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

Abstract

ObjectivesTo perform fi ne 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 identifi ed two independent genetic effects with functional consequences: one represented by two tightly linked associated haplotype blocks signifi cantly 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 confi rmed. 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.

Genetics of systemic lupus erythematosus and Sjögren's syndrome: an update

“…In this regard, few examples exist of rare genetic variants in SLE related to the genes identified in GWAS studies. One example is BLK, where a rare mutation (a nonsynonymous change from alanine to threonine) was identified [41]. We recently observed that such mutation could modify the protein-protein interaction of BLK with BANK1 through alteration of the SH3 domain of BLK [42].…”

The Plasmacytoid Dendritic Cell as the Swiss Army Knife of the Immune System: Molecular Regulation of Its Multifaceted Functions

“…Reducing BLK levels in mouse pDC increased TLR9-induced IFNα production(105). Given that genetic variants in the BLK locus are identified in SLE patients by genome-wide association studies, it is notable that certain polymorphisms correlate with reduced BLK levels(106). Consequently, this may elevate IFNα secretion and hence contribute to SLE predisposition.…”

Concordance of Increased B1 Cell Subset and Lupus Phenotypes in Mice and Humans Is Dependent on BLK Expression Levels

“…BLK is also associated with other autoimmune disorders, such as rheumatoid arthritis (RA) (6), systemic sclerosis (SSc) (7), Sjögren’s syndrome (8, 9), primary anti-phospholipid syndrome (APS) (10), dermatomyositis (11) and Kawasaki disease (12). The SNP risk alleles found in regulatory regions have been shown to associate with reduced mRNA levels of BLK and reduced protein expression (3, 13–15). …”

“…BLK is mainly expressed by B lymphocytes but also, to a lesser extent by non-B-cell lineages, such as plasmacytoid dendritic cells (pDCs), pancreatic β-cells, and γδT cells (13, 16–19). Though Blk phosphorylation is detectable upon anti-IgM stimulation (20–22), BLK expression is downregulated upon BCR stimulation (15), suggesting that BLK may play a dual role downstream of BCR signaling.…”