Basic and translational research Figure 4 Genotyping of the Ala71Thr substitution in exon 4 of BLK. (A) Schematic diagrams showing the procedure for genotyping the single-nucleotide polymorphism (SNP) rs55758736 in exon 4 of BLK. Genomic DNA was ampliﬁed with primers ﬂanking exon 4 (In3-F: 5′AGAAGCCTGTCCTCCTTGGTAGC 3′ and In4-R: 5′GGAAAGATTTTGGAGAGGAAGACA 3′) and the PCR products were digested with the restriction enzyme AciI. The A allele disrupts the restriction site generating only two fragments of 48 and 322 bp while the G allele produces three fragments of 48, 80 and 242 bp. (B) PCR products of four subjects showing a unique band of 370 bp and (C) the subsequent AciI digestion of the ampliﬁcation products. Subjects 2 and 3 are heterozygotes (G/A) and subjects 1 and 4 are homozygotes for the wild type allele (G/G). (D) Sequence chromatographs showing one subject heterozygous for the BLK mutation (above) and one subject homozygous for the wild type allele (below). RESULTS Fine mapping and conditional analysis of BLK reveals two independent effects A total of 158 SNPs with MAF>0.005 were tested for asso- ciation in 1163 European patients with SLE and 1482 age- and country-of-origin-matched controls. A meta-analysis demonstrated that the strongest association signal was in the BLK promoter region (rs998683, PCMH-corrected=1.05×10−4 and OR 1.38 (1.21 to 1.58)) (online table S1). However, even after correction for multiple testing, multiple association sig- nals across the gene remained signiﬁcant. To reﬁne the BLK association, we tested sliding windows, which narrowed it down to three blocks (denominated B1, B3 and B5 in ﬁgure 1). The best model of association was observed for a 1.2 kb haplotype window between SNPs rs2248932 and rs9329246 located at the end of BLK’s ﬁrst intron (p=1.58×10−10, omni- bus haplotype test). The ﬁrst block (B1) covered BLK 5′ upstream region, exon 1 (untranslated-UTR) and beginning of intron 1, which corre- spond to the promoter. This block comprised a set of highly cor- related SNPs including all variants previously associated with SLE (rs2409780, rs2618444, rs2736336, rs2736337, rs2736338, rs2736340, rs13277113) and our strongest hit (rs998683) (ﬁgure 2A and online ﬁgure S1). Haplotype-speciﬁc analysis demon- strated a common, risk haplotype tagged by the minor alleles of these SNPs, including the rs13277113-A variant (omnibus test p value=1.61×10−5, OR=1.32 (1.16 to 1.49)) (table 1). The second block (B3) was at the end of intron 1 (ﬁgure 1) and displayed moderate LD with B1 (ﬁgure 2A). Consistent with Ann Rheum Dis 2012;71:1219–1226. doi:10.1136/annrheumdis-2011-200987 this observation, a risk haplotype correlated with the promoter risk haplotype and with similar haplotype frequency was over- represented in cases, although only with nominal association (p=0.013) (table 1). In addition, an independent and low-frequency-haplotype within B3 (1.1% in controls) was strongly associated and had a large effect size (p=5.16×10−7, OR=2.75 (1.80 to 4.19)) (table 1). This haplotype correlated completely with a risk haplotype observed on the third associated block B5 (p=1.57×10−4, OR=2.48 (1.54 to 4.00)), which covered the protein-coding exons 4, 5 and 6 (ﬁgure 1). The minor alleles of six low-frequency variants occurred in this haplotype: rs55758736, a non-synonymous SNP in exon 4 resulting in an alanine to threonine substitution at amino acid posi- tion 71 (Ala71Thr; GCT>aCT); chr8:11443817 (intron 4); chr8:11444706 (intron 5), chr8:11446229 (intron 5); and chr8: 8-11446791 and rs2255227 (intron 6) (ﬁgure 2A). Conditional analysis using multivariate logistic regression demonstrated that the association of SNPs in B3 disappeared after adjusting by the strongest hit located in B1 (rs998683) sug- gesting that the B3 and the B1 association was not independent (online table S2). In contrast, the low-frequency associated SNPs located in B5 remained associated after adjustment to rs998683 supporting two independent effects in BLK. Further, we observed that SNP rs55758736 had an r2≈0 with SNPs tag- ging B1 and B3 (figure 2B,C and online table S2), although this may be an underestimation due to the low frequency of the SNP. 1223