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Genetic Mutations in a Spanish Population with Chronic Pancreatitis
Original Paper Pancreatology 2009;9:644–651 DOI: 10.1159/000181177
Received: April 22, 2008 Accepted after revision: October 29, 2008 Published online: August 4, 2009
Genetic Mutations in a Spanish Population with Chronic Pancreatitis Josefina Mora a Laia Comas a Elia Ripoll a Patricia Gonçalves b Josep M. Queraltó a Francesc González-Sastre a Antoni Farré b Departments of a Clinical Chemistry and b Gastroenterology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
Key Words Genetic mutations ⴢ Chronic pancreatitis ⴢ Idiopathic chronic pancreatitis ⴢ Alcohol-related pancreatitis ⴢ Hereditary pancreatitis
Abstract Background/Aims: Mutations in the PRSS1 and the SPINK1 genes have variably been associated with alcohol-related, idiopathic and hereditary chronic pancreatitis (CP). The aim of this study was to determine for the first time the significance of PRSS1, SPINK1 mutations and genetic variants of AAT in a group of Spanish patients with CP. Methods: 104 consecutive patients with CP were included, as well as 84 healthy control subjects. The R122H and N29I mutations in the PRSS1 gene, the N34S mutation in the SPINK1 gene and PiS and PiZ mutations in the AAT gene were analyzed by RFLP-PCR methods. Results: No R122H mutation was found in the PRSS1 gene, and N29I mutation was detected in 7.7% of CP patients. A N29I mutation was observed in 3.9% of patients with alcohol-related pancreatitis (ACP). A total of 5.8% of CP patients were identified with the N34S mutation. Genotype MS, SS and MZ were detected in 18.3, 3.8 and 1.3% of CP patients, respectively. Conclusion: The percentage of N29I mutations in ACP patients was higher than that reported in other studies, while the percentage of N34S and AAT mutations in ACP and idiopathic CP patients was similar. Copyright © 2009 S. Karger AG, Basel and IAP
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Introduction
Chronic pancreatitis (CP) is an ongoing disease characterized by chronic inflammation and progressive fibrosis associated with loss of exocrine and/or endocrine function. Among the etiological factors involved in its pathogenesis, alcohol is considered the main risk factor in Western countries, accounting for 70–90% of all cases [1]. However, only 10–20% of alcoholics develop the disease [2]. In approximately 10–30% of CP patients no association is found with alcohol or other risk factors and the disease is classified as idiopathic chronic pancreatitis (ICP). In recent years, epidemiological and clinical studies in several countries suggest a possible genetic basis in CP. In 1996 the discovery of a mutation in the cationic trypsinogen gene (PRSS1) as a cause of hereditary CP contributed greatly to our knowledge of CP pathogenesis [3]. The search to characterize genes that may render individuals susceptible to pancreatic disease, including alcohol-related pancreatitis (ACP) and ICP, has since been extended to other possible candidates. The first is the serine protease inhibitor, Kazal type 1 (SPINK1), which provides a defense mechanism against premature activation of trypsinogen [4]. Other candidates include the cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible for cystic fibrosis [5, 6], genes implicated in pancreatic enzymatic activity such as the genetAntoni Farré, MD Department of Gastroenterology, Hospital de la Santa Creu i Sant Pau Av. Antoni M. Claret 167, ES–08025 Barcelona (Spain) Tel. +34 93 291 9141, Fax +34 93 291 9278 E-Mail [email protected]
Table 1. Characteristics of the CP patients and controls
Patients Sex, M/F Age, years Age at onset, years
ACP
ICP
HP
Others
Total CP
Controls
78 66/12 52 (35–76) 45 (30–76)
16 10/6 59.5 (19–83) 57 (14–82)
3 1/2 35 (13–72) 16 (13–43)
7 4/3 50 (24–73) 36 (18–69)
104 81/23 52 (13–83) 44.5 (8–82)
84 37/47 46 (18–87) –
Age values are expressed as median and range.
ic variants of ␣1-antitrypsin, and genes involved in alcohol metabolism [7]. Some of the aforementioned genes have undergone genetic testing in both ACP and ICP patients. PRSS1 mutations [8] have a low incidence in ICP patients [9, 10], and constitute a minor finding in ACP [11, 12]. In the case of SPINK1, since a N34S mutation was first described in 18 out of 96 (19%) unrelated children and adolescents in an ICP series in Germany [13], other studies have confirmed a high frequency – ranging from 6.4 to 28.6% – of this mutation in cohorts of ICP patients [14, 15]. In contrast, the frequency of mutations in ACP patients is about 5– 6%. The small differences compared to controls suggest that this genetic variant does not play a major role in these patients. Furthermore, approximately 1–2.5% of the normal population is heterozygous for the N34S mutation [16, 17]. All genetic variants of PRSS1 and SPINK1 genes are described in a continuously updated database [18]. With regard to ␣1-antitrypsin (AAT), the genetic variants of this enzyme do not appear to play a predominant role in chronic non-alcoholic pancreatitis [19, 20] or in ACP [21]. However, little is known about its possible impact on the pathogenesis of CP. The aim of this study was to determine the significance of PRSS1, SPINK1 mutations and genetic variants of AAT in a group of Spanish patients with CP. This is the first such study in the CP population in Spain.
Material and Methods Patients Between April 2000 and February 2005, 104 consecutive Caucasian patients with CP (81 men, 23 women; median age 52 years, range 13–83 years) were included. CP was diagnosed in accordance with the standard diagnostic criteria consisting of a typical history of pain, suggestive imaging tests (CT scan, MR and/or endoscopic ultrasonography), impaired pancreatic exocrine function when assessed and histological analysis in cases in which
Genetic Mutations in a Spanish Population with Chronic Pancreatitis
surgical resection was indicated. In this series, alcohol abuse, 1 60 g/day for at least 2 years, was considered the only etiological factor in 78 patients. An idiopathic etiology was considered in 16 patients. Three affected members of the same family were included in the diagnosis of hereditary pancreatitis (HP) based on criteria of 3 or more relatives with CP in two or more generations [22, 23]. CP was considered to be due to other causes in 7 patients (autoimmune CP in 3, obstructive CP in 2, familiar hypertriglyceridemia in 1 and chronic renal failure in 1). No ethnic differences were observed in this series. To analyze the possible contribution of genetic alterations in the onset and course of the disease, the most frequent CP complications (calcifications, pseudocyst and biliary obstruction) were taken into consideration. The onset of CP was defined as the initial manifestation of either the first episode of acute pancreatitis or the start of symptomatic complications. The age threshold to consider an early onset was 20 years (table 1). Tobacco smoking was also taken into account. Patients consuming 110 cigarettes/ day were considered smokers. The majority of the smokers included in this study exceeded this figure. The control group was made up of 84 healthy volunteers from the same institution. The study had the approval of the Ethics Committee at Hospital de la Santa Creu i Sant Pau and written informed consent was obtained from all the patients. DNA Extraction Genomic DNA was extracted from 3 ml of peripheral venous blood samples using EDTA tubes according to QIAamp쏐 DNA Blood Mini Kit (Qiagen, Basel, Switzerland) instructions. Detection of PRSS1 Mutations Two mutations were analyzed in the cationic trypsinogen (PRSS1) gene: the R122H in exon 3 and the N29I in exon 2. The R122H mutation was analyzed by means of a restriction fragment length polymorphism method in products amplified by PCR (RFLP-PCR), using the primers listed in table 2 and the Pml I enzyme (New England BioLabs, Ipswich, Mass., USA). This enzyme has one restriction site [CACGTG] in wild-type amplified samples and two restriction sites in mutant R122H samples [24]. After polyacrylamide gel electrophoresis (9%) and ethidium bromide staining, the bands which depicted the R122H mutant allele (208 and 20 bp), wild-type allele (228 bp) and control digestion (327 bp) were visualized (fig. 1). A RFLP-PCR method which uses the primers listed in table 2 and the restriction enzyme HpyCH4 III (New England BioLabs)
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Table 2. PCR primers used in the study
PRSS1 Exon 2 Exon 3
Forward Reverse Forward Reverse
5ⴕ-CGC CAC CCC TAA CAT GCT AT-3ⴕ 5ⴕ-CTC TCC CAG GCA GAC TGG CC-3ⴕ 5ⴕ-GGT CCT GGG TCT CAT ACC TT-3ⴕ 5ⴕ-GTA ATG GGC ACT CGA AAT GT-3ⴕ
SPINK1 Exon 3 Forward Reverse AAT Exon 3 Exon 5
5ⴕ-AGT TTC AGA AGG GCC ATA GGA-3ⴕ 5ⴕ-GTT TGC TTT TCT CGG GGT GAG-3ⴕ
Forward (SF) Reverse (SR) Forward (ZF) Reverse (ZR)
5ⴕ-AGG GGA AAC TAC AGC ACC TCG-3ⴕ 5ⴕ-TGG GTA CTG TTC TCC TCA TCG AGC ATG-3ⴕ 5ⴕ-GGC TGT GCT GAC CAT CGT C-3ⴕ 5ⴕ-AAC TCT TCT TTA ATG TCA TCG AGG-3ⴕ
Uncut 555 bp
Uncut 266 bp 181 bp
327 bp 102 bp 85 bp 79 bp
228 bp 208 bp Neg. 1 R122R
H
2 H 3
H 4
H 5 Pos. R122H
Uncut Neg. 1 N29N
2
3
4
H Pos. N29I
Fig. 1. Detection of R122H mutation in the PRSS1 gene by RFLP/ PCR using PmI I enzyme. Negative control (228-bp wild-type band, R122R). Negative samples (1–4). Control samples without DNA (H). Positive control (208-bp mutant band, R122H). Control digestion (327-bp band) and Uncut (555-bp band). Molecular weight marker (⌽ ! 174, Hae III digested).
Fig. 2. Detection of N29I mutation in the PRSS1 gene by RFLP/ PCR using HpyCH4 III enzyme. Negative control (102- and 79-bp wild-type bands, N29N). Negative samples (1–4). Control sample without DNA (H). Positive control (181-bp mutant band, N29I). Control digestion (85-bp band) and Uncut (266-bp band). Molecular weight marker (⌽ ! 174, Hae III digested).
was adapted to detect the N29I mutation [25]. Using this strategy, a 266-bp fragment was obtained. Wild-type alleles showed two restriction sites [ACTGT] for the enzyme: the control digestion site and the restriction site lost when the N29I mutation is present. Therefore, after polyacrylamide gel electrophoresis (12%), three fragments were obtained for wild-type samples (102, 79 and 85 bp) and two fragments for the N29I mutant samples (181 and 85 bp) (fig. 2).
were generated from the wild-type allele (298, 116, and 20 bp) and two additional fragments (189 and 109 bp) when the N34S mutation was present. The 116-bp fragment depicted the control digestion band (fig. 3).
Detection of the SPINK1 Mutation The N34S mutation in exon 3 of the gene SPINK1 was detected by RFLP-PCR, with the primers listed in table 2. We used a modified version of the method described by Teich et al. [26]. The restriction enzyme HpyCH4 III (New England BioLabs) has two restriction targets [ACTGT] in the wild-type samples and three restriction targets in the mutant N34S samples. The digested products were then run on a polyacrylamide gel electrophoresis (12%) followed by ethidium bromide staining. Three fragments
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Detection of the AAT Mutations The PiS and PiZ mutations were detected by a RFLP-PCR method, using the mutated primers listed in table 2. These primers are able to introduce an artificial restriction site for Taq I (Fermentas, Glen-Burnie, Md., USA) that is lost when the mutation is present. Taq I has two restriction targets [TCGA] in the wild-type genotype samples (MM) and one target in the mutant PiS and PiZ samples [27]. The PCR generated a 285-bp product in the study of PiS mutation and a 250-bp product in the study of PiZ mutation. The digested PCR samples were run on a polyacrylamide gel electrophoresis (8%) and stained with ethidium bromide. In the study of PiS mutation, three fragments were observed (245, 21 and 19 bp) for the wild-type and two fragments (264 and 21 bp) when the
Mora /Comas /Ripoll /Gonçalves / Queraltó /González-Sastre /Farré
Table 3. Distribution of the PRSS1, SPINK1 and AAT mutations in CP patients and controls
ACP (n = 78)
ICP (n = 16)
HP (n= 3)
Others (n = 7)
Total CP (n = 104)
Controls (n = 84)
PRSS1 mutations R122H N29I
0 3/78 (3.9%)
0 1/16 (6.3%)
0 3/3*** (100%)
0 1a/7 (14.3%)
0 8/104** (7.7%)
0 0
SPINK1 mutation N34S
3/78 (3.9%)
3/16** (18.8%)
0
0
6/104* (5.8%)
0
3/16 (18.8%) 2/16 (12.5%) 0
2/3 (66.7%) 1/3 (33.3%) 0
1b/7 (14.3%) 0 0
AAT mutations MS SS MZ
13/78 (16.9%) 1/78 (1.3%) 1/78 (1.3%)
19/104 (18.3%) 4/104 (3.8%) 1/104 (0.96%)
14/84 (16.6%) 1/84 (1.2%) 1/84 (1.2%)
Percentages refer to the proportion of patients within each given group. * p < 0.05 vs. control; ** p < 0.01 vs. control; *** p < 0.001 vs. control. a Obstructive. b Autoimmune cause.
mutations were analyzed by the Mann-Whitney U test (continuous variables) and Fisher’s exact test (categorical data). p ! 0.05 was considered significant. Uncut 434 bp 298 bp
Results
189 bp 116 bp 109 bp Neg. 1 2 H 3 4 N34N
H 5 6 H Pos. N34S
Fig. 3. Detection of N34S mutation in the SPINK-1 gene by RFLP/
PCR using HpyCH4 III enzyme. Negative control (298-bp wildtype bands, N34N). Negative samples (1, 3–6). Positive sample (2). Control samples without DNA (H). Positive control (189- and 109-bp mutant band, N34S). Control digestion (116-bp band) and Uncut (434-bp band). Molecular weight marker (⌽ ! 174, Hae III digested).
PiS mutation was present. In the study of PiZ mutation, three fragments (209, 23 and 18 bp) were observed for the wild-type and two fragments (227 and 23 bp) when the PiZ mutation was present. Statistical Analysis The two-sided Fisher’s exact test was used to analyze differences in the frequency of PRSS1, SPINK1 and AAT mutations among ACP, ICP and HP patients as well as in controls. The association of various clinical parameters (age at onset, exocrine insufficiency, calcifications, etc.) and the presence or absence of
Genetic Mutations in a Spanish Population with Chronic Pancreatitis
Distribution of PRSS1, SPINK1 and AAT Mutations in CP Patients The frequency of mutations of the PRSS1 and SPINK1 genes and ␣1-antitrypsin deficiency alleles is summarized in table 3. All controls were negative for the R122H and the N29I mutations of the PRSS1 gene. The R122H mutation was not detected in any of the four CP groups studied. The N29I mutation was detected in 8/104 of the studied patients (7.7%; p = 0.007 vs. controls). According to the etiology, 3 patients (3.9%) in the ACP group, 1 patient (6.3%) in the ICP group, 3 out of 3 patients (100%) in the HP group (p ! 0.001 vs. controls) and 1 patient with an obstructive cause of CP had this N29I mutation. No family history of pancreatitis was ascertained in the ACP, ICP and obstructive causes of pancreatitis with this mutation. The 3 affected HP patients were homozygous for this mutation while the remaining 5 patients were heterozygous. In the analysis of the SPINK1 gene, no N34S mutation was observed in the control group. A total of 6 patients (5.8%; p = 0.027 vs. controls) were identified with the N34S mutation, 3 patients (3.9%) belonging to the ACP group and 3 patients (18.8%) belonging to the ICP group Pancreatology 2009;9:644–651
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Table 4. Complications of pancreatitis and tobacco smoking in CP patients
Patients Age at onset, years Diabetes mellitus Exocrine insufficiency Calcifications Biliary obstruction Pseudocysts Surgical procedures Tobacco smoking
PRSS1 mutation: N29I
SPINK1 mutation: N34S
positive
negative
positive
negative
8 41 (13–68) 1 (12.5%) 4 (50%) 6 (75%) 2 (25%) 2 (25%) 2 (25%) 4 (50%)
96 45 (8–82) 52 (54.2%) 44 (45.8%) 69 (71.8%) 34 (35.4%) 41 (42.7%) 36 (37.5%) 78 (81.2%)
6 41.5 (14–64) 3 (50%) 5 (83.3%)* 5 (83.3%) 0 1 (16.7%) 0 5 (83.3%)
98 44.5 (8–82) 50 (51%) 43 (43.9%) 70 (71.4%) 36 (36.7%) 42 (42.9%) 38 (38.9%) 77 (78.6%)
Age is expressed as median and range. Percentages refer to the proportion of patients within each given group. * p < 0.05.
(p = 0.003 vs. controls). The 6 patients were heterozygous for N34S mutation. The combined analysis of common mutations of PRSS1 and SPINK1 genes detected at least one mutation in 6/78 patients (7.7%) in the ACP group (p = 0.011 vs. controls) and in 4/16 patients (25%) in the ICP group (p ! 0.001 vs. controls). The mutations N29I and N34S did not coexist in any patient of the ACP or ICP group. In the study of the AAT deficiency alleles, genotype MS was detected in 14 controls (16.6%) and in a total of 19 patients (18.3%); 13 patients in the ACP group, 3 patients in the ICP group, 2 patients in the HP group and 1 patient with autoimmune pancreatitis. The genotype SS was identified in 1 control (1.2%) and in a total of 4 patients (3.8%); 1 patient in the ACP group, 2 patients in the ICP group and 1 patient in the HP group. The genotype MZ was found in 1 control (1.2%) and in 1 patient (0.96%) belonging to the ACP group. No significant differences were observed between patients and controls in the frequency of AAT deficiency alleles (p = 0.1). Nevertheless, we detected AAT mutations in the 3 members of the family with HP (SS mutation in 1 member and MS mutation in the other 2 members). Mutation Status and Clinical Outcome in CP Patients As shown in table 1, the median age at onset of the symptoms was 45 years (range 30–76) in patients with ACP, 57 years (range 14–82) in patients with ICP, and 16 years (range 13–43) in patients with HP. The presence of the N29I or N34S mutations in the total group of CP patients was not associated (p = 0.325 and p = 0.590, respectively) with early onset of the disease, 648
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except for N29I in HP patients (p = 0.045). The age at onset in 2/3 ICP patients with N34S mutation was !20 years, but the correlation was not statistically significant (p = 0.071). Table 4 shows the association of N29I or N34S mutations and aspects of disease severity and complications (diabetes mellitus, exocrine insufficiency, calcifications, biliary obstruction, pseudocysts or surgical procedures) and tobacco smoking. No significant association was found except for the detection of the N34S mutation and the presence of exocrine pancreatic insufficiency (p = 0.043). In addition, this correlation was maintained in ACP patients when evaluated separately (p = 0.036).
Discussion
The significance of PRSS1, SPINK1 mutations and genetic variants of ␣1-antitrypsin has not been reported previously in a Spanish population. While PRSS1 mutations have revealed an association with HP, they can also be found in patients with ICP, though the incidence is very low. In this study, no R122H mutation of the PRSS1 gene was observed in any of the groups evaluated. The N29I mutation, the second most prevalent mutation in the PRSS1 gene [8], was detected in the 3 HP patients and in 6.3% of ICP, patients. Several large studies focused on the association of ICP, and the most common trypsinogen mutations (R122H and N29I) have shown a frequency of mutations ranging from 0.2 to 10% of patients [28– 32]. PRSS1 mutations are rarely found in ACP patients [33–35]. O’Reilly et al. [11] described the R122H mutaMora /Comas /Ripoll /Gonçalves / Queraltó /González-Sastre /Farré
tion in 1/36 ACP patients (2.8%) and Bernardino et al. [12] reported a PRSS1 mutation in 1/64 ACP patients (1.5%). In the present study, the percentage of mutations in the PRSS1 gene was higher in the ACP group of patients; the N29I mutation was detected in 3/78 of ACP patients (3.9%). Furthermore, the N29I mutation was found in 1 patient with obstructive CP. The presence of the N29I mutation was significantly associated with an early onset of the disease in HP patients. Several large studies [36] have reported that symptoms in HP patients harboring the N29I mutation appear at a younger age, such as 14 years. No significant association was found with any aspects of disease severity, or tobacco smoking. In the case of the SPINK1 gene, Witt et al. [13], Chen et al. [37], and Pfützer et al. [38] have recently reported the presence of N34S mutations in patients with CP. Whether these mutations induce pancreatitis by themselves or together with other causative factors remains controversial. N34S mutations have been associated with the occurrence of pancreatitis [13] and observed in both Caucasian and Japanese pancreatitis patients [39], but other authors have suggested that even though there was no association between SPINK1 mutations and pancreatitis in HP patients [37, 38], this association was confirmed in ICP patients [14, 38]. Pfützer et al. [38] suggested that mutations in this gene might promote a predisposition to pancreatitis, possibly by lowering the threshold when other factors are present, and they concluded that SPINK1 mutations act as disease modifiers changing the phenotypic expression of the disease [40]. In the study of our population, the N34S mutation was found in 3/16 patients (18.8%) with ICP. This frequency is higher than that observed in several reports: 5% [41], 10.3% [9] and 13% [42]; similar to the range acknowledged in other studies: 18.6% [13] and 23.1% [43] and lower than that described in some series: 25.9% [38] and 28.6% [15]. Chen et al. [14] found that the frequency of N34S mutations in ICP patients differed according to age groups, changing from 6.4% in the total ICP group to 14.7% in their ^20-year-old ICP group. In our study, two thirds of the ICP patients with a N34S mutation were !20 years of age when diagnosed, supporting the association of the N34S mutation with an early-onset ICP [44]. These patients were heterozygous for the N34S mutation; in previous studies [13, 14, 38] no phenotypic differences between heterozygous and homozygous N34S ICP patients were detected. In the Spanish population, as in other industrialized countries, alcohol is the major etiological factor for CP. The N34S mutation in the SPINK1 gene was
detected in 3.9% of patients with ACP, similar to the range of frequencies (0–6%) reported from other European countries and Japan [16, 17, 35, 42]. In a recent study from Finland a higher frequency (10%) was reported [45]. Concerning disease severity, the presence of N34S mutations was statistically associated with pancreatic exocrine insufficiency in total CP patients and in the ACP group. The N34S mutation was not detected in controls, although in other reports based on a higher number of controls the mutation was described in about 0.8% (4/540) [16]. A protease-to-protease inhibitor imbalance in patients with AAT deficiency was previously thought to contribute to the development of CP. Some authors supported an association between CP and deficient AAT alleles [46] while others did not [21]. 23 of 104 patients (22%) with CP were hetero- or homozygous (19 and 4, respectively) for S allele, and 1/104 CP patients (0.96%) was heterozygous for Z allele. The PiZ mutation was found heterozygously in 1 patient with ACP (1.3%) and was absent in the ICP and HP groups. The frequency for AAT deficiency alleles in CP patients did not significantly differ from those reported in a control Spanish population [47]. Like ourselves, Witt et al. [20] found no significant differences between AAT deficiency allele in CP patients and controls. The PiS mutation frequency in CP patients in Spain (22%) was higher than that reported in Germany (4.1%), while the PiZ frequency was lower (0.96 vs. 2%) [19]. In conclusion, in the present study the percentage of N29I mutations in the PRSS1 gene in ACP patients was higher than that reported in published data, while the percentage of N34S mutations in the SPINK1 gene in ACP and ICP patients was similar to findings in other studies and populations. The N34S mutation was associated with exocrine insufficiency in total CP and ACP patients. Although PiS mutation frequency in the AAT gene in our CP population was elevated, no significant differences were observed with respect to reported controls.
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Acknowledgments This study was supported in part by a grant from the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo of Spain. Redes de Investigación Cooperativa, G03/156. The authors thank Assumpta Antonijuan for technical assistance and Carolyn Newey for language editing support.
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27 Braun A, Meyer P, Cleve H, Roscher AA: Rapid and simple diagnosis of the two common ␣1-proteinase inhibitor deficiency alleles Pi*Z and Pi*S by DNA analysis. Eur J Clin Chem Biochem 1996; 34:761–764. 28 Simon P, Weiss FU, Zimmer KP, Rand S, Brinkmann B, Domschke W, et al: Spontaneous and sporadic trypsinogen mutations in idiopathic pancreatitis. JAMA 2002; 288: 2122. 29 Creighton J, Lyall R, Wilson DI, Curtis A, Charnley R: Mutations of the cationic trypsinogen gene in patients with chronic pancreatitis. Lancet 1999;354:42–43. 30 Chen JM, Piepoli Bis A, Le Bodic L, Ruszniewski P, Robaszkiewicz M, Deprez PH, et al: Mutational screening of the cationic trypsinogen gene in a large cohort of subjects with idiopathic chronic pancreatitis. Clin Genet 2001;59:89–193. 31 Truninger K, Köck J, Wirth HP, Muellhaupt B, Arnold C, von Weizsäcker F, et al: Trypsinogen gene mutations in patients with chronic or recurrent acute pancreatitis. Pancreas 2001;22:18–23. 32 Teich N, Bauer N, Mössner J, Keim V: Mutational screening of patients with nonalcoholic chronic pancreatitis: identification of further trypsinogen variants. Am J Gastroenterol 2002;97:341–346. 33 Teich N, Mössner J, Keim V: Screening for mutations in the cationic trypsinogen gene: are they of relevance in chronic alcoholic pancreatitis? Gut 1999; 44:413–416. 34 Monaghan KG, Jackson CE, Kukuruga DL, Feldman GL: Mutation analysis of the cystic fibrosis and cationic trypsinogen genes in patients with alcohol-related pancreatitis. Am J Med Gen 2000;94:120–124. 35 Perri F, Piepoli A, Stanziale P, Merla A, Zelante L, Andriulli A: Mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the cationic trypsinogen (PRSS1) gene, and the serine protease inhibitor, Kazal type 1 (SPINK1) gene in patients with alcohol chronic pancreatitis. Eur J Hum Genet 2003;11:687–692. 36 Howes N, Lerch MM, Greenhalf W, Stocken DD, Ellis I, Simon P, et al: Clinical and genetic characteristics of hereditary pancreatitis in Europe. Clin Gastroenterol Hepatol 2004;2:252–261. 37 Chen JM, Mercier B, Audrezet MP, Ferec C: Mutational analysis of the human pancreatic secretory trypsin inhibitor gene in hereditary and sporadic chronic pancreatitis. J Med Genet 2000;37:67–69. 38 Pfützer RH, Barmada MM, Brunskill AP, 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.
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39 Nishimori I, Onishi S: Hereditary pancreatitis in Japan: a review of pancreatitis-associated gene mutations. Pancreatology 2001; 1: 444–447. 40 Pfützer RH, Whitcomb DC: SPINK1 mutations are associated with multiple phenotypes. Pancreatology 2001; 1:457–460. 41 Ockenga J, Dörk T, Stuhrmann M: Low prevalence of SPINK1 gene mutations in adult patients with chronic idiopathic pancreatitis. J Med Genet 2001;38:243–244.
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42 Kume K, Masamune A, Mizutamari H, Kaneko K, Kikuta K, Satoh M, et al: Mutation in the serine protease inhibitor Kazal type 1 (SPINK1) gene in Japanese patients with pancreatitis. Pancreatology 2005; 5: 354– 360. 43 Noone PG, Zhou Z, Silverman LM, Jowell PS, Knowles MR, Cohn JA: Cystic fibrosis gene mutations and pancreatitis risk: relation to epithelial ion transport and trypsin inhibitor gene mutations. Gastroenterology 2001;121:1310–1319. 44 Truninger K, Witt H, Köck J, Kage A, Seifert B, Ammann R, et al: Mutations of the serine protease inhibidor, Kazal type 1 gene, in patients with idiopathic chronic pancreatitis. Am J Gastroenterol 2002; 97:1133–1137.
45 Lempinen M, Paju A, Kemppainen E, Smura T, Kylänpää ML, Nevanlinna H, 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 2005; 40:225–230. 46 Novis BH, Young GO, Bank S, Marks IN: Chronic pancreatitis and ␣1-antitrypsin. Lancet 1975;ii:748–749. 47 Hutchison DC: Alpha-1-antitrypsin deficiency in Europe: geographical distribution of Pi types S and Z. Respir Med 1998;92:367– 377.
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Received: April 22, 2008 Accepted after revision: October 29, 2008 Published online: August 4, 2009
Genetic Mutations in a Spanish Population with Chronic Pancreatitis Josefina Mora a Laia Comas a Elia Ripoll a Patricia Gonçalves b Josep M. Queraltó a Francesc González-Sastre a Antoni Farré b Departments of a Clinical Chemistry and b Gastroenterology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
Key Words Genetic mutations ⴢ Chronic pancreatitis ⴢ Idiopathic chronic pancreatitis ⴢ Alcohol-related pancreatitis ⴢ Hereditary pancreatitis
Abstract Background/Aims: Mutations in the PRSS1 and the SPINK1 genes have variably been associated with alcohol-related, idiopathic and hereditary chronic pancreatitis (CP). The aim of this study was to determine for the first time the significance of PRSS1, SPINK1 mutations and genetic variants of AAT in a group of Spanish patients with CP. Methods: 104 consecutive patients with CP were included, as well as 84 healthy control subjects. The R122H and N29I mutations in the PRSS1 gene, the N34S mutation in the SPINK1 gene and PiS and PiZ mutations in the AAT gene were analyzed by RFLP-PCR methods. Results: No R122H mutation was found in the PRSS1 gene, and N29I mutation was detected in 7.7% of CP patients. A N29I mutation was observed in 3.9% of patients with alcohol-related pancreatitis (ACP). A total of 5.8% of CP patients were identified with the N34S mutation. Genotype MS, SS and MZ were detected in 18.3, 3.8 and 1.3% of CP patients, respectively. Conclusion: The percentage of N29I mutations in ACP patients was higher than that reported in other studies, while the percentage of N34S and AAT mutations in ACP and idiopathic CP patients was similar. Copyright © 2009 S. Karger AG, Basel and IAP
© 2009 S. Karger AG, Basel and IAP 1424–3903/09/0095–0644$26.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com
Accessible online at: www.karger.com/pan
Introduction
Chronic pancreatitis (CP) is an ongoing disease characterized by chronic inflammation and progressive fibrosis associated with loss of exocrine and/or endocrine function. Among the etiological factors involved in its pathogenesis, alcohol is considered the main risk factor in Western countries, accounting for 70–90% of all cases [1]. However, only 10–20% of alcoholics develop the disease [2]. In approximately 10–30% of CP patients no association is found with alcohol or other risk factors and the disease is classified as idiopathic chronic pancreatitis (ICP). In recent years, epidemiological and clinical studies in several countries suggest a possible genetic basis in CP. In 1996 the discovery of a mutation in the cationic trypsinogen gene (PRSS1) as a cause of hereditary CP contributed greatly to our knowledge of CP pathogenesis [3]. The search to characterize genes that may render individuals susceptible to pancreatic disease, including alcohol-related pancreatitis (ACP) and ICP, has since been extended to other possible candidates. The first is the serine protease inhibitor, Kazal type 1 (SPINK1), which provides a defense mechanism against premature activation of trypsinogen [4]. Other candidates include the cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible for cystic fibrosis [5, 6], genes implicated in pancreatic enzymatic activity such as the genetAntoni Farré, MD Department of Gastroenterology, Hospital de la Santa Creu i Sant Pau Av. Antoni M. Claret 167, ES–08025 Barcelona (Spain) Tel. +34 93 291 9141, Fax +34 93 291 9278 E-Mail [email protected]
Table 1. Characteristics of the CP patients and controls
Patients Sex, M/F Age, years Age at onset, years
ACP
ICP
HP
Others
Total CP
Controls
78 66/12 52 (35–76) 45 (30–76)
16 10/6 59.5 (19–83) 57 (14–82)
3 1/2 35 (13–72) 16 (13–43)
7 4/3 50 (24–73) 36 (18–69)
104 81/23 52 (13–83) 44.5 (8–82)
84 37/47 46 (18–87) –
Age values are expressed as median and range.
ic variants of ␣1-antitrypsin, and genes involved in alcohol metabolism [7]. Some of the aforementioned genes have undergone genetic testing in both ACP and ICP patients. PRSS1 mutations [8] have a low incidence in ICP patients [9, 10], and constitute a minor finding in ACP [11, 12]. In the case of SPINK1, since a N34S mutation was first described in 18 out of 96 (19%) unrelated children and adolescents in an ICP series in Germany [13], other studies have confirmed a high frequency – ranging from 6.4 to 28.6% – of this mutation in cohorts of ICP patients [14, 15]. In contrast, the frequency of mutations in ACP patients is about 5– 6%. The small differences compared to controls suggest that this genetic variant does not play a major role in these patients. Furthermore, approximately 1–2.5% of the normal population is heterozygous for the N34S mutation [16, 17]. All genetic variants of PRSS1 and SPINK1 genes are described in a continuously updated database [18]. With regard to ␣1-antitrypsin (AAT), the genetic variants of this enzyme do not appear to play a predominant role in chronic non-alcoholic pancreatitis [19, 20] or in ACP [21]. However, little is known about its possible impact on the pathogenesis of CP. The aim of this study was to determine the significance of PRSS1, SPINK1 mutations and genetic variants of AAT in a group of Spanish patients with CP. This is the first such study in the CP population in Spain.
Material and Methods Patients Between April 2000 and February 2005, 104 consecutive Caucasian patients with CP (81 men, 23 women; median age 52 years, range 13–83 years) were included. CP was diagnosed in accordance with the standard diagnostic criteria consisting of a typical history of pain, suggestive imaging tests (CT scan, MR and/or endoscopic ultrasonography), impaired pancreatic exocrine function when assessed and histological analysis in cases in which
Genetic Mutations in a Spanish Population with Chronic Pancreatitis
surgical resection was indicated. In this series, alcohol abuse, 1 60 g/day for at least 2 years, was considered the only etiological factor in 78 patients. An idiopathic etiology was considered in 16 patients. Three affected members of the same family were included in the diagnosis of hereditary pancreatitis (HP) based on criteria of 3 or more relatives with CP in two or more generations [22, 23]. CP was considered to be due to other causes in 7 patients (autoimmune CP in 3, obstructive CP in 2, familiar hypertriglyceridemia in 1 and chronic renal failure in 1). No ethnic differences were observed in this series. To analyze the possible contribution of genetic alterations in the onset and course of the disease, the most frequent CP complications (calcifications, pseudocyst and biliary obstruction) were taken into consideration. The onset of CP was defined as the initial manifestation of either the first episode of acute pancreatitis or the start of symptomatic complications. The age threshold to consider an early onset was 20 years (table 1). Tobacco smoking was also taken into account. Patients consuming 110 cigarettes/ day were considered smokers. The majority of the smokers included in this study exceeded this figure. The control group was made up of 84 healthy volunteers from the same institution. The study had the approval of the Ethics Committee at Hospital de la Santa Creu i Sant Pau and written informed consent was obtained from all the patients. DNA Extraction Genomic DNA was extracted from 3 ml of peripheral venous blood samples using EDTA tubes according to QIAamp쏐 DNA Blood Mini Kit (Qiagen, Basel, Switzerland) instructions. Detection of PRSS1 Mutations Two mutations were analyzed in the cationic trypsinogen (PRSS1) gene: the R122H in exon 3 and the N29I in exon 2. The R122H mutation was analyzed by means of a restriction fragment length polymorphism method in products amplified by PCR (RFLP-PCR), using the primers listed in table 2 and the Pml I enzyme (New England BioLabs, Ipswich, Mass., USA). This enzyme has one restriction site [CACGTG] in wild-type amplified samples and two restriction sites in mutant R122H samples [24]. After polyacrylamide gel electrophoresis (9%) and ethidium bromide staining, the bands which depicted the R122H mutant allele (208 and 20 bp), wild-type allele (228 bp) and control digestion (327 bp) were visualized (fig. 1). A RFLP-PCR method which uses the primers listed in table 2 and the restriction enzyme HpyCH4 III (New England BioLabs)
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Table 2. PCR primers used in the study
PRSS1 Exon 2 Exon 3
Forward Reverse Forward Reverse
5ⴕ-CGC CAC CCC TAA CAT GCT AT-3ⴕ 5ⴕ-CTC TCC CAG GCA GAC TGG CC-3ⴕ 5ⴕ-GGT CCT GGG TCT CAT ACC TT-3ⴕ 5ⴕ-GTA ATG GGC ACT CGA AAT GT-3ⴕ
SPINK1 Exon 3 Forward Reverse AAT Exon 3 Exon 5
5ⴕ-AGT TTC AGA AGG GCC ATA GGA-3ⴕ 5ⴕ-GTT TGC TTT TCT CGG GGT GAG-3ⴕ
Forward (SF) Reverse (SR) Forward (ZF) Reverse (ZR)
5ⴕ-AGG GGA AAC TAC AGC ACC TCG-3ⴕ 5ⴕ-TGG GTA CTG TTC TCC TCA TCG AGC ATG-3ⴕ 5ⴕ-GGC TGT GCT GAC CAT CGT C-3ⴕ 5ⴕ-AAC TCT TCT TTA ATG TCA TCG AGG-3ⴕ
Uncut 555 bp
Uncut 266 bp 181 bp
327 bp 102 bp 85 bp 79 bp
228 bp 208 bp Neg. 1 R122R
H
2 H 3
H 4
H 5 Pos. R122H
Uncut Neg. 1 N29N
2
3
4
H Pos. N29I
Fig. 1. Detection of R122H mutation in the PRSS1 gene by RFLP/ PCR using PmI I enzyme. Negative control (228-bp wild-type band, R122R). Negative samples (1–4). Control samples without DNA (H). Positive control (208-bp mutant band, R122H). Control digestion (327-bp band) and Uncut (555-bp band). Molecular weight marker (⌽ ! 174, Hae III digested).
Fig. 2. Detection of N29I mutation in the PRSS1 gene by RFLP/ PCR using HpyCH4 III enzyme. Negative control (102- and 79-bp wild-type bands, N29N). Negative samples (1–4). Control sample without DNA (H). Positive control (181-bp mutant band, N29I). Control digestion (85-bp band) and Uncut (266-bp band). Molecular weight marker (⌽ ! 174, Hae III digested).
was adapted to detect the N29I mutation [25]. Using this strategy, a 266-bp fragment was obtained. Wild-type alleles showed two restriction sites [ACTGT] for the enzyme: the control digestion site and the restriction site lost when the N29I mutation is present. Therefore, after polyacrylamide gel electrophoresis (12%), three fragments were obtained for wild-type samples (102, 79 and 85 bp) and two fragments for the N29I mutant samples (181 and 85 bp) (fig. 2).
were generated from the wild-type allele (298, 116, and 20 bp) and two additional fragments (189 and 109 bp) when the N34S mutation was present. The 116-bp fragment depicted the control digestion band (fig. 3).
Detection of the SPINK1 Mutation The N34S mutation in exon 3 of the gene SPINK1 was detected by RFLP-PCR, with the primers listed in table 2. We used a modified version of the method described by Teich et al. [26]. The restriction enzyme HpyCH4 III (New England BioLabs) has two restriction targets [ACTGT] in the wild-type samples and three restriction targets in the mutant N34S samples. The digested products were then run on a polyacrylamide gel electrophoresis (12%) followed by ethidium bromide staining. Three fragments
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Detection of the AAT Mutations The PiS and PiZ mutations were detected by a RFLP-PCR method, using the mutated primers listed in table 2. These primers are able to introduce an artificial restriction site for Taq I (Fermentas, Glen-Burnie, Md., USA) that is lost when the mutation is present. Taq I has two restriction targets [TCGA] in the wild-type genotype samples (MM) and one target in the mutant PiS and PiZ samples [27]. The PCR generated a 285-bp product in the study of PiS mutation and a 250-bp product in the study of PiZ mutation. The digested PCR samples were run on a polyacrylamide gel electrophoresis (8%) and stained with ethidium bromide. In the study of PiS mutation, three fragments were observed (245, 21 and 19 bp) for the wild-type and two fragments (264 and 21 bp) when the
Mora /Comas /Ripoll /Gonçalves / Queraltó /González-Sastre /Farré
Table 3. Distribution of the PRSS1, SPINK1 and AAT mutations in CP patients and controls
ACP (n = 78)
ICP (n = 16)
HP (n= 3)
Others (n = 7)
Total CP (n = 104)
Controls (n = 84)
PRSS1 mutations R122H N29I
0 3/78 (3.9%)
0 1/16 (6.3%)
0 3/3*** (100%)
0 1a/7 (14.3%)
0 8/104** (7.7%)
0 0
SPINK1 mutation N34S
3/78 (3.9%)
3/16** (18.8%)
0
0
6/104* (5.8%)
0
3/16 (18.8%) 2/16 (12.5%) 0
2/3 (66.7%) 1/3 (33.3%) 0
1b/7 (14.3%) 0 0
AAT mutations MS SS MZ
13/78 (16.9%) 1/78 (1.3%) 1/78 (1.3%)
19/104 (18.3%) 4/104 (3.8%) 1/104 (0.96%)
14/84 (16.6%) 1/84 (1.2%) 1/84 (1.2%)
Percentages refer to the proportion of patients within each given group. * p < 0.05 vs. control; ** p < 0.01 vs. control; *** p < 0.001 vs. control. a Obstructive. b Autoimmune cause.
mutations were analyzed by the Mann-Whitney U test (continuous variables) and Fisher’s exact test (categorical data). p ! 0.05 was considered significant. Uncut 434 bp 298 bp
Results
189 bp 116 bp 109 bp Neg. 1 2 H 3 4 N34N
H 5 6 H Pos. N34S
Fig. 3. Detection of N34S mutation in the SPINK-1 gene by RFLP/
PCR using HpyCH4 III enzyme. Negative control (298-bp wildtype bands, N34N). Negative samples (1, 3–6). Positive sample (2). Control samples without DNA (H). Positive control (189- and 109-bp mutant band, N34S). Control digestion (116-bp band) and Uncut (434-bp band). Molecular weight marker (⌽ ! 174, Hae III digested).
PiS mutation was present. In the study of PiZ mutation, three fragments (209, 23 and 18 bp) were observed for the wild-type and two fragments (227 and 23 bp) when the PiZ mutation was present. Statistical Analysis The two-sided Fisher’s exact test was used to analyze differences in the frequency of PRSS1, SPINK1 and AAT mutations among ACP, ICP and HP patients as well as in controls. The association of various clinical parameters (age at onset, exocrine insufficiency, calcifications, etc.) and the presence or absence of
Genetic Mutations in a Spanish Population with Chronic Pancreatitis
Distribution of PRSS1, SPINK1 and AAT Mutations in CP Patients The frequency of mutations of the PRSS1 and SPINK1 genes and ␣1-antitrypsin deficiency alleles is summarized in table 3. All controls were negative for the R122H and the N29I mutations of the PRSS1 gene. The R122H mutation was not detected in any of the four CP groups studied. The N29I mutation was detected in 8/104 of the studied patients (7.7%; p = 0.007 vs. controls). According to the etiology, 3 patients (3.9%) in the ACP group, 1 patient (6.3%) in the ICP group, 3 out of 3 patients (100%) in the HP group (p ! 0.001 vs. controls) and 1 patient with an obstructive cause of CP had this N29I mutation. No family history of pancreatitis was ascertained in the ACP, ICP and obstructive causes of pancreatitis with this mutation. The 3 affected HP patients were homozygous for this mutation while the remaining 5 patients were heterozygous. In the analysis of the SPINK1 gene, no N34S mutation was observed in the control group. A total of 6 patients (5.8%; p = 0.027 vs. controls) were identified with the N34S mutation, 3 patients (3.9%) belonging to the ACP group and 3 patients (18.8%) belonging to the ICP group Pancreatology 2009;9:644–651
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Table 4. Complications of pancreatitis and tobacco smoking in CP patients
Patients Age at onset, years Diabetes mellitus Exocrine insufficiency Calcifications Biliary obstruction Pseudocysts Surgical procedures Tobacco smoking
PRSS1 mutation: N29I
SPINK1 mutation: N34S
positive
negative
positive
negative
8 41 (13–68) 1 (12.5%) 4 (50%) 6 (75%) 2 (25%) 2 (25%) 2 (25%) 4 (50%)
96 45 (8–82) 52 (54.2%) 44 (45.8%) 69 (71.8%) 34 (35.4%) 41 (42.7%) 36 (37.5%) 78 (81.2%)
6 41.5 (14–64) 3 (50%) 5 (83.3%)* 5 (83.3%) 0 1 (16.7%) 0 5 (83.3%)
98 44.5 (8–82) 50 (51%) 43 (43.9%) 70 (71.4%) 36 (36.7%) 42 (42.9%) 38 (38.9%) 77 (78.6%)
Age is expressed as median and range. Percentages refer to the proportion of patients within each given group. * p < 0.05.
(p = 0.003 vs. controls). The 6 patients were heterozygous for N34S mutation. The combined analysis of common mutations of PRSS1 and SPINK1 genes detected at least one mutation in 6/78 patients (7.7%) in the ACP group (p = 0.011 vs. controls) and in 4/16 patients (25%) in the ICP group (p ! 0.001 vs. controls). The mutations N29I and N34S did not coexist in any patient of the ACP or ICP group. In the study of the AAT deficiency alleles, genotype MS was detected in 14 controls (16.6%) and in a total of 19 patients (18.3%); 13 patients in the ACP group, 3 patients in the ICP group, 2 patients in the HP group and 1 patient with autoimmune pancreatitis. The genotype SS was identified in 1 control (1.2%) and in a total of 4 patients (3.8%); 1 patient in the ACP group, 2 patients in the ICP group and 1 patient in the HP group. The genotype MZ was found in 1 control (1.2%) and in 1 patient (0.96%) belonging to the ACP group. No significant differences were observed between patients and controls in the frequency of AAT deficiency alleles (p = 0.1). Nevertheless, we detected AAT mutations in the 3 members of the family with HP (SS mutation in 1 member and MS mutation in the other 2 members). Mutation Status and Clinical Outcome in CP Patients As shown in table 1, the median age at onset of the symptoms was 45 years (range 30–76) in patients with ACP, 57 years (range 14–82) in patients with ICP, and 16 years (range 13–43) in patients with HP. The presence of the N29I or N34S mutations in the total group of CP patients was not associated (p = 0.325 and p = 0.590, respectively) with early onset of the disease, 648
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except for N29I in HP patients (p = 0.045). The age at onset in 2/3 ICP patients with N34S mutation was !20 years, but the correlation was not statistically significant (p = 0.071). Table 4 shows the association of N29I or N34S mutations and aspects of disease severity and complications (diabetes mellitus, exocrine insufficiency, calcifications, biliary obstruction, pseudocysts or surgical procedures) and tobacco smoking. No significant association was found except for the detection of the N34S mutation and the presence of exocrine pancreatic insufficiency (p = 0.043). In addition, this correlation was maintained in ACP patients when evaluated separately (p = 0.036).
Discussion
The significance of PRSS1, SPINK1 mutations and genetic variants of ␣1-antitrypsin has not been reported previously in a Spanish population. While PRSS1 mutations have revealed an association with HP, they can also be found in patients with ICP, though the incidence is very low. In this study, no R122H mutation of the PRSS1 gene was observed in any of the groups evaluated. The N29I mutation, the second most prevalent mutation in the PRSS1 gene [8], was detected in the 3 HP patients and in 6.3% of ICP, patients. Several large studies focused on the association of ICP, and the most common trypsinogen mutations (R122H and N29I) have shown a frequency of mutations ranging from 0.2 to 10% of patients [28– 32]. PRSS1 mutations are rarely found in ACP patients [33–35]. O’Reilly et al. [11] described the R122H mutaMora /Comas /Ripoll /Gonçalves / Queraltó /González-Sastre /Farré
tion in 1/36 ACP patients (2.8%) and Bernardino et al. [12] reported a PRSS1 mutation in 1/64 ACP patients (1.5%). In the present study, the percentage of mutations in the PRSS1 gene was higher in the ACP group of patients; the N29I mutation was detected in 3/78 of ACP patients (3.9%). Furthermore, the N29I mutation was found in 1 patient with obstructive CP. The presence of the N29I mutation was significantly associated with an early onset of the disease in HP patients. Several large studies [36] have reported that symptoms in HP patients harboring the N29I mutation appear at a younger age, such as 14 years. No significant association was found with any aspects of disease severity, or tobacco smoking. In the case of the SPINK1 gene, Witt et al. [13], Chen et al. [37], and Pfützer et al. [38] have recently reported the presence of N34S mutations in patients with CP. Whether these mutations induce pancreatitis by themselves or together with other causative factors remains controversial. N34S mutations have been associated with the occurrence of pancreatitis [13] and observed in both Caucasian and Japanese pancreatitis patients [39], but other authors have suggested that even though there was no association between SPINK1 mutations and pancreatitis in HP patients [37, 38], this association was confirmed in ICP patients [14, 38]. Pfützer et al. [38] suggested that mutations in this gene might promote a predisposition to pancreatitis, possibly by lowering the threshold when other factors are present, and they concluded that SPINK1 mutations act as disease modifiers changing the phenotypic expression of the disease [40]. In the study of our population, the N34S mutation was found in 3/16 patients (18.8%) with ICP. This frequency is higher than that observed in several reports: 5% [41], 10.3% [9] and 13% [42]; similar to the range acknowledged in other studies: 18.6% [13] and 23.1% [43] and lower than that described in some series: 25.9% [38] and 28.6% [15]. Chen et al. [14] found that the frequency of N34S mutations in ICP patients differed according to age groups, changing from 6.4% in the total ICP group to 14.7% in their ^20-year-old ICP group. In our study, two thirds of the ICP patients with a N34S mutation were !20 years of age when diagnosed, supporting the association of the N34S mutation with an early-onset ICP [44]. These patients were heterozygous for the N34S mutation; in previous studies [13, 14, 38] no phenotypic differences between heterozygous and homozygous N34S ICP patients were detected. In the Spanish population, as in other industrialized countries, alcohol is the major etiological factor for CP. The N34S mutation in the SPINK1 gene was
detected in 3.9% of patients with ACP, similar to the range of frequencies (0–6%) reported from other European countries and Japan [16, 17, 35, 42]. In a recent study from Finland a higher frequency (10%) was reported [45]. Concerning disease severity, the presence of N34S mutations was statistically associated with pancreatic exocrine insufficiency in total CP patients and in the ACP group. The N34S mutation was not detected in controls, although in other reports based on a higher number of controls the mutation was described in about 0.8% (4/540) [16]. A protease-to-protease inhibitor imbalance in patients with AAT deficiency was previously thought to contribute to the development of CP. Some authors supported an association between CP and deficient AAT alleles [46] while others did not [21]. 23 of 104 patients (22%) with CP were hetero- or homozygous (19 and 4, respectively) for S allele, and 1/104 CP patients (0.96%) was heterozygous for Z allele. The PiZ mutation was found heterozygously in 1 patient with ACP (1.3%) and was absent in the ICP and HP groups. The frequency for AAT deficiency alleles in CP patients did not significantly differ from those reported in a control Spanish population [47]. Like ourselves, Witt et al. [20] found no significant differences between AAT deficiency allele in CP patients and controls. The PiS mutation frequency in CP patients in Spain (22%) was higher than that reported in Germany (4.1%), while the PiZ frequency was lower (0.96 vs. 2%) [19]. In conclusion, in the present study the percentage of N29I mutations in the PRSS1 gene in ACP patients was higher than that reported in published data, while the percentage of N34S mutations in the SPINK1 gene in ACP and ICP patients was similar to findings in other studies and populations. The N34S mutation was associated with exocrine insufficiency in total CP and ACP patients. Although PiS mutation frequency in the AAT gene in our CP population was elevated, no significant differences were observed with respect to reported controls.
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Acknowledgments This study was supported in part by a grant from the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo of Spain. Redes de Investigación Cooperativa, G03/156. The authors thank Assumpta Antonijuan for technical assistance and Carolyn Newey for language editing support.
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