Hereditary pancreatitis in a family of Aboriginal ... - Wiley Online Library

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Genetic testing revealed a pathogenic mutation in the cationic trypsinogen gene in the proband, her father and her paternal grandmother. As far as we are aware ...

J. Paediatr. Child Health (2004) 40, 487–489

Hereditary pancreatitis in a family of Aboriginal descent JM McGaughran,1,2 R Kimble,2 J Upton,3 and P George3 1Queensland Clinical Genetics Service, Royal Children’s Hospital, 2University of Queensland, Department of Paediatrics and Child Health, Royal Children’s Hospital, 3Health District, Brisbane, Queensland, Australia and 3Molecular Pathology Laboratory, Canterbury Health Laboratories, Christchurch Hospital, Christchurch, New Zealand

Abstract: Hereditary pancreatitis is an autosomal dominant condition characterized by recurrent episodes of acute pancreatitis, usually starting in childhood. We present a family who was ascertained when an 11-year-old girl presented with an episode of acute pancreatitis. Her father and other family members had also had recurrent bouts of acute pancreatitis. Genetic testing revealed a pathogenic mutation in the cationic trypsinogen gene in the proband, her father and her paternal grandmother. As far as we are aware, this is the first Aboriginal kindred with mutation-proven hereditary pancreatitis. Hereditary pancreatitis is an important differential diagnosis to consider in a patient with recurrent episodes of acute pancreatitis with no obvious precipitating cause. This family is of Aboriginal descent and the implications of the family’s background are also discussed when considering the aetiology of the condition. We emphasize the need to ascertain a full family history from patients with a history of repeated episodes of acute pancreatitis and also emphasize the need to avoid ethnic stereotypes when assessing patients. Key words: Aboriginal; hereditary pancreatitis.

The first reported description of hereditary pancreatitis (HPC) was by Comfort and Steinberg (1952).1 They reported the case of a 26-year-old man from a family in which 6 of 34 members had confirmed pancreatitis and an additional three members had suspected pancreatitis. By 1994, more than 50 families with hereditary pancreatitis had been described.2 It is an autosomal dominant condition with a penetrance of 80%, often presenting in childhood.3 The major gene for the condition, the cationic trypsinogen gene on 7q, was identified in 1996.4 A common mutation in the cationic trypsinogen gene has been found in many families with hereditary pancreatitis. This was originally referred to as the 117 Arg-to-His (R117H) mutation and was found initially in eight unrelated families with hereditary pancreatitis. This has since been re-designated the 122 Arg to His mutation or R122H and other mutations in the same gene have been identified in other families with hereditary pancreatitis.5,6 Mutations in the serine protease inhibitor, Kazal type 1 (PSTI), a pancreatic trypsin inhibitor, have also been identified in patients with chronic pancreatitis. Witt et al. in 2000 demonstrated mutations in the PSTI gene in children and adolescents with chronic pancreatitis.7 Eighteen of 96 patients had a missense mutation in codon 34 (N34S). A review of the incidence of this mutation looked at three studies where a normal population had been tested. The incidences were 0.18%, 0.79% and 0.75%.8 Whilst the mutation is present in less than 1% of the population, the incidence of chronic pancreatitis is lower, suggesting other factors are needed for the development of pancreatitis. We present a family of Aboriginal descent who was ascertained when an 11-year-old girl presented with acute pancreatitis.

CASE REPORT The proband was an 11-year-old female who presented with acute abdominal pain. An appendicectomy was carried out but as there was no resolution of her symptoms, a laparotomy was carried out. Acute pancreatitis was diagnosed clinically and confirmed with serum lipase levels. She had a number of investigations carried out to investigate the cause of her pancreatitis including serum calcium and lipid profile and imaging with ultrasound, CT and Magnetic resonance cholangiopancreatography (MRCP). She did not have any of the eight common mutations for cystic fibrosis. During this admission, she had a course of relapsing episodes of pancreatitis eventually requiring a partial pancreatectomy. She developed a pancreatocutaneous fistula, which has now resolved. She has also required drainage of a pseudocyst, which was carried out endoscopically.8 Since her initial presentation, she has not had any further episodes of pancreatitis. She is not diabetic and does not require pancreatic enzyme supplements. A family history was taken. Her father had developed acute pancreatitis at the age of 18 years. He continues to have at least annual episodes requiring hospitalization. He has had a partial pancreatectomy, is an insulin-dependent diabetic and requires pancreatic enzyme supplements. There were no obvious precipitating factors and he did not drink alcohol. He was, like the proband’s mother, of Aboriginal ancestry. His mother is asymptomatic but her mother and sister both have insulin-dependent diabetes. Neither, apparently, has a history of pancreatitis. One of his mother’s nephews had developed episodes of acute pancreatitis at the age of 9 years. He was also said to have insulin-dependent diabetes and require pancreatic enzyme supplementation. Another cousin

Correspondence: Dr JM McGaughran, Queensland Clinical Genetics Service, Royal Children’s Hospital, Herston, Brisbane 4029, Qld, Australia. Fax: +61 7 3636 1987; email: [email protected] Accepted for publication 10 July 2003.

488 was believed to have had pancreatitis but no clear information was available. Given the family history of recurrent acute pancreatitis, it was felt the family, clinically, had hereditary pancreatitis. DNA was obtained for mutation analysis. Investigations In view of the family history, the proband was first screened for the two common mutations associated with pancreatitis, the R122H mutation in cationic trypsinogen and the missense mutation in codon 34 (N34S) of the PSTI gene. Informed consent was obtained for the DNA testing.

METHODS Genomic DNA was extracted from peripheral whole blood using the method of Ciulla et al.10 Primer sequences used for PCR amplification were as follows:4,7 Cationic trypsinogen exon 3: 5′-CgATAggTCCTgggTCTCATACCTT-3′ and 5′-gTAAATAgTTTgCTTTTCTCggggTgAg-3′ Pancreatic secretory trypsin inhibitor (PSTI) exon 3: 5′-TCTCCCAATCACAgTTATTCCCCAgAg-3′ and 5′-gTAAATAgTTTgCTTTTCTggggTgAg-3′ PCR was performed using Platinum Taq polymerase (0.5 U: Invitrogen), approximately 50 ng DNA template, deoxynucleoside triphosphates (200 µmol/L) and primer (0.5 µmol/L) in a total volume of 50 µL. Cycle conditions were, for the cationic trypsinogen exon 3 mutation, initial denaturation for 2 min at 96°C followed by an initial annealing for 15 s at 60°C; 30 cycles of 2 min at 70°C (anneal/extend) and 30 s at 96°C, with a final extension of 10 min at 70°C. Conditions for the PSTI exon 3 mutation were; initial denaturation for 2 min at 96°C followed by an initial annealing for 15 s at 60°C; 35 cycles of 30 s extension at 72°C, 30 s denaturation at 96°C and 15 s annealing at 67°C, with a final extension of 10 min at 72°C. The presence of PCR product was confirmed by electrophoresis on 2% agarose gel. Restriction digestions The Arg to His (nt 133 283 g- > A) mutation in exon 3 of cationic trypsinogen creates a restriction site for Afl III (New England Biolabs, MA). Digestion was carried out at 37°C for 3 h (or overnight) using 10 µL of PCR product and 5 U of Afl. The 34 Asn to Ser (nt 101 A- > g) mutation in exon 3 of PSTI creates a restriction site for TspR1 (NEB). Digestion was carried out a 65°C for 2 h using 40 µL of PCR product and 10 U of TspR1. All digestion products were separated by electrophoresis on 2% agarose at 100 V and stained with ethidium bromide.

RESULTS The proband was found to have the common exon 3 R122H mutation in the cationic trypsinogen gene. Her father, and his mother, were subsequently tested and found to have the same mutation. Both the proband and her father were tested for the PSTI 34 Asn- > Ser mutation. The mutation was not present.

JM McGaughran et al. DISCUSSION Hereditary pancreatitis was first described in 1952.1 It is an autosomal dominant condition with 80% penetrance. The major gene involved in its aetiology was localized by linkage analysis in 1996. Whitcomb et al.11 and Pandya et al.12 performed a genomewide linkage analysis on families extensively affected with hereditary pancreatitis from Kentucky, Western Virginia, Virginia, and Tennessee. They established linkage between the hereditary pancreatitis phenotype and 7q. Several genes previously mapped to 7q were considered candidates for HPC because they were known to be expressed in the exocrine pancreas and to encode enzymes that could potentially activate digestive enzymes within the pancreas. At least eight trypsinogen genes are located on 7q35. Trypsinogen is an inactive proenzyme for trypsin, which becomes active when an 8-amino acid N-terminal peptide is removed. Mutational screening analyses for each of the exons from two of the genes, the cationic and anionic trypsinogen genes, was carried out in multiple affected and unaffected family members. Whitcomb et al. in 1996 identified a single G-to-A transition in the third exon of cationic trypsinogen in all affected members and obligate carriers in 1 family.4 The mutation was predicted to result in an Arg (CGC)-to-His (CAC) substitution at amino acid residue 122 of trypsin. This mutation has since been found in a number of other families, including the one reported here. Other causes of pancreatitis have been ruled out in both the proband and her father and the presence of the mutation with the striking family history makes it likely that the mutation is the cause of their pancreatitis. Although this family is of Aboriginal descent, a number of their ancestors were European. The mutation seen in them is well reported in Europeans and is believed by family members to have been inherited from the side of the family of European descent. The pathogenesis of the condition has also been considered. The hypothesis that pancreatitis results from inappropriate activation of pancreatic proenzymes was first promulgated by Chiara in 189613 and subsequently demonstrated to be an experimental model for pancreatitis.14 Whitcomb et al. provided a diagram of a model of the trypsin self-destruct mechanism designed to prevent pancreatic autodigestion.4 Active trypsin is inhibited normally by a limited supply of trypsin inhibitor. If trypsin activity exceeds the inhibitory capacity of PSTI, then proenzymes, including mesotrypsin and enzyme Y, are activated. The activation of these enzymes is postulated to be part of a feedback mechanism for inactivating wildtype trypsinogen, trypsin, and other zymogens. When the Arg122 cleavage site for mesotrypsin, enzyme Y, and trypsin is replaced by histidine, trypsin continues to activate trypsinogen and other zymogens unabated, leading to autodigestion of the pancreas and pancreatitis. A number of treatments have been suggested to decrease the frequency and severity of episodes of acute pancreatitis. These include taking vitamin E, selenium and antioxidants. Small carbohydrate rich meals should be eaten and alcohol and tobacco avoided. However, there are only limited data suggesting these may have some effect.15 Another long-term complication of this condition is an increased risk of pancreatic malignancy, up to 40% by the age of 70 years.16 In this family, the paternal grandmother of the proband is a non-penetrant carrier of the condition, demonstrating the variability of the condition. The family history of the proband suggested the possibility of HP as the diagnosis. The father of the proband had required numerous hospital admissions over the years. He gave no history of precipitating factors for his pancreatitis. He has not consumed alcohol since his first

Hereditary pancreatitis in Aboriginal family

episode at aged 18 years. However, because of his Aboriginal background, it was considered that inappropriate alcohol ingestion was the most likely cause even though he strongly denied this. On occasion it was suggested to him by nursing staff that he was drinking but not admitting to it. It has been suggested that excessive drinking is associated with negative racial stereotypes in Aboriginals.17 This emphasizes the need to ascertain a full family history from patients with a history of repeated episodes of acute pancreatitis and also emphasizes the need to avoid ethnic stereotypes when assessing patients.

REFERENCES 1 Comfort MW, Steinberg AG. Pedigree of a family with hereditary chronic relapsing pancreatitis. Gastroenterology 1952; 21: 54–63. 2 Rumenapf G, Kamm M, Rupprecht H, Scheele J. Surgical management of hereditary pancreatitis: report of a case and presentation of a new family. (Letter). Pancreas 1994; 9: 398–9. 3 Keim V, Bauer N, Teich N et al. Clinical characterization of patients with hereditary pancreatitis and mutations in the cationic trypsinogen gene. Am. J. Med. 2001; 111: 622–6. 4 Whitcomb DC, Gorry MC, Preston RA et al. Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nature Genet. 1996; 14: 141–5. 5 Ferec C, Raguenes O, Salomon R et al. Mutations in the cationic trypsinogen gene and evidence for genetic heterogeneity in hereditary pancreatitis. J. Med. Genet. 1999; 36: 228–32. 6 Gorry MC, Gabbaizedeh D, Furey W et al. Mutations in the cationic trypsinogen gene are associated with recurrent acute and chronic pancreatitis. Gastroenterology 1997; 113: 1063–8.

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7 Witt H, Luck W, Hennies HC et al. Mutations in the gene encoding the serine protease inhibitor, Kazal type 1 are associated with chronic pancreatitis. Nature Genet. 2000; 25: 213–16. 8 Chen JM, Mercier B, Audrezet MP, Raguenes O, Quere I, Ferec C. Mutations of the pancreatic secretory trypsin inhibitor (PSTI) gene in idiopathic chronic pancreatitis. Gastroenterology 2001; 120: 1061–4. 9 Kimble RM, Cohen R, Williams S. Successful endoscopic drainage of a pancreatic pseudocyst in a child. J. Pediatr. Surg. 1999; 34: 1518–20. 10 Ciulla TA, Sklar RM, Hauser SL. A simple method for DNA purification from peripheral blood. Anal. Biochem. 1988; 174: 485–8. 11 Whitcomb DC, Preston RA, Aston CE et al. A gene for hereditary pancreatitis maps to chromosome 7q35. Gastroenterology 1996; 110: 1975–80. 12 Pandya A, Blanton SH, Landa B et al. Linkage studies in a large kindred with hereditary pancreatitis confirms mapping of the gene to a 16-cM region on 7q. Genomics 1996; 38: 227–30. 13 Chiara H. Ueber Selbstverdauung des menschlichen Pankreas. Ztschr. Heilkunde 1896; 17: 70–96. 14 Steer ML, Meldolesi J. The cell biology of experimental pancreatitis. N. Eng. J. Med. 1987; 316: 144–50. 15 Uomo G, Talamini G, Rabitti PG. Antioxidant treatment in hereditary pancreatitis. A pilot study on three young patients. Digestive Liver Dis. 2001; 33: 58–62. 16 Lowenfels AB, Maisonneuve P, Whitcomb DC. Risk factors for cancer in hereditary pancreatitis. International Hereditary Pancreatitis Study Group. Med. Clinics North Am. 2000; 84: 565–73. 17 Healy B, Turoin T, Hamilton M. Aboriginal drinking: a case study in inequality and disadvantage. Aust. J. Social Issues 1985; 20: 191–208.

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