▶ Additional ﬁgures and tables are published online only. To view these ﬁles please visit the journal online (http://ard.bmj.com/ content/71/7.toc). For numbered afﬁliations see end of article Correspondence to Marta E Alarcón-Riquelme, Centro de Genómica e Investigación Oncológica Pﬁzer-Universidad de Granada- Junta de Andalucía (GENYO), Avenida de la Ilustración 114, Granada, 18007, Spain; marta.alarcon@genyo.es Received 31 October 2011 Accepted 31 January 2012 Basic and translational research EXTENDED REPORT Fine mapping and conditional analysis identify a new mutation in the autoimmunity susceptibility gene BLK that leads to reduced half-life of the BLK protein Angélica M Delgado-Vega1, Mikhail G Dozmorov2*, Manuel Bernal Quirós3, Ying-Yu Wu2, Belén Martínez-García3, Sergey V Kozyrev4, Johan Frostegård5, Lennart Truedsson6, Enrique de Ramón7, María F González-Escribano8, Norberto Ortego-Centeno9, Bernardo A Pons-Estel10, Sandra D’Alfonso11, Gian Domenico Sebastiani12, Torsten Witte13, Bernard R Lauwerys14, Emoke Endreffy15, László Kovács16, Carlos Vasconcelos17, Berta Martins da Silva18, Jonathan D Wren2, Javier Martin19, Casimiro Castillejo-López3, and Marta E Alarcón-Riquelme2,3 ABSTRACT Objectives To perform ﬁne mapping of the autoimmunity susceptibility gene BLK and identify functional variants involved in systemic lupus erythematosus (SLE). Methods Genotyping of 1163 European SLE patients and 1482 controls and imputation were performed covering the BLK gene with 158 single-nucleotide polymorphisms. Logistic regression analysis was done using PLINK and conditional analyses using GENABEL’s test score. Transfections of BLK constructs on HEK293 cells containing the novel mutation or the wild type form were analysed for their effect on protein half-life using a protein stability assay, cycloheximide and western blot. CHiP-qPCR for detection of nuclear factor κ B (NFkB) binding. Results Fine mapping of BLK identiﬁed two independent genetic effects with functional consequences: one represented by two tightly linked associated haplotype blocks signiﬁcantly enriched for NFκB-binding sites and numerous putative regulatory variants whose risk alleles correlated with low BLK mRNA levels. Binding of NFkBp50 and p65 to an associated 1.2 Kb haplotype segment was conﬁrmed. A second independent genetic effect was represented by an Ala71Thr, low-frequency missense substitution with an OR=2.31 (95% CI 1.38 to 3.86). The 71Thr decreased BLK protein half-life. Conclusions These results show that rare and common regulatory variants in BLK are involved in disease susceptibility and both, albeit independently, lead to reduced levels of BLK protein. INTRODUCTION The BLK gene located on chromosome 8p23-p22 encodes a tyrosine kinase of the SRC family of proto-oncogenes. A genome-wide association study identiﬁed a single-nucleotide polymorphism (SNP) rs13277113 in the 5′ upstream region associ- ated with systemic lupus erythematosus (SLE),1 a chronic autoimmune disease with complex inheri- tance. To date, multiple studies have conﬁrmed the association of BLK with SLE in European and Asian populations as well as with other autoimmune disorders.2–10 BLK protein has 505 amino acids with SH3 (58–118), SH2 (124–220) and protein kinase domains (241–494). Although BLK is expressed in various tissues such as pancreatic β cells, thymus, hair follicles and salivary ducts,11 12 it is preferen- tially expressed in B lymphoid cells and has a role in B cell receptor signalling and B cell development.13 The BLK-rs13277113 SNP is part of a group of common variants across several genes that have been associated with SLE through genome-wide association study.14 Together, they account for 15% of the disease’s familial aggregation and few have been demonstrated to be functional.14 The SLE- risk allele rs13277113-A correlated with decreased expression of BLK mRNA1 but the mechanism by which this or another SNP in linkage disequilib- rium (LD) with it affects gene expression has not been characterised yet. To address which and if common and/or low frequency functional variants are involved in SLE susceptibility at the BLK locus, we performed ﬁne mapping by genotyping tag SNPs aided by imputation. METHODS Patients and controls After quality control of the data, the study sam- ple consisted of 1163 patients with SLE and 1482 ethnicity-matched healthy control subjects from the European multicentre collaboration network BIOLUPUS (supported by the European Science Foundation), comprising individuals from Argentina (60 patients and 109 controls), Belgium (70 patients and 59 controls), Germany (284 patients and 176 controls), Hungary (38 patients and 35 controls), Italy (264 patients and 320 controls), Portugal (163 patients and 165 controls), Spain (206 patients and 227 controls) and Sweden (78 patients and 391 con- trols). A completely new and independent set of 1103 healthy Spanish controls was also used. All patients fulﬁlled at least four of the American College of Rheumatology 1982 criteria for the classiﬁcation of SLE.15 Subjects with an individual genotyping rate lower than 90% or duplicated and/or related Ann Rheum Dis 2012;71:1219–1226. doi:10.1136/annrheumdis-2011-200987 1219