EDITORIALS

How to Think About SPINKand PancreatitisGenetics has clearly become important in understanding thepancreas. The discovery that mutations in the cationictrypsinogen gene (PRSS1) lead to an 80% likelihood ofdeveloping recurrent acute and/or chronic pancreatitis stim-ulated intense investigation into genetic factors predisposingto pancreatic diseases. Mutations in two other genes encod-ing pancreatic proteins—the cystic fibrosis transmembraneconductance regulator (CFTR) gene (1–3) and the pancre-atic secretory trypsin inhibitor (serine protease inhibitorKazal type 1 [SPINK1]) gene (4–7)—clearly contribute tothe burden of pancreatic diseases.

In this issue Truningeret al. (8) describe a genetic asso-ciation study of SPINK1 mutations in patients with idio-pathic chronic pancreatitisversus a control population. Thisgroup first described the associations between mutations inthe SPINK1 gene and idiopathic chronic pancreatitis inchildren in June, 2000 (4). Two closely associated articleswere also published in the year 2000 demonstrating thatSPINK1 mutations were present in the general populationbut were not associated with typical hereditary pancreatitis(9), and that SPINK1 mutations were associated with famil-ial pancreatitis and idiopathic pancreatitis developing beforethe age of 20 yr (5). Since then several studies confirmed theassociations between SPINK1 mutations and various otherforms of pancreatitis in diverse populations, including about6% of patients with alcoholic pancreatitis (6) and over athird of patients with a subtype of tropical pancreatitis (10).In the current report a highly significant association wasidentified between the SPINK1 N34S mutation and early-onset chronic pancreatitis relative to a control population.What does this mean in clinical practice, and how should thephysician think about SPINK1 mutations?

Understanding the clinical impact of SPINK1 mutationsrequires answers to several questions. First, how do SPINK1mutations predispose to pancreatitis? Second, is the mildSPINK1 N34S mutation clinically important? Third, doSPINK1 mutations cause hereditary pancreatitis or familialpancreatitis? And finally, should patients be geneticallyscreened for SPINK1 mutations?

How do SPINK1 mutations predispose to pancreatitis?Currently we believe that the SPINK1 (the pancreatic se-cretory trypsin inhibitor) molecule is capable of inhibitingup to 20% of the potential trypsin within the pancreas in theevent trypsinogen becomes prematurely activated (11).SPINK1 therefore forms the first line of defense againstprematurely activated trypsinogen. The second line of de-fense is the fail-safe autolysis step whereby trypsin or me-sotrypsin cleaves the trypsin side chain that connects the twohalves of the trypsin molecule exactly at the key site mu-

tated in some cases of hereditary pancreatitis [PRSS1R122H (12) and R122C (13)]. Therefore, aloss of SPINK1function should increase free trypsin activity inside thepancreas by allowing active trypsin to overcome the firstline of defense more easily (4, 5).

The second question is whether the SPINK1 N34S mu-tation is clinically important. It turns out that the simplemechanistic model of SPINK1 being “on” or “off” could notbe directly extended to the common SPINK1 N34S muta-tions. The first surprise came with the discovery thatSPINK1 N34S mutations are very common in nearly allcontrol populations, with a prevalence of up to�2% ofpatients (1% alleles frequency) (4, 5, 7, 9). This means thatthe SPINK1 N34S mutation ismany times more commonthan chronic pancreatitis (1/16,000, or 0.006% of the pop-ulation) and especially more common than idiopathicchronic pancreatitis in children. The second surprise is thatpatients with idiopathic pancreatitis may have genotypesthat are heterozygous, compound heterozygous, or homozy-gous for SPINK1 N34S mutations without a clear differencein age of onset or disease severity (5). This may mean thatthe SPINK1 N34S mutation is an important risk factor forpancreatitis, but the effect may be more of a disease mod-ifier, potentiating the harmful effects of other genetic orenvironmental insults to the pancreas that actually initiateepisodes of pancreatitis (5, 14, 15).

Determining whether or not a mutation is clinically im-portant requires understanding several interacting factors,including the severity of the mutation, the prevalence of themutation in specific populations, and the role of the mutatedgene in normal physiology. Theseverity of a mutationreflects a change in the function of the encoded protein byaltering the amount or location of expression, the proteinfunction, or the capacity to be regulated by or to interactproperly with other factors. In some cases a mutation maycause a gain-of-function, as in the hereditary pancreatitis–associated trypsinogen gene mutations (16). In general,more severe mutations are more likely to cause disease inindividual patients with those mutations. However, at thepresent time the SPINK1 N34S mutation does not seem tobe a severe mutation.

Next, theprevalence of a mutation (or DNA sequencevariance) impacts the question of clinical importance. Onecan envision a large variety of very severe gene mutationsthat disrupt the function of key proteins, but if they are neverseen in large populations of patients, then they are of lessgeneral clinical importance. In the case of the SPINK1N34S mutation the underlying DNA sequence variant isvery common, as noted above. As an interesting side note,clues exist as to why the N34S mutation is so common. Itturns out that the N34S polymorphism is very close to four

THE AMERICAN JOURNAL OF GASTROENTEROLOGY Vol. 97, No. 5, 2002© 2002 by Am. Coll. of Gastroenterology ISSN 0002-9270/02/$22.00Published by Elsevier Science Inc.

other DNA sequence variants surrounding the SPINK1gene, and they are usually inherited together (completelinkage disequilibrum). Because it would be nearly impos-sible for these five mutations to occur together in unrelatedpeople by random chance, it appears that the worldwidedistribution of the SPINK1 N34S mutation came from asingle individual or disease founder. Indeed, these five se-quence variants are present in patients from France (17),Germany (4), the United States (5, 14), Japan (18), andBangladesh (10). Another interestingly point is that Witt etal. (4) described a functionally severe SPINK1 M1T muta-tion that interrupts the expression of exon 1. However, thisis a very rare mutation and therefore of less general clinicalsignificance. Thus, the prevalence of a DNA sequence vari-ance in the population is an important consideration.

The physiological function of the gene product plays acritical role in determining whether the mutation is clini-cally important. In other words, does the gene play a pivotalrole in a critical process? Are there adaptive or redundantmechanisms in place to lessen the impact of a mutation?Does the disease seem to have an autosomally dominant,autosomally recessive, or complex genetic pattern, and doesit require environmental factors or mutations in a secondgene to become clinically evident? Growing experiencewith gene “knockout” mice reveals that many genes we onceconsidered to be critical fail to cause disease when knockedout. SPINK1 is thought to play an important but incompleterole as the first line of defense against excess trypsinogenactivation. So partially bypassing the first line of defensealone may be insufficient to cause pancreatitis. However,the mutation clearly increases the risk of pancreatitis.

The SPINK1 N34S mutation is likely mild and veryprevalent, and alters an important but not critical role inprotecting the pancreas from pancreatitis. This mutation isclearly clinically important because of the high incidence ofthis mutation in idiopathic chronic pancreatitis. The unan-swered question is whether and which additional genetic orenvironmental factors must be present for pancreatitis todevelop in a person with the common SPINK1 N34S mu-tation.

The principles outlined for determining the clinical im-portance of SPINK1 mutations can also be applied to otherpancreatitis-associated gene mutations. The first example,noted at the beginning of this article, encompasses muta-tions in the cationic trypsinogen gene (PRSS1). The PRSS1R122H (19), N29I (20), R122C (13, 21, 22), and N29T (13)mutations are severe, they are relatively uncommon in gen-eral populations [although they may be observed in �10%of some populations with idiopathic pancreatitis (23, 24)],but the functional role for trypsin appears central, so that thedisease appears as an autosomally dominant disorder. An-other example is cystic fibrosis. The CFTR �F508 mutationis severe, it is very common in white populations, and theCFTR molecule plays a central role in epithelial cell phys-iology. The homozygous CFTR �F508 mutation produces aclear autosomally recessive disorder, cystic fibrosis (25).

However, there are about 1000 other mutations and DNAsequence variations in the CFTR gene, so many compoundheterozygous genotypes appear. These sequence variationshave been classified into six groups depending on the impactof the mutation in the function of the CFTR protein (26).Recent evidence suggests that many patients with idiopathicchronic pancreatitis harbor a combination of one severemutation, such as �F508, and a milder mutation, such asCFTR R117H, which allows enough CFTR activity for mostorgans to function (1). However, this combination of severeand mild CFTR mutations clearly predisposes individuals torecurrent acute and chronic pancreatitis (1). Thus, in con-sidering the clinical impact of mutations, one must considerwhether or not the mutation itself is severe, whether or notit is common, and whether or not it disrupts a physiologi-cally critical process.

The third major question about SPINK1 is whether theSPINK1 N34S mutation causes hereditary pancreatitis orfamilial pancreatitis. To answer this question one must firstdefine the two. In pancreatic disease, hereditary pancreatitisis a narrow, specific term, whereas familial pancreatitis is abroad, general term that encompasses hereditary pancreati-tis. The foundation of the definition of hereditary pancre-atitis rests on the observation that the pancreatitis phenotypeappears to be inherited in an autosomally dominant pattern(27). This implies an underlying genetic factor. The termfamilial pancreatitis reflects the observation that more pan-creatitis is present in a family then would be expected, giventhe frequency of pancreatitis within a defined population(e.g., more than one case of pancreatitis in a typically sizedfamily). Familial pancreatitis may or may not be caused bya genetic defect. Therefore, this broader definition clearlyencompasses hereditary pancreatitis. Clarifications of thesedefinitions are becoming critical because of the strong med-ical, ethical, and potentially legal implications associatedwith the diagnosis of “hereditary pancreatitis” (28–30).Indeed, the diagnosis of hereditary pancreatitis has powerfulimplications for the patient’s medical future or insurability,and for the patient’s descendants and other relatives. Aprecise definition is also important from a public health andepidemiological perspective. The current difficulty withthese definitions arises with the identification of pancreati-tis-associated mutations and the use of genetic testing. Forexample, we recently demonstrated that the observed inher-itance pattern of pancreatitis within many families did notpredict the underlying “hereditary pancreatitis” causing mu-tations in the PRSS1 gene (31). Even the strongest heredi-tary pancreatitis–associated mutations (PRSS1 R122H andN29I) were detected in families that appeared to inherit thepancreatitis trait in an autosomally dominant, autosomallyrecessive, familial, or sporadic pattern (31). Furthermore,about 40% of large families with a clear autosomally dom-inant inheritance pattern do not have identifiable genespredisposing to these mutations (31). Therefore, the role ofgenetic testing in the definition and diagnosis of hereditary

1086 Editorials AJG – Vol. 97, No. 5, 2002

pancreatitis is uncertain. Until an international consensus isreached on the definition of hereditary pancreatitis, weusually diagnose an individual with it only when multiplemembers of the family have otherwise unexplained pancre-atitis that appears in an autosomally dominant inheritancepattern, or when a patient is diagnosed with a PRSS1R122H, N29I, or related mutation. SPINK1 mutations thatusually do not cause pancreatitis appear in an autosomallydominant pattern (5, 9), although they are often present infamilies defined as having familial pancreatitis (5) or spo-radic idiopathic pancreatitis (4, 5, 32).

Thus, our group does not consider the finding of aSPINK1 N34S mutation diagnostic of hereditary pancreati-tis, but we do consider it a risk factor contributing to familialpancreatitis.

In light of the above discussion, should patients withconfirmed or suspected pancreatitis or at risk of it be testedfor SPINK1 mutations? Genetic test results might have adeep impact on lifestyle decisions and, possibly, insurabil-ity, as chronic pancreatitis and, especially, hereditary pan-creatitis are associated with pancreatic exocrine insuffi-ciency and diabetes mellitus (31, 33) and a lifetimepancreatic cancer risk of approximately 40% (34–36). InApril, 2001, a consensus conference was held during theThird International Symposium on Inherited Diseases of thePancreas (37) to help establish guidelines for genetic testingin patients with pancreatitis (30, 38, 39). It was the consen-sus that, at present, only testing for PRSS1 R122H or N29Imutations can be clearly justified as having a clinical benefitfor symptomatic patients. The published guidelines recom-mended testing for PRSS1 mutations in patients with unex-plained recurrent acute pancreatitis, unexplained chronicpancreatitis, or family histories of pancreatitis or in a childsuffering an unexplained episode of pancreatitis. They fur-ther recommended that genetic testing for all other genesassociated with pancreatitis, including SPINK1 mutations,should only be performed in research ethics committee–approved protocols (30). However, there may be clinicalcircumstances in which genetic testing for the SPINK1N34S mutation is justified (e.g., to determine if a cause otherthan alcohol abuse may be contributing to chronic pancre-atitis in a young patient), and it is currently available insome commercial laboratories (e.g., Molecular DiagnosticsLaboratory, Department of Pathology, University of Pitts-burgh). Thus, because of the high prevalence of the mutationin the general population and the uncertainty of the role ofother factors, this test should probably be limited to patientswith unexplained existing pancreatitis in an effort to identifycontributing risk factors. Waiting until additional studies arecompleted to better interpret any SPINK1 genetic test re-sults appears to be the consensus opinion of most experts.

Recognition of the role and importance of SPINK1 mu-tations in pancreatic disease is likely to grow. The informedclinician must be prepared to respond to this new informa-

tion in an intelligent manner. The article by Truninger et al.(8) represents a great start.

David C. Whitcomb, M.D., Ph.D.Division of Gastroenterology, Hepatology and Nutrition

Departments of Medicine, Cell Biology & Physiology andHuman Genetics

University of PittsburghPittsburgh, Pennsylvania

REFERENCES

1. Noone PG, Zhou Z, Silverman LM, et al. Cystic fibrosis genemutations and pancreatitis risk: Relation to epithelial ion trans-port and trypsin inhibitor gene mutations. Gastroenterology2001;121:1310–9.

2. Cohn J, Friedman K, Silverman L, et al. CFTR mutationspredispose to chronic pancreatitis without cystic fibrosis lungdisease. Gastroenterology 1997;114(4):A434 (abstract).

3. Sharer N, Schwarz M, Malone G, et al. Mutations of the cysticfibrosis gene in patients with chronic pancreatitis. N EnglJ Med 1998;339:645–52.

4. Witt H, Luck W, Hennies HC, et al. Mutations in the geneencoding the serine protease inhibitor, kazal type 1 areassociated with chronic pancreatitis. Nat Genet 2000;25:213–6.

5. Pfutzer RH, Barmada MM, Brunskil APJ, et al. SPINK1/PSTIpolymorphisms act as disease modifiers in familial and idio-pathic chronic pancreatitis. Gastroenterology 2000;119:615–23.

6. Witt H, Luck W, Becker M, et al. Mutation in the SPINK1trypsin inhibitor gene, alcohol use, and chronic pancreatitis.JAMA 2001;285:2716–7.

7. Threadgold J, Greenhalf W, Ellis I, et al. The N34S mutationof PSTI (SPINK1) is associated with a familial pattern ofidiopathic chronic pancreatitis but does not cause the disease.Gut (in press).

8. Truninger K, Witt H, Kock J, et al. Mutations of the serineprotease inhibitor, Kazal type 1 gene, in patients withidiopathic chronic pancreatitis. Am J Gastroenterol 2002;97:1126 –30.

9. Chen J-J, Mercier B, Audrezet M-P, Ferec C. Mutationalanalysis of the human pancreatic secretory trypsin inhibitor(PSTI) gene in hereditary and sporadic chronic pancreatitis.J Med Genet 2000;37:67–9.

10. Rossi L, Pfutzer RL, Parvin S, et al. SPINK1/PSTI mutationsare associated with tropical pancreatitis in Bangladesh. Apreliminary report. Pancreatology 2001;1:242–5.

11. Rinderknecht H. Pancreatic secretory enzymes. In: Go VLW,DiMagno EP, Gardner JD, et al, eds. The pancreas: Biology,pathobiology, and disease, 2nd ed. New York: Raven, 1993:219–51.

12. Whitcomb DC, Gorry MC, Preston RA, et al. Hereditarypancreatitis is caused by a mutation in the cationic trypsinogengene. Nat Genet 1996;14:141–5.

13. Pfutzer RH, Myers E, Applebaum-Shapiro SE, et al. Novelcationic trypsinogen (PRSS1) N29T and R122C mutationscause autosomal dominant hereditary pancreatitis. Gut 2002;50:271–2.

14. Pfutzer RH, Finch R, Shapiro SE, et al. Mutations in theSPINK1 gene modify the phenotypic expression of hered-itary pancreatitis caused by cationic trypsinogen (PRSS1)mutations R122H and N29I. Gastroenterology 2001;120(5):A33.

15. Shimosegara T. Do point mutations in the PSTI (SPINK1)

1087AJG – May, 2002 Editorials

gene truly contribute to the pathogenesis of chronic pancre-atitis? J Gastroenterol 2001;36:645–7.

16. Whitcomb DC. Hereditary pancreatitis: New insights intoacute and chronic pancreatitis. Gut 1999;45:317–22.

17. Chen JM, Mercier B, Audrezet MP, et al. Mutations of thepancreatic secretory trypsin inhibitor (PSTI) gene in idiopathicchronic pancreatitis. Gastroenterology 2001;120:1061–4.

18. Kuwata K, Hirota M, Sugita H, et al. Genetic mutations inexons 3 and 4 of the pancreatic secretory trypsin inhibitorin patients with pancreatitis. J Gastroenterol 2001;36:612–8.

19. Whitcomb DC, Gorry MC, Preston RA, et al. A gene forhereditary pancreatitis maps to chromosome 7q35. Gastroen-terology 1996;110:1975–80.

20. Gorry MC, Gabbaizedeh D, Furey W, et al. Mutations in thecationic trypsinogen gene are associated with recurrentacute and chronic pancreatitis. Gastroenterology 1997;113:1063–8.

21. Simon P, Weiss FU, Sahin-Togh M, et al. Hereditary pancre-atitis caused by a novel PRSS1 mutation (Arg-122uCys) thatalters autoactivation and autodegradation of cationic trypsino-gen. J Biol Chem 2001;21:21.

22. Le Marechal C, Chen JM, Quere II, et al. Discrimination ofthree mutational evens that result in a disruption of the R122primary autolysis site of the human cationic trypsinogen(PRSS1) by denaturing high performance liquid chromatog-raphy. BMC Genet 2001;2(91):19.

23. Etemad B, Whitcomb DC. Chronic pancreatitis: Diagnosis,classification and new genetic developments. Gastroenterol-ogy 2001;120:682–707.

24. Creighton J, Lyall R, Wilson DI, et al. Mutations of thecationic trypsinogen gene in patients with chronic pancreatitis.Lancet 1999;354(9172):42–3 (letter).

25. Riordan JR, Rommens JM, Kerem B, et al. Identification ofthe cystic fibrosis gene: Cloning and characterization of com-plementary DNA. Science 1989;245(4922):1066–73.

26. Mickle JE, Cutting GR. Genotype-phenotype relationships incystic fibrosis. Med Clin North Am 2000;84:597–607.

27. Comfort M, Steinberg A. Pedigree of a family with hereditarychronic relapsing pancreatitis. Gastroenterology 1952;21:54–63.

28. Applebaum SE, O’Connell JA, Aston CE, Whitcomb DC.Motivations and concerns of patients with access to genetictesting for hereditary pancreatitis. Am J Gastroenterol 2001;96:1610–7.

29. Applebaum SE, Kant JA, Whitcomb DC, Ellis IH. Genetictesting: Counseling, laboratory and regulatory issues and theEUROPAC protocol for ethical research in multi-center stud-ies of inherited pancreatic diseases. Med Clin North Am2000;82:575–88.

30. Ellis I, Lerch MM, Whitcomb DC. Genetic testing for hered-itary pancreatitis: Guidelines for indications, counselling, con-sent and privacy issues. Pancreatology 2001;1:405–15.

31. Applebaum-Shapiro SE, Finch R, Pfutzer RH, et al. Hereditarypancreatitis in North America: The Pittsburgh-Midwest Multi-Center Pancreatic Study Group Study. Pancreatology 2001;1:439–43.

32. Witt H. Gene mutations in children with chronic pancreatitis.Pancreatology 2001;1:432–8.

33. Sossenheimer MJ, Aston CE, Preston RA, et al. Clinicalcharacteristics of hereditary pancreatitis in a large familybased on high-risk haplotype. Am J Gastroenterol 1997;92:1113–6.

34. Lowenfels AB, Maisonneuve P, Whitcomb DC, et al. Ciga-rette smoking as a risk factor for pancreatic cancer in patientswith hereditary pancreatitis. JAMA 2001;286:169–70.

35. Lowenfels AB, Maisonneuve P, Cavallini G, et al. Pancreatitis

and the risk of pancreatic cancer. International PancreatitisStudy Group. N Engl J Med 1993;328:1433–7.

36. Lowenfels A, Maisonneuve P, DiMagno E, et al. Hereditarypancreatitis and the risk of pancreatic cancer. J Natl CancerInst 1997;89:442–6.

37. Whitcomb DC, Ulrich DC, Learch MM, et al. Conferencereport: Third International Symposium on Inherited Diseasesof the Pancreas. Pancreatology 2001;1:423–31.

38. Whitcomb D. Pancreas.org: A site for information and help.Available at: http://www.pancreas.org. Accessed February 19,2002.

39. Pancreatology. Available at: http://www.karger.com/journals/pan/pan_jh.htm. Accessed February 19, 2002.

Reprint requests and correspondence: David C. Whitcomb,M.D., Ph.D., Professor of Medicine, Cell Biology & Physiologyand Human Genetics, University of Pittsburgh, UPMC Presbyte-rian, Mezzinine Level, C Wing, 200 Lothrop Street, Pittsburgh, PA15213.

Received Dec. 19, 2001; accepted Feb. 7, 2002.

Under Pressure to PrognosticateOver many years there has been a desire to understand andthen predict the natural history of alcoholic liver disease.This may be of enormous importance not only to screen,treat, and hopefully prevent common complications ofchronic liver disease, but also to identify those patients whomight benefit from liver transplantation. To these ends avariety of scoring systems have evolved, including theChild-Pugh classification and a Combined Clinical and Lab-oratory Index among others (1–4). As many of the compli-cations of chronic liver disease relate to portal hypertension,it might at first glance seem logical that the hepatic venouspressure gradient (HVPG) would hold some prognosticvalue.

It is in this context that Deltenre et al. in this issue (5)have reevaluated the prognostic significance of a one-offmeasurement of HVPG in a group of patients undergoingtransvenous liver biopsy. They conclude that this measure-ment is not helpful in predicting survival. This is in contrastto other studies in this area, which have indicted a predictivethreshold of 12 mm Hg for subsequent variceal hemorrhageand of 15–16 mm Hg for subsequent mortality (6, 7). Thisdifference might be explained by some of the limitations ofDeltenre and colleagues’ study, which was performed witha highly select group of patients with significant coagulopa-thy and/or tense ascites, and therefore does not represent thealcoholic cirrhotic population as a whole. The study isfurther compromised by the poverty of follow-up, as infor-mation was available on only just over half of the originalpopulation assessed. This information consisted purely ofwhether the patient was dead or alive. There was no infor-mation relating to the cause of death or to whether any othercomplication of chronic liver disease occurred. It is alsoclear that this study, probably like many others that haveevaluated the prognostic value of HVPG in chronic liver

1088 Editorials AJG – Vol. 97, No. 5, 2002