Genetics of chronic pancreatitis

0 2000 l?dltlon$

sclentlfiques

Blamed & Pharmacother 2000 ; 54 : 394-9 et mtdicales Elsevier SAS. All rights reserved

Dossier: Gastroenterology

Genetics of chronic pancreatitis C. Arduinol*, ’ Umtci Operativa 2 Unit6 Operativa

dl Genetica Medlca, di Gastrornterologia,

E. Gaia’

Azlenda Ospedallera S. Ghmni Battista Dipurtimento dl Medmna Generule, Ovhcrssano, Torrno, Itch

di Torino. Via Sontrrm 19 10126, Torino; Aziendu Ospedulrera S. Lugi Gorwga,

Summary - Chronic pancreatitis is an inflammatory disease causmg structural and progressive damage resultmg m permanent deficit of both the exocrine and endocrme components. Although a few risk factors for the disease are known, of which the primary one is alcohol consumption, the actual mechamsms responsible for the initial steps and evolution of the disease are not. The discovery of mutations m the catlonic trypsmogen gene in patients with hereditary pancreatltls and a high mcldence of mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) m patients with chronic pancreatitis might be Important clues to understanding the molecular mechamsms of this disease 0 2000 Editions scientifiques et medIcale\ Elsevler SAS chronic

pancreatitis

/ cystic

fibrosis

/ trypsinogen

Chronic pancreatitis is an inflammatory disease causing structural and progressive damage resulting in permanent deficit of both the exocrine and endocrine components. Alcohol is one of the main causes of the disease: in industrialized countries between 50 and 70% of chronic pancreatitis is due to alcohol abuse. Duct obstruction is another cause of pancreatitis in 5-10s of cases. This rare form of chronic obstructive pancreatitis can be caused by scars formed by acute pancreatitis, tumour tissue expansion. by a trauma or by congenital alteration such as pancreas divisum. There are, however, other forms of chronic pancreatitis: tropical. hereditary, autoimmune, and those caused by hypercalcemia; there is also a certain number of cases in which the cause is not evident. These are defined as idiopathic

[Il. A proper understanding of what actually triggers pancreatitis has encountered a series of obstacles: the pancreas is not accessible for observation, the onset of the disease is unpredictable, pain at onset has no specific characteristic, pancreatic biopsy is not always feasible and the difficulty of examining tissue that destroys itself during the disease. Quite apart from aetiology and onset causes, at an advanced stage the disease is characterized by the

* Correspondence

and reprmts

degeneration of acinar cells, which are replaced by fibrotic tissue. Two recent discoveries have provided the elements that explain the mechanism responsible for initial pancreatic damage and throw some light on why the disease becomes chronic: I) the discovery of the gene responsible for hereditary pancreatitis; and 2) the involvement of the cystic fibrosis (CF) gene in chronic pancreatitis. HEREDITARY

PANCREATITIS

The first description of a family with autosomaldominant pancreatitis datesback to Comfort and Steinberg 121.The only distinction between this and other acute and chronic formsof the diseaseis the early ageof onset and the autosomaldominant transmission. The pancreatitisgenewasmappedon the long arm of chromosome7 [3. 41 and later identified by analysing the mutation of a candidategene: a point mutation was found in the cationic trypsinogen gene in which histidine took the place of arginine in the 117 position (RI l7H) 151.This mutation was present in all the patients with hereditary pancreatitis in the families investigatedbut was not found in 140healthy subjects. Another mutation of the cationic trypsinogen gene was discoveredin two families with hereditary pancreatitis 161.A point mutation in exon 2 was identified in

Genetics

of chronic

these families. This substituted asparagine with one isoleucine (N211) in position 21. Disease onset is at a later age in patients with this mutation and its clinical picture is not as marked as that observed for R 117H. So far, these two mutations have been found in families of Italian [.5], French [7], German [8,9], American [ 10, 111, and Japanese [ 121 origin. Numerous elements would seem to indicate that these mutations in the trypsinogen gene protect trypsin from inactivation by autolysis [5]. Trypsin is formed by two domains joined by a chain where the active site is at the interface of the two domains. Mutation R117H is found on the external face of the trypsin molecule, opposite the bond site for the pancreatic secretory trypsin inhibitor (PSTI) and far from the lateral chain of the trypsinogen activation peptide (TAP). Therefore, it seems unlikely that the substitution of arginine with histidine has any influence on trypsin enzyme activity, on PST1 inhibition or on trypsinogen activation. Trypsin recognizes arginine and lysine residues of the peptide chains as hydrolytic sites and arginine 117 acts as an initial hydrolysis site of trypsin by trypsin itself, which is followed by further molecule degradation as new internal sites become exposed. As arginine substitutes histidine, it eliminates the initial hydrolytic site and makes trypsinogen and trypsin resistant to autolysis, and consequently to inactivation. Therefore, if trypsinogen is activated within the pancreas at a quantity which exceeds PSTI-inhibiting capacity (it can only inhibit 20% of trypsin activity potential [ 13]), and if the remaining trypsin stays active because mutation RI 17H protects it from inactivation, then trypsin can activate all the other digestive proenzymes, start pancreas selfdigestion and cause pancreatitis. It would seem, then, that mutation R117H in the cationic trypsinogen leads to this autosomal dominant disease through a ‘gain of function’, which eliminates a decisive inhibitory mechanism. The mechanism with which mutation R117H causes pancreatitis seems clear, while it is only possible to hypothesize how mutation N2 1I acts. When asparagine is substituted by isoleucine, the amino acid sequence of the cationic trypsinogen is the same as that of the anionic trypsinogen for the tract between amino acids 15 and 63. Substitution does not, however, modify the enzyme cleavage sites, but it alters the original secondary ‘turn’ structure into a ‘sheet’ structure in the mutated protein [ 121. Human trypsin is very different from that of other animals in the tract between glutamic acid residue in position 23 (E23) and serine 26 (S26) residue and N21I substitution adds a hydrophobic residue of isoleucine on the surface of the cationic

39.5

pancreatitis

trypsin next to this tract. Foreseeable changes in conformation, though slight, may lead glutamic acid 24 (E24) residue near enough to R117 to create a saline bridge. This interaction modifies the position in space on the chain formed by alanine 112-leucine 123 residues, decisive for the bond with another trypsin molecule and the lysis of the peptide bond in R117 [6, 141. The discovery that cationic trypsinogen is responsible for hereditary pancreatitis has led to understanding the events that cause acute pancreatitis. The first step requires the activation of trypsinogen in the pancreas: this step is reached when mutations are present in the gene or, in the case of the wild-type trypsinogen, when there are high concentrations of calcium. In these conditions cationic trypsin stays active and resistant to autolysis [ 15,161. It has been suggested [ 171 that during hyperstimulation, i.e., when intercellular calcium concentration is very high [18201, trypsinogen self-activates and remains active, triggering a cascade activation of the digestive enzymes, which causes autodigestion of the pancreatic acinar cells, leading to pancreatitis. The succession of events by which trypsinogen activation leads to digestion of the pancreas depends, therefore, on the defence mechanisms of the acinar cell, that is, the ability to control oxidative stress, prevent intracellular hypercalcemia, inhibit trypsin and repair damage. CFTR GENE AND CHRONIC

PANCREATITIS

Hereditary pancreatitis, however, is but a fraction (< 1%) of the causes of chronic pancreatitis: about 30% of the cases of this disease do not have a known cause and are defined as idiopathic. In patients with chronic pancreatitis the disease may present characterisitics similar to those found in cystic fibrosis patients. Cystic fibrosis (CF) is the most frequent recessive autosomal genetic disease found in Caucasians. Its characteristic features are chronic pulmonary pathology, pancreatic fibrosis which usually leads to exocrine insufficiency, obstructive azoospermia in males due to atresia of the vas deferens, and an increase in chloride concentration in the sweat. CFTR (cystic fibrosis transmembrane conductance regulator) is the gene responsible for CF and is located on chromosome 7q3.1 [21]. CFTR is a chloride channel activated by ATP [22]: the ions move through the electrochemical gradient, e.g., in the exocrine glands the ions are secreted in the lumen. The channel’s activity is regulated by other proteins and by CAMP-dependent phosphorylation.

396

C. Arduino,

Since the cystic fibrosis genewasdiscoveredin 1989 [21, 23, 241,over 800 mutations have been identified. (A complete list of the mutations can be found at the web-site address: http://www.genet.sickkids.on.ca/ cftr/.) The mostfrequent mutation, AF508, is presentin about 70% of the mutated chromosomes.All the other mutations are rare, though their frequency varies from one population to another. The mutations have been classifiedaccording to their effect on the phenotypeand to the effect on CFTR protein function. The following five classesof mutation are basedon the mechanism with which they influence CFTR protein expression [25]: I. Absence of or reduced protein synthesis; II. Maturation and intracellular translocation defect; III. Altered regulation of the CFTR ionic channel; IV. Altered conductanceof the CFTR ionic channel;and V. Reducedconcentration of wild-type CFTR protein. The severity of the diseaseis very variable, in part as a consequenceof the large numberof mutationsresponsible for the disease. About 85% of cystic fibrosis patients show progressive pancreatic damagecausedby early obstruction of ducts along with maldigestion caused by pancreatic insufficiency; the remaining 15% still have residual pancreatic function which is sufficient to allow normal digestion. Although the latter are considered to have pancreatic sufficiency, all present alteration of acinar and tubular secretion [261. This subgroupusually presentslessseriousrespiratory symptoms,ageat diagnosis is greater and chloride concentration in sweat is lower. Pancreaticalteration (slight or severe)dependson the type of mutation [27]. Patientswith pancreatic insufficiency have two alleles of classesI, II or III, while patients with pancreatic sufficiency have one or two alleles of classesIV or V. This suggeststhat only one allele with residualchloride channel function is sufficient for the diseaseto be lesssevere(dominant effect of the allele coupled with a slight mutation). Someof the patientsin the secondgroup may present relapsing attacks of acute pancreatitis that in the end becomes chronic. This does not arise in the case of patients with insufficiency sincethe gland is destroyed. The ability to identify the mutationsin the CFTR gene hasrevealeda much wider clinical spectrumof the diseasethan wasthought previously. Many subjects present an atypical or monosymptomatic picture of the diseasesuch aschronic bronchitis, sinusitis with nasal polyps, pancreatitis and, in males, infertility caused by a congenital bilateral absenceof vasdeferens(CBAVD). Numerousdata indi-

E. Gaia

cate that cystic fibrosis attacks different organsin different ways. but is foreseeableaccording to the CFTR genotype [28, 291. Genotypesthat reduce CFTR protein function to 1c/r of its normal value causetypical cystic fibrosischaracterized by pulmonary pathology, pancreatic insufficiency, CBAVD and sweat test alteration. Genotypes that reduceCFTR protein function to 5% of normalvalues cause cystic fibrosis without pancreatic insufficiency, while those that reduce it to 10% causeonly CBAVD. Variant 5T of intron 8 is an example of a CBAVD causingCFTR allele without CF pulmonary pathology. There are three known variants: 5T, 7T and 9T, which differ for the numberof pyrimidines. Variant 5T is present in 5% of the chromosomesof the generalpopulation and it differs from alleles 7T and 9T becauseit reducesthe splicingefficiency of exon 9. causing,therefore. CBAVD by reducing the concentration of functioning CFTR protein. Patientswith a AF508 allele and a 5T allele do not have cystic fibrosis becausethese produce sufficient quantities of CFTR to prevent pulmonary disease,but they do not produce enough to protect the vasadeferentia [30]. In light of the above, it could be presumed that chronic pancreatitis, too. may representan atypical or monosymptomaticform of CF. Indeed, a higher incidence of mutation in the CFTR gene in patients with chronic pancreatitis has been observedby Shareret al. [311and by Cohn et al. [32] and, with the exception of Pallares-Ruizet al. [33], confirmed by other authors(table I). Of the 363 patients with chronic pancreatltis, 55 (15.2%) have at least one mutation, a frequency over seventimes greaterthan that of the population. Mutation frequency is greater still in patients with idiopathic pancreatitis(18.7%). Frequency of mutation 5T is not different in the patients as comparedto the population. In the majority of the patients only one mutation of the gene was found, and two mutations were found in only ten subjects:four by Sharer [31], three by Cohn [32] andthree by Castellani[35j. A number of theseten patients,though, presentedsomealteration (chronic pulmonary pathology, CBAVD, sweat test or pathological nasalpotential). therefore it can be thought that the diseaserepresentsan atypical form of cystic fibrosis.Most of the patients,however, presented only one mutation. This result may be the consequence of the methodsof investigationused.In fact, only a limited numberof mutationswerebeing investigated,more specifically thosemorefrequently found in CFpatlents, namelymutationsprevalently belongingto the classesI,

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Table I. Patients in whom at least one mutation creatitis

(column

of the CFTR gene or the 5T variant was found among patients with idiopathic chronic 1) or due to other cause (column 2) or among all chronic pancreatitis patients (column 3). n.d. = not determined.

Patients with idiopathic chronic pancreatltrs (N = 251)

Patients

with chronic pancreatitis due to other cause (N = 112)

Total patients wtth chronic pancreatitis (N = 363)

Reference

CFTRMut.

5T

CFTRMut.

5T

12160

5160

6174

5174

18/134

101134

31

II27

5127

-

-

7121

5127

32

19/104

n.d.

-

-

19/104

n.d.

34

6130

2130

2119

2119

8149

4149

35

3120

5120

0119

l/19

3139

6139

36

o/10

2110

-

-

0110

2/10

47125 1

191147

8/l 12

8/l 12

551363

211259

18.7%

12.9%

7.1%

7.1%

13.9%

10.4%

II or III. In pancreatic patients at least one mutation shouldbe mild and would not, therefore, be identified in this examination. Consequently, investigation was expectedto find only onemutation (if severe)in patients with chronic pancreatitis. Thus it will be necessaryto wait until all the CFTR gene has been investigated to identify all the possiblemutations to be able to establish whether chronic pancreatic patientsare compound heterozygotes for two mutations, of which the second has not yet been identified, or whether only one mutation is responsiblefor the disease.Theseresults,therefore, confirm the CFTR gene’sinvolvement in chronic pancreatitis, but with modalities and mutations which are different as comparedto other CFTR genecorrelated diseases,suchas CBAVD. How can a mutation in the CFTR protein cause pancreatitis? CFTR protein hasanimportant role in exocrine secretion in the pancreas.This protein is mainly found on the apical plasma membrane of the cells that line the intralobular ductsand mediatesthe secretionof an alkaline fluid rich in bicarbonatesthat maintainsthe solubility of secreted enzymes. Reduced function of the CFTR reduces bicarbonate secretion and causesthe endoluminarcontent of the pancreatic acinusto acidify [37]. Endoluminar acidity due to the lack of chloridebicarbonate exchangedoes not provide proper protein solubility and leadsto consequentobstruction at secretion outflow and thick protein accumulation at acinus lumen [38]. This obstruction is responsiblefor enzyme activation and acute pancreatitis. It is presumed that when CFTR function is completely abolishedby two severemutations, then pan-

CFTR

Mut.

pan-

5T

33 Total

creatic duct obstruction, rapid pancreatic atrophy and insufficiency must take place at an early stage, in the very first years of life. CONCLUSIONS Recent studieson chronic pancreatitis have provided new insight into the pathogeneticmechanismof the disease.Since a genetic predispositionis a necessaryprelude to the disease,for it to occur it requiresmutations either in the cationic trypsinogen or cystic fibrosis genes.Diseaseonset,however, is triggered when other factors, most likely diet-connected, hyperstimulate the pancreaticcells andcausean acuteattack. Repetition of suchattacks leadsto chronic pancreatitis. There are still many unanswered questions, of which: - Only in someof the genetically predisposedsubjects doespancreatitisactually occur: about 80% of the patients with genemutation of the cationic trypsinogen and to a lesserextent in individuals with mutations in the CFTR gene.What is the explanation for this ‘resistance’to the disease?Do thesesubjectshave some‘protective’ genes? - Is the high incidenceof pancreatictumoursin these patientsin someway connectedto the samefactors that causechronic pancreatitis,and if so, in what way? - So far it is not clear whether a singlemutant CFTR allele associatedto other pathogenetic factors, as yet unknown, is sufficient to predisposepancreatitis. -Does the disease progression in patients with chronic pancreatitis and CFTR gene mutations differ from those without CFTR mutations?

398

C. Arduino,

- Do subjects, heterozygous for one CFTR gene mutation, undergo a higher risk of pancreatitis? Only further research in this field will hopefully reveal new genes connected to the disease and then it will be possible to understand why some individuals with mutations do not have pancreatitis. Update: Another gene responsible for idiopathic pancreatitis has been identified after this manuscript was submitted. Witt et al. 1391 analysed 96 unrelated children and adolescents for mutations in the gene encoding the pancreatic secretory trypsin inhibitor PSTI. The authors found mutations in 23% of the patients. In 18 patients they detected a missense mutation at codon 34 (asparagine to serine), which was homozygous in six patients. They also found four different sequence variants. These results highlight the key role of trypsin inhibition in autodigestion and inflammation in the pancreas. ACKNOWLEDGEMENTS This work was supported by a grant from the Italian Ministry

of Health

and Regione

Piemonte.

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