Pancreatitis risk in primary hyperparathyroidism: relation to mutations in the SPINK1 trypsin inhibitor (N34S) and the cystic fibrosis gene

OBJECTIVE: Primary hyperparathyroidism (pHPT)-related hypercalcemia is considered to represent a risk factor for the development of pancreatitis. We therefore explored whether mutations in genes that were previously identified to increase the risk for pancreatitis coexist in a cohort of 826 patients with pHPT prospectively studied between 1987 and 2002. METHODS: Among 826 patients with pHPT, 38 patients were identified with pancreatitis (4.6%). DNA was available from 25 patients (13 women/12 men, 16 acute pancreatitis/9 chronic pancreatitis). These individuals and 50 patients with pHPT without pancreatitis were analyzed for mutations in the serine protease inhibitor Kazal type I (SPINK1) gene (N34S) and the cationic trypsinogen gene (PRSS1) (N29I, R122H) by melting curve analysis and DNA sequencing. Sequence analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene was carried out for the detection of 36 mutations and the Tn polymorphism. RESULTS: Four of 25 patients with pHPT and pancreatitis carried the N34S missense mutation in the SPINK1 gene (16%), while all 50 controls (pHPT without pancreatitis) showed no mutation in SPINK1 or PRSS1 genes (P < 0.05 vs controls, P < 0.001 vs general population). CF-causing CFTR mutations were present in four patients (P < 0.05 vs general population), while one patient carried a 5T allele. One patient was transheterozygous (SPINK1: N34S/CFTR: R553X). Mean serum calcium levels in pancreatitis patients (3.1 mmol/L) did not differ significantly from the mean of the entire cohort (3.0 mmol/L) or pHPT patients without pancreatitis (3.1 mmol/L). CONCLUSION: Pancreatitis risk is approximately 10-fold elevated in pHPT, but pancreatitis occurs infrequently. This indicates an existing but minor impact of pHPT-related hypercalcemia. If pancreatitis occurs, it seems associated with genetic risk factors such as mutations in the SPINK1 and CFTR genes. In contrast, a combination of both hypercalcemia and genetic variants in SPINK1 or CFTR increases the risk to develop pancreatitis in patients with pHPT.     

Absence of PRSS1 mutations and association of SPINK1 trypsin inhibitor mutations in hereditary and non-hereditary chronic pancreatitis

BACKGROUND AND AIMS: Mutations in the cationic trypsinogen (protease, serine, 1 (trypsin 1); PRSS1) gene are causally associated with recurrent acute and chronic pancreatitis. We investigated whether mutations in the PRSS1 gene are associated with hereditary and non-hereditary pancreatitis. As a modifier role has been proposed for trypsin inhibitor (serine protease inhibitor, Kazal type I; SPINK1) mutations, the role of SPINK1 mutations in these patients was also analysed. SUBJECTS AND METHODS: The coding regions of PRSS1 and SPINK1 genes were sequenced in 290 controls and 198 patients, of whom 120 were diagnosed as idiopathic (ICP), 41 as alcoholic (ACP), and 37 as hereditary pancreatitis (HP). Twenty four unaffected relatives of HP probands were also analysed and genotype-phenotype correlations and statistical analyses were performed. RESULTS: No mutations in the PRSS1 gene were detected in any of the patients, including HP patients, while the N34S mutation was observed in the SPINK1 gene in the majority of HP patients (73%). Similarly, 26.8% of ACP (11 of 41) and 32.5% (39 of 120) of ICP patients also had SPINK1 mutations. The N34S mutation was observed in both homozygous and heterozygous conditions. In comparison, only 2.76% of the control population had the N34S allele (p<0.001). The P55S mutation was observed in one ICP and one ACP patient, and in three normal individuals. Genotype-phenotype correlations did not suggest any significant difference in the age of onset, severity of disease, or pancreatic endocrine insufficiency in patients with or without mutated SPINK1 and irrespective of the allelic status of N34S SPINK1. CONCLUSIONS: Irrespective of the aetiology, mutations in the PRSS1 gene are not associated with chronic pancreatitis, including HP. In contrast, the N34S mutation in the SPINK1 gene shows a significant correlation in these patients. A comparable phenotype in terms of age of onset, diabetes mellitus, and other phenotypic features in patients with or without SPINK1 mutations and N34S homozygotes and heterozygotes suggests that there may still be involvement of other genetic or environmental factors.     

SPINK1/PSTI mutations are associated with tropical pancreatitis and type II diabetes mellitus in Bangladesh

BACKGROUND & AIMS: Tropical pancreatitis, including tropical calcific pancreatitis and fibrocalculous pancreatic diabetes, is endemic in parts of Asia and Africa. In a preliminary study, we found serine protease inhibitor, Kazal type 1 (SPINK1) mutations in 6 of 8 patients with fibrocalculous pancreatic diabetes in Bangladesh. A more extensive investigation of patients with pancreatic diseases in Bangladesh, including non-insulin-dependent diabetes mellitus, was undertaken. METHODS: Patients with fibrocalculous pancreatic diabetes (n = 22), tropical calcific pancreatitis (n = 15), and non-insulin-dependent diabetes mellitus (n = 43) and controls (n = 76) from Bangladesh were studied. DNA was extracted, and the SPINK1 gene was sequenced in all patients and 50 controls. Exon 3 was sequenced in an additional 26 controls. RESULTS: SPINK1 N34S mutations appeared in 1 of 76 controls (1.3%), 12 of 22 patients with fibrocalculous pancreatic diabetes (55%; odds ratio, 83; P < 0.00001), 3 of 15 with tropical calcific pancreatitis (20%; odds ratio, 11.2; P = 0.04), and 6 of 43 with non-insulin-dependent diabetes mellitus (14%; odds ratio, 11.9; P = 0.009). P55S was present in 2 of 76 controls (3%) and in 1 of 22 patients with fibrocalculous pancreatic diabetes (5%; P = not significant). A novel Y54H (160T>C) mutation was identified in 1 of 15 tropical calcific pancreatitis patients. CONCLUSIONS: In Bangladesh, the SPINK1 N34S mutation increases the risk of several forms of pancreatic disease, including fibrocalculous pancreatic diabetes, tropical calcific pancreatitis, and non-insulin-dependent diabetes mellitus.     

Divergent Roles of SPINK1 and PRSS2 Variants in Tropical Calcific Pancreatitis

Background/Aims: Tropical calcific pancreatitis (TCP) refers to a type of idiopathic pancreatitis prevalent in Asia. The trypsin inhibitor (SPINK1) N34S variant partially explains the genetic susceptibility to TCP. As anionic trypsinogen (PRSS2) G191R protects against chronic pancreatitis in Europeans, we investigated whether this variant protects from TCP in Indians. Methods: We enrolled 174 patients and 794 controls from two Indian tertiary care referral hospitals. We analyzed PRSS2 and SPINK1 variants by melting curve analysis, allele-specific discrimination assay, and sequencing. Results: G191R was detected in 1 TCP patient (0.6%) compared to 13 controls (1.6%; OR 0.27, 95% CI 0.03-2.1; p = 0.33). SPINK1 N34S was enriched in the TCP population 67/174 (38.5%) compared to controls 10/234 (4.3%; OR 14, 95% CI 6.9–28.3; p < 0.001). Conclusion: G191R PRSS2 is a rare allele in the Indian population and the data suggest a nonsignificant trend towards a protective effect. N34S SPINK1 represents the major genetic risk factor in TCP.     

Mutations in serine protease inhibitor Kazal type 1 are strongly associated with chronic pancreatitis

BACKGROUND: Although chronic pancreatitis is associated with risk factors such as alcoholism, hyperparathyroidism, and hypertriglyceridaemia, little is known of the actual aetiology of the disease. It is thought that inappropriate activation of trypsinogen causes pancreatitis, and indeed in cases of hereditary pancreatitis mutations of cationic trypsinogen (PRSS1) have been described. As serine protease inhibitor Kazal type 1 (SPINK1) is a potent natural inhibitor of pancreatic trypsin activity, we hypothesised that SPINK1 mutations would be more common than expected among an unselected cohort of adult chronic pancreatitis patients. AIMS: To detect the prevalence of SPINK1 mutations in a cohort of chronic pancreatitis patients. METHODS: DNA was isolated from a cohort of 115 adult patients with chronic pancreatitis of alcoholic (n=72), hereditary (n=10), idiopathic (n=24), and miscellaneous (n=9) origin. We performed mutational analysis for two PRSS1 mutations (R122H, N29I) and four specific SPINK1 gene mutations (M1T, L14P, N34S, P55S) and compared the results with a control group of 120 healthy Dutch subjects. RESULTS: In six of the 10 patients that fulfilled the criteria for hereditary pancreatitis, but in none of the control subjects, mutations in the PRSS1 gene were found. In 14 patients we detected a SPINK1 mutation. Eleven patients were heterozygous for the N34S mutation and sequencing confirmed the homozygous state of N34S in a brother and sister. Two patients carried the P55S mutation, one as a compound heterozygote with N34S. The M1T and L14P SPINK1 mutations were not found in our cohort. The N34S mutation was detected in only two of 120 controls, while the P55S, M1T, and L14P mutations were absent in the same group. Patients with the N34S allele had a later onset of disease than those with PRSS1 gene mutations but earlier onset compared with the mutation negative group. CONCLUSION: Identification of SPINK1 mutations in 12.2% of patients with adult alcoholic and idiopathic chronic pancreatitis suggests an important role for SPINK1 as a predisposing factor in adult chronic pancreatitis.     

Limited contribution of the SPINK1 N34S mutation to the risk and severity of alcoholic chronic pancreatitis: a report from the United States

Mutations in the SPINK1 gene (e.g. N34S) have been reported in patients with idiopathic, familial, tropical, and alcoholic pancreatitis. The prevalence of SPINK1 N34S differs between different patient populations, and its contribution to the risk and the severity of alcoholic chronic pancreatitis has not been defined in the United States. Mutational analysis of the exon 3 was performed in 32 patients with alcoholic chronic pancreatitis, 39 patients with nonalcoholic chronic pancreatitis or recurrent acute pancreatitis, and 190 previously studied healthy controls. The course of alcoholic chronic pancreatitis with and without N34S was compared in age of onset- and sex-matched patients. All SPINK1 gene sequence variations were heterozygous. SPINK1 N34S was present in 3/190 (1.6%) and P55S was found in 2/190 (1.1%) of controls. In alcoholics, the N34S mutation was identified in 2/32 patients (6.3%, P < 0.05). In nonalcoholics, N34S and P55S were identified in 6/39 patients (15.4%, P < 0.005, N34S N = 4, P55S N = 1, N34S/P55S N = 1). The clinical course of alcoholic chronic pancreatitis was similar between patients with and without the N34S mutation. The N34S mutation is uncommon in patients with alcoholic chronic pancreatitis in the United States; its prevalence is similar to other countries and appears not to alter the onset or the severity of alcoholic chronic pancreatitis.     

The SPINK1 N34S variant is associated with acute pancreatitis

OBJECTIVE: Acute pancreatitis (AP) is a disease whose pathogenesis remains largely obscure. Genetic research has focussed attention upon the role of the pancreatic protease/protease inhibitor system. The aim of this study was to investigate the prevalence of genetic variants of the trypsin inhibitor, SPINK1, in acute pancreatitis. METHODS: We genotyped 468 patients with AP and 1117 healthy controls for SPINK1 alterations by single-strand conformation polymorphism analysis and by melting curve analysis using fluorescence resonance energy transfer probes. RESULTS: The c.101A>G (p.N34S) variant was detected in 24/936 alleles of patients and in 18/2234 alleles of healthy controls (odds ratio=3.240; 95% confidence interval: 1.766-5.945; P<0.001). In the UK patients, the mean age of patients with N34S was 11.9 years younger compared with N34S negative patients (P=0.023), but this was not apparent in the German patients. Allele frequencies for the c.163C>T (p.P55S) variant did not differ between patients and controls. CONCLUSION: The SPINK1 N34S variant is associated with acute pancreatitis. This supports the importance of premature protease activation in the pathogenesis of AP and suggests that mutated SPINK1 may predispose certain individuals to develop this disease.     

The SPINK1 N34S mutation is not associated with Type 2 diabetes mellitus in a population of the USA.

AIMS: Mutations in the serine protease inhibitor (SPINK1) gene have been associated with all forms of chronic pancreatitis. Recently, an association of SPINK1 mutations with early-onset Type 2 diabetes mellitus has been reported in patients from Bangladesh. Therefore, we determined the frequency of SPINK1 N34S mutations in patients with Type 2 diabetes mellitus from the USA. METHODS: The study population of Hispanic and non-Hispanic white people consisted of 387 patients with Type 2 diabetes and familial clustering of the disease, 232 family members without diabetes, 259 patients with Type 2 diabetes without a family history, and 302 ethnically matched healthy controls as part of the San Luis Valley Diabetes Study. We performed linkage- and association-analysis in 82 multiplex families with Type 2 diabetes mellitus. RESULTS: No significant linkage or allele sharing was detected between Type 2 diabetes mellitus and the SPINK1 locus. The frequency of the N34S mutation was determined by fluorescence polarization and was similar between patients (n = 14/387 patients with familial clustering; n = 2/259 patients without family history) and controls (n = 5/232 family members without diabetes; n = 10/302 individuals). Variables such as ethnicity, age of diabetes onset and percentage of individuals with impaired glucose tolerance did not differ significantly between carriers and homozygous normal individuals. CONCLUSION: The SPINK1 N34S mutation appears not to predispose Hispanic or non-Hispanic white people from the USA to the development of Type 2 diabetes mellitus.     

Association of cathepsin B gene polymorphisms with tropical calcific pancreatitis

BACKGROUND AND AIMS: Tropical calcific pancreatitis (TCP) is a type of chronic pancreatitis unique to countries in the tropics. Mutations in pancreatic secretory trypsin inhibitor (SPINK1) rather than cationic trypsinogen (PRSS1) explain the disease in only 50% of TCP patients. As cathepsin B (CTSB) is known to activate cationic trypsinogen, we attempted to understand the role of CTSB mutations in TCP. Evidence of epistatic interaction was investigated with the previously associated N34S SPINK1 allele, a variant considered to be a modifier rather than a true susceptibility allele. Subjects and METHODS: We sequenced the coding region of CTSB gene in 51 TCP patients and 25 controls and further genotyped 89 patients and 130 controls from the same cohort for Leu26Val, C595T, T663C, and Ser53Gly polymorphisms. The positive findings observed in the earlier cohort were re-examined in an ethnically matched replication cohort comprising 166 patients and 175 controls. Appropriate statistical analyses were performed and Bonferroni correction for multiple testing was applied. RESULTS: We found a statistically significant association of the Val26 allele at Leu26Val polymorphism with an odds ratio (OR) of 2.15 (95% confidence interval (CI) 1.60-2.90 (p = 0.009)), after Bonferroni correction (corrected p value = 0.025). This significant association of Leu26Val with TCP was replicated in another cohort (OR 2.10 (95% CI 1.56-2.84); p = 0.013). Val26 allele also showed significantly higher frequency in N34S positive and N34S negative patients than in controls (p = 0.019 and 0.013, respectively). We also found significant differences in the mutant allele frequencies at Ser53Gly and C595T single nucleotide polymorphisms between N34S positive patients and controls (p = 0.008 and 0.001, respectively). Although haplotype analysis did not complement the results of allelic association, it did uncover a unique haplotype protective for TCP (p = 0.0035). CONCLUSION: Our study suggests for the first time that CTSB polymorphisms are associated with TCP. As PRSS1 mutations are absent in TCP and the N34S SPINK1 mutation is proposed to play a modifier role, these variants may be critical as a trigger for cationic trypsinogen activation.     

Mutational screening of patients with nonalcoholic chronic pancreatitis: identification of further trypsinogen variants

OBJECTIVES: Mutations of the cationic trypsinogen (CT) and the serine protease inhibitor, Kazal type 1 (SPINK 1) are associated with chronic pancreatitis. After mutational screening of a cohort of patients with nonalcoholic chronic pancreatitis, we report three novel variants of the trypsinogen molecule and the clinical characteristics of the carriers. METHODS: The coding region of the exon 2 and 3 of the CT gene of 523 patients with chronic nonalcoholic pancreatitis (108 patients with suspected hereditary pancreatitis (HP) and 415 patients with "idio pathic" pancreatitis [IP]) and 82 controls was analyzed after polymerase chain reaction amplification. Clinical characteristics were obtained by questioning the patients and their relatives and physicians. HP was suspected when two members of a family had chronic pancreatitis. A restriction digestion was used to analyze the N34S mutation SPINK1. RESULTS: The mutation R122H of the cationic trypsinogen was found in 21 index patients, N291 in six index patients, and A16V and D22G in one index patient, all from HP families. The N34S mutation of SPINK1 was found in two index patients with a family history of HP. In three patients, the novel point mutations L104P, R116C, and C139F of the cationic trypsinogen were found. A clear autosomally dominant inheritance of chronic pancreatitis was not present in these families. In 75 index patients from HP families (69.4%), no mutation could be found. The SPINK 1-mutation N34S was detected in only one patient carrying a CT mutation, and was found in 68 (16.4%) of patients with IP. CONCLUSIONS: The R122H and N291 mutations of CT are the most common disease-associated mutations in HP; the N34S mutation of SPINK I is the most frequent genetic risk factor associated with IP. The CT gene carries several variations that could be associated with chronic pancreatitis. To avoid overestimating the pathogenetic impact of novel trypsinogen variants, a detailed clinical characterization of all patients with early onset chronic pancreatitis is mandatory.     

SPINK1/PSTI polymorphisms act as disease modifiers in familial and idiopathic chronic pancreatitis

BACKGROUND & AIMS: Gain-of-function trypsin mutations cause acute pancreatitis and chronic pancreatitis. Loss of trypsin inhibitor function may have similar effects. We investigated the prevalence of SPINK1 (PSTI) mutations in familial pancreatitis, idiopathic chronic pancreatitis, and controls. METHODS: Genetic-linkage studies were performed in 5 familial pancreatitis families. The entire SPINK1 gene was sequenced in 112 affected individuals and 95 control DNA samples, and exon 3 was sequenced in 95 additional controls. X-ray crystallography-based model building and statistical studies were performed. RESULTS: Significant linkage between pancreatitis and 5q31.1-2 was excluded. Novel SPINK1 mutations, one D50E mutation, one IVS3+125 C>A, and five IVS3+184 T>A intronic polymorphisms were identified. The N34S and P55S mutations were observed in 29 of 112 patients (25%) as N34S/N34S (n = 7), N34S/wt (n = 19), N34S/P55S (n = 2), and N34S/D50E (n = 1). Three hundred eighty control alleles revealed 3 N34S (0.77%), 2 P55S (0.53%), and no D50E mutations. Age of disease onset and severity were similar between homozygous and heterozygous patients. Structural modeling revealed several possible pathophysiologic mechanisms for the N34S mutation. CONCLUSIONS: SPINK1 mutations are common in the population (approximately 2%), but are clearly associated with pancreatitis. The mutation-associated risk is low. Modeling and familial clustering suggest that SPINK1 mutations are disease modifying, possibly by lowering the threshold for pancreatitis from other genetic or environmental factors, but by themselves do not cause disease.     

Presence of SPINK-1 variant alters the course of chronic pancreatitis

Background and Aims: There is growing evidence that genetic mutations/variants increase susceptibility to the development and progression of chronic pancreatitis (CP). Several mutations have been identified that have a direct and indirect role in events leading to CP. Mutations in the serine protease inhibitor, Kazal type‐1 (SPINK‐1) gene have been reported to lower the threshold for pancreatitis in the presence of other genetic or environmental factors. The prevalence and impact of SPINK‐1 mutations on the clinical course and outcomes of CP remains unclear. This study was conducted to assess the prevalence of the SPINK‐1/N34S variant in patients with CP, and to understand the impact of the SPINK‐1 mutation on the natural history of CP. Methods: A retrospective‐prospective analysis of 239 patients with CP was performed. A detailed history, including duration of symptoms, type of pain (intermittent flares or chronic continuous pain), number of flares requiring hospital admission, alcohol and smoking history, and family history was obtained. The baseline morphological stage of CP was categorized by Cambridge classification. Clinical outcome variables included frequency and severity of pain episodes, presence of exocrine failure (defined by presence of steatorrhea and/or fecal elastase < 200 ug/g), and diabetes. The genetic tests included the cationic trypsinogen gene‐1 mutation, cystic fibrosis gene mutations (Genzyme assay), and the SPINK‐1/N34S mutation. Results: Of the 239 patients with CP, 13 (5.4%) were positive for the SPINK‐1/N34S mutation. There were 35 (14.6%) patients with idiopathic pancreatitis (IP) in this cohort. Most of the patients who were positive for the SPINK‐1/N34S mutation had IP and were Caucasian (69.2%). The patients with the SPINK‐1/N34S mutation had a younger age of onset (32.9 ± 10.2 vs 40.1 ± 13.6 years; P = 0.108) than those with IP and no mutation. Over a median follow up of 9.6 years, the patients with the SPINK‐1/N34S mutation had a significantly greater number of acute flares each year, as compared to those without the mutation (11.8 ± 1.5 vs 4 ± 0.98; P = 0.0001). Conclusions: The prevalence of the SPINK‐1/N34S mutation in patients with CP is 5.4%, and is approximately 37.1% in patients with IP. These mutations are more prevalent in Caucasian patients with CP. The SPINK‐1/N34S mutation predisposes to early onset IP and more frequent acute flares of pancreatitis that might ultimately lead to pancreatic insufficiency. The patients with IP and borderline alcohol history should be considered for testing for genetic analysis, including SPINK‐1 mutations, initially restricted to clinical trials.     

Do genetic variants in the SPINK1 gene affect the level of serum PSTI?

Background

The serine protease inhibitor Kazal type 1 (SPINK1), also known as pancreatic secretory trypsin inhibitor (PSTI), is a peptide secreted by pancreatic acinar cells. Genetic studies have shown an association between SPINK1 gene variants and chronic pancreatitis or recurrent acute pancreatitis. The aim of this study was to clarify whether the SPINK1 variants affect the level of serum PSTI.

Methods

One hundred sixty-three patients with chronic pancreatitis or recurrent acute pancreatitis and 73 healthy controls were recruited. Serum PSTI concentrations were determined with a commercial radioimmunoassay kit.

Results

Ten patients with the p.N34S variant, 7 with the IVS3+2T>C variant, two with both the p.N34S and the IVS3+2T>C variants, and one with the novel missense p.P45S variant in the SPINK1 gene were identified. The serum PSTI level in patients with no SPINK1 variants was 14.3?±?9.6?ng/ml (mean?±?SD), and that in healthy controls was 10.7?±?2.2?ng/ml. The PSTI level in patients carrying the IVS3+2T>C variant (5.1?±?3.4?ng/ml), but not in those with the p.N34S variant (8.9?±?3.5?ng/ml), was significantly lower than that in the patients without the SPINK1 variants and the healthy controls. The serum PSTI level in the patient with the p.P45S variant was 4.9?ng/ml. Low levels of serum PSTI (<6.0?ng/ml) showed sensitivity of 80?%, specificity of 97?%, and accuracy of 96?% in the differentiation of IVS3+2T>C and p.P45S carriers from non-carriers.

Conclusion

Serum PSTI levels were decreased in patients with the IVS3+2T>C and p.P45S variants of the SPINK1 gene.     

Genetic basis of chronic pancreatitis in Asia Pacific region

Chronic pancreatitis (CP) is a disease characterized by irreversible destruction and fibrosis of the parenchyma, leading to pancreatic exocrine insufficiency. In developed countries, the etiology for 60% to 70% of CP amongst male patients is alcohol and 25% are classified as idiopathic chronic pancreatitis (ICP). The genetic predisposition to CP could be an inappropriate activation of trypsinogen in the pancreas. Two common haplotypes, c.101A>G (p.N34S) and c.-215G>A, and four intronic alterations of the serine protease inhibitor Kazal type 1 (SPINK1) gene have been found to increase the risk for CP in the Asia Pacific region. Hence, SPINK1 is thought to be a candidate gene for pancreatitis. A loss-of-function alteration in chymotrypsinogen C (CTRC) gene has been shown to be associated with tropical calcific pancreatitis (TCP). Cathepsin B (CTSB) is also found to be associated with TCP. However mutations in cationic and anionic trypsinogen gene do not play an important role in causing CP in Asia Pacific region.     

PRSS1 and SPINK1 mutations in idiopathic chronic and recurrent acute pancreatitis

AIM: To identify gene mutations in PRSS1 and SPINK1 in individuals with early onset idiopathic chronic or recurrent acute pancreatitis.METHODS: The cationic trypsinogen gene (PRSS1; exons 2 and 3) and the serine protease inhibitor Kazal 1 gene (SPINK1; exon 3) were selectively amplified and sequenced from blood samples of 19 patients admitted to the Pancreas Clinic at our institution with chronic pancreatitis and/or idiopathic recurrent acute pancreatitis that were diagnosed or with onset before age 35. Fifty healthy volunteers served as controls. Whole blood samples were collected and gene specific sequences were amplified by polymerase chain reaction (PCR). All PCR products were subsequently sequenced in order to identify the presence of any mutations.RESULTS: Nineteen patients with pancreatitis (14 males; median age 24 years, range 15-48 years) were included in this study, of which five showed the presence of gene mutations. Direct sequencing results indicated the presence of two previously unidentified mutations in exon 2 of PRSS1 (V39E and N42S) in two patients with recurrent acute pancreatitis. Two cases had the N34S SPINK1 mutation. Analysis of the relatives of one patient homozygous for this mutation showed that five of the six family members carried the N34S SPINK1 mutation. Of these members, three were healthy heterozygous carriers and two were homozygotes (one sibling had diabetes, the other was healthy). Another patient was heterozygous for a novel SPINK1 mutation located on exon 3 (V46D). All members from this patient’s family had normal genotypes, indicating that it was a de novo mutation. No mutations in either gene were present in the control subjects.CONCLUSION: Two novel PRSS1 mutations and one novel SPINK1 mutation were identified in Mexican patients with early onset idiopathic recurrent acute pancreatitis.     

Mutations of the serine protease inhibitor,Kazal type 1 gene,in patients with idiopathic chronic pancreatitis

OBJECTIVE: The pathogenesis of chronic pancreatitis (CP) is poorly understood. Genetic studies revealed mutations in the cationic trypsinogen gene and an increased frequency of cystic fibrosis gene mutations in patients with CP. Recently, a point mutation (N34S) in the gene encoding the serine protease inhibitor, Kazal type 1 (SPINK1), was found in approximately 20% of patients with CP. The aim of our study was to determine the frequency of the N34S SPINKI gene mutation in a well-defined patient cohort with idiopathic CP (ICP) and to compare the incidence with healthy controls. In addition, we investigated the impact of this mutation on the long-term course of CP. METHODS: Fourteen patients with early-onset and four patients with late-onset CP of our well-defined pancreatitis cohort were enrolled in the present study, and 397 healthy individuals served as a control population. Coding exonic and the flanking intronic sequences of SPINK1 were investigated by direct DNA sequencing. The mutations found were confirmed by melting curve analysis. In addition, the N34S mutation was detected by analyzing the DNA fragments generated by digestion with restriction enzyme TspR I. Clinical data of patients with the N34S mutation were compared with those without mutations. RESULTS: The N34S mutation was detected in six of 14 (43%) patients with early-onset ICP. One patient was homozygous, and five patients were heterozygous for this mutation. The N34S mutation in a heterozygous state was found in four of 397 healthy controls (1.0%). The different allele frequency observed (seven of 28 vs four of 794) was significant (odds ratio = 66, 95% CI = 18-242, p < 0.0001). The clinical course was similar in patients with a mutation compared with those without a mutation. No other SPINKI mutations were detected. The N34S mutation was not found in patients with late-onset ICP. CONCLUSIONS: Our results indicate that the N34S mutation in the SPINKI gene is strongly associated with ICP, especially with the early-onset type. The natural course is similar in patients with mutations compared with SPINK1 mutation-negative patients. The N34S mutation may easily be screened for by restriction digestion with TspR I.     

Mutations in the serine protease inhibitor Kazal Type 1 (SPINK1) gene in Japanese patients with pancreatitis.

BACKGROUND/AIMS: Recent studies have shown an association between the N34S mutation in the serine protease inhibitor Kazal type 1 (SPINK1) gene and chronic pancreatitis (CP). We here examined the prevalence of SPINK1 mutations in Japanese patients with pancreatitis. METHODS: Genomic DNA was prepared from 80 Japanese patients with CP, 36 patients with acute pancreatitis (AP), and 165 healthy controls. All exons and the promotor region of the SPINK1 gene were amplified by the polymerase chain reaction, and directly sequenced. RESULTS: We found four types of mutation (N34S, IVS1-37T>C, -215G>A, and IVS3 + 2T>C) and two types of polymorphism (-253T>C and 272C>T). The N34S mutation cosegregated with IVS1-37T>C, and was present in 8 CP and 1 AP patients. The -215G>A mutation was in a complete linkage with IVS3 + 2T>C, and was present in 8 CP and 1 AP patients. The prevalences of [N34S; IVS1-37T>C] and [-215G>A; IVS3 + 2T>C] were significantly higher in patients with familial pancreatitis (38 and 13%, respectively) and with idiopathic CP (13 and 16%) than normal subjects (0.6 and 0%). In addition, the frequency of [N34S; IVS1-37T>C] mutation was higher in patients with autoimmune CP (33%). CONCLUSION: The SPINK1 gene mutations were associated with pancreatitis also in Japan.     

Missense mutations in pancreatic secretory trypsin inhibitor (SPINK1) cause intracellular retention and degradation

BACKGROUND/AIMS: Mutations of the SPINK1 gene encoding pancreatic secretory trypsin inhibitor have been identified in association with chronic pancreatitis. The vast majority of patients carry the N34S variant, whereas other genetic variants are relatively rare and their disease association is uncertain. The aim of this study was to characterise and compare the functional defects caused by the six published missense mutations that affect mature SPINK1-namely, N34S, D50E, Y54H, P55S, R65Q, and R67C. METHODS: Wild type and mutant SPINK1 were expressed in human embryonic kidney 293T cells via transient transfection. SPINK1 expression was characterised by RT-PCR, activity assays, and western blots. RESULTS: Mutations N34S and P55S did not alter secretion of SPINK1 from HEK 293T cells, whereas mutation R65Q decreased secretion about twofold. Remarkably, mutations D50E, Y54H, and R67C abolished or markedly diminished secretion, but all three mutants were detected in cell extracts, indicating intracellular retention and degradation. CONCLUSIONS: The results identify intracellular folding defects as a novel mechanism of SPINK1 deficiency associated with chronic pancreatitis. The dramatic effects of the D50E and Y54H mutations indicate that the interaction between Asp50 and Tyr54 is critical for proper folding of the inhibitor. The disease-causing biochemical defect in the N34S mutant is unrelated to secretion or trypsin inhibitory activity and remains enigmatic. Finally, the patent functional defects in mutants D50E, Y54H, and R67C suggest disease association of these rare SPINK variants.