Basic and translational research Figure 1 Systemic lupus erythematosus-associated single-nucleotide polymorphisms (SNPs) and haplotype blocks and genomic features of the BLK locus. From top to bottom: –log10 of the p value corrected by false discovery rate for 158 SNPs. Below, the association for haplotype windows of two, three and four SNPs is displayed; the horizontal black line marks the threshold for Bonferroni correction (p=0.05/323 windows tested). Only SNP windows contained within block B1 (5′ upstream, exon 1 – UTR – and beginning of intron 1), B3 (end of intron 1) and B5 (exons 4, 5 and 6) were associated after correction. The exon/intron structure of BLK is displayed next and then NFkBp65 (blue), IRF4 (yellow) and PAX5 (green) Chip-Seq identiﬁed binding sites and promoter-associated histone marks (H3K4Me3) according to ENCODE project. Recombination rate (cM/Mb) is displayed at the bottom. samples were excluded. We removed individuals with <90% of European ancestry, as estimated with STRUCTURE v2.3.3 and 350 ancestry informative markers. Tag SNP selection From the LD structure of the BLK locus in the HapMap CEU population (data release 27; available at http://hapmap.ncbi. nlm.nih.gov/), tag SNPs selection capturing more than 90% of common variation at the gene locus (minor allele frequency of greater than or equal to 10%) including 20 kb upstream and downstream of the gene, at a r2 threshold of greater than or equal to 0.8, were selected using the tagger algorithm implemented in Haploview, V.4.1.16 After quality control of the data, 27 tag SNPs were used for genotyping and to guide the imputation. Genotyping All samples were genotyped at the Feinstein Institute for Medical Research (Manhasset, New York, USA) using a GoldenGate Custom Genotyping Assay and a BeadXpress Reader from Illumina (San Diego, California, USA). Two SNPs (rs1478895 and rs2248932) were genotyped by TaqMan 5′-exonuclease assay (ABI, Foster City, California, USA). SNP rs55758736 was genotyped by RFLP and sequencing for veriﬁcation in 178 indi- viduals. An Illumina custom BeadArray was used for genotyping this SNP in 1103 healthy controls. Imputation We used IMPUTE217 across a 250 kb genomic region (chr8:11,288,930-11,559,517) of the BLK locus. As reference panels we used haplotypes from all HapMap Consortium18 19 1220 populations together (phase 3, release 2) and haplotypes of the CEU population from the pilot phase of 1000 Genomes Project20 (downloaded from http://mathgen.stats.ox.ac.uk/impute/ impute_v2.html#ﬁltered_1kg_hm3_haps on May 13th, 2011). HapMap haplotypes duplicated with 1000 Genomes data were removed. Default parameters were used and the Ne argument set to 15 000. Only imputed genotypes with a probability equal to or greater than 0.9, a minor allelic frequency (MAF) equal to or greater than 0.005, following Hardy–Weinberg equilibrium (p>0.001), and a genotyping rate equal to or greater than 0.95 were retained for association. Statistical analysis Genetic association analysis was done using PLINK v1.0721 and GenABEL v1.6-5.22 All SNPs with a genotype call rate <95% or not in Hardy–Weinberg equilibrium (p<0.001) were excluded. For single-marker tests, we performed a Cochran-Mantel-Haenszel (CMH) meta-analysis, using the country of origin as the stratiﬁca- tion variable, and a Breslow-Day test to assess homogeneity of the ORs. p Values were adjusted using false discovery rate. We also performed an omnibus haplotype test for all windows of 2, 3 and 4 SNPs across the gene. In this test, the alternate hypothesis of each haplotype having a unique effect is compared with the hypothesis (df=number of haplotypes-1).21 Omnibus as well as haplotype-speciﬁc tests for haplotype blocks were conducted by testing each haplotype against all others pooled together (1 df). A Bonferroni correction using the number of windows tested was applied (p<0.05/323 windows tested). Conditional analysis was done by logistic regression using GenABEL’s score test. By specifying the country of origin as Ann Rheum Dis 2012;71:1219–1226. doi:10.1136/annrheumdis-2011-200987