We first carried out two independent case-control association studies of CTLA4
SNPs, in two independent Romanian case-control cohorts. The first Romanian cohort consisted of 66 unrelated CEU patients with generalized vitiligo (18 with other concomitant autoimmune diseases) and 85 unrelated CEU controls with no auto-immune diseases; details of this case-control cohort have been published previously (Jin et al., 2007
). The second Romanian case-control cohort consisted of 101 unrelated CEU patients and 100 unrelated healthy CEU controls from the same geographical region. Among the 101 patients in the second cohort, 90 had vitiligo vulgaris, 9 acrofacial vitiligo, and 2 vitiligo universalis, and 32 (31.8%) also had other concomitant autoimmune diseases, including autoimmune thyroid disease (17), rheumatoid arthritis (5), adult-onset insulin-dependent diabetes (5), psoriasis (3), systemic lupus erythematosus (1), and alopecia areata (1). There were no signifi-cant differences of gender or age distributions among the patients (48.2 ± 18.5) versus controls (47.8 ± 18.7).
In the first case-control cohort, we genotyped seven SNPs spanning 77 kb in the CTLA4
region of chromosome 2; six SNPs (rs1863800, rs231775, rs3087243, rs11571302, rs11571297, and rs10932037) were genotyped directly and one (rs231806) was imputed based on LD patterns (Marchini et al., 2007
). None of these CTLA4
SNPs deviated from Hardy–Weinberg equilibrium among the controls (not shown). None of these SNPs showed statistically significant (defined as nominal P-value <0.05) allelic () or genotypic (not shown) association with generalized vitiligo in either the total patient group (n = 66) or in subgroups of patients who had (n = 18) or did not have (n = 48) other autoimmune diseases. No multiple testing correction was applied, given the lack of any apparant association. Six of the seven SNPs tested were in a single block of LD (D’ > 0.8), and we observed no significant association of CTLA4
SNP haplotypes with disease in any patient group (P = 0.15; not shown).
Allelic association of CTLA4 SNPs in Romanian vitiligo cases and controls
In the second case-control cohort we genotyped only SNP rs231775, and we again found no significant allelic or genotypic association with vitiligo overall. Nevertheless, in the subgroup of patients who had other concomitant autoimmune diseases (n = 32) allelic association approached statistical significance (P = 0.06), at an OR of 1.69 (95% CI: 0.87−2.05).
Blomhoff et al. (2005)
reported significant association (P < 0.03) of CTLA4
SNPs in that subgroup of vitiligo patients who had other concomitant autoimmune diseases at ORs > 2.1. However, initial reports of genetic association tend to over-estimate ORs, resulting in over-estimates of statistical power of subsequent studies (Goring et al., 2001
). The two present studies had estimated 80% power to detect significant association (P < 0.05) at ORs > 1.8−1.9. To enhance statistical power, we carried out a meta-analysis that combined data from these two present studies with those of the two previously-published studies of CTLA4
SNPs in CEU cohorts (Blomhoff et al., 2005
; Laberge et al., 2008
). SNP rs231775 was genotyped in all four studies. SNPs rs231806, rs3087243, rs11571302, and rs11571297 were genotyped by Blomhoff et al. (2005)
, Laberge et al. (2008)
, and the first of the two present studies. Two other published studies (Itirli et al., 2005
; Kemp et al., 1999
) analyzed an un-annotated (AT)n CTLA4
microsatellite; each reported association with a different allele, and thus cannot be reconciled with each other and furthermore cannot be statistically combined with SNP-based studies in a meta-analysis.
Considering the four studies of vitiligo in CEU patients that utilized SNPs and which thus can be combined in a meta-analysis, we found no significant heterogeneity across these studies based on Cochran Q-tests of heterogeneity (P > 0.1) and based on I2
tests of inconsistency (I2
< 50%) applied to each comparison and to each SNP (). Meta-analysis combining data from these four studies provided greatly enhanced statistical power. For SNP rs231775, the only SNP genotyped in all four studies, the meta-analysis provided 80% power for all comparisons at threshold P < 0.05 and OR 1.4, but still showed no association of rs231775 with vitiligo in either the total patient group or the subgroup of patients having only vitiligo, versus controls (). However, rs231775 showed significant association in the comparison of the subgroup of vitiligo patients who had other concomitant autoimmune diseases, versus controls (P = 0.01), and in the comparison of vitiligo patients who had other concomitant diseases, versus patients who had only vitiligo (P = 0.03), under the random-effects model. Cumulative meta-analysis of the allelic contrast (G versus A) for rs231775 showed progressive chronological decline in random effect pooled ORs in the comparison of vitiligo patients who had other concomitant autoimmune disease, versus controls, from 1.7 in the earliest study (Blomhoff et al., 2005
) to 1.4 in the meta-analysis that combined all four studies. These results are indicative of the ‘winner's curse’, the tendency of initial significant genetic association studies to over-estimate the corresponding OR (Goring et al., 2001
Pooled ORs for association of CTLA4 SNP alleles with vitiligo under fixed- and random-effects models
SNPs rs231806, rs3087243, rs11571302, and rs11571297, all of which are in LD (D’ > 0.8) with SNP rs231775, were tested in three studies. Meta-analysis combining these data provided >80% power to detect association at ORs ranging from 1.25 comparing all vitiligo patients versus controls, to 1.5 comparing the subgroup of vitiligo patients with other concomitant autoimmune diseases versus controls or versus patients with isolated vitiligo. Nevertheless, meta-analysis showed no significant association of any of these SNPs in any comparison under the more conservative random-effects model (), but all showed trends toward significance (P-values 0.05−0.10) in the comparison of vitiligo patients with other concomitant autoimmune disease versus controls. Similarly, SNPs rs11571302 and rs11571297 showed trends toward significant association in the comparison of vitiligo patients with other concomitant autoimmune diseases versus those patients having only vitiligo.
These results of the meta-analysis, showing signifi-cant association of CTLA4
SNP rs231775 with vitiligo only in the subgroup of patients with vitiligo plus other concomitant autoimmune diseases, versus controls, and near-significant association of other SNPs in LD with rs231775 in the same patient subgroup, are generally consistent with one of two microsatellite-based studies that could not be combined with the SNP-based studies in a meta-analysis. Kemp et al. (1999)
found that the 106-bp allele of a CTLA4
microsatellite was marginally associated (uncorrected P = 0.02) with vitiligo in the total patient group, but showed much stronger association (uncorrected P = 0.0001) in the subgroup of patients with other concomitant autoimmune diseases. The 106-bp allele of this microsatellite is in strong LD with the high-risk G allele of SNP rs231775 (Holopainen and Partanen, 2001
), and both of these variants have been genetically associated with a number of other autoimmune diseases.
The high-risk G allele of SNP rs231775 (+49A/G) results in the substitution of threonine by alanine at codon 17 of the leader sequence of the CTLA4 polypeptide, possibly affecting conformation of the leader peptide resulting in altered intracellular CTLA4 trafficking. This variant has been correlated with decreased negative regulation of T-cell proliferation, and is thereby thought to predispose to the development of autoimmune diseases (Brand et al., 2005
; Gough et al., 2005
). Our meta-analysis showed no apparent association of CTLA4
SNPs with generalized vitiligo overall, but showed significant association of the high-risk G allele of SNP rs231775 in that subgroup of vitiligo patients who had other concomitant autoimmune diseases. The OR for this association was 1.4 (95% CI: 1.05−1.90), comparable to the ORs from meta-analyses for other autoimmune diseases that have primary genetic association with CTLA4
SNP rs231775 (Brand et al., 2005
; Gough et al., 2005
). Furthermore, SNP rs231775 showed a significant difference in the comparison of patients with vitiligo and other concomitant autoimmune diseases versus patients who had only vitiligo, indicating that there is a clear difference between these two patient subgroups with respect to CTLA4
. Overall, these results suggest that CTLA4
is not genetically associated with vitiligo per se, and that the weak association of CTLA4
with vitiligo is secondary, reflecting epidemiologic association of vitiligo with other autoimmune diseases that have primary genetic association with CTLA4