Mapping of a disease susceptibility locus in chromosome 6p in Japanese patients with ulcerative colitis

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Abstract

Ulcerative colitis (UC) is a multifactorial disorder with both genetic and environmental factors. HLA-B*52 and DRB1*1502 are reported to be strongly associated with UC in Japan. However, the actual susceptible gene has not been identified yet. In this study, to map precisely the susceptible locus for UC, we performed association mapping in the chromosome 6p using 24 microsatellite markers distributed over 16 Mb. A total of 183 patients with UC and 186 healthy controls (HC) were included in this study. In all, 15 markers around the human leukocyte antigen (HLA) region showed statistical significance in the genotypic differentiation test concerned with the allelic distribution between the UC and HC. Especially, the markers between the centromeric region of HLA class I and the telomeric region of class III showed remarkably low P-values and the allele239 of C2-4-4 in class I marker showed the strongest association (Pc=2.9 × 10−9: OR=3.74, 95% CI=2.50–5.60). Furthermore, we found strong linkage disequilibrium (LD) between the allele239 of C2-4-4 and HLA-B*52 in haplotype analysis. These results provide evidence that, in Japanese, important determinants of disease susceptibility to UC may exist in HLA, especially between the centromeric region of class I and the telomeric region of class III, under the strong LD with HLA-B*52.

Introduction

Little is known about the pathogenesis of ulcerative colitis (UC) and Crohn’s disease (CD), which are collectively referred to as inflammatory bowel diseases (IBD). Epidemiological data including familial aggregation, increased concordance rates in monozygotic twins compared with dizygotic twins, ethnic differences in disease prevalence, and an association with recognized genetic syndromes have suggested that a genetic background of susceptibility is important in the pathogenesis of IBD.1, 2, 3, 4, 5, 6, 7, 8, 9, 10 Previous genome-wide linkage studies in Western Europe, North America and Australia have demonstrated several loci relevant to IBD.11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 These studies have used panels of multiply affected relative pairs and hypothesis-free, nonparametric methods of linkage analysis to detect genomic regions showing significant linkage. Putative susceptibility loci have been found on chromosomes 1, 5, 6, 12, 14, 16, and 19. The chromosome 6p is named IBD3 and overlaps with the human leukocyte antigen (HLA) region. It has been reported that HLA class I genes are associated with UC in many populations, such as B52 in Japanese,23 B35 in Ashkenazi Jews,24 Cw7 in Finns,25 and A33 in Asian Indians.26 It has also been reported that HLA class II genes are associated with UC, such as DRB1*1502, DQB1*0601 and DPB1*0901 in Japanese,27, 28, 29 DR2 in ethnically matched patients in California30 and DRB1*0103 in European Caucasian patients with UC.11 The discrepancies in the results between the different ethnic groups may be attributed to genetic heterogeneity, the effect of recombination on an ancestral haplotype structure, and differences of allele frequencies in each population.

It is known that a conserved haplotype of HLA A*24-B*52-DRB1*1502-DQB1*0601-DPB1*0901 is strongly associated with UC in Japan,29 which includes almost the entire HLA region. Therefore, we considered that the susceptible gene for UC in Japanese is located within or near the HLA region and that it would be important to map the short arm of chromosome 6 adequately to elucidate the susceptible locus of UC.

Recently, a large number of new polymorphic microsatellite markers have been identified throughout the HLA region.31, 32, 33, 34 They provide valuable information for focusing on the critical regions of diseases, such as Behçet disease,35 psoriasis vulgaris,36 type I diabetes,37 rheumatoid arthritis 38 and nonmelanoma skin cancer.39

In the present study, we employed a two-stage approach for association mapping using microsatellite markers spanning 16 Mb of chromosome 6p including the HLA region to elucidate precisely the susceptible locus for Japanese UC.

Results

Hardy–Weinberg proportion test

This study was performed using 24 microsatellite markers (20 markers and an additional four markers for fine mapping) in patients with UC and the healthy controls (HC) in stages 1 and 2. No markers deviated from the Hardy–Weinberg equilibrium (HWE) (P>0.05) in HC in either stage. However, three markers (C1-2-A, DQ.CAR, TNFa) in the UC of stage 1 and four markers (DQ.CAR, C1-2-A, D6S1568, D6S1629) in the UC of stage 2 significantly deviated from the HWE (Figure 1).

Figure 1
figure1

Analysis of the Hardy–Weinberg proportion test for UC. The vertical axis indicates the exact P-value estimated by the simulations based on the Markov chain method with the following parameters: Dememorization number 3000, number of batches 300, iterations per batch 3000. P-values <0.05 are regarded as statistically significant. The horizontal axis indicates the nominal position of the microsatellite markers distributed over 16 Mb including HLA region.

Genotypic differentiation test

A genotypic differentiation test for the allelic distribution between the UC and HC groups was performed. Eight markers (C1-2-A, D6S2444, C3-2-11, D6S1629, D6S2445, D6S273, TNFa, DQ.CAR) showed significantly different distributions of alleles between UC and HC in stage 1. In stage 2, nine markers (C1-2-A, D6S273, C3-2-11, TNFa, DQ.CAR, D6S1629, D6S2444, D6S1683, D6S306) showed significantly different allelic distributions between UC and HC (Figure 2). The allelic distributions in UC at markers between the centromeric region of class I and the telomeric region of class III were clearly different from those in HC, whereas the allelic distributions in UC at markers outside of the HLA region were not strongly different from those in HC in either stage. C1-2-A in class I showed the lowest P-value in both stages (Pstage 1=0.00104, Pstage 2=0.00039).

Figure 2
figure2

Analysis of the genotypic differentiation test. The vertical axis, the horizontal axis and the parameters are the same as in Figure 1.

We replicated similar results in the two-stage study, suggesting that the susceptible gene to UC was most likely localized in the HLA region, especially around the C1-2-A.

Allelic association test

Each allele of the microsatellite markers was named according to the size of the amplified fragment and the allele frequencies were compared between UC and HC in each marker. Among alleles in the 20 markers, the strongest association was found in allele234 of C1-2-A in both stages 1 and 2 (allele234: Pcstage 1=0.000014, Pcstage 2=0.000003, Pctotal=3.9 × 10−9: ORtotal=2.99, 95% CItotal=2.13–4.19) (Table 1, Figure 3).

Table 1 Alleles statistically significantly associated with UC in a combined cohort of stages 1 and 2
Figure 3
figure3

Analysis of the allelic association test. P-values are obtained in the alleles most associated with UC in each marker.

Fine mapping with additional microsatellite markers

The results of the genotypic differentiation test and allelic association test at C1-2-A showed the lowest P-value among markers tested in stage 1 and were replicated in stage 2, suggesting that a susceptible gene for UC is likely to exist near C1-2-A. Therefore, we performed further fine mapping with the additional four markers (C1-4-1, C1-2-5, C1-3-1, C2-4-4) near C1-2-A. Final evaluations using the 20 and the additional four markers were made in a combined cohort of both stages 1 and 2. The four markers did not deviate from the HWE in HC. In contrast, C1-2-5 and C2-4-4 deviated from the HWE in UC (Figure 1). The allelic distributions at the additional four markers in UC significantly deviated from those in HC on the basis of the genotypic differentiation test (Figure 2). The strongest association was found in allele239 of C2-4-4 with the allelic association test (allele239: Pc=2.9 × 10−9: OR=3.74, 95% CI=2.50–5.60) (Table 1, Figure 3).

Linkage disequilibrium analysis

Because a remarkable association between HLA-B*52 and Japanese patients with UC was reported in a previous study,29 we performed a linkage disequilibrium (LD) analysis between HLA-B*52 and each allele of all the microsatellite markers used in this study. All alleles that were positively associated with UC showed LD with HLA-B*52 (data not shown). A tight LD was observed between HLA-B*52 and allele239 of C2-4-4 (R=0.858, P=1.1 × 10−35, Pc=6.8 × 10−34).

Analysis in the subgroups of UC

When patients with UC were classified according to disease phenotypes (proctitis, left-sided colitis, total colitis or segmental type) and the allelic association test was performed in a combined cohort of both stages 1 and 2, the frequency of allele234 of C1-2-A was higher in the left-sided colitis than in proctitis (P=0.0149, Pc=0.0447). No significant differences were found in the other alleles (data not shown).

Discussion

Previous association studies carried out using modern molecular genotyping methods provided strong evidence that genes within or near the HLA region are important determinants of susceptibility and disease behavior in IBD. In the present study, we performed an association mapping using microsatellite markers spanning 16 Mb in the IBD3 to narrow the susceptible locus in Japanese patients with UC. These markers were densely distributed in the HLA region at appropriate distances. The average number of alleles at these microsatellite markers was 10.2 and these had been selected as informative polymorphic genetic markers.35, 36, 37, 38, 39 Microsatellite markers are generally considered to be useful for association mapping, because of the higher levels of heterozygosity and longer extent of useful levels of LD than single-nucleotide polymorphisms (SNPs).41

In this study, several markers in the HLA region showed significant deviations from the HWE in UC. These markers also showed significant results in the genotypic differentiation and allelic association tests. Among these markers, C1-2-A showed the strongest association with UC on the basis of odds ratio, and all significant results of C1-2-A in stage 1 were replicated in stage 2, suggesting that these results had high reliability and that the susceptible gene was near C1-2-A. Then further mapping was performed in a combined cohort of stages 1 and 2. We set up an additional four markers near the C1-2-A, and found that allele239 of C2-4-4 (282 kb telomeric from the HLA-B) showed the strongest association (Pc=2.9 × 10−9: OR=3.74, 95% CI=2.50–5.60). These results strongly suggest that candidate susceptibility gene for UC is likely to exist between the centromeric region of class I and the telomeric region of class III. Interestingly, this genomic region corresponds with psoriaris vulgaris36 and nonmelanoma skin cancer.39 However, it is likely that the ancestral haplotypes for UC, psoriaris vulgaris and nonmelanoma skin cancer population groups are different, because the associated alleles for the same markers are not concordant among these diseases. Moreover, we found strong LD (R=0.858) between the allele239 of C2-4-4 and HLA-B*52 which has been reported to be associated with UC. Because 90% of HLA-B*52 form the DRB1*1502-HLA-B52* haplotype in the Japanese population,42 it is thought that the allele239 of C2-4-4 also shows strong LD with DRB1*1502 which has been also reported to be associated with UC. Thus, the associations of the allele239 of C2-4-4, HLA-B*52, and DRB1*1502 with UC appear to reflect the LD with an identical risk allele of a true susceptibility gene for UC.

It has been hypothesized that UC is not a single, homogenous disorder, but consists of heterogeneous groups that may be produced by extensive genetic heterogeneity. Therefore, it is necessary to investigate the allele frequencies in subgroups of UC stratified according to the clinical phenotypes. When UC was classified by the location of the lesions, only the allele234 of C1-2-A was higher in the left-sided colitis than in proctitis. No significant differences were found in other markers in the allele or carrier frequencies between the respective subgroups. Considering these data, it seems that the gene near C1-2-A may affect the disease location to some extent.

The genetic heterogeneity is just one of the factors that will confound attempts to isolate complex disease genes. UC possesses a complex genetic trait wherein several genes interact with different environmental factors and determine the clinical phenotype of the disease. Moreover, there is a possibility that combinations of alleles may interact on the same or the opposing haplotype to cause disease. Therefore, the mapping of complex trait loci is a difficult task and requires a large sample size and a dense genetic map. Further analysis is needed to narrow the susceptible locus using the SNPs for high-resolution mapping in the centromeric region of class I to the telomeric region of class III.

In conclusion, the susceptible gene of Japanese UC is localized in HLA, especially between the centromeric region of class I and the telomeric region of class III, under the strong LD with HLA-B*52.

Materials and methods

Subjects

Blood samples in stage 1 were obtained from 92 HC and 91 patients with UC who visited Tohoku University Hospital from May 1998 to December 1999, and samples in stage 2 were obtained from 94 HC and 92 patients who visited the hospital from January 2000 to January 2002. There was no overlap of subjects between stages 1 and 2. All patients and HC were Japanese. The characteristics of the subjects are summarized in Table 2. No differences were found in the age of onset, mean age, or clinical and analytical characteristics between the patients of stages 1 and 2. The diagnosis of UC was made on the basis of clinical symptoms and endoscopic, radiographic, and histological findings according to conventional criteria. The study protocols were approved by the Ethics Committee of Tohoku University School of Medicine. All patients and HC gave written and informed consent to participate in this study.

Table 2 Demographic characteristics of patients with UC and HC

Genotyping for microsatellite alleles

Genomic DNAs were obtained from peripheral blood mononuclear cells by standard phenol–chloroform extraction and ethanol precipitation or by utilizing the NA-1000 Automated Nucleic Acid Extraction Machine (Kurabo, Osaka, Japan). Microsatellite markers and primer sequences used in this study have been previously reported.31, 32, 33, 34, 35, 36, 37, 38, 39 In all, 24 microsatellite markers were distributed over a 16 Mb including the HLA region. To determine the number of repeat units of the microsatellite loci exhibiting polymorphisms, we synthesized forward primers by labeling the 5′-ends with the fluorescent reagents 6-FAM, HEX, and TET (PE Applied Biosystems, Foster City, CA, USA). The PCR reaction mixture contained 12.5 ng of genomic DNA, 1 μl of dNTP (2.5 mM each), 1 μl of 10 × PCR buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2), 5 pmol of forward and reverse primers and 0.25 U of AmpliTaq Gold (PE Applied Biosystems) in a total volume of 10 μl. Amplification was carried out using a PTC-200 DNA Engine (MJ Research, Inc. Waltham, MA, USA) under the following conditions: 40 cycles of denaturing at 94°C for 30 s, annealing at 53–67°C for 30 s, extension at 72°C for 30 s with a final incubation at 72°C for 10 min. The amplified products were denatured for 2 min at 96°C, mixed with formamide containing stop buffer, applied with a size standard marker of GS500 TAMRA (PE Applied Biosystems) to each lane and run on a 4% polyacrylamide denaturing sequencing gel containing 8 M urea in an automated DNA sequencer. The fragment sizes of PCR products were determined automatically using GeneScan software (PE Applied Biosystems).

Patient classification

Patients with UC were classified according to the location and the extent of the inflammatory lesions (proctitis, left-sided colitis, total colitis, or segmental type), need for intensive intravenous steroid therapy, and need for surgical treatments. Steroid therapy was administered according to an intensive intravenous regimen described previously.40 Surgical operation was carried out in patients with severe UC who did not respond to the steroid therapy.

Statistical analysis

A two-stage approach for association analysis was performed. The results of stage 1 were reconfirmed in stage 2 in order to exclude false-positive results. Allele and carrier frequencies (proportion possessing one or two copies of an allele) were estimated by direct counting. The exact test of Hardy–Weinberg proportion (HWP) for multiple alleles and the genotypic differentiation test for the allelic distribution between the UC and HC groups were performed by the Markov chain method using the Genepop software package.43, 44, 45 The Markov chain method has the advantage of providing a complete enumeration for testing HWP in cases where the number of alleles and the sample size is small. A P-value of less than 0.05 was regarded as statistically significant. Subsequently, an allelic association test was performed by comparing the allele frequencies of each marker between the UC and the HC groups with the χ2-test using a 2 × 2 contingency table. The P-value was corrected by multiplying the number of alleles observed in each locus tested (corrected P-value: Pc-value). A Pc-value of less than 0.05 was regarded as statistically significant. The strength of association was estimated by the odds ratio (OR). Moreover, an additional four microsatellite markers (C1-4-1, C1-2-5, C1-3-1, C2-4-4) were estimated in the same way for further investigation. Comparisons within each stratum (classified according to the clinicopathological features) were made by the χ2-test for independence.

The LD analysis with HLA-B was performed using haplotype frequency estimation software (EH program). This program uses an expectation–maximization algorithm to estimate the haplotype frequencies based on a maximum likelihood approach.46, 47 HLA-B genotyping was performed by the PCR-SSP (sequence–specific primer) method.48 Each haplotype was analyzed using a 2 × 2 contingency table with the χ2-test, and P was corrected by the number of haplotypes observed between each marker and HLA-B (Pc). The degree of LD (R) was calculated in HC by the EH program and assessed by the χ2-test. This measure of R has a range of −1 to 1, and is equal to 0 when there is no LD.

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Nomura, E., Kinouchi, Y., Negoro, K. et al. Mapping of a disease susceptibility locus in chromosome 6p in Japanese patients with ulcerative colitis. Genes Immun 5, 477–483 (2004) doi:10.1038/sj.gene.6364114

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Keywords

  • ulcerative colitis
  • human leukocyte antigen
  • microsatellite marker
  • susceptible gene

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