Preston RA, et al. Hereditary pancreatitis
is caused by a mutation in the cationic trypsinogen gene. Nat Genet
2. Pho-Iam T, Thongnoppakhun W, Yenchitsomanus PT, et al. A Thai family with hereditary pancreatitis
and increased cancer risk due to a mutation in PRSS1 gene. World J Gastroenterol
3. Pfutzer RH, Barmada MM, Brunskill AP, et al. SPINK1/PSTI polymorphisms act as disease modifiers in familial and idiopathic chronic pancreatitis
4. Novis BH, Young GO, Bank S, et al. Chronic pancreatitis
and alpha-1-antitrypsin. Lancet
5. Sharer N, Schwarz M, Malone G, et al. Mutations of the cystic fibrosis gene in patients with chronic pancreatitis
. N Engl J Med
6. Cohn JA, Friedman KJ, Noone PG, et al. Relation between mutations of the cystic fibrosis gene and idiopathic pancreatitis
. N Engl J Med
7. Sarner M, Cotton PB. Definitions of acute and chronic pancreatitis
. Clin Gastroenterol
8. Sarner M, Cotton PB. Classification of pancreatitis
9. Van de Kamer JH, Ten Bokkel Huinink H, Weyers HA. Rapid method for the determination of fat in feces. J Biol Chem
10. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res
11. Witt H, Luck W, Becker M. A signal peptide cleavage site mutation in the cationic trypsinogen gene is strongly associated with chronic pancreatitis
12. Ferec C, Raguenes O, Salomon R, et al. Mutations in the cationic trypsinogen gene and evidence for genetic heterogeneity in hereditary pancreatitis
. J Med Genet
13. Simon P, Weiss FU, Sahin-Toth M, et al. Hereditary pancreatitis
caused by a novel PRSS1 mutation (Arg-122→Cys) that alters autoactivation and autodegradation of cationic trypsinogen. J Biol Chem
14. Plendl H, Siebert R, Steinmann D, et al. High frequency of the N34S mutation in the SPINK1 gene in chronic pancreatitis
detected by a new PCR-RFLP assay. Am J Med Genet
15. Henry MT, Cave S, Rendall J, et al. An alpha1
-antitrypsin enhancer polymorphism is a genetic modifier of pulmonary outcome in cystic fibrosis. Eur J Hum Genet
16. Dork T, Macek M, Mekus F, et al. A novel 21 kilobase deletion, CFTR dele2,3 (21 kb), in the CFTR gene: a cystic fibrosis mutation of Slavic origin common in Central and East Europe. Hum Genet
17. Chillon M, Casals T, Mercier B, et al. Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deference. N Engl J Med
18. Rommens J, Kerem BS, Greer W, et al. Rapid nonradioactive detection of the major cystic fibrosis mutation. Am J Hum Genet
19. Comfort M, Steinberg A. Pedigree of a family with hereditary chronic relapsing pancreatitis
20. Audrezet MP, Chen JM, Le Marechal C, et al. Determination of the relative contribution of three genes-the cystic fibrosis transmembrane conductance regulator gene, the cationic trypsinogen gene, and the pancreatic secretory trypsin inhibitor gene-to the etiology of idiopathic chronic pancreatitis
. Eur J Hum Genet
21. Aznarez I, Bal J, Casals T, et al. Analysis of mutations in the CFTR gene in patients diagnosed with cystic fibrosis in Poland. Med Wieku Rozwoj
22. Durno C, Corey M, Zielenski J, et al. Genotype and phenotype correlations in patients with cystic fibrosis and pancreatitis
23. Oppenheimer EH, Esterly JR. Pathology of cystic fibrosis: review of the literature and comparison with 146 autopsied cases. Perspect Pediatr Pathol
24. Lempinen M, Paju A, Kemppainen E, et al. Mutations N34S and P55S of the SPINK1 gene in patients with chronic pancreatitis
or pancreatic cancer and in healthy subjects: a report from Finland. Scand J Gastroenterol
25. Witt H, Luck W, Hennies HC, et al. Mutations in the gene encoding the serine protease inhibitor, Kazal type 1 are associated with chronic pancreatitis
. Nat Genet
26. Witt H, Luck W, Becker M, et al. Mutation in the SPINK1 trypsin inhibitor gene, alcohol use, and chronic pancreatitis
27. Rinderknecht H. Pancreatic secretory enzymes. In: Go VLW, DiMagno EP, Gardner JD, et al, eds. The Pancreas: Biology, Pathobiology and Disease
, 2nd ed. New York: Raven, 1993:219-51.
28. Sahin-Toth M, Toth M. Gain-of-function mutations associated with hereditary pancreatitis
enhance autoactivation of human cationic trypsinogen. Biochem Biophys Res Commun
29. Kukor Z, Toth M, Pal G, et al. Human cationic trypsinogen. Arg(117) is the reactive site of an inhibitory surface loop that controls spontaneous zymogen activation. J Biol Chem
30. Sibert JR. Hereditary pancreatitis
in England and Wales. J Med Genet
31. Howes N, Lerch MM, Greenhalf W, et al. Clinical and genetic characteristics of hereditary pancreatitis
in Europe. Clin Gastroenterol Hepatol
32. Lowenfels AB, Maisonneuve P, DiMagno EP, et al. Hereditary pancreatitis
and the risk of pancreatic cancer. International Hereditary Pancreatitis
Study Group. J Natl Cancer Inst
33. Ulrich CD, Consensus Committees of the European Registry of Hereditary Pancreatic Diseases, Midwest Multi-Center Pancreatic Study Group, International Association of Pancreatology. Pancreatic cancer in hereditary pancreatitis
: consensus guidelines for prevention, screening and treatment. Pancreatology
34. Braxel C, Versieck J, Lemey G, et al. Alpha 1-antitrypsin in pancreatitis
35. Larsson C. Natural history and life expectancy in severe alpha1-antitrypsin deficiency, PiZ. Acta Med Scand
36. Teich N, Walther H, Bodeker J, et al. Relevance of variants in serum antiproteinases for the course of chronic pancreatitis
. Scand J Gastroenterol
37. Witt H, Kage A, Luck W, et al. Alpha1-antitrypsin genotypes in patients with chronic pancreatitis
. Scand J Gastroenterol