Figure 5 Reduced half-life of BLK protein in the presence of the 71Thr mutation. (A) Plasmids expressing each of the two BLK isoforms fused with V5 were transfected into HEK293T cells. Twenty-four hours after transfection, the cells were treated with 20 µg/ml cycloheximide (CHX) for various times as indicated. The recombinant proteins were detected by western blot using anti-V5 antibody. Tubulin was used as loading control. (B) Quantiﬁcation of the blots from three independent experiments; the SD at each time point is represented by bars. After 8 h of inhibition of protein synthesis, the level of BLK 71Thr was reduced to half while the wild type isoform Ala71 remained highly stable. and there is evidence that while phosphorylation sites are important for activation of proteins in signal transduction path- ways phosphorylation sites can act at the same time as signals for degradation by ubiquitin ligases.27 28 Protein stability has been extensively studied in the cycling protein family. The abundance of cycling E is controlled by phosphorylation-triggered ubiquitin-dependent proteolysis.29 30 Phosphorylation of the Thr380 allows cycling E to be recogn- ised by Fbw7, an F box protein substrate of related E3 ubiquitin ligases.31 Mutation of this Thr380 to Ala prevents degradation of cyclin E.32 We are currently investigating if the reduction of the half-life of the BLK 71Thr protein is due to an active process of degradation mediated by phosphorylation or just the result of a general decrease in protein stability. As the LD within the promoter blocks of BLK was very tight, it was not possible for us to deﬁne a single variant that could explain the correlation between low BLK levels and a polymor- phism. Instead, we observed a clear correlation between associ- ated blocks and transcription factor binding sites for NFκB as experimentally determined by ChIP-Seq and contained in the ENCODE site. However, the database contains only NFkBp65 experiments. NFkBp65 is known to have a after BcR activation. Alternatively, NFkBp50 homodimers that lack a transactivating domain bind to the human BLK promoter, as has been shown Ann Rheum Dis 2012;71:1219–1226. doi:10.1136/annrheumdis-2011-200987 Basic and translational research in mice.33 Here, homodimers of NFκB/p50 were shown to com- pete with Pax5 and reduce Blk gene expression upon mature B cell activation and development to antibody-producing plasma cells,33 opposite to what occurs during early B cell differentiation, when Blk expression plays an important role.33 We also do not know the role of IRF4 in this context and a much more detailed analysis of the regulatory landscape of BLK is required to under- stand the correlation between the promoter risk haplotypes and lowered BLK gene expression. Also, due to the strong LD within the promoter haplotypes, it has also not been possible to discard the region proximal to the C8orf13 or FAM167A region lying 5′ of BLK. In this context, the same associated SNPs correlate with increased levels of FAM167A gene expression.1 The mean- ing of this is currently unknown and there is no knowledge that this gene would have any role in immunity in general. In this context, we chose not to include the FAM146A gene in our ﬁne mapping, as ours and the actual evidence in other autoimmune diseases support BLK as the susceptibility gene in this locus. We propose that subtle quantitative differences between risk and protective haplotypes occur during mature B cell stimula- tion leading to lower BLK levels, and that epigenetically several sites show a variegated effect of NFκBp50 homodimers on the promoter region of BLK. Nevertheless, our genetic analysis did show a potentially unique location within the highly associ- ated 1.2 Kb haplotype window where a single binding site for NFκBp65 was shown to be particularly strong in the ENCODE data and conﬁrmed here for NFκBp50 as well. In summary, we identiﬁed two independent effects within the BLK locus one of which covers the promoter region of the gene where the associated haplotype blocks are enriched for transcription factor binding sites for NFκB and IRF4, providing a potential mechanism for the correlation of the risk alleles and reduced levels of BLK expression. We also identiﬁed a novel mutation where the SLE-associated 71Thr leads to the reduced half-life of BLK protein. What effect reduced BLK expression has on the function of human B lymphocytes is at present under investigation. Contributors AMDV performed all the genetic analyses and wrote the initial draft of the manuscript; MGD and JDW performed the bioinformatic analyses; YYW performed the qPCR-CHIP; BMG did the genotyping for the Ala71Thr; CCL designed the protein degradation experiments on the Ala71Thr and the RFLP; MBQ performed the degradation experiments; JF, LT, EdR, MFGE, BAPE, SD’A, GDS, TW, BRL, EE, LK, CV, BMdaS and JM provided samples; MEAR directed the work; MEAR, ADG and CCL wrote the manuscript. AMDV, MEAR, CCL and MGD contributed equally to this work. Acknowledgements The authors would like to thank all SLE patients who con- sented to their recruitment in this project. Funding for the project was provided by The Swedish Research Council, the Gustaf Ve:80-Års Fond, the Swedish Association against Rheumatism, the Instituto de Salud Carlos III (PS09/00129) that coﬁnanced partly through FEDER funds of the European Union, the Research Network BIOLUPUS with support from the European Science Foundation, the Consejería de Salud de Andalucía (PI0012) and grants from the NIH RR020143, the Alliance for Lupus Research and OCAST to MEAR. Funding Funding provided by FCT (Fundação Ciencia e Tecnologia, Portugal), DFG WI 1031/6-1 (Germany), Grupo Italiano LES and the CVDIMMUNE Project supported by the European Commission. Competing interests None Ethics approval Approval provided by the IRB from each participating institution. Provenance and peer review Not commissioned; externally peer reviewed. Data sharing statement We will share data related to this article. Author afﬁliations 1Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, 751 85, Sweden 2Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, 73104, United States of America 3Centro de Genómica e Investigación Oncológica GENYO Pﬁzer-Universidad de Granada-Junta de Andalucía, Granada, 18007, Spain 1225