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Original Article: Pancreatology

Genetic and Electrophysiological Characteristics of Recurrent Acute Pancreatitis

Werlin, Steven*; Konikoff, Fred M.; Halpern, Zamir§; Barkay, Olga; Yerushalmi, Baruch||; Broide, Efrat; Santo, Erwin§; Shamir, Raanan#; Shaoul, Ron**; Shteyer, Eyal; Yaakov, Yasmin; Cohen, Michael; Kerem, Eitan; Ruszniewski, Philippe††; Masson, Emmanuelle‡‡; Ferec, Claude‡‡; Wilschanski, Michael

Author Information
Journal of Pediatric Gastroenterology and Nutrition: May 2015 - Volume 60 - Issue 5 - p 675-679
doi: 10.1097/MPG.0000000000000623
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Abstract

Acute pancreatitis (AP) in adults is the leading cause of hospitalization for gastrointestinal disorders in the United States (1). AP in children is an increasingly recognized clinical diagnosis (2,3). Until recently, the diagnosis of pancreatitis in children was hampered by the lack of accepted definitions. The International Study Group of Pediatric Pancreatitis In Search for a Cure consortium recommended that the diagnosis of AP meet at least 2 of the following 3 parameters: typical abdominal pain, an elevated amylase/lipase >3 times normal range, and/or confirmatory radiological findings (4). These are similar to the criteria used in adults.

The etiologies and management of an acute attack of pancreatitis are well known, but the occurrence of recurrent attacks of AP (defined as 2 or more episodes of pancreatitis) requires a different differential diagnosis including anatomic, environmental, metabolic, autoimmune, and genetic factors.

Chronic alcohol use and cholelithiasis are the most common causes of acute recurrent pancreatitis (ARP) in adults. The most common causes of ARP and chronic pancreatitis (CP) in children have been believed to be anatomic abnormalities, trauma, metabolic disorders, and drug toxicity (2). In a large study of patients of all ages, no etiology could, however, be found in 51% of patients (5). Improved imaging techniques and genetic testing have allowed the identification of patients at high risk for developing recurrent and CP (6). Advances in the field of genetics led to the discovery that mutations in the cationic trypsinogen (PRSS1), cystic fibrosis transmembrane conductor regulator (CFTR), and chymotrypsin C (CTRC) genes can predispose patients to develop both ARP and CP in both children and adults (7). There is growing evidence that a significant proportion of patients with “idiopathic” ARP and CP actually have mutations in 1 of the above-mentioned genes (8). Data from France in a group of 253 patients with onset of disease before age 20 revealed a genetic cause in 23.7% of individuals and a strong susceptibility factor in another 24.5% of patients. There has also been study of the serine protease inhibitor Kazal type 1 (SPINK1). Although mutations in SPINK1 may not predispose to pancreatitis, it is likely that a patient with a SPINK1 mutation who develops pancreatitis is at higher risk for recurrence (7,8). There has been no study relating abnormal electrophysiology (sweat test [ST], nasal potential difference [NPD]) to mutations in the CFTR or other genes. The aims of our study were to describe a single tertiary center experience of idiopathic ARP in Israeli children and adults and to assess the relations of electrophysiology (ST, NPD), mutations in the PRSS1, CFTR, SPINK, and CTRC genes.

METHODS

Patients were referred to the Pediatric Gastroenterology Department at Hadassah Hebrew University Medical Center. Nineteen patients were local, and 48 patients were referred from other Israeli centers. Patients included in the study answered a questionnaire including questions on alcohol use, and all of the patients had normal fasting lipids, immunoglobulin G4, and normal cholangiopancreatograms (endoscopic retrograde cholangiopancreatography or magnetic resonance cholangiopancreatography). The following genes were tested: PRSS1 (exons 1–3), SPINK1 (exons 1–4), CTRC (exons 3, 5, 6, 7), and CFTR (30 mutations as previously reported (8). If a CFTR mutation was found on 1 allele, full sequencing (exons 1–24) was carried out. STs were performed in the clinical laboratory of Hadassah Hospital or at the referring hospital. NPD was performed in the Electrophysiology Laboratory at Hadassah as previously reported (9). This study was approved by the Helsinki Committee of the Hadassah Hebrew University Medical Center and by the institutional review board of the Medical College of Wisconsin.

RESULTS

Sixty-seven patients were referred for the evaluation (34 men) with a mean age of 23 ± 17 years (median 17.0 years, range 1.5–72 years), and 35 patients were <18 years of age. All of the participants had genetic analysis for SPINK1, PRSS1, CTRC, and CFTR(10,11).

The number of episodes of pancreatitis ranged from 2 to >20. Two patients required temporary stenting. There were no reports of pseudocysts, and no patient required surgery. Other comorbidities included asthma in 5 patients and sinusitis in 4 patients. None of these 9 patients had mutations in the CFTR gene, abnormal STs, or abnormal nasal PD.

Ethnicity

The ethnicity was Ashkenazi Jewish, 23 (34%); Sephardic Jewish, 20 (30%); mixed Ashkenazi/Sephardic Jewish, 9 (14%); Georgian Jewish, 7 (10%); Arab, 7 (10%); and mixed Ashkenazi Jewish/non-Jewish, 1 (2%).

Electrophysiology

Sweat Chloride Test

Sweat chloride tests were performed on 54 patients. The ST was normal (sweat chloride <40 mmol/L) in 26, borderline (sweat chloride 40–60 mEq/L) in 23, and abnormal (sweat chloride >60 mmol/L) in 5.

Nasal Potential Difference Test

NPD was determined in 56 patients and was abnormal in 4. Three of the 4 patients with abnormal NPD had elevated sweat chloride tests (68, 82, and 100 mmol/L), and 1 had a borderline ST (54 mmol/L). The CFTR genetic analysis of these 4 patients revealed W1282X/Y1014C (1), R117H/Y1014C (1), and R117H/− (1). None of these 4 patients had pulmonary disease, but they were referred to specialist CF centers for follow-up.

Forty-nine patients underwent both ST and NPD, and are presented in Table 1 and Figure 1. These shows that 2 patients with elevated sweat chloride (>60 mmol/L) had normal NPD, 3 patients with sweat chloride >60 mEq/L had abnormal NPD and 1 patient with a borderline sweat chloride had an abnormal NPD.

TABLE 1
TABLE 1:
ST and NPD results for patients who underwent both tests
FIGURE 1
FIGURE 1:
ST versus NPD exponent index in patients who underwent both tests. The horizontal dashed line represents the cutoff value for NPD results: value <0.7 is normal NPD; value >0.7 is abnormal NPD. The vertical dashed line represents the cutoff value for sweat chloride level: value <60 is normal sweat chloride level; value >60 is abnormal sweat chloride level. NPD = nasal potential difference; ST = sweat test.

Genetics

Twenty-three of the 67 patients tested had mutations in 1 or more of the 4 genes tested (Table 2).

TABLE 2
TABLE 2:
Genetic analysis results of the genesPRSS1, SPINK1, CTRC, and CFTR *

PRSS1

All of the 67 patients had testing for mutations in PRSS1 with mutations found in 10. K23R was found in 7. R122H and D21A were found in 2 and 1 patients, respectively. Six patients with K23R had at least 1 Georgian parent. Seven of our patients were children <14 years of age, who already had had ARP. No patient had developed pancreatic cancer.

CFTR

Genetic testing for CFTR mutations revealed that 6/67 patients were compound heterozygotes and 4 carried a single mutation (Table 2). One patient with F508del/L997F had an ST of 40 mmol/L. L997F has previously been associated with pancreatitis. A second patient was a 4-year-old girl with STs of 45 and 52 mmol/L. On CFTR analysis she had W1282X/5T (12TG). Her NPD was normal. A third patient with F508del/5T had an ST of 55 mmol/L and a normal NPD. This patient, a 2-year-old-boy of Ashkenazi/Sephardic Jewish extraction, also had a SPINK1 mutation (I42M) and a CTRC mutation (V235I). A fourth patient with W1282X/Y1014C had elevated sweat chloride level (100 mmol/L) and abnormal NPD. We found Y1014C in 2 patients. Previously, it has been reported in a single patient but is not listed in the CFTR2 database (12). A fifth patient with F508del/R31C had normal sweat chloride level (25 mmol/L) and also normal NPD. R31C is considered to be a non–CF-causing mutation (12). A sixth patient with R117H/Y1014C had borderline sweat chloride level (54 mmol/L) and abnormal NPD.

SPINK1

Two of 67 tested had mutations in the SPINK1 gene. One was an Arab, heterozygote for R67H, also heterozygote for CTRC mutation (V235I). The second had CFTR and CTRC mutations.

CTRC

Four of the 67 patients tested for CTRC mutations were positive. One is heterozygous for K172E, and 3 are heterozygous for V235I. Two patients with V235I are also heterozygous for SPINK1 mutations.

Combined Mutations

One single patient had mutations in CFTR, SPINK1, and CTRC genes each of which is known to be associated with pancreatitis. This was the 2-year-old boy described above. Because there was only 1 patient with a single mutation in CFTR alone, SPINK1 alone, and CTRC alone, the clinical presentations of the groups cannot be described.

DISCUSSION

This study shows that in Israel pediatric as well as adult ARP and CP are often associated with PRSS1, SPINK1, CTRC, or CFTR mutations. Twenty-three of 67 patients (34%) with ARP and/or CP were found to have a mutation in 1 or more of these genes. The median age at testing was 17.0 years (range 1.5–72 years). As in previous publications from other parts of the world, those patients with genetic mutations were younger than the cohort; all but 2 were <18 years of age. This is a lower percentage than found in other recent studies (13,14)

PRSS1, the gene that is responsible for classical hereditary pancreatitis, is the most common gene responsible for ARP/CP (12,14). A PRSS1 mutation was present in 10 patients (15%). The median age at diagnosis was 11.3 years (range 2–41 years). Our patients had rare and unusual mutations. Only 2 had R122H, the most common mutation worldwide. Worldwide, 99% of patients have mutations in R122H (70%), N21 (25%), and A16V (4%). Seven of our patients had the K23R missense mutation, and 1 patient (Ashkenazi Jewish/non-Jewish Georgian) had the D21A mutation. K23R and D21A directly affect the trypsinogen activation bond (15). The activation peptides of these variants are believed to be released at a higher rate than from wild-type trypsinogen leading to increased amount of trypsin in the pancreas. The physiological activator enteropeptidase activates both the D21A and K23R mutants normally. These mutations have only rarely been reported. The high prevalence of unusual mutations likely reflects the varied genetic roots of the Israeli population. The 2 patients who required stents had R122H and K23R mutations.

CF is the most common inherited disease of the exocrine pancreas among whites. The association of CFTR mutations with idiopathic CP was initially described in 1998 (16–19). Symptomatic pancreatitis occurs in 22% of patients with CF and preserved pancreatic function but rarely occurs in patients with pancreatic insufficiency (20–22). A spectrum of pancreatic abnormalities occurs in patients with CF, especially in those with mild CF mutations. The mild-variable and borderline mutations, when combined with a severe CFTR mutation, are associated with atypical CF (21–24). The mild-variable mutations account for the residual pancreatic function that may protect these patients from lung disease caused by CF but are required for pancreatitis (24). Pancreatitis in these patients is believed to result from the viscid secretions producing pancreatic duct obstruction, ineffective clearing of secretions from the pancreatic duct, and autodigestion of the pancreas by activated proteolytic enzymes. In a recent study by Ooi et al (24), patients with pancreatic sufficient CF, 277 with pancreatitis and 215 without pancreatitis, were analyzed. Patients with pancreatitis were more likely to have genotypes associated with mild (70%) than moderate–severe (30%) disease based on the pancreatic insufficiency prevalence score. A recent study suggested a lower rate of CFTR mutations in patients with pancreatitis than had been previously thought (12).

The NPD test was abnormal in 4 patients, 2 had STs >60 mEq/L, and of the 22 patients with STs 40 to 60 mEq/L, 21 NPD tests were normal. All of these patients with borderline STs will need to be followed up, but only the patients with the abnormal NPD were referred to a CF center.

5T, 7T, and 9T are the polythymidine polymorphisms in IVS8 in intron 8 of the CFTR gene. Compared with the other IVS8 polymorphisms 7T (50%–100% normal-length transcript) and 9T (>95% normal length), in 5T there is little normal mRNA left and thus less functional CFTR protein is synthesized. When associated with a CF-causing mutation, 5T may have various consequences, from no health issues at all, congenital bilateral absence of the vas deferens, CP, or even CF with pancreatic insufficiency (25–27).

The role of the L997F mutation in CF is disputed (28). Lucarelli et al also found that genotype–phenotype variability is attributable to the formation of a complex allele (R117H/L997F) in those patients with the highest STs. Patients without this complex allele have wide variability in clinical findings. Strom et al (29) reported that L997F is a mild CF mutation. They detected the (R117H/L997F) complex allele in the 4 subjects with the highest ST values and CF. The 8 subjects without the complex allele showed the most varied biochemical and clinical outcome and were diagnosed as having mild CF, CFTR-related disorders, or even no disease. The new complex allele partially explains the variable phenotype in subjects with CF with the L997F mutation. CFTR complex alleles are likely to have a role in the definition of the genotype–phenotype relation in CF. We have also seen this mutation in 1 patient with CP in our study of Wisconsin patients (30). In the CFTR2 database it is classified as a non–CF-causing mutation (13). R31C is considered to be a non–CF-causing mutation when combined with a known CF causing mutation; however, there are 13 patients with this mutation in the CFTR2 database (13).

We found Y1014C in 3 patients. Previously it has been reported in a single patient with congenital absence of the vas deferens, but is not listed in the CFTR2 database (13,30). In our series, at least 1 CFTR mutation was found in only 4/58 (7%) patients. This is lower than previously published by us from Wisconsin and by others (5,19). It is undoubtedly an underestimate because full genome analysis was not performed. In Wisconsin, we found CFTR mutations in 14/29 children with idiopathic CP (30). Of 381 patients with a primary diagnosis of chronic or recurrent pancreatitis, Keiles and Kammesheidt (5) identified CFTR mutant alleles in 32% of 381 patients. Similarly, Bishop et al (31) found at least 1 CFTR mutation or variant in 18/40 patients (45%) with idiopathic CP and in 6/16 patients (38%) with idiopathic recurrent AP. In addition, Cohn et al (16) studied 27 patients referred for an evaluation of idiopathic pancreatitis. DNA was tested for 17 CFTR mutations and for the 5T variants; 10 patients (37%) had at least 1 abnormal CFTR allele. In the Cohn et al study the association between CFTR mutations and pancreatitis may have been underestimated as DNA was only tested for 17 CFTR mutations.

CTRC is a trypsin-degrading enzyme. Mutations that decrease CTRC activity promote the development of pancreatitis (32). Our series included only 4 patients with CTRC mutations, 2 of whom had mutations in multiple genes.

SPINK1 mutations are more common in recurrent pancreatitis but not in sentinel AP (33). We found SPINK1 mutations in 2 patients (3%), which is within the 0% to 6.5% published background rate for whites. One of our patients was, however, Arab and the other was Jewish Iraqi/Ashkenazi, and the background rate of this gene is not known for these populations. Neither of these mutations are listed in the Leipzig University database (34). Although R67H is not found in the Leipzig database, R67C is. Kuwata et al (35) reported a family in which the mother and a maternal uncle were homozygotes for the N34S mutation, whereas the father and brother were compound heterozygotes for the N34S and R67C mutations. One patient with multiple mutations had an I42M mutation in SPINK1. This mutation has not previously been described and is not listed in the Leipzig database.

Most of the mutations found in our patients were unusual. Eighteen of our patients had mutations in 1 or more of the 4 genes tested that are considered uncommon or rare based on studies of white populations from Europe or North America. Additional studies are required to further elucidate the mutations leading to genetic pancreatitis in patients from relatively isolated populations, such as Bedouin Arabs and Georgians.

One of the strengths of this study is the unusual population studied. K23R is a rare missense mutation in the white population. In this study 6 among the 7 patients carrying the K23R were of Georgian origin; this is probably the result of a founder effect in this population of this rare amino acid change, and this is a real confirmation that the K23R is a defective PRSS1 mutation.

Weaknesses of this study include the fact that because many patients were referred only for electrophysiological studies, not all the relevant clinical data were available for all of the patients. In some patients CFTR analysis was limited to the most common mutations. It is known that in approximately 5% of Ashkenazi Jewish and 40% of Sephardic Jewish and Arab patients with CF no CFTR mutations are detected (8). Similarly, not all of the patients had the full battery of genetic and electrophysiological testing. Despite the relatively large number of positive findings, the “idiopathic” group is still dominant. Future research including other genes will be needed to elucidate the etiology of this group of patients.

In conclusion, this is the first study in Israel on recurrent pancreatitis. We found a relatively high prevalence of hereditary pancreatitis mutations and a relatively small number of patients with atypical CF. NPD is a useful test to rule out CF in borderline cases.

Acknowledgments

The authors thank Ayala Yaron, MD (Souraski Medical Center), Chagit Nagar, MD (Shaare Zedek Medical Center), Yosef Klauzner, MD (Souraski Medical Center), Miri Berenshtein, MD (Meir Medical Center), Vered Pinsk, MD (Soroka Medical Center), Zvi Akerman, MD (Hadassah Hebrew University Medical Center), Amiel Migdal, MD (Hadassah Hebrew University Medical Center), Malena Cohen, MD (Hadassah Hebrew University Medical Center), Eran Goldin, MD (Shaare Zedek Medical Center), Harold Jacobs, MD (Hadassah Hebrew University Medical Center), Lea Bentur, MD (Meyer Children's Medical Center), Hanna Blau, MD (Schneider Children's Medical Center), Shmuel Goldberg, MD (Shaare Zedek Medical Center), Dan Turner, MD (Shaare Zedek Medical Center), Husein Shamaly, MD (French Hospital), and Michal Kori, MD (Kaplan Medical Center) for their support.

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Keywords:

acute recurrent pancreatitis; cftr; nasal potential difference

© 2015 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,