HLA

Our meta-analysis confirmed a positive association of ulcerative colitis with DR2 and allele 1502 of its split antigen DR15. Interestingly, an association between HLA-DR9 and ulcerative colitis was also found. In addition, HLA-DR4 seemed to be protective against ulcerative colitis. HLA-DR7, DRB3*0301 and DQ4 were positively associated with Crohn’s disease, and negative associations with DR2 and DR3 were noted.

In order to maximally extract information on HLA-disease associations, several concessions were made. Summing the number of patients that are DR15 and 16, DR11 and 12, and DR13 and 14 positive does not necessarily yield the number of DR2, DR5, and DR6 positives respectively. If many individuals were heterozygous for these split antigens it could lead to an overestimation of DR2, DR5, or DR6. Because DR12 and DR16 are rare, this would have only a minor effect on the outcome of our analysis. DR13 and DR14 are more frequent and therefore their combined incidence may be greater than the real DR6 phenotype frequency. However, omitting the studies in which we summed DR13 and DR14 positives did not yield an overall association with DR6 (data not shown).

Another potential confounder of meta-analyses is disease heterogeneity. Ulcerative colitis and Crohn’s disease may not encompass defined diseases but represent rather heterogenic disorders with different genetic backgrounds. In this case, in one population a certain HLA-DR allele could confer disease susceptibility, whereas in another population other genes may play a role. Hence, when overall associations are found in a meta-analysis that includes studies with different ethnic, racial, and geographical factors, a strong argument for a transethnic analysis of these phenotypes in relation to disease subgroups is provided. Moreover, when Japanese studies and studies on white populations were analysed separately, no gross differences in the outcome were observed.

The association of DR215 with ulcerative colitis was frequently noted in the homogeneous Japanese population.16-18 ,30 In other homogeneous populations such as the Sicilians19 and the Finns,21increased frequencies were also found. However, studies in more heterogeneous white populations gave conflicting results. Some studies confirmed the increased frequency,6 ,15 ,20 ,23 ,31 ,32whereas others only found equal frequencies13 ,22 ,33 or even a lower frequency.34 In the meta-analysis, DR2 is firmly associated with ulcerative colitis and when the Japanese studies are left out of the meta-analysis the association remains significant (OR = 1.51, CI = 1.20–1.90, table 4). It has been suggested that the DR2 association was mainly determined by a subspecificity of DR15, DRB1*1502 which is frequently found among the Japanese.30However, a subsequent study in whites showed that the frequencies of both subspecificities of DR15, DRB1*1501 and DRB1*1502, were increased in patients with ulcerative colitis when compared with controls.20 Five studies have looked at these subspecificities and our meta-analysis shows a positive association for DRB1*1502 but not for DRB1*1501 (fig 3D–E). However, this result is biased by the high frequency of this allele in the Japanese study included in this analysis and the association is no longer positive when the Japanese study is omitted from the meta-analysis (data not shown). Taken together, these data suggest that in white populations, alleles other than 1502 are responsible for the association of DR2 with ulcerative colitis.

Associated phenotypes in Japanese and white populations analysed separately

Several studies have indicated that HLA-DR4 protects against ulcerative colitis,6 ,16-18 ,21 and this association is also apparent in the meta-analysis.

The meta-analysis found a novel association with HLA-DR9. The fact that this association was not noted before is probably a consequence of the low frequency of this antigen in most populations. Indeed, 10 of 12 studies reported an increased frequency of DR9 in patients with ulcerative colitis, but this increase failed to reach statistical significance. In the Japanese population, the frequency of HLA-DR9 is relatively high, and may thus be a more important factor for disease susceptibility compared with other populations. When the three Japanese studies were analysed separately an odds ratio of 1.72 was obtained (CI = 1.06–2.78), which corresponds with an aetiological fraction of 0.15.

Finally, the HLA-DRB1*0103 allele is associated with ulcerative colitis. Three studies (one not yet published) have assessed the frequency of this rare allele in IBD patients and controls, and all reported a higher frequency in patients with ulcerative colitis.13 ,22 Others have found that this allele also predisposes to extensive disease.35 ,36

Studies on HLA-DR frequencies in Crohn’s disease have reported associations with DR1,6 ,7 DR4,8-10 and recently DR7.7 ,11 The latter studies also reported that DR3 conferred disease resistance.7 ,11 The meta-analysis confirmed only the positive association with HLA-DR7 and a negative association for HLA-DR3. It should be noted that HLA-DR7 is in linkage disequilibrium with HLA-B44 and it remains unclear whether the association with DR7 is indirectly due to this allele.

DRB3*0301 has been associated with Crohn’s disease26 and we have identified three studies (including one not yet published) that report on the phenotypic frequency of this allele.26 ,37Meta-analysis of these data shows a significant association between Crohn’s disease and B3*0301. This allele is in tight linkage disequilibrium with DRB1*1302 in whites.38 Therefore the association may either be due to one of these alleles or due to another allele in linkage disequilibrium with the DRB1*1302–B3*0301 haplotype.

The negative association of DR3 with Crohn’s disease is intriguing. Three studies have shown that the DR3 frequency is particularly low in patients with severe disease (as indicated by the need for azathioprine treatment),39 and in patients with perianal fistulas.40 In Crohn’s disease, this association seems independent from linkage of DR3 with the infrequent allele of the –308 restriction fragment length polymorphism in the TNF-α promoter, because the frequency of this allele was not reduced in the patients with Crohn’s disease with perianal fistulas.40

The association of Crohn’s disease with DQ4 is no longer significant when the two Japanese studies are omitted from the analysis. The DQ4 phenotype is common among the Japanese and may therefore constitute a more important risk factor in this population.

The calculations of the aetiological and preventive fractions should be interpreted with caution, as the HLA-DR phenotype frequencies vary among different populations and data on the age distribution of the groups that were studied are lacking.28 ,29 However, these calculations can serve as an indicator for the relative contribution of the specific HLA-DR molecules to disease susceptibility. Thus, the contribution of HLA-DR to disease susceptibility for ulcerative colitis is relatively high (aetiological fraction of 0.2 for DR2, 0.05 for DR103, and 0.03 for DR9), whereas for Crohn’s disease the contribution for DR molecules is smaller (0.06 for DR7 and 0.11 for DRB3*0301). These findings are in agreement with data from recent linkage analyses: for ulcerative colitis the attribution of the HLA region may determine most of the genetic aetiological fraction, whereas for Crohn’s disease the role of the HLA region seemed limited.22 The aetiological fractions that we found cannot account for the total genetic risk on IBD. Genome scans indicate that non-MHC genes may play a role as well. Linkage of disease susceptibility to loci on chromosomes 1, 2, 4, 3, 7, 12, 1641-43 has been reported, but replication of linkage has only been obtained for the loci on chromosomes 12 and 16.44-47

The frequency of HLA class II alleles varies between different populations and selection bias may underlie some of the associations found by the meta-analysis. For example, the association with DR7 is not noted when the white and Japanese populations are analysed separately (table 4). This is not surprising because DR7 is very infrequent among the Japanese population and only two studies have assessed its frequency. Therefore, the aetiological fraction of DR7 is very low for the Japanese population (due to the low prevalence of DR7). Conversely, the DR9 association with ulcerative colitis and the DQ4 association with Crohn’s disease are not detected when only white populations are included in the analysis. Most likely, this is also a consequence of the low frequency of these phenotypes in the white population.

In conclusion, this meta-analysis indicates that ulcerative colitis is associated with DR2,15 DR9, and DRB1*0103 and that DR4 confers protection. For Crohn’s disease, an association with DR7, DRB3*0301, and DQ4 and a negative association with DR2 and DR3 were found. The contribution of HLA-DR molecules to the pathogenesis of ulcerative colitis may be threefold larger when compared with Crohn’s disease. None of the aetiological fractions associated with these phenotypes can account for the total genetic contribution to disease susceptibility.