Advances in the etiology of chronic pancreatitis

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1 Advances in the etiology of chronic pancreatitis 550 Jahre Universität Greifswald 1456 European Postgraduate School in Gastroenterology Prague, April 2010 Markus M. Lerch Department of Medicine A Ernst-Moritz-Arndt Universität Greifswald Medizinische Klinik der königlichen Universität Greifswald 1859

2 Chronic Pancreatitis - Etiology Reasons to discriminate between different etiologies: Specific treatment options Inherent co-morbidities Different cancer surveillance strategies Alcohol Idiopathic Metabolic Anatomical Hereditary Autoimmune

3 Incidence of calcifications Propabililty in % Chronic Pancreatitis Alcohol Cessation Time after onset of symptoms Non-Alcoholic CP Alcoholic-CP Layer et al, Gastroenterology 1994;107:

4 Chronic Pancreatitis - Alcohol Cessation Strum et al, 1971 Gastard et al, 1973 Alcohol Stop Alcohol Leger et al, 1974 Prinz et al, 1974 Bornman et al, 1980 Marks et al, 1980 Miyaka et al, 1987 Hayakawa et al, 1989 Strum et al, Patients with pain [%]

5 Cumulative Incidence of calcifications, % Chronic Pancreatitis Cessation of Smoking Maisoneuve P, Gut 2005; 54: Log rank p< OR 2.0 [CI ] Smokers Non-Smokers years after diagnosis of chronic alcoholic pancreatitis

6 Hereditary Pancreatitis Hereditary pancreatitis is clinically indistinguishable from other forms and varities of pancreatitis. 14 year old girl with chronic pancreatitis and R122H-mutation 48 year old women with chronic pancreatitis and R122H-mutation

7 Hereditary pancreatitis in Germany 1952, first description of hereditary pancreatitis (autosomal dominant trait) Discovery of the first mutation associated with hereditary pancreatitis in the cationic trypsinogen gene (PRSS1) by Whitcomb et al. Haplotype-Analysis of 10 unrelated families with a R122H mutation from an area of 100 km resulted in 7 different haplotypes. This precludes a Founder-Effect Chr. 7: 142, ,183 kb PRSS1 PRSS2 Münster Greifswald Block A (17 kb) Block B (62 kb) Weiss F.U. Am J Gastroenterol. 2008;103:

8 Cationic trypsin C139F D100H L104P K92N P36R R116C Activation site V123M R122H/C K23R D22G N29I/T E79K G83E

9 Cumulative age at onset, % Cumulative incidence of diabetes, % Clinical course of Hereditary Pancreatitis Alcoholic CP age, years R122H negativ N29I A16V age, years male female Howes N, Lerch MM et al Clinical Gastroenterology and Hepatology 2004; 2:

10 Pancreatic Cancer in Hereditary Pancreatitis age at diagnosis of pancreatic cancer 50-70% increased risk for pancreatic cancer in patients with hereditary pancreatitis. 40% cumulative risk until 70. years of age. Elimination/Treatment of causal factors: Smoking, Alcohol Hyperlipidemia, Hypercalcemia, Gallstones, Duct stricture, Drugs and Medications cumulative pancreatic cancer risk, % age, years p < 0.01 Non-smokers smokers Howes N, Lerch MM et al Clinical Gastroenterology and Hepatology 2004; 2: ; Lowenfels AB, JAMA 2001; 286:

11 Indications for Genetic Hereditary Pancreatitis testing Recurrent (2 or more) episodes of acute Pancreatitis without identifyable cause or etiology or Idiopathic chronic Pancreatitis - especially in children and young adults under the age of 25 years or Pancreatitis in a patients with a positive family history of Pancreatitis (one or more first or second degree relatives) I. Ellis, M.M. Lerch, D.C. Whitcomb: Pancreatology 2001:1:

12 Trypsin activity (%) Pathophysiological role of two mutant trypsinogens: Decreased autoactivation Trypsin activity (%) PRSS1 Wild-Type PRSS2 Wild-Type R122C G191R Time (h) Time (min) ph 8.0, 5 mm Ca 2+ Simon et al. J. Biol. Chem. 2002; 277: Witt et al Nature Genetics 2006; 38:

13 SH I CH 2 I H C =COO - 3 N + - I H Cystein kda 60 PRSS1 PRSS2 insertion of a new cleavage site EGGKD / ERGKD Wild type G191R min 42 wt R122C wt R122C Trypsinogen Trypsin reducing non-reducing Simon P. et al. J. Biol. Chem. 2002; 277: Witt et al Nature Genetics 2006; 38:

14 The phenotype of two novel trypsinogen mutations Symbole o. A. Pancreatitis carrier test for R122C I:1 69y diab. PRSS1 I:1 87y PRSS2 Screening of Exon 4 for G191R mutations: ICP/HP 17/1414 (1.2%) II:1 II:2 62y II:3 54y II:4 II:5 52y II:6 II:7 45y ACP 4/405 (1.0%) III:1 31y III:2 30y III:3 25y III:4 24y III:5 17y III:6 12y Simon P. et al. J. Biol. Chem. 2002; 277: CONTROL 157/4581 (3.5%) Witt et al Nature Genetics 2006; 38:

15 Trypsin Activity (%) R116C Trypsin Retention and ER Stress I R116C Wild type II 63y 62y 60 III 48y 44y 40y 38y 36y 40 IV 17y 15y 20 0 ph 8.0, 5 mm Ca time (h) Kereszturi et al. Hum Mutation 2009; 30: kda Expression in 293T cells Homogenate Pellet Supernatant wt R116C wt R116C wt R116C wt R116A R116C BiP (78 kda)

16 PRSS-Mutations: gain or loss of function? Autoactivation Autolysis Cathepsin B- ind. activation A16V??? D19A D22G K23R N29I E79K R122C R122H XXX R116C G191R? intracellular processing intracellular processing

17 Sporadic point mutations in the PRSS1- Gen in idiopathic chronic Pancreatitis In 5 of 50 Patients with idiopathic Pancreatitis (10%) mutations in the cationic Trypsinogen gene were found. 10% 65% 90% Affected Patients represented 35% of all patients under 25 years. Simon P, Weiss F.U. et al JAMA. 2002;288: %

18 Sporadic point mutations in the PRSS1- The French Cohort Rebours V Am J Gastroenterol 2008; 103: , Rebours AJG 2009; 104: 2312 Masson E et al Clin Gastroenterol Hepatol 2008; 6: In 78 families with hereditary pancreatitis and 200 individuals (6673 patients years) PRSS1 mutations were detected in 68%. R122H: 78%, N29I: 12% and others 10%. Cumulative risk of pancreatic cancer was 11% at the age of 50 and 49% at the age of 75. Smoking and diabetes mellitus are the main risk factors. Trypsinogen copy number variations are present in 6% of idiopathic chronic pancreatitis cases but are unrelated to familial chronic or tropical calcifying pancreatitis.

19 Chymotrypsin C (CTRC) variants that diminish activity or secretion are associated with chronic pancreatitis Rosendahl et al Nature Genetics 2008; 40: Chymotrypsin C is a trypsin-degrading enzyme. Alterations in the CTRC Gene at position p.r254w and p.k247_r254del are present in 30 out of 901 (3.3%) pancreatitis individuals but only in 21 of 2804 controls (0.7%). Functional analysis showed impaired activity or reduced secretion indicating that loss of function alterations in CTRC predispose to pancreatitis.

20 Pancreatic secretory Trypsin Inhibitor (PSTI, SPINK-1) Witt et al, (Nat. Genet., 2000) Mutations in 23% of children with idiopathic chronic Pancreatitis autosomal-recessive disorder Pfützer et al, (Gastroenterology, 2000) Mutations in idiopathic chronic Pancreatitis (25%), hereditary Pancreatitis and in the healthy population (2%). Modifier - Gene, risk of pancreatitis < 1% Model by A. Brunskil & W. F. Furey Bhatia et al, Schneider et al, (Gastroenterology, 2002) Mutations in tropical calcifying pancreatitis (up to 44%) and in Fibrocalculous Pancreatic Diabetes mellitus (55%). Risk factor for tropical Pancreatitis and Diabetes mellitus

21 SPINK1-Mutations in Patients with Hereditary Pancreatitis Symbols Empty symbol chronic Pancreatitis I. I:1 54 I:2 81 Asymptomatic carrier positiv for PRSS1 Mutation II:1 64 II:2 II:3 61 II:4 69 II:5 60 II:6 68 II:7 50 II:8 46 wt positiv for N34S Mutation II. III. II:1 50 I:1 I:2 87 II:2 59 II:3 59 II:4 62 III:1 IV:1 8 III:2 36 IV:2 5 III:3 35 III:4 I:1 45 II:1 I:2 45 III:5 16 III:6 13 III:1 28 III:2 26 III:3 30 III:4 32 SPINK1 Mutations (N34S) are found among Pancreatitis patients as well as among healthy carriers of Trypsinogen mutations. Weiss F.U. et al. J. Med. Gen. 2003;40:e40,1-5

22 Phenotype, % of total SPINK1 Mutations in Hereditary Pancreatitis Cumulative Incidence of Pancreatitis PRSS1, SPINK wt PRSS1, SPINK N34S mild Pancreatitis severe Pancreatitis Age at disease onset, years No Diabetes Diabetes SPINK1 Mutations have no influence on the severity of clinical disease course of hereditary pancreatitis Weiss F.U. et al. J. Med. Gen. 2003;40:e40,1-5

23 Association of CTSB Polymorphisms with Tropical Calcific Pancreatitis Hyderabad Calicut Total Patients/Controls Patients/Controls Patients/Controls (n=140) (n=155) (n=166) (n=175) (n=306) (n=330) Leu26Val * 0.29* * p= Ser53Gly * 0.04* N34S positive N34S negative Controls (n=134) (n=172) (n=330) Leu26Val * p= E64d ethnic origin: Dravidian His111 His110 Mahurkar et al. Gut :

24 CTSB Val26 mutation in German ICP Patients n C/C C/G G/G pg pg (Mahurkar) Patients ,398 (51/128) 0,46 Controls ,480 (96/200) 0,29 X 2 p-value 0,428 0,578 0,117 0,147 0,013 OR 0,764 0,836 1,897 0,718 2,09 95% CI 0,372-1,569 0,424-1,648 0,792-4,621 0,45-1,15 1,55-2,81 Weiss et al. Gut (9):

25 Cathepsin B mutation Leu26Val in pancreatitis N34S positive N34S negative Controls (n=134) (n=172) (n=330) Leu26Val Mahurkar et al. Gut : Population Ethnic origin (n) pg (Val26) CEPH Mixed Caucasian 92 0,320 Pooled_CEPH Caucasian 94 0,323 HapMap-CEU European 55 0,355 SC_95_C Caucasian 45 0,367 HapMap-YRI Sub-Saharan African 52 0,394 TSC_42_C Caucasian 41 0,430 JBIC-allele Japanese 732 0,493 HapMap-JPT Asian 44 0,523 HapMap-HCB Asian 43 0,547 Weiss et al. Gut : ,452 ± 0,042 see refsnp ID: rs12338 at /SNP/snp_ref.cgi?rs=12338

26 Chronic Pancreatitis and CFTR Mutations One third of patients (n=27) with idiopathic pancreatitis carry CFTR-Mutations (Risk x 80). J. Cohn et al. New Engl J Med 1998;339: Kerem et al. Science 1989; 245: F 508 Mutation CFTR Mutations represent a risk factor for chronic pancreatitis in patients without a history of alcohol abuse (19% of n = 60), but not for those with alcoholic pancreatitis (8.5% of n = 72).N. Sharer et al. New Engl J Med 1998;339: Weiss FU Gut 2005; 54: CFTR allele frequency 12/66 Patients with CFTR Mutations, 8/66 Patients with T5 Allels

27 Prevalence of gene mutations in chronic pancreatitis Chymotrypsin C mutations Idiopathic pancreatitis Trypsinogen mutations 45.5% SPINK-1 mutations 18.2% 12.1% 15.2% 10% T5 Allels CFTR mutations

28 Metabolic Chronic Pancreatitis Causal treatment Hyperlipidemia (apocii deficiency, lipoprotein lipase deficiency) Serum triglyercide levels > 1000 mg/dl Incidence extremely low Treatment which maintains TG below 500 mg/dl leads to resolution of symptoms. Toskes PP, Gastroenterol Clin North Am 1990; 19: Hyperparathyroidism: Hyperthyreoidims leads to increased serum calcium levels, what is associated with an increased risk of pancreatitis. The incidence of chronic pancreatitis is between 1.5-7%. Early parathyreoidectomy leads to resolution of symptoms. Bess MA JAMA 1980; 243: ; Russel CF Br. J. Surg. 1982; 69: ;

29 Summary and Conclusion Hereditary chronic and idiopathic chronic pancreatitis are associated with mutations in the trypsinogen gene, the SPINK-1 Gene, the chymotrypsin C gene and the CFTR gene. More genes will surely be identified. The pathophysiological impact of these gene mutations are not completety understood and further experiments are warranted. Etiologies of pancreatitis already treatable (such as autoimmune pancreatitis etc. ) need to be distinguished from pancreatitis varieties that are not yet treatable but may become so. In hereditary pancreatitis (trypsin mutations) smoking cessation may reduce the risk of pancreatic cancer. Other surveillance strategies are, however, urgently needed.

30 Julia Mayerle Uli Weiss Peter Simon Ali Aghdassi Gabriele Sauter

31 Output in % of initial Chronic Pancreatitis - Alcohol Cessation Stop Alcohol Alcohol Bicarbonate Lipase Chymotrypsin n= 18 vs. 14 pt 4-11 years p<0.01 Gullo et al, Gastroenterology 1988;95:

32 P. Simon, F.U. Weiss et al. J Biol Chem. 2002;277: Diagnosis and Screening for Hereditary Pancreatitis A C G C C N G C G T G T C T Today, restriction enzyme digest with BstU I represents the most extensive initial screening test for hereditary pancreatitis. Sequence in Exon 3 Arg RSS1 wt AAC-GCC-CGC-GTG-T Cys R-122-C AAC-GCC-TGC-GTG-T Afl III BstU I

33 Natural Course of Alcoholic Chronic Pancreatitis - Stage 1 (early) Stage 2 Stage 3 Pain Serum Enzyme Elevation Exocrine Pancreatic Function years Modified according to R Ammann, Schweiz Rundsch Med Prax. 1970; 59: