Genetics of IBD. Stefan Schreiber. Institute for Clinical Molecular Biology and Department of General Internal Medicine,

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1 Genetics of IBD Stefan Schreiber Institute for Clinical Molecular Biology and Department of General Internal Medicine, Christian-Albrechts-University, Kiel

2 Inflammatory Bowel Disease Epidemiology: The Phenotype Manifestation in early adulthood years Steep rise in incidences after World War II North-South-gradient Current prevalence 0.5 1%, Incidence 15-50/100,000 Severe impact on quality of life Diarrhea, pain, systemic symptoms and extraintestinal manifestations Complications include fistulae, stenoses, colorectal cancer Genetics: Familial clustering λ s (relative sibling risk): clustering of disease characteristics in families Twin concordance 4% among dizygotic twins 56% among identical twins sub-phenotype concordance in identical twins

3 NOD2 insertion mutation: Casecontrol NC CD UC Genotype N (frequency) N (frequency) RR (95% CI) N (frequency) (91.2%) 272 (77.9%) 1 61 (93.8%) (8.8%) 57 (16.4%) (6.2%) (5.7%) Total Hampe et al., Lancet 2001

4 NOD2 (CARD15, IBD1) Albrecht et al., FEBS Lett. 2003

5 A Homeostasis of mucosal flora B Dysbalance in mucosal flora Release of antimicrobial peptides MDP Cytoinvasion Diminished release of antimicrobial peptides MDP Cytoinvasion NOD2 NOD2 3020insC RIP2 RIP2 Deficient MDP sensing? NF-κB activation Intraepithelial bactericidal action NF-κB activation Increased intraepithelial bacterial load Chemokine secretion Chemokine secretion Immune cell recruitment Bacterial translocation & Overactivation of the mucosal immune system Schreiber et al., Nat Gen Rev 2005

6 Crohn s Disease and NOD2 NOD2 contributes NOD2 major factor No contribution of NOD2

7 Conclusions I Detection of NOD2 as a disease gene for CD through positional cloning (Hugot 2001) and by candidate gene evaluation (Cho 2001, Hampe 2001). High odds ratio associated with NOD2 variants in the LRR, almost autosomal recessive risk constellation NOD2 variants involved in a minority (20-30%) of CD cases.

8 NOD/CATERPILLER protein family characterized by central NOD-domain (nucleotide binding and oligomerization domain = NACHT + NAD)

9 Other Mechanisms with same pathophysiological consequences

10 NOD2 Splice Variants bp cdna STP bp cdna CARD1 tcard2 NOD2 - S

11 NOD2 S is an Endogenous Inhibitor of NOD2 Signalling Rosenstiel et al., PNAS 2006

12 LIGAND-INDUCED PROXIMITY AND ACTIVATION OF NOD PROTEINS LRR NOD Contact with Ligands Signalling CARD Contact with Ligands No Signalling

13 A Homeostasis of mucosal flora B Dysbalance in mucosal flora Release of antimicrobial peptides MDP Cytoinvasion Diminished release of antimicrobial peptides MDP Cytoinvasion NOD2 NOD2 3020insC RIP2 RIP2 Deficient MDP sensing? NF-κB activation Intraepithelial bactericidal action NF-κB activation Increased intraepithelial bacterial load Chemokine secretion Chemokine secretion Immune cell recruitment Bacterial translocation & Overactivation of the mucosal immune system Schreiber et al., Nat Gen Rev 2005

14 Swidsinski et al. 1998, 2002

15 Genomic Techniques to Type Bacterial Flora Most bacteria can not be cultivated genetic identification is possible using 16SrDNA and gyrase B sequences

16 Approach 16S rdna bp V1 V2 V3 V4 V5 V6 V7 V8 V9 conserved regions variable regions 16S gene Features/applications: - culture-independent - identification/quantification - taxonomy/phylogeny

17 Results II: Investigation of the bacterial flora of IBD Patients vs. Controls Intestinal Clone Libraries Bacterial species n % Gram-positive anaerobic bacteria Actinomyces sp Clostridium irregularis Eubacterium halii Lactobacillus acidophilus Ruminococcus obeum Clostridium leptum Clostridium methoxybenzovorans Clostridium ramosum Clostridium symbiosum Peptostreptococcus productus Clostridium indolis Clostridium sp Clostridium glycolicum Clostridium orbiscindens Clostridium nexile Butyrate-producing bacterium Ruminococcus gnavus Ruminococcus hansenii Gram-positive facultative anaerobic bacteria Staphylococcus epidermidis Gram-positive aerotolerant anaerobic bacteria Streptococcus cremoris Streptococcus intermedius Streptococcus thermophilus Lactococcus lactis Streptococcus sanguis Streptococcus anginosus Streptococcus mitis Streptococcus salivarius Streptococcus sp Streptococcus parasanguis Bacterial species n % Gram-negative aerobic bacteria Alcaligenes sp Gram-negative anaerobic bacteria Veillonella dispar Veillonella sp Fusobacterium nucleatum Dialister sp Dialister pneumosintes Fusobacterium sp Veillonella atypica 20 2 Gram-negative facultative anaerobic bacteria Enterobacter aerogenes Morganella morganii Salmonella typhi Serratia marcescens Shigella flexneri Shigella sonnei Klebsiella sp Pantoea sp Klebsiella pneumoniae Enterobacter cancerogenes 10 1 Escherichia coli Uncultured bacteria Different clones Diversity n = 1019 Control 41 Ulcerative colitis 40 Crohn s disease 17 Total 86

18 controls Ulcerative Colitis SSCP

19 Conclusions II NOD2 dysfunction as a pathophysiological mechanism with similar global function as etiologically relevant sequence variants? Reduced diversity of intestinal flora may be key for understanding of IBD trigger factors

20 Two competing approaches

21 OCTN1 (5q31) Nature Genetics (2004), Peltekova et al. SLC22A5: G C transversion in promoter SLC22A4: missense substitution exon 9 L503F (leucine phenylalanine), 11th transmembrane domain Specifically expressed in intestinal cell types affected by Crohn s disease Polytopic transmembrane Na + -dependent carnitine and Na + -independent organic cation transporter Activity and expression is impaired

22 2,5 2 Chromosome 10 Crohn's Disease Ulcerative colitis IBD LOD score 1,5 1 0, Distance (cm) Hampe et al. Am. J. Hum. Genet. 1999

23 Linkage Disequilibrium across DLG5 and flanking region KCNMA1 DLG5 RPC155

24 DLG5 (10q23) Nature Genetics (2004) Stoll et al. Identified by positional cloning Encodes a scaffolding protein involved in the maintenance of epithelial integrity Non-synonymous mutation 113G A (R30Q aa substitution) Mutation probably impedes scaffolding Stratification for CARD15 variants showed a significant difference in association Rather weak effect with OR = 1.5

25 Association of DLG5 variants in an independent study sample (case/control study design) SNP marker Cases Controls χ2 P value O.R. (95% C.I.) R30Q (CI ) P1371Q (CI ) DLG5_e (CI ) 2, CI for genotypes 1-1 and 1-2 combined (allele 1 as risk allele) 2 CI for genotypes 1-2 and 2-2 combined (allele 1 as risk allele) 3 inverse O.R. for undertransmitted allele 2: 0.74

26 DLG5 in different ethnicities Scottland Noble 2005 Germany Torok 2005 Norway Medici 2005 Germany Buning 2006 England Tremelling 2006 Japan (+/-) Yamazaki 2004 Italy/Canada Daly 2005 Scottland (Children) Satsangi 2005 Belgium (modifyer gene) Vermeire 2005 Canada Newman 2006

27 Allele frequencies for the DLG5 R30Q variant for both gender groups in the German, Italian, Quebec cohort and in the unified cohort males females 0 CD (Germany) Controls (Germany) CD (Italy) Controls (Italy) CD (Quebec) Controls (Quebec) CD (all) Controls (all) Allele frequency [%] of DLG5 R30Q variant Friedrichs et al., Hum Genet Apr;119(3):305-11

28 Conclusions IV Detection of coding SNPS within a large haplotype block on 5q31. Detection of a positional signal that locates genetic risk that has suggested by linkage to chromosome 10q to a haplotype block spanning DLG5. No definition of the causative variant in DLG5. The SNP rs (113A) is an interesting variant leading to an amino-acide-exchange (R30Q). Low impact on phenotype (95%CI OR between ), heterogenous replication. Significant population differences between male and females at birth and throughout populations. Further, phenotype relevant asssociations (arthritis!) within the HLA genes on chromosome 6

29 Major Acknowledgements University Kiel, Germany Philip Rosenstiel Jochen Hampe Andreas Till Almut Nebel Georg Waetzig Susanna Nikolaus The Broad Institute, Boston John D. Rioux Mark J. Daly Sonia Brescianini University Muenster, Germany Monika Stoll Frauke Friedrichs Guys Hospital London Chris Matthew AstraZeneca, Mölndal Maria Lagerström-Fermer Peter Brodin Britt Corneliussen Stephan Pierrou San Giovanni Rotondo, Italy Vito Annese Anna Latiano