Genomic investigation of chronic intestinal inflammation (GENETICS OF IBD)

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Objectives - Only 60% - 70 % of patients with IBD respond to treatment with glucocorticoids making non-responsiveness a major clinical problem. Similarly, treatment with monoclonal antibodies (infliximab) directed against tumour-necrosis-factor alpha leads to a substantial improvement in 2/3 of steroid and/or azathioprine refractory patients with Crohn's disease. The molecular basis for failing therapeutic response in both situations is unknown. Pharmacogenomics offers an interesting concept for a more precise and targeted use of therapeutics. - We will use the SNPs generated in WP1, 2 and novel SNPs established in the novel inflammation genes identified in WP5-7 to investigate them as response predictors in pharmacogenetic cohorts. Description of Work Cohorts used: - A cohort from an open label study of the infliximab anti-TNF monoclonal antibody by Essex GmbH (Munich/Germany), in refractory Crohn's disease (approx. 120 patient DNAs available). - A cohort from the ACCENT I study of Centocor, Inc., in which 450 patients with steroid refractory Crohn's disease receive the infliximab anti-TNF monoclonal antibody. For this second cohort, DNA sample collection is expected to be completed by 10/00. All patients have been extensively characterised and are followed through prospective, monitored clinical trials. DNA samples will be processed in 96 well microtiter plates for semi-automated PCR and TaqMan analysis. Established single nucleotide polymorphisms ( SNPs ) will be tested in a cohort of patients (untreated, steroid treated/responsive, steroid treated/unresponsive, controls) employing TaqMan technology as described in WP3. The additional cost for the genotyping of existing SNPs is expected to be low, because the respective assays have already been established in the positional cloning efforts. It is expected that the use of high throughput typing (WP3) as well as the necessary bioinformatics infrastructure will generate a high level of synergism with the positional cloning projects. Analysis of data will involve case-control cohort statistics as well as multiple regression models for the identification of statistically independent predictors of drug response. Confirmatory samples from independent trials will be used for re-testing in order to eliminate false positives. The completion of this task will eventually need to lead to prospective clinical trials using the new markers for stratification. Current Status: A large, comprehensive analysis of variants in the TNF/TNF-R system and in the NOD2 gene has been completed. Both pathways do not predict anti-TNF response. Two publications have appeared in late 2002. A further analysis of combined effects of variants in different genes in the pathways was currently performed. Deliverables: Public report of predictors of anti-TNF (DL14) - delivered Reference to Milestones and Expected Results: The project is based on WP1-3. It will complement the identification of an IBD susceptibility gene. Further susceptibility genes for IBD were successfully tested in the pharmacogenetic cohorts.

Description of Work: The single nucleotide polymorphisms obtained from the public databases or identified during mutation detection (see WP1 and WP2) will be typed in the families (basic set 450 affected sibling pairs from 350 multiplex families) as well as in the triplets if appropriate. High throughput TAQman typing technology will be used in microtiter plates for both families and triplets. The use of this method in conjunction with microtiter plates with a fixed layout is in the participants experience the best option to achieve the necessary throughput and reliability. Since the DNA is dried in the plates, setup of experiments is streamlined, because the master mix essentially just has to be aliquoted into the prepared plates. PCR will be performed in ABI 9700 96 well or 384well PCR machines and the data acquired in a 'single point' read in the ABI 7700 or in a standard fluorometer (for 384well plates). Detailed methodology: The typing of SNPs can be achieved by a variety of different methods. Restriction enzymes are a robust possibility if the recognition sequence of the enzyme is altered by the base alteration. A PCR product spanning the polymorphism can be digested with the relevant enzyme and the resulting products be analysed on an agarose gel. A number of other methods, including allele specific oligonucleotide hybridisation (ASO) (100-102), allele specific PCR and solid state mini-sequencing (103), the oligonucleotide ligation assay (OLA) (104, 105) and the allele specific ligase chain reaction (LCR) (106, 107) are very labor intensive. Another method that is based on using the 5' exonuclease activity of TAQ polymerase uses a combination of PCR and competitive hybridisation (108-110). This method was commercially developed and is available as the 'TAQman' method from ABI (Sequence Detector 7700). Depending on the genotype, different amounts of two fluorescent dyes are freed from oligonucleotide probes in the essay. The TAQman method is established in the participant's 1 and 2 laboratories. Currently the process is transferred to 384well plates to meet the throughput requirements. The reading of the Taqman plates is performed in a standard fluorometer (TECAN Spectrafluor) for 384well plates using software developed by the applicants. In order to allow the automated process to work, DNA are arrayed on 96 or 384 well plates, thus providing a fixed assignment of patients to plate coordinates. The plates for the project will be centrally produced at participant 1. The TAQman PCR plates are read in the ABI Prism Sequence Detector 7700 and the files are imported into the integrated database established by the applicants. Given the correct plate code, genotypes can be assigned to all the respective individuals. Additionally, the genotypes of the control individuals on each plate are used for quality control. Status: 2.3 Mill. SNP genotypes have been generated since project start. A technology transition to multiplexed 'SNPlex' genotyping has been performed. This will lead to a cost reduction of a factor of 10 for future experiments of the consortium. This will ensure a competitive position for the next years. Deliverables: DL3: > 500.000 SNP genotypes to be delivered to collaborators has been achieved. However, due to scientific progress in the field it appears that the number of SNPs needed, will be much higher. Therefore, WP3 was continued. The consortium has reacted to these new challenges by implementing new and more cost-efficient genotyping technologies. Reference to Milestones and expected results: The SNP typing platform will be a key element in the identification of genetic mutations associated with a disease risk or treatment response in pharmacogenomic studies.

Objectives DNA stocks for some of the British affected sib pairs used for positional cloning are running low in quantities Description of Work - Patients, who have already been previously characterized and bled, will be recalled/revisited. EDTA blood will be drawn and DNA will be prepared by standard precipitation techniques. The DNA from about 200 affected sib pairs will be re-stocked. There is also now a substantial effort to build a large DNA and phenotype database from British IBD cases to provide sufficient power for large-scale association studies and genotype/phenotype analysis. - Final Status: The genotype and phenotype database of IBD patients and families is now well established and is being used to assess genotype/phenotype correlations for all relevant IBD loci, including CARD15, IBD5, MDR1 and DLG5 Deliverables: DNA from 200 ASP (DL17) and sampling from 1000 IBD patient completed. Reference to Milestones and Expected Results: Enhancements of activities in WP1-3. Support of search for susceptibility gene.

Objectives The objective is to establish full-length sequence, genomic and promoter structure of 20 novel inflammation genes. Description of Work - It is anticipated, that the hundreds of differentially expressed transcripts will be reduced to a the most interesting approx. 20 transcripts in WP5. The consolidated set of ESTs, for which a consistent expression pattern has been established, will be characterised in detail. - Using the resources available at participant 4, the corresponding clones to the UNIGENE cluster will be pulled from the libraries. Full-length cDNA will be established through the use of techniques which are described more detailed in the final report. The full-length sequence cDNAs will be cloned into expression vectors (pQE30) in order to allow the further characterization in WP7. Status and progress In the contract we negotiated for 20 novel cDNAs from inflammation genes to be cloned and sequenced. This task was modified during the project. As part of this workpackage we also aimed to amplify predicted promoter regions of selected candidate genes, which are associated with inflammatory bowel disease. It was planned to amplify these regions, which then should be subcloned, verified by sequencing and subsequently could be used for printing onto a promoter-specific microarrays or alternatively, cloned into reporter vectors for a more detailed promoter analysis. This then also could provide the background work for future experiments, e.g. chromatin immunoprecipitation (ChIP) analysis, which can be used for defining transcriptional regulatory networks of selected genes. This part of the project is not completely finished and will be extended within the German National Genome Research Network (NGFN-2). We currently filed 45 genes for the amplification of the regulatory regions followed by cloning into reporter vectors for a detailed promoter analysis. In collaboration with the Dept. of Computational Molecular Biology we analysed and characterised promoter regions of about 45 inflammatory related genes as described in the third year report. A bioinformatic analysis and prediction of promoter regions was performed using the CORG-, DBTSS- and EPD-databases. Suitable primer pairs were designed for the amplification of regulatory regions. We have implemented preliminary experiments during the reporting period and tested the use of human genomic DNA as the template for PCR amplification. PCRs were performed and the conditions were optimised. The amplification products were visualised on agarose gels, and then subcloned using TOPO TA cloning kit. In this workpackage we observed difficulties in designing suitable PCR primers because most of the selected regions contained repeats. The main observed experimental problems were also the highly repetitive elements in the regulatory region and the distribution all over the human genome, e.g. see sequencing results for TLR7. Although careful primer design was carried out, none of the selected promoter region could be amplified from human genomic DNA. For further experiments we plan to use the corresponding BAC clone as templates for amplification. As mentioned above we will continue the generation of 45 promoter-reporter constructs within the German NGFN-2 network. A high-throughput primer design and amplification protocol will be established to generate about 3,000 promoter-reporter constructs using BAC clones as templates. Currently we developed a test set ob 33 human an 44 mouse promoters amplified from BAC clones with a success rate of about 50 percent for the selected human and about 80 percent for the selected mouse promoters. A detailed analysis showed that a correct alignment of some of the BAC clones with the genomic DNA was difficult, because many of the BAC clones have been used only for filling the sequence gaps of the human genome and a correct localisation is not know. A further problem is that some of the BAC clones did not grow and had to be replaced. Deliverables: DL8: public report on the sequence and expression of novel inflammation genes, DL9: promoter constructs for further characterization DL13: full-length cDNA in expression vectors.

Objectives Independent and critical validation of molecular variants, including an estimation of population based predictive values, sensitivity and specificity of the genetic variations. Description of Work Participants 1 and 2 have access to the following, clinically well characterized cohorts: - extensive phenotype characterization of the IBD family and trio cohort. Additional information like disease severity, steroid use, age of onset, extraintestinal manifestations, family size etc. have been collected. - a cohort of 6000 patients with presenting with general GI symptoms prospectively recruited from general practitioners - a population based cohort of newly diagnosed cases with IBD from Norway (IBSEN project) - additional cohorts (400 patients with H. pylori positive gastritis, 400 patients with hepatitis C) to control for the specificity of the findings. Participant 1 has established a database integrating phenotypic and genotyping information (manuscript submitted) that can be used as a basis for the projected analyses. Scientific program: In order to understand the biological implications of the findings on an individual level, an investigation of the interrelations of structural genetic information (i.e. genomic polymorphisms), expression and protein patterns and the complex phenotype characterisation will be performed. This will be based on the (existing) extensive phenotype characterization of the investigated cohorts. The existing integrated database of phenotype and genetic information (manuscript submitted) will be extended. It will be important to cluster and analyse phenotype characteristics in the same detail as functional and structural molecular information. Additional cohorts will be introduced into the analysis as described above. IBD disease model: The most important cohorts are the 6000 patients with GI symptoms recruited from general practitioners and the population based cohort of newly diagnosed cases with IBD (IBSEN project). Here, actual population based relative risks - also in relation to the established environmental factors predisposing to IBD can be derived. We will use logistic regression to quantitatively assess the relative contributions of these factors and to approach a general risk model for IBD. Specificity controls: Additional cohorts (400 patients with H. pylori positive gastritis, 400 patients with hepatitis C) will be used to control for the specificity of the findings. A database integrating phenotype-genotype-expression data will form the basis of the proposed investigations. To this end, the existing expression and phenotype-genotype database will be merged. Final status: The described cohorts are now available on the applicants platform. Several published results are based on the integrated use of independent European verification cohorts (e.g. Norwegian patients - Hampe et al., 2002) Deliverables: DL13: Public report describing the validated variants including population relative risks and an integrated susceptibility model for IBD. Reference to Milestones and Expected Results: Validation of IBD disease susceptibility genes, the project progressed as expected. Multiple verification findings have been published (Medici et al; IBSEN group 2006). In fact, the availability of a diverse range of population samples was one of the key competitive edges of this consortium.

Objectives Interaction partners and posttranslational modifications of novel proteins will be identified and characterized using established mass spectrometry based methods. This includes a detailed characterization of cellular signalling pathways regulated by phosphorylation or glycosylation and their role in the inflammatory cascade. Description of Work: Mass spectrometry provides the sensitivity, selectivity and specificity required for analysis of complex mixtures of biological origin for determination of modification sites in proteins. Sample preparation methods are crucial for successful MS analysis. The combination of affinity-based sample preparation methods, capillary HPLC and mass spectrometry for characterization of post-translationally modified proteins allow investigation of signalling pathways. NOD2 protein was purified by immuno-precipitation/affinity-chromatography, using the expression vectors provided in WP6. Protein components were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by silver staining. In pilot-studies, western blotting was used to optimise purification conditions for known proteins components and their interaction partners. Protein bands of interest were cut out from the gel, enzymatically digested and the derived peptides were analysed by MALDI TOF MS (peptide mass mapping) and by ESI tandem MS (peptide sequencing). Mass spectrometry data generated from the peptides was used for sequence database queries to retrieve the cognant amino acid and gene sequences. Progress Status: - The main focus was the characterization of protein phosphorylation by mass spectrometry. One protein called Nod2 was found in the genomic screens and expressed as epitope tagged protein by the Kiel group. Phosphorylation of affinity purified Nod2 was characterized by partner 6 in Odense by in-gel digestion, specific enrichment of phosphopeptides by metal affinity chromatography (IMAC) and TiO2 and mass spectrometry. Mass spectrometry was performed with a LTQ-FT instrument, a combination of a linear ion trap and a Fourier transform ion cyclotron resonance mass spectrometer, which provides the opportunity to employ a phosphoserine and threonine specific neutral loss scan for identification of phosphopeptides with high specificity and increased efficiency. Combined with the high mass accuracy of the FTICR instrument, this method facilitates the identification of phosphopeptides and the annotation of the phosphorylation site with high confidence. One phosphorylation on serine-116 was identified for Nod2. Biological follow-up experiments with a mutant form of Nod2 are in progress in the Kiel group. - A second objective of this work package was the detailed characterization of cellular signalling pathways regulated by phosphorylation. We have developed and improved methods for the enrichment of phosphopeptides from complex mixtures and their quantitative analysis by highly sensitive modification-specific peptide sequencing by mass spectrometry and successfully applied them to the characterization of the pheromone response pathway, a prototypic MAP kinase signalling pathway in yeast (Gruhler at al., 2005). Pheromone-treated yeast cells were mixed with control cells labelled with arginine-13C6 and lysine-13C6 prior to extraction of proteins and enzymatic cleavage by trypsin. Phosphopeptides were enriched employing a combination of strong cation exchange (SCX) and immobilized metal affinity chromatography (IMAC) and analyzed by LC-MS using a linear ion trap Fourier Transform ion cyclotron resonance mass spectrometer, which allowed the identification of phosphopeptides with exquisite mass accuracy and high confidence. This work lead to the identification and quantitation of more than 700 unique phosphopeptides, 139 of which were differentially regulated by pheromone. Among them were many key kinases and downstream effector proteins of the pheromone signalling pathway. The results obtained in this work demonstrated for the first time that the activation of a MAP kinase signalling pathway can be examined by phosphoproteomic techniques. The methods developed here can be directly transferred to the examination of signalling pathways in other organisms, including human cells. In addition, we have received a new NOD2 sample, which was immuno-precipitated from stimulated cells. Deliverables: NOD2 was shown to be phosphorylated on Ser-116 by IMAC and mass spectrometry. Quantitative proteomic strategies were developed for large-scale analysis of protein expression, protein phosphorylation and signalling networks. Reference to Milestones and Expected Results: Pathophsysiological role of IBD susceptibility genes.

Objectives: Disease-associated balanced chromosome rearrangements (DBCRs) have been instrumental in the isolation of many disease genes by positional cloning or related strategies. Participant 3 has previously shown that ~1% of the mutations associated with many genetic disorders may be DBCR, e.g. translocations or inversions, that truncate or otherwise inactivate specific genes. The advantages of DBCRs as research tools are, that 1) they can be detected (and screened for) in sporadic as well as in familial cases, 2) they provide a microscopically visible link between genotype and phenotype, and 3) a specific breakpoint can directly identify the precise region/gene involved at the DNA level. Via co-ordination of a global network, Mendelian Cytogenetics Network (MCN), aimed at the systematic identification, mapping and cloning of DBCRs, participant 3 has detected a 10;12-translocation associated with Crohn's disease in a Danish patient. The breakpoint on chromosome 12 is within a region containing a previously mapped IBD susceptibility locus. The objective of this workpackage is therefore the mapping and cloning of the 12q12-q14 breakpoint, which may lead to the isolation of the first IBD-susceptibility gene. Secondary objectives are the identification of other loci by systematic screening of IBD-patients. The applicants are aware, that the link of chromosomal abnormalities with a complex phenotype is a novel and not generally appreciated phenomenon. The chromosomal rearrangements observed may also be due to coincidence not bear any functional relevance. However, given the experimental nature of all mapping efforts in complex diseases, we think that this project is a valuable addition to the positional cloning effort in IBD. Status: Completed 2004: Two candidate genes at one of the breakpoints are being investigated. We have carried out two different approaches to identify candidate susceptibility genes for IBD2: - Identifying candidate genes at 12q13-q14 and screening a panel of patients for mutations in the candidate genes: The potential susceptibility locus for inflammatory bowel disease (IBD2) was localized at 12q13-14 in the vicinity of the DNA marker D12S83 by several independent studies. The genes in the vicinity of this marker with a biological plausibility are therefore strong candidates for IBD susceptibility. We have screened GeneMap'99 (http://www.ncbi.nlm.nih.gov/genemap/) for genes that might be putative candidates for the IBD2 locus. One of the genes within an interval of 4 Mb distal to the microsatellite anchor marker D12S83 is a promising candidate gene for IBD2. We have screened 23 patients for mutations in this gene, which is organised in 20 exons, by sequencing the whole coding region and detected 2 SNPs. Currently haplotype association test of this region is being carried out by partner Kiel. - Mapping the 12q translocation breakpoint of a patient with an apparently balanced translocation t(10;12) and Crohn's disease. To map the 12q13-q14 chromosomal region, we have carried out fluorescence in situ hybridisation (FISH) using more than 80 BAC clones mapping to this region according to UCSC Human Genome Project Working Draft (http://genome.ucsc.edu/). For a long time the 12q breakpoint of the patient was within a gap, but this gap has been closed meanwhile and we have been able to identify a BAC spanning the breakpoint. At the breakpoint region there is an mRNA of an unknown gene, which is currently being characterised. Two SNPs from the translocation breakpoint region chromosome 12q were genotyped in British cases and controls, but thus far no evidence of association has been found. Deliverables: Addition of IBD specific breakpoints to the breakpoint map which is constructed by the global mendelian cytogenetics network ( published both for internet access at (MCNdb): http://mcndb.imbg.ku.dk.) and in peer reviewed journals) this has been completed.

Objectives - Partner Berlin has developed and tested a highly multiplexed microarray manufacturing system capable of producing high-density cDNA arrays. Based on this EST-chip containing 38,000 non-redundant sequences, a database of normal gene expression in the GI tract has been established. The database and bioinformatics infrastructure for analysis and data curation are in place. The database of normal gene expression in the GI tract is currently being published. - In this work package, a global assessment of differential gene regulation in inflamed GI tissue will be used to identify novel inflammation genes, that are generic or specific for certain conditions. Description of the work The methodology for expression screening from GI tissues using biopsy material is established. An average of two biopsies is needed for a duplicate experiment. The mRNA is reversely transcribed and labelled radioactively. The available expression database allows a description of normal variability of gene expression within the GI tract.. In addition, the presently used UniGene cluster will be replaced/enriched by an inflammation specific set of clones, which are sequence and gel verified. Status and progress Resources from work-package 5 were re-directed for the development of a chromosome 6-specific microarray, enriched with additional probes for inflammation related genes from other chromosomes. Resources from work-package 6 were re-directed into the development of a novel and improved nano-dispensing technology platform for microarray production and nano-well applications. Furthermore, resources from work-package 8 were used to establish protein microarrays for serum screening. As mentioned earlier the decision was made to re-direct resources from this work-package for the development of a chromosome 6-specific microarray, enriched with additional probes for inflammation related genes from other chromosomes. This decision was made because expression levels of genes located in the linkage regions of IBD on chromosome 6 have not yet been systematically analysed. Therefore partner 1 and partner 4 agreed to develop a chromosome 6 specific cDNA microarray. This new type of microarray includes predicted genes, splices variants and predicted splice variants. One pathway (TNF) with about 100 associated genes has been designed across all chromosomes. The microarray contains approximately 3000 features including internal and external controls and enables us to investigate the interplay of genes located on chromosome 6p, including differential splicing, and their implication in several functional clusters (see below in more details). For providing high quality microarrays we earlier optimised the process of spotting high-density microarrays (>25,000 spots per array) and additional technical advances have been made in order to provide excellent spotted microarrays. Additionally, partner Berlin has established a protein microarray platform for serum screening experiments. In the third period the generation and application of IBD protein chips in screening IBD patient serum was proposed. Initially it was planned to express about 100 IBD related proteins and to spot on glass slides, which then could be applied for screening with IBD patient serum. The technology platform and application of protein chips for serum screening has been established in this workpackage for quantitative screening and analysis with control and patient serum. Major delays occurred in the throughput of the generation of the proteins for spotting. The extended chromosome 6 microarray The rational for developing a chromosome 6-specific microarray was that former epidemiological, clinical and molecular studies provided strong evidence that inherited predisposition is important in the pathogenesis of chronic inflammatory diseases. Multiple linkage scans have demonstrated that a region on chromosome 6p contributes to susceptibility for Crohn's Disease and Ulcerative colitis. Moreover chromosome 6p also contributes to other chronic inflammatory diseases like Psoriasis, Sarcoidosis, Atopic dermatitis, Asthma etc. To identify disease specific genes and to investigate their functional interplay a microarray representing known genes and ESTs, which -map to chromosome 6p has been developed and produced at the Max Planck Institute for Molecular Genetics in Berlin (partner 4) in co-operation with the Mucosal Immunology Group Kiel (partner 1). Deliverables: - DL6: Glass microarray with a dense inflammation gene content (initially restricted, then publicly available), DL7: List of consistently differentially regulated novel transcripts. - Reference to Milestones and Expected Results: Discovery of susceptibility genes in IBD - a list of genes that are differentially expressed has been generated and was verified on a population level.

Description of Work: The applicants were part of the European group, which originally established linkage of IBD to chromosome 16, and we have since replicated and extended this analysis with the Kiel group. Gene identification has been attempted in a transcript oriented disequilibrium mapping approach. A marker density of 0.05 - 0.1 cM is attempted in the linkage interval of approximately 30cM, corresponding to around 400 SNPs. Sources of SNPs: As a transcript-based mutation detection has been used for efficient discovery of novel SNPs. More than 600 SNPs have been typed until today. Genotyping of SNPs: The SNPs are genotyped using the TAQMAN based high throughput platform under WP3. We will use a core combined family cohort of approx. 450 affected sibling pairs, plus 500 trios and 300 normal controls for each SNP. Data analysis: The applicants have established an integrated database, that allows the export of genetic linkage data into the relevant file formats (LINKAGE, CRIMAP etc.). Genetic data analysis is performed jointly between the statisticians of participants 1 and 2. Final Status: Studies on mutation and evolution of the NOD2/CARD15 susceptibility gene on chromosome 16 have been completed. A paper on the rare mutations and evolutionary conservation of CARD15 has been published (King et al, 2006). A follow-up study of the CARD15 promoter region and the expression of CARD15 in primates (in collaboration with Dr Rosenstiel, University of Kiel) is also completed. Genetic studies of other loci of relevance to IBD have been finalised and published or submitted for publication. These include replication of the DLG5 gene association (first described by the Kiel group) on chromosome 10 (Daly, Pearce et al, 2005), a meta-analysis and genotype/phenotype analysis of MDR1 (Onnie et al, 2006), non-replication of an association of the NFKB1 gene promoter (Mirza et al, 2005), and a detailed haplotype analysis of the IBD5 locus on chromosome 5q31 (Fisher, Hampe et al. 2006) Deliverables: A comprehensive cSNP resource of inflammatory-related genes in the peri-centromeric region of chromosome 16, including novel SNPs, their frequencies and LD relationships has been completed. A first susceptibility gene has been delivered. A further susceptibility gene on chromosome 10q has been delivered.

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