The etiology of the chronic inflammatory bowel diseases. Evidence for Inflammatory Bowel Disease of a Susceptibility Locus on the X Chromosome

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1 GASTROENTEROLOGY 2001;120: Evidence for Inflammatory Bowel Disease of a Susceptibility Locus on the X Chromosome SEVERINE VERMEIRE,* JACK SATSANGI, MARC PEETERS,* MILES PARKES, DEREK P. JEWELL, ROBERT VLIETINCK, and PAUL RUTGEERTS* Gastroenterology Unit, Radcliffe Infirmary, Oxford, England; and Department of Human Genetics and *Gastroenterology Unit, University Hospital Gasthuisberg, Leuven, Belgium Background & Aims: The technique of genomewide scanning has been applied successfully in inflammatory bowel disease (IBD). A number of putative susceptibility loci have been identified through genomewide searches including replicated regions of linkage on chromosomes 12, 16, 6 (the HLA region), and 14. We have investigated the contribution of the X chromosome in 145 Belgian affected relative pairs. Methods: In the first stage of the study, 79 (68 CD, 11 mixed) sibling pairs were genotyped at 12 microsatellite markers covering the X chromosome. In the second stage, 10 additional markers in the X-pericentromeric region were studied in the families involved in stage 1 together with 62 additional families (52 sibling pairs, 14 second-degree relative pairs). Results: In the first stage, evidence for linkage was found over a 30-cM pericentromeric region spanning dxs991, dxs990, and dxs8096 (multipoint maximum LOD score in the CD subgroup, 2.5; P ). The remainder of the X chromosome was excluded (exclusion under LOD-2) for a locus with s 2. Fine mapping in the second stage confirmed linkage, and narrowed and shifted the linked region to Xq21.3 around dxs1203 (nonparametric linkage [NPL], 2.90; P ). The NPL-1 interval around the linkage peak comprises 19.7 cm. Conclusions: These data provide suggestive evidence for the presence and chromosomal location of an X-linked susceptibility gene in IBD. The etiology of the chronic inflammatory bowel diseases (IBDs), Crohn s disease (CD) and ulcerative colitis (UC), is unknown. The relationship between CD and UC remains under investigation. With a combined prevalence of /100,000 in Europe and North America, IBD has a young age at onset and a chronic often complicated pattern. 1,2 Concordance rates in twins and siblings suggest that important genetic factors contribute to the disease pathogenesis. 3 6 It is now generally accepted that neither CD nor UC follow a simple mendelian mode of inheritance and that the epidemiologic data are more consistent with a complex model. The number of genes involved and the way the genes interact with each other is still not known. Many autosomal susceptibility loci have already been suggested from the results of genomewide scans (Table 1) and candidate gene studies The regions of linkage on chromosomes 12 and 16 have been widely, although not universally, replicated More recently, loci on chromosomes 6 (the HLA region) and 14 have also been confirmed in independent data sets Epidemiologic data suggest that there may be a sexlinked susceptibility gene in IBD. Of particular interest is the association between IBD and the Turner syndrome. The association is most strong with isochromosomal Turner syndrome in which an abnormal (46XiXq) rather than an absent X chromosome is present. 25 In addition, CD is more frequent in females than males with a ratio of 1.3/1. Furthermore, Akolkar et al. 26 have suggested that imprinting may be pertinent in IBD. They reported a striking predominance of mother/child affected pairs compared with father/child affected pairs (28 vs. 5; P ). 26 The contribution of the X chromosome in IBD has not yet been extensively studied using molecular methods. A genomewide scan performed in a large multicenter European data set reported nominal evidence for linkage between UC and X with a maximum logarithm of odds (LOD) score of 1.71 (P 0.005, 2-tailed). 10 In the present study, molecular analysis of the X chromosome was performed in 145 Belgian IBD-affected relative pairs. The Belgian data set is of particular interest because no linkage has been found in these families with the regions on chromosomes 3, 7, 12, or 16, implicated elsewhere in Europe. 24 A 2-stage study was carried out and has provided evidence that an important susceptibility locus might be located on the long arm of the X chromosome. Abbreviations used in this paper: CD, Crohn s disease; IBD, inflammatory bowel disease; LOD, logarithm of odds; MLS, maximum LOD score; NPL, nonparametric linkage; PCR, polymerase chain reaction; UC, ulcerative colitis by the American Gastroenterological Association /01/$35.00 doi: /gast

2 March 2001 IBD SUSCEPTIBILITY AND THE X CHROMOSOME 835 Table 1. Genomewide Scans Involving Affected Sibling Pair/Relative Pair Analysis in IBD Study No. of affected sibpairs Linkage region MLS Comments Hugot et al cen 3.17 IBD1 Satsangi et al IBD2 7q p Cho et al p 2.65 Epistasis 1p- IBD1 (297 relative pairs) 3q 2.29 (37% Ashkenazi Jews) 4q 1.71 In mixed, Ashkenazi families 16cen 1.69 CD only Hampe et al CD only (European Multicenter Study) 1q p 2.07 IBD CD only X 1.71 UC only CD only UC only Ma et al q (All CD affected) 17q Jewish only 5q Jewish only Duerr et al q (127 relative pairs, all CD affected) Rioux et al p q Early-onset CD 3p p 2.30 NOTE. Table shows the strongest linkages (MLS) reported by each genome search in decreasing order of MLS. Materials and Methods Patients All patients were seen at the Gastroenterology Unit of the University Hospital Gasthuisberg Leuven. Diagnosis was established using conventional clinical, radiologic, endoscopic, and histologic criteria, as described previously. 27 In the first stage, 56 families including 79 white sibling pairs (68 CD, 11 mixed) were studied (Table 2). A family was called mixed if both CD and UC occurred within 1 family. Both parents were genotyped in 25 of 56 families (44.6%); in another 10 families (17.9%), 1 parent was Table 2. (Top) Number of Families and Relative Pairs Studied in Stages 1 and 2 and (Bottom) Number of Relative Pairs According to Sibship Size and Sex No. of families No. of relative pairs CD UC Mixed Total 1st stage (all sibpairs) 2nd stage (52 sibpairs, 14 2nd rel pairs) Total Sibship size Brother/brother Brother/sister Sister/sister Total NOTE. All families were white. available. If the parents were absent, one or more unaffected siblings were genotyped to infer the missing parental genotype. The female-male ratio was equal in both populations. In the second stage, 62 families were added, involving 52 additional sibling pairs and 14 second-degree relative pairs (2 aunt/nephew, 2 aunt/niece, 4 uncle/nephew, 1 uncle/niece, and 5 second-cousin pairs), in total giving 145 relative pairs studied (129 CD, 16 mixed). Breakdown of sibling pairs according to sex is listed in Table 2. There were no affected nonsibling pairs of an affected male related through his father (because this is noninformative for chromosome X). Ethical agreement was given by the University of Leuven. All patients gave informed consent. Genotyping DNA was extracted from venous blood using a saltingout procedure. 28 In stage 1, genotyping was done by polymerase chain reaction (PCR) using 12 fluorescence-labeled microsatellite markers (Applied Biosystems) spanning 140 centimorgan (cm) (96%) of the X chromosome at an average spacing of 10.2 cm. In the second stage, 10 additional microsatellite markers (dinucleotide repeats) in the 30-cM pericentromeric region together with the 3 markers in this defined region from stage 1 were studied in the same way (average spacing, 2.19 cm) (Figure 1). Average spacing between the markers was calculated from the published Genethon human linkage map. 29 Each primer was optimized before amplification over a range of annealing temperatures (55 60 C) and MgCl concentrations (

3 836 VERMEIRE ET AL. GASTROENTEROLOGY Vol. 120, No. 4 and for CD and mixed families individually. Stage 1 of the study was performed at the Wellcome Trust Centre for Human Genetics in Oxford, U.K. Stage 2 was done by the same investigator at the Gastroenterology Unit and Center for Human Genetics in Leuven, Belgium. Figure 1. Diagram of the X chromosome showing markers studied in (A) stage 1 and (B) stage 2. mmol). The 15- L PCR mixture contained 5 L DNA (10 ng/ L), 0.04 L Taq polymerase (5 U/ L), 0.6 L of each primer (66 ng/ L), 1.5 L KCl buffer, L dntps (concentration, 100 mol/l), and MgCl in sterile water. After PCR amplification, products were pooled, electrophoresed on 6% acrylamide gels, and run on 373A DNA sequencers (Applied Biosystems). Genotyping was done using the Genescan/ Genotyper System 2.1 (Applied Biosystems). Statistics Nonparametric linkage (NPL) was assessed based on proportions of alleles shared identity-by-descent at each location, and a maximum LOD score (MLS) was calculated. An excess of allele sharing in affected relative pairs was taken as evidence for linkage. Allele frequencies for each marker were calculated from the unrelated parents in the samples. For each marker, single-point analysis was performed with the SIB- PAIR 30 (stage 1) and GENEHUNTER 31 (stage 2) program with a special extension to the X chromosome. Multipoint analysis and exclusion mapping were done using a sex-linked program of MAPMAKER/SIBS (stage 1) 32 and GENE- HUNTER (stage 2). The GENEHUNTER program provides an NPL score, 31 from which the level of significance (P value) may be derived directly. The exclusion map was constructed using a s of 2. A locus was excluded for linkage if the LOD score was less than 2 for this value of s. For each marker, the statistical probability of linkage was calculated for IBD overall Results Stage 1 Twelve markers were analyzed in the first stage, covering 140 cm (96%) of the X chromosome. Results of the single-point analysis are shown in Table 3. Distortion of allele sharing was seen for 2 adjacent markers spanning a 4-cM region on the short arm of the X chromosome, dxs538 and dxs1068. This distortion of allele sharing was seen both in the whole data set (LOD, 1.52; P ; and LOD, 1; P 0.016, respectively), as well as in the CD subgroup (LOD, 1.40; P 0.006; LOD, 1.14; P 0.011, respectively). This is not surprising because our data set comprises nearly all CD siblings. The multipoint analysis using a sex-linked version of MAPMAKER/SIBS is shown in Figure 2. The peak of linkage was in the pericentromeric region with a multipoint MLS of 2.4 (P ) around dxs991, dxs990, and dxs8096. In the CD subgroup, this multipoint MLS reached 2.5 (P ) in the same region. Exclusion mapping (Figure 3) further implicates this region. Stage 2 Ten additional markers were analyzed in the families involved in stage 1 together with a further 66 related pairs to confirm the linkage and to refine the region. Single-point analysis showed significant distortion of allele sharing at 5 markers (Table 4) with a maximum NPL of 2.13 (P 0.015) at dxs1203. Table 3. Single-Point Analysis of First Data Set (SIBPAIR) Marker cm LOD (P value), overall LOD (P value), CD only DXS (0.50) 0.00 (0.50) DXS (0.50) 0.00 (0.50) DXS (0.16) 0.03 (0.36) DMD (0.19) 0.13 (0.22) DXS (0.0041) 1.40 (0.006) DXS (0.016) 1.14 (0.011) MAOB (0.22) 0.10 (0.25) DXS (0.11) 0.90 (0.02) DXS (0.23) 0.26 (0.14) DXS (0.41) 0.10 (0.25) DXS (0.50) 0.00 (0.50) DXS (0.50) 0.00 (0.50) NOTE. Twelve markers spanning the X chromosome were studied in 79 affected sibling pairs. Nominal evidence for linkage (P 0.05) is seen with 2 adjacent markers, dxs538 and dxs1068.

4 March 2001 IBD SUSCEPTIBILITY AND THE X CHROMOSOME 837 Table 4. Single-Point Analysis of Stage 2 (GENEHUNTER) Marker cm NPL (P value), overall NPL (P value), CD only DXS (0.048) 0.56 (0.284) DXS (0.150) 0.22 (0.41) DXS (0.373) 0.42 (0.335) DXS (0.287) 0.17 (0.569) DXS (0.020) 1.60 (0.050) DXS (0.020) 0.52 (0.298) DXS (0.051) 1.17 (0.116) DXS (0.037) 1.21 (0.109) DXS (0.015) 1.90 (0.027) DXS (0.35) 0.11 (0.45) DXS (0.072) 1.10 (0.139) DXS (0.168) 0.04 (0.483) DXS (0.55) 0.51 (0.70) Figure 2. Multipoint analysis for the 12 markers studied in the first stage (MAPMAKER/SIBS). LOD scores are shown for the overall data set, CD only, and for the different groups according to sex. A multipoint MLS of 2.4 (P ) (2.5 in CD subgroup; P 003) was observed in the pericentromeric region around dxs990 and dxs8096 (peak of linkage). Highest NPL scores were seen in the whole IBD data set. When the CD subgroup or the mixed subgroup were analyzed, results were less significant. Multipoint analysis (GENEHUNTER sex-linked) allowed calculation of the NPL peak 1 interval around the linkage peak 33 and narrowed this region to 19.7 cm located around dxs1203 with a maximum NPL of 2.90 (P ) (Figure 4). Discussion This is the most detailed molecular study on the contribution of the X chromosome in IBD yet reported. Figure 3. Exclusion mapping using the criterion of LOD-2 for a disease locus s 2 in the first stage and data set of 79 affected sibling pairs (MAPMAKER/SIBS). Except for the pericentromeric region of the X chromosome, the remainder of X may be excluded as containing a susceptibility gene, of s 2. NOTE. Ten additional markers within the previously defined region of linkage together with dxs990, dxs991, and dxs8096 from the first stage were studied in 145 affected relative pairs. Maximum NPL was seen at dxs1203 (NPL, 2.13; P 0.015). Previous studies have mainly concentrated on the autosomal regions where statistical methods are much more straightforward. Recent modifications of existing programs of NPL have allowed ourselves and other investigators 10 to dissect the contribution of the X chromosome. The present data provide suggestive evidence for the presence of an X-linked susceptibility locus in IBD. Exclusion mapping has implicated a 30-cM pericentromeric region; the use of additional markers within this region allowed to refine the region to 19.7 cm around Xq21. The threshold for defining linkage in polygenic diseases is a matter of debate. Lander and Kruglyak 34 have suggested a classification based on the number of times that one would expect to see a result at random in a whole genome scan. According to their guidelines, results can be classified as suggestive linkage (LOD 2.2; P ), significant linkage (LOD 3.6; P ), or highly significant linkage (LOD 5.4; P ) (1-tailed P values). They encourage investigators to report any linkage, which falls into one of these categories. Using these significance threshold criteria strictly, the present linkage results may be classified as suggestive. Our data are strengthened by the fact that markers showing evidence for linkage are clustered together over a rather broad region. Terwilliger et al. 35 showed that true-positive linkage intervals are broader than false-positive peaks. The present data may be compared with previously reported data. Of all studies and genomewide scans that included analyses on chromosome X, only Hampe et al. 10 reported nominal evidence for linkage on the X chromosome. Two linkage peaks were seen at opposite

5 838 VERMEIRE ET AL. GASTROENTEROLOGY Vol. 120, No. 4 Figure 4. Multipoint analysis in stage 2 investigation involving 145 affected relative pairs (GENEHUNTER). The NPL-1 linkage support interval around the peak is shown and spans 19.7 cm. Maximum linkage is seen around dxs1203 (NPL score, 2.90; P ). ends of the X chromosome. An MLS of 1.59 (P 0.007) was reported for IBD overall at markers dxs1202 and dxs1214 and for UC only at markers dxs1001 and dxs1047 (MLS, 1.71; P 0.005). Furthermore, the reported linkages were located on the opposite ends of the X chromosome (Xp21 and Xq25, 86.9 and 35 cm, respectively, away from our linkage peak). Our results are all the more notable in view of the fact that the linkages with chromosomes 3, 7, 12, and 16 suggested in other European populations have not been replicated in this population of Belgian families. 24 There may be statistical, clinical, or genetic explanations for these differences between the Belgian results and elsewhere. From a clinical point of view, it is striking that CD (both familial and sporadic) is more common in Belgium than UC. This contrasts with epidemiologic data from elsewhere in Europe From a statistical point of view, replication of linkage for the X chromosome is likely to be even more difficult than the autosomes. More families are required because the number of informative meioses is smaller. It is likely that any susceptibility locus, autosomal or X-linked, will interact with other genes and with environmental factors. Stratification according to genotypes and association of genotypes may help us to define certain subgroups of patients. A recent study on insulin-dependent diabetes mellitus showed evidence that the linkage on Xp13-p11 interacts with the IDDM-1 locus. 39 Cho et al. 9 suggested that, in CD, there may be epistasis between the susceptibility locus on chromosome 1p and the IBD1 locus on chromosome 16. Population-based prevalence studies examining these kinds of association are now necessary. It is unclear how the presence of a genetic determinant of susceptibility on the X chromosome to IBD may explain all the epidemiologic observations cited above including the association with Turner syndrome, female predominance in IBD, and Akolkar s observations of predominantly maternal inheritance. 26 However, the chance of an association of Turner syndrome and IBD on a random bias has been estimated to 1:500 million total live female births. 40 Because there are now more than 20 reported cases of this association in literature, this strongly indicates that these 2 disorders do not occur only by chance. Similarly, other immune-mediated or autoimmune diseases including giant cell hepatitis, Hashimoto thyroiditis, diabetes mellitus, rheumatoid arthritis, pernicious anemia, and Addison disease have all been associated with Turner syndrome, and implicated an immunologic link with this chromosomal abnormality. How a disorder with a chromosomal abnormality such as Turner syndrome is linked to immune-mediated or autoimmune disease is unknown. An imbalance of X- linked immunoregulatory genes may be included and may influence the development of immune tolerance rendering affected patients more susceptible to autoimmune stimuli. 41 Within the pericentromeric region on the X chromosome there are a number of potentially relevant candidate genes involving the regulation of the immune response, including interleukin 2 receptor and interleukin 13 receptor 2. It is also of interest that a translocation t(1,x) between chromosome 1 and the X chromosome which predisposes to renal cell carcinoma is localized within this region. 42

6 March 2001 IBD SUSCEPTIBILITY AND THE X CHROMOSOME 839 Future work must first include replication of these data, now regarded as an essential step in dissecting complex traits. 34 As discussed, the implication of X- linked loci in complex diseases may prove to be even more difficult than the replication of autosomal regions in IBD. 43 Narrowing this region by studying additional markers is necessary. Candidate gene analysis may be the most practicable approach, as applied to the loci on chromosomes 6, 16, and 12, and is likely to be helped by the data from the Human Genome Project. References 1. Binder V, Both H, Hansen PK, Hendriksen C, Kreiner S, Torp- Pedersen K. Incidence and prevalence of ulcerative colitis and Crohn s disease in the county of Copenhagen, Gastroenterology 1982;83: Calkins BM, Mendelhoff AI. The epidemiology of idiopathic inflammatory bowel disease. In: Kirsner JB, Shorter RG, eds. Inflammatory bowel disease. 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