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Original Articles: Gastroenterology

Analysis of CFTR, SPINK1, PRSS1 and AAT Mutations in Children With Acute or Chronic Pancreatitis

Sobczyńska-Tomaszewska, Agnieszka*; Bąk, Daniel*†; Oralewska, Beata; Oracz, Grzegorz; Norek, Aleksandra; Czerska, Kamila*†; Mazurczak, Tadeusz*; Teisseyre, Mikołaj; Socha, Jerzy; Zagulski, Marek; Bal, Jerzy*

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Journal of Pediatric Gastroenterology and Nutrition: September 2006 - Volume 43 - Issue 3 - p 299-306
doi: 10.1097/01.mpg.0000232570.48773.df
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Abstract

INTRODUCTION

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.

TABLE 1
TABLE 1:
Possible causes of pancreatitis in examined patients

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.

Biochemical Analysis

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).

Molecular Analysis

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.

Statistical Analysis

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.

RESULTS

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).

TABLE 2
TABLE 2:
Characteristics of CP and ARP patients

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).

TABLE 3
TABLE 3:
Frequency of mutated alleles of CFTR, PRSS1, SPINK1 and AAT in pancreatitis patients

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).

TABLE 4
TABLE 4:
Characteristics of pancreatitis patients with identified mutations in the analysed genes

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).

TABLE 5
TABLE 5:
Characteristics of severity of disease course according to Cambridge classification system (6,7) in pancreatitis patients

DISCUSSION

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.

Acknowledgments

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.

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Keywords:

Pancreatitis; AAT; CFTR; PRSS1; SPINK1

© 2006 Lippincott Williams & Wilkins, Inc.