Basic and translational research Table 1 Haplotype analysis of the BLK-associated blocks Block (SNPs)/Start-End (Size) No. SNPs Omnibus test Haplotype Frequency Cases Frequency Controls OR (95% CI) B1 (rs2409780-rs2618478) 11374997-11393411 (18.4 kb) B3 (rs58373594-8-11433666) 11428036-11433666 (5.6 kb) Strongest window (rs2248932-rs9329246) / 11429059-11430289 (1.2 kb) B5 (rs2244931-rs17744726) 11441178-11449232 (8.1 kb) 21 17 4 15 1.75E-04 3.85E-07 1.58E-10 2.56E-04 TACGGTAGCTGTAGGGGCCGA CCCGTCCGTTATAGGAGCCGA TACGGTAGCTGTAGGGGCGGA TATAGTAACCGCCCAGAGCAA TATAGTAACCGCCCAGAGCAC ATCGCATAGAGTAAAAG TTGGCATCCAATAGAAG AACACACAGGACAGTAG AACACACAGGACGGTAG AACACATAGGACAGTAG CAAGAGTACCGTTTA CGAGGGCACCGTATG GAAGAGTACCGTATG CAAGAGTACCGTATG GGAGGGCACCGTATG GAAGGGTACCGTATG CAAGGGTACCGTATG GGAGGGTACCGTATG CAAAAATATTTCATG 0.079 0.299 0.504 0.011 0.108 0.489 0.113 0.045 0.323 0.030 0.291 0.134 0.014 0.061 0.370 0.077 0.012 0.019 0.023 0.073 0.245 0.559 0.014 0.109 0.541 0.113 0.045 0.290 0.011 0.328 0.110 0.010 0.067 0.362 0.078 0.010 0.024 0.010 ns 1.32 (1.16-1.49) 0.81 (0.73-0.91) ns ns 0.80 (0.72-0.89) ns ns 1.13 (1.01-1.27) 2.75 (1.80-4.19) 0.84 (0.75-0.95) 1.26 (1.06-1.49) ns ns ns ns ns ns 2.48 (1.54-4.00) P-value 0.4517 1.61E-05 7.56E-05 0.3503 0.9182 2.70E-04 0.9928 0.9493 0.013 5.16E-07 3.94E-03 0.011 0.2331 0.3859 0.5478 0.8688 0.6186 0.236 1.57E-04 The ﬁrst column indicates the block for which the haplotype analysis corresponds to. For each block, the ﬁrst and the last SNP, the starting and ending positions, as well as the size in kilobases (kb) are also presented. The Omnibus P value indicates the overall signiﬁcance of the association of the block. Only haplotypes with frequencies equal to or greater than 0.01 were tested. For the haplotype-speciﬁc analyses, the odds ratio (OR) with 95% conﬁdence interval (95% CI) and P-values were determined for each haplotype tested against all of the others pooled together. In block B3, the SNPs comprising the strongest associated window are underlined and the omnibus test for this window is presented. All analyses were performed using PLINK software, version 1.07. ns: non-signiﬁcant Analysis of epigenetic marks and transcription factor binding sites shows an over-representation of NFkB and IRF4 along the promoter region associated blocks We ﬁrst attempted the identiﬁcation of functional regulatory vari- ants across the promoter blocks B1 and B3. Bioinformatic analysis revealed that B1 was signiﬁcantly enriched for H3K4me3 sites, a hallmark of active promoters (ﬁgure 1, online ﬁgure S2 and online table S3). Furthermore, SNPs in B1 and B3, when compared with randomly chosen nucleotides, were enriched for binding sites for the transcription factor NFκBp65 and IRF4 but not PAX5 (using data from ENCODE project Chip-Seq for lymphoblastoid cell lines), while SNPs in the non-associated blocks B2 and B4 were not (ﬁgure 1, online ﬁgure S2 and online table S3). However, we were unable to single out a unique variant to explain the correla- tion between low BLK gene expression and risk alleles of SNPs in B1 and B3 due to the strong LD. We hypothesised that mul- tiple sites would therefore explain a coordinated regulatory effect on the expression of BLK. Nevertheless, the 1.2 Kb haplotype window with the strongest genetic association and within B3 contained SNP rs2248932 that overlapped with a DNAse hyper- sensitive cluster and with a strong NFκB binding site (online ﬁgure S3 and online table S4). To conﬁrm if NFκB binds to this segment of BLK we performed a CHiP-qtPCR experiment in a human Daudi cell line (ﬁgure 3A–C). We observed that NFκBp50 binds somewhat earlier than NFκBp65 (30 min post-BcR stimulation). The IkBa promoter served as control (ﬁgure 3C). Simultaneously, expression of BLK is reduced after activation (ﬁgure 3A). The 71Thr mutation leads to reduced half-life of BLK protein The independent Ala71Thr substitution is located on the SH3 (Src homology 3) domain of the BLK protein, which is of importance 1224 in protein–protein interactions. The SIFT algorithm25 26 predicted this substitution to be potentially damaging. As rs55758736 was identiﬁed through imputation, we veriﬁed the presence of the mutation in 178 selected samples of cases and controls. Of these, we genotyped all of those individuals predicted by imputation to have the mutation and a group of individuals predicted not to have it. Veriﬁcation was done using PCR, RFLP and sequenc- ing (ﬁgure 4) reaching 92% concordance. The mutation was observed in 2.17% of SLE patients and 0.92% of controls, all in the heterozygous state (OR=2.31; 95% CI 1.38 to 3.86 with the Pearson’s goodness-of-ﬁt χ2 (df=1)). Adding 1103 new controls, the OR was adjusted to 1.79 (95% CI 1.15 to 2.51). At the translational level, the substitution of alanine for threo- nine implies the formation of a putative phosphorylation site that could lead to the reduction of the half-life of the protein. We investigated if this was the case. Using constructs containing each allele of rs55758736 in transfection experiments we observed that, indeed, 71Thr had an accelerated degradation after treat- ment with the protein synthesis inhibitor cycloheximide as com- pared with the Ala71 allele (ﬁgure 5 A,B and ﬁgure S4). DISCUSSION We have herein shown that blocks of associated SNPs within the promoter regions of the BLK gene were signiﬁcantly enriched for NFκB and IRF4 binding sites, potentially involved in regulat- ing levels of BLK mRNA and the related reduction in BLK gene expression levels correlating with the risk haplotypes. Also, through conditional analysis we have identiﬁed a potentially deleterious, low-frequency mutation leading to reduced BLK protein half-life. Unlike the wild type allele, the threonine resi- due of the mutated variant is capable of being phosphorylated, Ann Rheum Dis 2012;71:1219–1226. doi:10.1136/annrheumdis-2011-200987