The study population characteristics for those subjects with successful genotyping are summarised in Table . Despite attempts to match cases and controls there were significant differences observed and adjustments were made by logistic regression to take this into account in the statistical analysis. The characteristics of recruited COPD patients across the GOLD severity categories are shown in Table . Out of 989 duplicate genotyping reactions there were 4 discordant results (0.4%). The samples genotyped by alternative methods were 100% concordant, satisfying criteria for the assays to be accepted for further analysis.
Characteristics of Controls and COPD subjects
Characteristics of COPD patients according to GOLD classification of disease severity
The values for linkage disequilibrium (LD) between the SNPs are shown in Figure . This reveals very strong levels of linkage disequilibrium between groups of SNPs in both genes. Within TGFB1 there is strong LD between SNPs 1 and 2 (rs2241712 and rs1800469; r2 = 0.97), which is reflected in the very similar odds ratios calculated as part of the published meta-analysis. Within IREB2, it is clear that the seven SNPs studied fall into three main groups. SNPs 2 and 5 are in linkage disequilibrium (rs2656069 and rs10851906; r2 = 0.99), as are SNPs 3, 4, 6 and 7 (rs1964678, rs12593229, rs965604 and rs13180; all pairwise r2 = 0.99). SNP 1 (rs2568494) is not in linkage disequilibrium with any of the other IREB2 SNPs (r2 <0.34). For this reason, subsequent tables show information for TGFB1 SNPs 2, 3 and 4, and IREB2 SNPs 1, 2 and 4 only. There are 51 SNPs in the region of IREB2 in HapMap, and using the 3 tagSNPs as presented here captures 64% of the variation (33/51 variants). These calculations are harder to do for TGFB1 as two of the SNPs we have used are not in HapMap, so we cannot calculate the LD between these SNPs and other variants in HapMap. There are only 8 SNPs in the TGFB1 region in HapMap, and rs1800469 captures information about one additional variant, ie 2 out of 8 SNPs (25%). It is likely that we have captured more variation than this, but are unable to quantify it more precisely due to the lack of information about our other two tagSNPs in HapMap.
Pairwise linkage disequilbrium between SNPs in the TGFB1 gene (a) and IREB2 gene (b). Values shown are for r2.
HWE analysis showed that all SNPs are in HWE (p > 0.01 for all SNPs) (Table ). The allele frequencies for SNPs in cases and controls are shown in Table . None of the TGFB1
SNPs showed significant associations with COPD. This is in contrast to the significant associations observed in the recent meta-analysis, which indicated that the minor alleles of rs2241712, rs1800470 and rs1800469 were associated with a reduced risk for COPD, whilst rs6957 was associated with increased risk [2
]. Haplotype analysis also failed to demonstrate any significant association with COPD (Additional file 1
: Table S1).
TGFΒ1 and IREB2 Allele frequencies and odds ratios in Controls and Cases
Each of the three IREB2 SNPs showed a significant difference in allele frequency between cases and controls (Table ). A Sidak correction for multiple testing of 6 SNPs shows that a single p-value ≤ 0.0085 corresponds to an overall p-value ≤ 0.05 [12
]. Thus the significant effects remained after adjustment for multiple comparisons. The genotype frequencies and associated odds ratios for the IREB2
SNPs are shown in Table . Again the genotype differences between cases and controls are significant after the Sidak correction. This is in agreement with previously published work [6
]. Haplotype analysis using IREB2 SNPs 1, 2 and 4 failed to yield any significantly stronger associations than the single SNPs alone (Additional file 1
: Table S2).
IREB2 Genotype frequencies and odds ratios in Controls and Cases.
We also analysed allele frequency differences between each of the GOLD Stages II, III and IV and controls. No significant differences or trends were observed for any of the TGF1 SNPs. IREB2 SNPs 2 and 4 showed differences between severity groups and controls (not significant after correction for multiple testing), but there were no trends with increasing severity and the results just reflect the overall case-control relationship (Table ). Whilst there appears to be a trend for an increase in SNP 1 minor allele frequency with increasing severity, this fails to reach statistical significance. Confining the analysis to cases only we examined the relationship between the allele distributions of the six SNPs and severity of disease using the quantitative trait FEV1 as a severity measure. We found no significant relationships between FEV1 and the alleles.
Minor Allele frequencies by case, control and severity status for IREB2 SNPs 1, 2 and 4
Though there were significant differences between centres for the allele distribution of some of the SNPs, our results are unlikely to be due to population stratification since cases and controls were matched within centres. We also examined the genotype odds ratios for all six SNPs in each centre (Additional file 1
: Table S3). The within-centre odds ratios for the three TGFB1
SNPs were all non-significant, showed no particular pattern and fluctuated around the overall (all-centre) odds ratios. The within-centre analysis for the IREB2
SNPs showed that, though non-significant due to the smaller sample size, the direction of the relationship was the same in each centre as the all-centre results. Thus each centre is showing the same IREB2 SNP relationships with COPD.