The autoimmunity-associated BLK haplotype exhibits cis-regulatory effects on mRNA and protein expression that are prominently observed in B cells early in development

Abstract

The gene B lymphocyte kinase (BLK) is associated with rheumatoid arthritis, systemic lupus erythematosus and several other autoimmune disorders. The disease risk haplotype is known to be associated with reduced expression of BLK mRNA transcript in human B cell lines; however, little is known about cis-regulation of BLK message or protein levels in native cell types. Here, we show that in primary human B lymphocytes, cis-regulatory effects of disease-associated single nucleotide polymorphisms in BLK are restricted to naïve and transitional B cells. Cis-regulatory effects are not observed in adult B cells in later stages of differentiation. Allelic expression bias was also identified in primary human T cells from adult peripheral and umbilical cord blood (UCB), thymus and tonsil, although mRNA levels were reduced compared with B cells. Allelic regulation of Blk expression at the protein level was confirmed in UCB B cell subsets by intracellular staining and flow cytometry. Blk protein expression in CD4(+) and CD8(+) T cells was documented by western blot analysis; however, differences in protein expression levels by BLK genotype were not observed in any T cell subset. Blk allele expression differences at the protein level are thus restricted to early B cells, indicating that the involvement of Blk in the risk for autoimmune disease likely acts during the very early stages of B cell development.

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

“…Allele T of SNP rs2736340 in the BLK promoter region is associated with SLE risk and with low expression of BLK mRNA (3). In agreement with previous reports (3, 13), carriers of the CC-allele showed relatively higher mRNA expression levels of BLK than CT- or TT-risk allele carriers (Figure 5A). According to Griffin et al (33) and recommended modifications (31, 32), we characterized human peripheral blood B1-like cells as CD19 + CD3 − CD20 + CD43 + CD27 + (Figure 5B).…”

“…Fluorescence in QuantStudio™ 3D Digital PCR 20K Chips was measured by QuantStudio™ 3D Digital PCR Instrument and data were analyzed by QuantStudio™ 3D AnalysisSuite™ Cloud Software (Life Technologies). As BLK mRNA expression in CD19 + B cells is at least 30-fold higher than in other BLK-expressing leukocytes (13), we have considered that the measured BLK expression comes mainly from B lymphocytes. Thus, the quantity of BLK mRNA measured by dRT-PCR was estimated using the formula: BLK expression in B lymphocytes = 100* BLK expression in PBMC / % CD19 + cells in PBMCs.…”

“…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.…”

GWAS implicates a role for quantitative immune traits and threshold effects in risk for human autoimmune disorders

“…In this context, we have recently reported on patterns of expression and allelic regulation of Blk (B lymphocyte kinase), a gene which has been associated with both SLE and RA. Although published studies in B cell lines have emphasized the cis-regulation of Blk by the autoimmune disease associated haplotypes, we have shown that the cis-regulatory effects on Blk expression in native B cells are largely restricted to transitional B cells[19]. …”