HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients

Transcription

1 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Boukje de Vries Student from the VUmc Amsterdam The Netherlands September 2002 January 2003 Supervisors: EG de la Concha, AS Peña, L Fernandez Research conducted at the department of Immunology at the Hospital Clinico San Carlos, Madrid, Spain

2 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients - Abstract Abstract Ulcerative Colitis (UC) is an Inflammatory Bowel Disease (IBD) with a yet unknown aetiology. Intensive research for genetic factors has made it clear that they play an important role in the disease pathology; however the relevant genes have not been identified yet. Genes of the major histocompatibility complex (MHC) are interesting candidate genes because of their role in the immune response and the strong associations between alleles at this locus and other immune related diseases. In this study we investigated the effect of the IKBL+738(C) allele and the effect of HLA- DRB1*1501 on Dutch ulcerative colitis patients and controls. Patients (n=189) and controls (n=115) were typed and subtyped for DR using gene amplification and sequence-specific oligonucleotide probes. IKBL and the microsatellites TNFα, TNFβ and MICA were also determined. HLA-DRB1*1501 did not have an effect on the overall disease susceptibility in ulcerative colitis patients, but it was found to be significantly increased in patients with extensive colitis (p=0.007, OR=2.33). HLA-DRB1*1501 was only found to be increased in extensive colitis in the absence of the 7.1 haplotype, which is defined by the presence of TNF a11,b4 and MICA 5.1 (p=0.0003, OR=5.36). We think that something in the 7.1 haplotype neutralises the effect of HLA-DRB1*1501 on extensive colitis. In our study IKBL+738(C) had no effect on overall susceptibility or disease extension. A previously published study from a Spanish group reported a significant association between IKBL+738(C) with extensive and severe disease, independent of DRB1*0103, which is known to confer susceptibility to UC. It might be possible that conflicting results are due to differences in the studied populations. The effect of IKBL+738(C) might be secondary to the effect of another gene that is linked to the IKBL+738(C) allele in the Spanish population, but not in the Dutch population. 2

3 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients - Introduction Contents Introduction Ulcerative Colitis (UC) Pathology of the disease Aetiology of the disease Epdemiology of the disease Environmental factors Genetical factors Treatment possibilities Major Histocompability Complex (MHC) MHC haplotypes Candidate genes for UC HLA class II genes and UC Other investigated markers in relation to UC IKBL and UC Aim of the study Materials and methods Patients and controls DNA isolation, HLA Class II genotyping and subgenotyping DNA isolation and genotyping DR1 and DR2 subgenotyping IKBL+738 Genotyping Microsatellite Genotyping Statistical analysis Results HLA-DRB1*1501 and ulcerative colitis IKBL+738(C) and ulcerative colitis Linkage disequilibrium between IKBL+738(C) and HLA-DRB1* HLA-DRB1*1501 in patients subgroups IKBL+738(C) in patients subgroups HLA-DRB1*1501 and IKBL+738(C) and patients subgroups haplotype in patients and controls HLA-DRB1*1501 and the 7.1 haplotype in patients with extensive colitis Discussion HLA-DRB1*1501 and ulcerative colitis DRB1*1501 results from other groups compared to our results IKBL and UC Acknowledgements References

4 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients - Introduction Introduction Ulcerative Colitis (UC) Ulcerative Colitis is an Inflammatory Bowel Disease (IBD) and was first described by Wilks in 1859 [1]. The disease has its mean peak of onset between 15 and 30 years of age and has a smaller second peak between 55 and 80 years [2, 3]. Men and women are equally affected [2, 4, 5]. Pathology of the disease Ulcers and continuous uniform inflammation in the mucosa and submucosa of the large intestine are observed in UC. Inflammation involves the rectum and can extend in a continuous manner to a part of the proximal colon. Disease severity is characterised by the extent of colonic involvement [6, 7]. Patients can be categorised by location of the inflammation. Inflammation limited to the rectum is called ulcerative proctitis and is found in 10% of the patients. When the disease is limited to the left side of the colon, rectum and the sigmoid it is called proctosigmoiditis, which is observed in 30-40% of the cases [8]. Pancolitis is inflammation of the entire colon and appears in 30% of the patients [7]. In the rest of the cases the disease is limited up to the splenic flexure and is called left sided colitis [8]. Patients with extensive colonic involvement and duration of disease of more than ten years have a higher risk for developing colon cancer [9]. One of the most common disease symptoms is diarrhoea, which is caused by the inflammation, the ulcers bleed and produce pus and mucus. Another common symptom of UC is abdominal pain and faecal urgency, but also weight loss, rectal bleeding, loss of body fluids and nutrients, fatigue and loss of appetite are often experienced by patients. Most patients experience a chronic and intermittent clinical disease course, which is unstable [10]. More than 50% of the patients experience increase of extent of disease [11]. Problems outside the colon are present in 25-30% of the UC patients. These extra intestinal manifestations (EIMs) include arthritis, inflammation of the eye, liver diseases, osteoporosis, kidney stones and mouth ulceration [7, 8]. It is thought that these 4

5 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients - Introduction complications occur as a result of inflammation in other parts of the body triggered by the immune response. Mostly, these problems are mild and disappear when the UC is medically treated [10]. Diagnosing UC is difficult, because the symptoms are very similar to other intestinal disorders, like Crohn s disease (CD) and irritable bowel syndrome. In about 10% of the cases it is not possible to classify if the patient has either CD or UC and these patients are designated as indeterminate colitis [12]. Etiology of the disease The etiology of UC is still unclear, but there are some theories. The most popular theory postulates that the immune system responds to virus or bacteria causing an ongoing inflammation in the intestinal wall. A strong argument for the bacterial theory is the fact that the side of the intestine, which is most frequently affected in UC, namely the distal ileum and the colon, are those containing the highest bacterial concentrations. It is thought that commensal enteric bacteria and their products are involved in the pathology of UC [13, 14]. A potential agent has thought to be Mycobacterium Tuberculosis, but this hypothesis has never been proved [7, 15]. A lot of other bacterial species have been investigated in relation to UC including Shigella, Campylobacter and Escherichia coli. Escherichia coli strains that express specialised adhesion molecules have been thought to play a role in the pathogenesis of UC, because of their increased ability to adhere and thereby damaging colonic mucosa [16]. Genetic and other environmental factors also play a role in the susceptibility to the disease and will be discussed below. Epdemiology of the disease It is well known that UC is distributed unevenly over the world and has different frequencies in populations. The disease is much more common in northern countries than in southern countries ( per 10 5 and per 10 5 respectively) [10]. This suggests an effect of environmental factors on pathogenesis of UC. Ashkenazi Jewish people have the highest risk for developing UC [17], which is 2-4 times higher compared to other ethnic groups, but it varies from region to region [18]. 5

6 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients - Introduction Environmental factors As mentioned above it is thought that environmental factors play a role in the disease pathology of UC. Smoking is one of the factors which decreases the risk of developing UC independent of background and gender. Former smokers have an increased risk for developing UC of approximately 1.7 times more than people who have never smoked [19-21]. Smoking has also effect on the disease course of UC; for example a decreased rate of pouchitis is observed in patients who have undergone colectomy [22, 23]. The use of non-steroidal anti-inflammatory drugs (NSAIDs) confers an increased risk for UC, which might be related to increased intestinal permeability to gut luminal antigens, which may play a role in triggering and perpetuating intestinal inflammation [24]. Another factor reported by Koletzko et al is the effect of breastfeeding on the risk of developing UC. They report that breastfed children have a decreased risk of developing UC and suggest that IgA might have an immunomodulatory effect on the child s intestinal mucosa and breast milk may also stimulate mucosal growth and development [25]. Appendectomy is also shown to be protective for developing UC. In patients 0.6% had appendectomy and in controls 25.4% had colectomy [26]. It might be possible that appendectomy changes the balance between T helper and suppressor cells in a way that is protective for developing UC, but more work is needed to see if this association is valid. Genetic factors Intensive genetic research results in strong evidence for the importance of genetic factors on disease pathology of UC [27, 28]. Data implicate that UC has no simple Mendelian pattern of inheritance, but may be a multifactorial heterogeneous disease [29]. An important argument for genetic influence on the disease is the fact that a family member with UC is a strong risk factor is of developing UC [30]. Patients with a positive family history show a more early age of disease onset and a more extensive disease form [31]. Other interesting results were found in twin studies. Higher concordance for monozygotic twins than for dizygotic twins is found for UC [28]. monozygotic twins share 100% of their genes and dizygotic twins share about 50% and data implicate that genetic factors are important to predisposition to UC. The fact that not 100% of the monozygotic twins are 6

7 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients - Introduction concordant for disease shows that also non-genetic factors must play a role in disease pathology. Those were described above. Treatment possibilities Mortality as a consequence of UC has decreased dramatically since the introduction of glucocorticosteroids in 1955 [32-34]. Treatment of the disease is still aspecific. Between patients a marked difference in severity is noted, regarding to presence of EIMs, response to medical treatment and prognosis. This heterogeneity of the disease makes it more difficult to treat [11] and for successful treatment the use of appropriate immune suppressive and anti-inflammatory drugs in relation to severity and localisation of the disease are needed. Major Histocompatibility Complex (MHC) The Human Leukocyte Antigen (HLA) complex is located on chromosome 6 p21 and contains more than 200 genes. The most important known functions of these genes are antigen processing and presentation to T cells and the production of cytokines that modulate the immune response, but the roles of many genes in this region still remains to be characterised. The production of molecules which are crucial for antigen recognition, makes the HLA complex important for the immune response and makes this region very interesting to look at with regard to immune related diseases, like UC. The HLA complex contains class I, class II and class III genes. Class I proteins are important for antigen presentation to CD8+ T cells. The peptides presented are derived from proteins synthesised in the cytosol. Class II genes are involved in presentation of antigens which are phagocytosed and are recognised by CD4+ T cells. The MHC class III genes include genes that encode immune related proteins like complement components, heat shock proteins and some cytokines, for example TNF-α and TNF-beta [35]. Cytokines have an important role in the initiation and amplification of the immune response in several immune diseases [36] and therefore are candidate loci for disease susceptibility. The HLA complex is polygenic and highly polymorphic, which means that there are different MHC class I and MHC class II genes in one individual and different alleles of the 7

8 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients - Introduction same gene in the population. There are three different class I genes, with polymorphisms in the α chain namely HLA-A, HLA-B and HLA-C and three pairs of class II genes with polymorphism in the α and β chain genes, HLA-DP, HLA-DQ and HLA-DR. In many cases, the HLA-DR cluster contains an extra beta-chain. The α-chain encoding genes of the class I molecules, the genes for the class II DQ α chain and the DR-, DP-, and DQ β-chains are highly polymorphic. This means that one individual is able to present a broad range of peptides, because more than one MHC molecule of each class is expressed at the cell surface. This makes it difficult for pathogens to evade the immune response [35]. For HLA-DR there is a non-polymorphic alpha-chain and there are 3 different highly polymorphic β chains. The β1 chain is the most polymorphic β -chain and is always present in all individuals. It is interesting to study the polymorphisms of the beta1 chain to see if there is a relation between HLA class II genes and a particular disease [37]. The HLA nomenclature is based on the molecular typing method. The names include the name of the molecule, the chain, the gene number by which it is encoded, an asterisk as an indication of molecular typing and the number of this allele [37]. MHC haplotypes It has become clear that different alleles of the MHC genes are strongly linked and highly polymorphic [37]. Many alleles of the MHC complex are in linkage disequilibrium (LD), this means that alleles of closely linked genes are not randomly associated, but occur more frequently together in a population than would be predicted by individual allele frequencies [30]. The particular combination of MHC alleles found on a single chromosome is known as a MHC haplotype. Some MHC alleles are conserved together in the population and are usually inherited as a block without recombination. These blocks are conserved in the population, because they probably have evolutionary advantage against other combinations. These haplotypes are called ancestral haplotypes (AHs). AHs are designated by their HLA- B allele [38]. As mentioned above MHC genes in AHs contain a lot of genes which are important for immune regulation, which make them interesting subjects to study in relation to susceptibility for immunopathological diseases [39]. 8

9 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients - Introduction An important AH for this study is the 7.1 haplotype. DRB1*1501 is presented in several haplotypes, but most frequently in the 7.1 AH, which is also the most conserved one. The 7.1 AH also includes HLA-B7, TNF a11,b4 and MICA 5.1. and is shown to be related with multiple sclerosis (MS) [40, 41]. For MS DRB1*1501 gives susceptibililty to MS, but more so in the absence of the 7.1 haplotype. Ancestral haplotypes do not have the same prevalence over the world, which means that some gene combinations are more frequent in some populations than in others. This makes it important to compare gene studies from different populations. Some immune diseases are more frequent in particular ethnic groups or in populations, which may implicate different influences of genetic and environmental factors. Candidate genes for UC A lot of studies have tried to find genetic factors which are related to an increased susceptibility for UC or an increased susceptibility for a particular subtype of UC [42-45]. Up to now, data are conflicting because of linkage disequilibrium in the HLA, which makes it difficult to know which gene or allele is responsible for a particular effect. Other factors that potentially could lead to problems are differences of used methods, differences in studied populations and heterogeneity of the disease itself. Because of the important role of the products of the HLA class II genes for immune response, they have been a subject of intensive research in relation to UC. HLA class II genes and UC A lot of groups have investigated the effect of HLA-DR2 on UC, but data are conflicting. DR2 can be split in DR15 and DR16. No association was found with DR16, but only with DR15. DR15 has two alleles DRB1*1501 and DRB1*1502. Several studies, especially from Japan, found a positive relation between DRB1*15 and UC [42, 46, 47]. In the Japanese studies the relation between DRB1*15 is found more often than in studies within Caucasian populations. It is thought that this might be due to the fact that in the Japanese population DRB1*1502 is much more common than DRB1*1501, whereas in the Caucasian population the DRB1*1501 is more common [48]. 9

10 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients - Introduction Studies in Caucasian populations were more variable. A Dutch study described a significant increase of HLA-DR2 frequency in UC patients and a Spanish study [49] also reported a significant association for HLA*DR2 and the subtype HLA-DRB1*1501 with UC, whereas other groups did not find an association with the alleles representing DR2 and UC [50, 51]. De la Concha and collaborators found DRB1*1501 modestly increased in patients overall and in patients with extensive disease, but not significant [52]. Others have reported positive associations for UC with HLA-DR6 [53], HLA-DR12 [50] and HLA-DR1*0103 [50]. Negative associations with UC were found with HLA-DR3 [54], HLA-DR4 [43, 46, 53, 55], HLA-DR6 [43] and HLA-DR7 [56], meaning they are more frequent in controls than in patients, which might implicate a protective role for these genes. There are also some studies, which investigated DQ genes in relation with UC. A positive relation was found for DQ2 [54] and negative for DQ3 [55]. The rare DRB1*0103 is found more often in patients with severe UC than in controls [50, 57]. It is thought to be related with extensive UC [27, 58]. Other investigated markers in relation to UC Cytokines are highly important for an appropriate immune response and genes involved in the regulation of these cytokines are candidates for disease pathology. Lot of research is done investigating the effect of tumor necrosis factor alpha (TNFα) and interleukin1 receptor antagonist (IL-1RA), which modulates the inflammation by blocking the interleukin 1 receptors. A relation is found between a polymorphism in the IL-1RA gene and susceptibility to UC, particularly for extensive colitis [59]. Another marker that has been investigated in relation to UC is a subset of autoantibodies reacting with the cytoplasma of human neutrophils; antineutrophil cytoplasmic antibody (ANCA). ANCA is found in most of the UC patients (84%), but not in with CD patients, therefore it can be used as a marker to distinguish between UC and CD. Significance of ANCA to the disease is not clear yet [60]. 10

11 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients - Introduction IKBL and UC The IKBL gene lies within the central major histocompatibility complex between HLA-B and -DR and carries a structural polymorphism at position The polymorphism involves a transition of a thymine (T) to cytosine (C) at position +738, which results in a cysteine to arginine substitution in a predicted protein kinase C phosphorylation site. The IKBL protein is most similar to IkBα, which regulates the nuclear localisation of the transcription factor NFkB. NFkB [61] regulates the transcription of TNFα and related cytokines [62]. The idea that IkBL might interact with NFkB makes IKBL polymorphisms interesting to study in relation with immunologic disorders, including UC. There is also strong linkage disequilibrium in this region and it might be possible that IKBL marks nearby genes with an effect on the immune response. The IKBL polymorphism was studied in a Spanish paper and they found that the IKLB+738(C) allele was more frequent in two haplotypes, namely one haplotype carrying HLA-DRB1*1501 (7.1 AH) and one carrying HLA-DRB1*0103 [52]. It is known that HLA-DRB1*0103 confers susceptibility for extensive disease [50] They reported a significant increase of IKBL+738 (C) frequency in UC patients (P<0.001), it seemed especially related with extensive UC. They found no statistical significant increase of HLA-DRB1*1501 in UC patients, only for HLA-DRB1*0103, which was associated with extensive disease and distal disease and with patients with intractability. It is still unclear if the IKBL+738 (C) itself has an effect or if it marks a closely linked susceptibility gene. 11

12 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Aim of the study Aim of the study UC is known to be a heterogeneous disorder of multifactorial aetiology. Many groups investigated candidate genes for UC. HLA genes are important candidates for a role in genetic susceptibility to UC, because their products play a central role in the immune response. Associations between MHC genes and UC have been described repeatedly [42, 43, 50]. Especially positive associations between HLA-DR2 UC [42, 46, 48, 55] and IKBL+738(C) [52] have been observed. A recent Spanish study investigated the effect of IKBL+738(C) on UC and they found that the IKBL+738(C) allele was more common in patients with severe disease than with nonsevere disease. This effect was independent of HLA-DRB1*0103, which is known to confer susceptibility for extensive colitis [52]. Several studies from Japan reported positive associations between HLA-DR2 and UC [42, 46, 48, 55], but results from studies in Caucasian populations were conflicting. [43, 50-52, 54, 57, 63]. Because of the clinical heterogeneous character of UC and the discordant results in HLA- DR2 studies, in this study we are interested in the effect of HLA-DRB1*1501 and IKBL+738(C) on Dutch patients subgroups in stead of patients overall. 12

13 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Material and methods Materials and methods Patients and controls Our study included one hundred and eighty-nine unrelated Dutch Caucasian ulcerative colitis patients and one hundred and fifteen unrelated Dutch controls. DNA of the patients and the controls was sent to Madrid by the Academic Hospital Vrije Universiteit Amsterdam. Controls were recruited from the same region as the patients. The diagnosis UC was based on conventional clinico-pathological criteria as described by Lennard-Jones [64]. Diagnosis was based on endoscopy and/or radiologic investigations. Localisation of the disease was defined as left-sided (gut involvement up to the splenic flexure), or as extensive (gut involvement beyond the splenic flexure). All patients and controls were genotyped for DR and were subgenotyped for HLA-DRB1, - DQA1 and DQB1. Also IKBL, TNFα, TNFβ and MICA were determined for patients and controls. Patients and controls were defined to have the 7.1 AH, when they were carrying HLA- DRB1*1501 in combination with the microsatellites TNF a11,b4 and MICA 5.1. DNA isolation, HLA Class II genotyping and subgenotyping DNA isolation and genotyping DNA was isolated from fresh peripheral blood leukocytes by the salting out method with 6 mol/l NaCl after overnight incubation with proteinase K [65]. Typing for HLA-DR and HLA-DQ was performed by amplification of the highly variable exons of the HLA-DR, - DQα and DQβ loci. DNA amplification by a polymerase chain reaction (PCR) was performed by using a PTC-100 Programmable Thermal Controller (MJ Research Inc., Watertown, MA, USA). The used PCR program which had the following conditions: starting with a 7 minutes preheating period at 95 C, then 39 cycles of annealing at 95 C for 20 seconds, extension at 55 C for 20 seconds and incubation at 72 C for 30 seconds, final step is cooling to 15 C. Primers used for the PCR were obtained from the 11 th international Histocompatibility Workshop and are presented in table 1a. 13

14 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Material and methods PCR mix per sample consisted of 1 µl buffer gold, 0.5 µl dntps and water to a final volume of 10 µl. Fifty µg of genomic DNA and 0.05 µl of amplitaq gold DNA polymerase was added to each sample. After amplification the quality of the PCR product was tested on agarose gel (1.5%) by electrophoresis and visualised by ethidium bromide staining and exposure to UV light. To perform the genotyping 10µl PCR products were slot blotted to the nylon membranes. Cross linkage of PCR products to membranes was achieved by exposure to UV light. Hybridisation was performed at specific temperatures with digoxygen-labeled allelespecific oligonucleotide probes, which were also obtained from the 11 th international Histocompatibility Workshop. Hybridisation temperatures and sequences of used probes are also shown in table 1b. The results were visualised by exposing the membranes to a radiographic film for 5-15 minutes. HLA-DR1 and HLA-DR2 subgenotyping For subgenotyping of HLA-DR1 we used amplification by PCR under the same conditions as described above. Used primers are shown in table 2a. After the amplification the Dotblot technique was used with specific probes for subtyping DRB1*0101, DRB1*0102 and DRB1*0103. For subgenotyping of HLA-DR2 we used amplification by an HLA-DR2 specific quantitative PCR. For analysis of the HLA-DR2 subtypes the Taqman 280 test was used, which is based on addition of an extra signal to the PCR mix which hybridises with the fragment that is amplified. The sequence of this signal contains on its 3` end a fluorescence marker and on its 5 end a quencher molecule. The presence of both substances gives rise to the transference of energy by resonance and the quencher molecule at the end lights up, emitting light of a low wave length. We used primers which were specific for one allele. When there is amplification the Taq polymerase by its 5-3 exonuclease activity liberates the fluorescence at the 5 end and the activity of the 3 end is detected as light emission. In a negative sample, no amplification takes place and no light emission will be detected. For each sample we did 2 quantitative PCRs at the same time. In one we used a specific primer for 1501 and in the other a primer which would recognize the other DR2 subtypes. This was to test if the amplification by PCR worked properly to avoid false negatives. Used 14

15 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Material and methods primers and probes are presented in table 2b. The machine used for the quantitative PCR is a Rotor gene 200 Real Time Cycler (Corbett Research, Sydney, Australia). IKBL+738 Genotyping IKBL+738 single nucleotide polymorphism genotyping was carried out by PCR-sequence specific priming. Primers for the human growth hormone (HGH-U and HGH-D) and for IKBL (MidR1 and CF) were used for amplification by PCR. For all the samples we used the primer that recognised the IKBL+738(C) polymorphism. Samples which were positive were repeated with the other primer which recognises IKBL+738(T) to see if they were homozygous or heterozygous for IKBL+738(C). We know that IKBL+738(C) is in linkage disequilibrium TNF 11/4 and by checking this false negatives are excluded. Primer sequences are presented in table 3. The 100bp long HGH product and the 144bp long IKBL PCR product were separated on agarose gel (2,5%) by electrophoresis and visualised by ethidium bromide staining and exposure to UV light. Microsatellite Genotyping TNF and MICA micosatellites genotyping was performed using PCR. Primers used for DNA amplification were IR2 and IR4 for TNF α and IR1 and IR5 for TNFβ (table 4a and 4b). The IR2 primer was 5 labelled with hexachloro-6-carboxyl fluorescein (HEX), and the IR5 primer was labelled with 6-carboxy fluorescein (FAM) (Applied Biosystems, Foster City, California). Primers for amplification of MICA were 5F and 5R. The MICA 5R primer was labelled with FAM. Amplification of DNA was done under the same conditions as above. An ABI PRISM 310 genetic analyser using Genescan software was used for analysing the PCR fragments for TNF α, TNFβ and MICA microsatellites. Tamra 500-bp was used as marker and Performance Optimised Polymer-4 (POP-4) as polymer, both compounds were obtained from the company Applied Biosystems. 15

16 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Material and methods Allele Primers Primer Sequence Pm DR DRB-AMP-A 5 -CCCCACAGCACGTTTCTTG-3 13 DR DRB-AMP-B 5 -CGGCTC\GCACTGTAAGCTCT-3 13 Dqalfa DQA-AMP-A 5 -ATGGTGTAAACTTGTACGAGT-3 13 Dqalfa DQA-AMP-B 5 -TTGGTAGCAGCGGTAGAGTT-3 13 Dqbeta DQB-AMP-A 5 -CATGTGCTACTTCACCAACGG-3 13 Dqbeta DQB-AMP-B 5 -CTGGTAGTTGTGTCTGCACAC 13 Table 1a: Sequences of primers used to type HLA class II Allele Probe DRB Primer Sequence Hybridization temperature ( C) TAAGTTTGAATGTCATTT , 6, GTACTCTACGTCTGAGTG GAGCAGGTTAAACATGAG , AGAAATAACACTCACCCG TGGCAGGGTAAGTATAAG GAAGCAGGATAAGTTTGA GAGGAGGTTAAGTTTGAG a 5 -CAGCAGGATAAGTATGAG GGTTACTGGAGAAGACACT b 5 -GCGAGTGTGGAACCTGAT GCCTGATGAGGAGTACTG-3 45 Table 1b: Sequences of probes used for DR subtyping and hybridization temperature 1 Does not recognize allele DRB1* Also recognizes DRB1*1404 a Probe 1009 recognizes gene DRB5 presented in haplotypes where allele DRB1 is DR2 b Probe 2810 recognizes gene DRB4 presented in haplotipos where DRB1 are DR4, -7, -9 16

17 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Material and methods Allele Probe DQA Primer Sequence Hybridization temperatures ( C) TGGGCAGTACAGCCATGA GAGATGAGGAGTTCTACG , 0103, GAGATGAGCAGTTCTACG , GAGACGAGCAGTTCTACG ACCTGGAGAAGAAGGAGA , 0102, TCAGCAAATTTGGAGGTT TCCACAGACTTAGATTTG , 0501, TCAGACAATTTAGATTTG ATCGCTGTCCTAAAACAT , 0401, CTTGAACATCCTGATTAA-3 37 Table 2aI: Sequences of probes used for DQA1 subtyping and hybridization temperatures Allele Probe Primer Sequence Hybridization temperatures ( C) 0603, CGTCTTGTAACCAGACAC , 0502, AGGAGTACGTGCGCTTCG GACGTGGAGGTGTACCGG GGTGTACCGGGCAGTGAC , GCGGCCTAGCGCCGAGTA GGCTGCCTGCCGCCGAGT , GGCCGCCTGACGCCGAGT GGCCGCCTGCCGCCGAGT GACCCGAGCGGAGTTGGA , GAGGGGACCCGGGCGGAG GAAACGGGCGGCGGTGGA-3 52 Table 2aII: Sequences of probes used for DQB1 subtyping and hybridization temperatures 17

18 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Material and methods Allele Probe DRB1 Primer Sequence Hybridization temperature ( C) AACTACGGGGCTGTGGAG , 0102, CCAAGAGGAGTCCGTGCG ACATCCTGGAAGACGAGC , AACTACGGGGTTGGTGAG-3 41 Table 2aIII: Sequences of probes used to subtype DRBI Allele Primers Primer Sequence Concentration used (pm) DR1 DRB 5 -CGGCTC\GCACTGTAAGCTCT-3 13 DR1 DR1specific 5 -TTCTTGTGGCAGCTTAAGTT-3 13 Table 2aIV: Sequences of primer used to subtype DR1 Allele Primers Primer Sequence Concentration (pm) Sense TCCTGTGGCAGCCTAAGAG-3 50 Antisense CTGCACTGTGAAGCTCTCCA-3 20 Antisense CTGCCACTGTGAACTCTCAC-3 20 Probe FAM 5 -TCCGTGCGCTTCGACAGCGAC3 PO 4 5 Table 2b: Sequences of oligonucleotides used to subtype DR2 Microsatellite Primer sequence IKBL HGH-U 5 -CAGTGCCTTCCCAACCATTCCCTTA-3 HGH-D 5 -ATCCACTCACGGATTTCTGTTGTGT-3 MidR1 5 -TCTCACGCAGCTCTTCCT-3 IRB788CF 5 -AGAAGCAGAGGGATCCCG-3 IRB788TF 5 AGAAGCAGAGGGATCCTG-3 Table 3: Primers for IKBL 18

19 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Material and methods Microsatellite Primer sequence TNFa: IR2 5 -GCCTCTAGATTTCATCCAGCCACA-3 IR4 5 -CCTCTCTCCCCTGCAACACACA-3 TNFb: IR1 5 -GCACTCCAGCCTAGGCCACAGA-3 IR5 5 -GTGTGTGTTGCAGGGGAGAGAG-3 Table 4a: Primers for TNFa and TNFb Microsatellite Primer sequence MICA: 5F 5 -CCTTTTTTTCAGGGAAAGTGC-3 5R 5 -CCTTACCATCTCCAGAACT-3 Table 4b: Primers for MICA Statistical analysis Allele frequencies of controls and UC patients were analyzed in 2 x 2 contingency tables and chi-square analyses with the Yates correction. When an expected cell value was <5 the Fisher exact test was used. Associations were considered to be significant when the p value was <

20 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Results Results HLA-DRB1*1501 and ulcerative colitis When frequencies of DRB1*1501 were determined (table 1), they seemed slightly increased in patients, but it appeared that there was no significant difference in DRB1*1501 frequency when patients were compared with controls (p=0.10, OR=1.55). Table 1 represents the numbers. Patients (n=189) Controls (n=115) P and OR values DRB1* P=0.10, OR=1.55 Percentages 31.2% 22.6% Table 1: DRB1*1501 distribution in patients and controls IKBL+738(C) and ulcerative colitis Table 2 shows the allele frequencies between patients and controls when compared for the distribution of the IKBL+738(C) allele. The frequency of IKBL+738(C) was not significantly different in patients compared to controls (p=0.78, OR=0.92). Patients (n=189) Controls (n=115) P and OR values IKBL+738(C) P=0.78, OR=0.92 Percentages 15.3% 16.5% Table 2: IKBL+738(C) distribution in patients and controls Linkage disequilibrium between IKBL+738(C) and HLA-DRB1*1501 We investigated if the IKBL+738(C) allele was in linkage disequilibrium (LD) with DRB1*1501, meaning that the alleles occur more often in combination than would be expected by random association. LD is always investigated in the control group and numbers showed that the IKBL+738(C) allele is significantly more frequent in controls that are positive for DRB1*1501 than in controls negative for DRB1*1501 (p= , OR =10.0). Numbers are shown in table 3. 20

21 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Results DRB1*1501 positive controls (n=26) DRB1*1501 negative controls (n=89) IKBL+738(C) positive 12 (46.2%) 7 (7.9%) IKBL+738(C) negative 14 (53.8%) 82 (92.1%) P and OR values P< ; OR=10.0 Table 3: IKBL*738(C) distribution in DRB1*1501 positive and negative controls We also found that there was no significant difference in the distribution of the IKBL+738(C) allele in DRB1*1501 positives between patients and controls (p=0.54, OR=0.75). Table 4 represents the numbers. Patients positive for DRB1*1501 Controls positive for DRB1*1501 (n=59) (n=26) IKBL+738(C) positive 23 (39.0%) 12 (46.2%) IKBL+738(C) negative 36 (61.0%) 14 (53.8%) P and OR values P=0.54, OR=0.75 Table 4: Distribution of IKBL+738(C) in DRB1*1501 positive patients and controls HLA-DRB1*1501 in patients subgroups Patients were grouped for disease extent. There were two groups of patients, patients with extensive colitis and patients with left sided colitis. We compared both patient groups with the group of controls to see if there is a difference in distribution of DRB1*1501. We found that patients with extensive colitis did have a significantly different frequency of DRB1*1501 compared with controls (p=0.007, OR=2.33). No significant difference was found between left sided colitis patients and controls (p=0.51, OR=1.24). Numbers are presented in table 5 and 6. Extensive colitis (n= 79) Controls (n=115) P and OR values DRB1*1501 positive 32 (40.5%) 26 (22.6%) P=0.007; OR=2.33 DRB1*1501 negative 47 (59.5%) 89 (77.4%) Table 5: Comparison of DRB1*1501 positives in extensive colitis group and in control group. 21

22 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Results Left sided colitis (n=98) Controls (n=115 ) P and OR values DRB1*1501 positive 26 (26.5%) 26 (22.6%) P=0.51, OR=1.24 DRB1*1501 negative 72 (73.5%) 89 (77.4%) Table 6: Comparison of DRB1*1501 positives in left sided colitis group and in control group. To see if both subgroups of UC were really different from each other we compared them and found that DRB1*1501 was significantly more frequent in patients with extensive colitis than in patients with left sided colitis (p=0.049, OR=1.89). Table 7 shows the numbers. Extensive colitis (n=79) Left sided colitis (n=98) P and OR values DRB1*1501 positive 32 (40.5%) 26 (26.5%) P=0.049; OR=1.89 DRB1*1501 negative 47 (59.5%) 72 (73.5%) Table 7: Distribution of the DRB1*1501 allele in patient subgroups IKBL+738(C) in patient subgroups As mentioned above no significant difference of the IKBL+738(C) allele frequency was found when patients overall were compared with controls. We also compared patients subgroups with regard to allelic distribution of IKBL+738(C). First, both patients groups were compared to controls, but no significant difference was found for any group (p=0.85, OR=1.09 for extensive colitis and p=0.81, OR=0.91 for left sided colitis). Numbers are shown in table 8 and 9. Extensive colitis (n=79) Controls (n=115) P and OR values IKBL+738(C) positive 14 (17.7%) 19 (16.5%) P=0.83, OR=1.09 IKBL+738(C) negative 65 (82.3%) 96 (83.5%) Table 8: Distribution of IKBL+738(C) in extensive colitis patients and controls Left sided colitis (n=98) Controls (n=115 ) P and OR values IKBL+738(C) positive 15 (15.3%) 19 (16.5%) P=0.81, OR=0.91 IKBL+738(C) negative 83 (84.7%) 96 (83.5%) Table 9: Distribution of the IKBL+738(C) in left sided colitis patients and controls 22

23 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Results To see if there was a difference between the two patients groups we compared these groups, but also between these groups no significant difference of IKBL+738(C) distribution was found (p=0.67, OR=1.19). Table 10 represents the numbers. Extensive colitis (n=79) Left sided colitis (n=98 ) P and OR values IKBL+738(C) positive 14 (17.7%) 15 (15.3%) P=0.67, OR=1.19 IKBL+738(C) negative 65 (82.3%) 83 (84.7%) Table 10: Distribution of the IKBL+738(C) in extensive colitis patients and left sided colitis patients HLA-DRB1*1501 and IKBL+738(C) and patient subgroups As DRB1*1501 and IKBL+738(C) are in LD, we investigated if the effect we found of HLA-DRB1*1501 on extensive colitis was independent of IKBL+738(C) or not. Patients with extensive colitis were divided in two groups of DRB1*1501 positive patients with and without IKBL+738(C). They were compared with controls. In the group of patients without IKBL+738(C) a significantly higher frequency of DRB1*1501 was found compared to controls (p=0.02, OR=2.45). In the group of patients with IKBL+738(C) no significant increase of DRB1*1501 was found compared to controls, but also in this case the OR was increased. Numbers are presented in table 11. Extensive colitis (n=79) Controls (n=115) P and OR values DRB1*1501 positive 12 (15.2%) 12 (10.4%) P=0.32, OR=1.54 IKBL+738(C) positive DRB1*1501 positive IKBL+738(C) negative 20 (25.3%) 14 (12.2%) P = 0.02, OR=2.45 Table 11: Distribution of DRB1*1501 in IKBL+738(C) negative and positive patients and controls 7.1 haplotype in patients and controls We wanted to see if there was a difference in frequency of the 7.1 haplotype in patients and controls. We compared both groups and saw that the 7.1 haplotype is significantly decreased in DRB1*1501 positive patients compared to DRB1*1501 positive controls (p=0.02, OR=0.31). Numbers are presented in table 12a. 23

24 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Results DRB1*1501 positive DRB1*1501 positive P and OR values Patients (n=59) Controls (n=26) TNF a11,b4 MICA (50.8%) 20 (76.9%) p=0.02, OR=0.31 positive TNF a11,b4 MICA (49.2%) 6 (23.1%) negative Table 12a: Distribution of the 7.1 haplotype in patients and controls To see which of the two groups was responsible for the decrease of 7.1 we found in DRB1*1501 positive patients we compared TNF a11, b4 MICA 5.1 positive patients (DRB1*1501 in 7.1 haplotype) with TNF a11,b4 MICA 5.1 positive controls (presented in table 12b) and we compared TNF a11,b4 MICA 5.1 negative patients (DRB1*1501 in other haplotypes) with TNF a11,b4 MICA 5.1 negative controls (presented in table 12c). DRB1*1501 in 7.1 AH Patients (n=189) Controls (n=115) P and OR value DRB1*1501 positive P=0.73, OR=0.90 TNF a11,b4 MICA 5.1 positive Percentages 15.9% 17.4% Table 12b: Comparison of DRB1*1501 positive and TNF a11,b4 MICA 5.1 positive patients with DRB1*1501 positive and TNF a11,b4 MICA 5.1 positive controls DRB1*1501 in other haplotypes Patients (n=189) Controls (n=115) P and OR value DRB1*1501 positive 29 6 P=0.007, OR=3.29 TNF a11,b4 MICA 5.1 negative Percentages 15.3% 5.2% Table 12c: Comparison of DRB1*1501 positive and TNF a11,b4 MICA 5.1 negative patients with DRB1*1501 positive and TNF a11,b4 MICA 5.1 negative controls We saw that when we compared DRB1*1501 positive, TNF a11,b4 MICA 5.1 positive patients with controls, that there was no significant increase in frequencies (p=0.73, OR=0.90), but when we compared DRB1*1501 positive, TNF a11,b4 MICA 5.1 negative patients with controls, a significantly increased frequency was found in the patients (p=0.007, OR=3.29). 24

25 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Results HLA-DRB1*1501 and the 7.1 haplotype in patients with extensive colitis We have already seen that only DRB1*1501 which was not in the 7.1 haplotype was increased in UC patients. Now we wanted to know if DRB1*1501 present in the not 7.1 haplotypes is also related to disease extension. This 7.1 haplotype is known to be associated with some immune diseases, including Multiple Sclerosis (MS) (Allcock et al, 1999; Madigand). The 7.1 haplotype includes TNF a11b4 and MICA 5.1 and we made two different patient groups, one positive for DRB1*1501 with TNF a11b4 and MICA 5.1. and one positive for DRB1*1501 without TNF a11b4 and MICA 5.1. Then we compared both groups with controls and we saw that the DRB1*1501 allele was only more frequent in extensive colitis compared with controls in absence of TNF a11b4 MICA 5.1 (p=0.0003, OR=5.36). In the group with TNF a11b4 MICA 5.1 no significant difference was found between the extensive colitis group and the controls (p=0.95, OR=1.02). Table 13a represents the numbers. Table 13b presents the numbers of the same comparison, but than with the left sided colitis group in stead of extensive colitis. No significant difference in DRB1*1501 frequency was found between the left sided colitis group and the controls (p=0.67, OR=0.85 and p=0.22, OR=1.91). Finally, we also compared both patient groups and found that there was also a highly significant difference between both patient groups, but only in the absence of TNF a11b4 MICA 5.1 (p=0.03, OR=1.53). When TNF a11b4 MICA 5.1 was included no difference between the groups was found (p=0.87, OR=1.07). Numbers are represented in table 13c. Extensive colitis (n=79 ) Controls (n=115 ) p and OR values DRB1*1501 positive 14 (17.7%) 20 (17.4%) P=0.95, OR=1.02 a11b4 MICA5.1 positive DRB1*1501 positive a11b4 MICA5.1 negative 18 (22.8%) 6 (15.2%) P=0.0003, OR=5.36 Table 13a: Distribution of DRB1*1501 with the 7.1 haplotype in extensive colitis and controls 25

26 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Results Left sided colitis (n=105) Controls (n=115 ) p and OR values DRB1*1501 positive 16 (15.2%) 20 (17.4%) P=0.67, OR=0.85 a11b4 MICA5.1 positive DRB1*1501 positive a11b4 MICA5.1 negative 10 (9.5%) 6 (5.2%) P=0.22, OR=1.91 Table 13c: Distribution of DRB1*1501 with the 7.1 haplotype in left sided colitis and controls Extensive colitis (n=79) Left sided colitis (n=105) p and OR values DRB1*1501 positive 14 (16.1%) 16 (15.2%) P=0.87, OR=1.07 a11b4 MICA5.1 positive DRB1*1501 positive a11b4 MICA5.1 negative 18 (20.7%) 10 (9.5%) P=0.03, OR=1.53 Table 13c: Distribution of DRB1*1501 with the 7.1 haplotype in extensive colitis and left sided colitis 26

27 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Discussion Discussion In this study we have found that HLA-DRB1*1501 is associated with extensive disease, especially in the absence of the 7.1 haplotype. IKBL+738(C) has no effect on susceptibility or extension of ulcerative colitis. HLA-DRB1*1501 and ulcerative colitis Several studies investigated susceptibility genes for UC and it has become clear that genetic factors play an important role in disease susceptibility for UC. A lot of groups investigated the effect of DR2 on UC and found that the DRB1*15 allele has an effect on UC, whereas the DRB1*16 allele has no effect [43, 44, 66, 67]. Especially in Japanese populations, which have a much higher frequency of the DRB1*1502 allele than the European populations, a clear effect of DRB1*15 was found on overall susceptibility to ulcerative colitis [42, 46, 55, 67]. Results from Europe in Caucasians were conflicting [49, 50, 54]. In this study HLA-DRB1*1501 is only modestly increased in frequency when we compared patients with controls (p=0.10, OR=1.55), but when patients were subgrouped in extensive colitis and left sided colitis HLA-DRB1*1501 was strongly associated with extensive colitis. This association was independent of the IKBL+738(C) allele, but not independent of TNF a11,b4 and MICA 5.1, which are markers of the 7.1 haplotype. We only found an association between extensive colitis and DRB1*1501 in the absence of the 7.1 haplotype (p= , OR=5.36). When we compared DRB1*1501 in combination with the 7.1 haplotype, no significant difference between patients with extensive colitis and controls was found (p=0.95, OR=1.02). This implicated that the 7.1 haplotype might have a protective effect on disease susceptibility for extensive colitis and that a gene in this haplotype neutralises the effect of DRB1*1501 on susceptibility. This would be comparable with the protective effect of the 7.1 haplotype on MS or Idiopathic achalasia [40, 41, 68]. Another explanation for the discordant behaviour of DRB1*1501 depending on the haplotype context, might be that it is not the DRB1*1501 allele that confers susceptibility, but something present in the other haplotypes containing DRB1*1501, but not present in the 7.1 haplotype. We prefer the first hypothesis, because we have seen that the non-7.1 DRB1*1501 positive haplotypes were very different from each other. 27

28 HLA-DR2 and IKBL+738(C) in Dutch Ulcerative Colitis Patients Discussion This effect of the non-7.1 DRB1*1501 positive haplotypes is restricted to extensive forms of the disease and it is not observed in the left sided forms of UC. HLA-DRB1*1501 results from other groups compared to our results To our knowledge this is the first time that a specific positive association between HLA- HLA-DRB1*1501 and extensive colitis is shown. Previously, in a number of papers [43, 51, 52, 54, 57, 63] a weak association DRB1*1501 with UC overall was observed, whereas other studies did not find any effect of DR2 on UC at all [50, 51]. An explanation for the conflicting results may be a different experimental design. A lot of studies did not subdivide patients in disease extent, whereas we found the effect only in patients with extensive colitis and as shown in this study the 7.1 haplotype has a protective effect on the susceptibility conferred by DRB1*1501, which makes the effect of DRB1*1501 more difficult to detect, as 7.1 is one of the most frequent haplotypes containing DRB1*1501. IKBL and UC In this study IKBL+738(C) seems not to be associated with UC overall (p=0.78, OR=0.92) or with disease subtype (p=0.67, OR=1.19). This result is conflicting with previous data reported by the group of De la Concha [52], which found a significant effect of IKBL+738(C) on extensive disease. The IKBL+738(C) is mostly found in only two different haplotypes, one containing DRB1*0103, which is known to be associated with extensive colitis and the other is the 7.1 haplotype. In their study De la Concha et al described that the effect of IKBL+738(C) was independent of DRB1*0103. They reported in their study that it is yet unknown if the observed effect of IKBL+738(C) might be an effect of the IKBL+738(C) allele itself or an effect of a nearby gene which is in LD with IKBL+738(C). A possible explanation for the conflicting results might be the difference of the studied populations. The possible effective gene might be linked to IKBL+738(C) in the Spanish population, but not in the Dutch population. 28