Overexpression of the Cytokine BAFF and Autoimmunity Risk

Abstract

Background: Genomewide association studies of autoimmune diseases have mapped hundreds of susceptibility regions in the genome. However, only for a few association signals has the causal gene been identified, and for even fewer have the causal variant and underlying mechanism been defined. Coincident associations of DNA variants affecting both the risk of autoimmune disease and quantitative immune variables provide an informative route to explore disease mechanisms and drug-targetable pathways.

Methods: Using case-control samples from Sardinia, Italy, we performed a genomewide association study in multiple sclerosis followed by TNFSF13B locus-specific association testing in systemic lupus erythematosus (SLE). Extensive phenotyping of quantitative immune variables, sequence-based fine mapping, cross-population and cross-phenotype analyses, and gene-expression studies were used to identify the causal variant and elucidate its mechanism of action. Signatures of positive selection were also investigated.

Results: A variant in TNFSF13B, encoding the cytokine and drug target B-cell activating factor (BAFF), was associated with multiple sclerosis as well as SLE. The disease-risk allele was also associated with up-regulated humoral immunity through increased levels of soluble BAFF, B lymphocytes, and immunoglobulins. The causal variant was identified: an insertion-deletion variant, GCTGT→A (in which A is the risk allele), yielded a shorter transcript that escaped microRNA inhibition and increased production of soluble BAFF, which in turn up-regulated humoral immunity. Population genetic signatures indicated that this autoimmunity variant has been evolutionarily advantageous, most likely by augmenting resistance to malaria.

Conclusions: A TNFSF13B variant was associated with multiple sclerosis and SLE, and its effects were clarified at the population, cellular, and molecular levels. (Funded by the Italian Foundation for Multiple Sclerosis and others.).

Figure 1. Regional Association Plots

Shown are association results in the TNFSF13B region for multiple sclerosis (Panel A), B-cell percentage with respect to lymphocytes (Panel B), level of soluble B-cell activating factor (BAFF) (Panel C), monocyte counts (Panel D), length of the 3′ untranslated region (3′ UTR) of TNFSF13B (Panel E), and TNFSF13B messenger RNA (mRNA) (Panel F). Each panel shows the association strength (expressed as negative log₁₀ P values) versus the genomic position (on the hg19/GRCh37 genomic build), with specifications on genes, exons, and direction of transcription shown in the box at the bottom of the column. In each plot, BAFF-var is the variant with the strongest association. Other variants in the region are color-coded to reflect their extent of linkage disequilibrium with BAFF-var (taken from pairwise r² values calculated for Sardinian haplotypes). Panel A shows results for 2273 patients with multiple sclerosis and 2148 controls and for the extended sample (diamonds) of 2934 patients with the disorder and 3392 controls. Panels B through F refer to the SardiNIA study cohort.

Figure 2. RNA Sequencing Coverage across the TNFSF13B Gene

RNA expression levels are plotted as the relative number of reads across the TNFSF13B locus, spanning the last exon (exon 6) and the 3′ UTR. The mean expression levels for the three alternative genotypes (wild type [WT]/WT, BAFF-var/BAFF-var, and WT/BAFF-var) are shown. The 3′ UTRs (long and short forms) are represented with dark gray bars, and the BAFF-var position is specified.

Figure 3. Post-transcriptional Regulation of BAFF Protein Expression by MicroRNAs

Panel A shows a box plot of soluble BAFF levels, as determined by enzyme-linked immunosorbent assay (ELISA), in serum specimens from 2733 genotyped SardiNIA volunteers, stratified according to genotype. Panel B shows the results of Western blot analysis of endogenous BAFF protein in THP1 cells 48 hours after transfection of the microRNA (miRNA) precursors. Panel C shows BAFF protein expression normalized to HSP90 levels and plotted relative to the scrambled miRNA control (miR-Ctr). Panel D shows relative miRNA expression in primary monocytes, quantified by reverse transcription and quantitative polymerase-chain-reaction (qPCR) analysis using U6 RNA as the endogenous control. Panel E shows quantification by qPCR of BAFF mRNA levels after miR-15a overexpression in THP1 cells, normalized to GAPDH mRNA levels. Soluble BAFF levels were measured by ELISA after transfection of the locked nucleic acid–anti–miR-15a oligonucleotide into THP1 cells, as shown in Panel F, and into primary monocytes, as shown in Panel G; results were normalized to locked nucleic acid–anti-miR control samples. Data in Panels C through G are the means of at least three independent experiments. I bars represent standard errors. Level of significance, calculated by t-test, is indicated.

Figure 4. Differential Regulation of BAFF Expression by miR-15a

Panel A shows a representation of luciferase reporter constructs. Wild-type (WT) BAFF 3′ UTR and variant (var) BAFF 3′ UTR sequences were cloned into the pmirGLO vector. Numbers 1 and 2 correspond to the miR-15a binding sites. Panel B shows the cotransfection of BAFF reporter constructs (WT or var) with miRNA-15a precursor or with a negative control into HeLa cells. “Mut. 1” and “Mut. 2” refer to the reporters mutated in the first and second miRNA-15a binding sites depicted in Panel A. Firefly luciferase levels were normalized to renilla luciferase activity for each sample, and all values were plotted relative to the WT construct. The graph is representative of four independent experiments. Level of significance, calculated by t-test, is indicated.

Figure 5. Effects of BAFF-var at the Levels of Transcript Type, Protein Production, and Cellular Response

Shown is a schematic depiction of the location of BAFF-var within TNFSF13B and its effects on the generation of mRNAs with different 3′ UTR lengths. The number and location of miRNA sites, production of soluble BAFF, endophenotypes, and autoimmunity risk are indicated. BAFF-var creates an alternative polyadenylation signal that generates a shorter 3′ UTR transcript lacking a miRNA binding site. In contrast to the wild-type allele, which is associated with long 3′ UTR only, BAFF-var leads to a mixed population of mRNAs with long and short 3′ UTRs, resulting in higher production of soluble BAFF. In turn, the increased levels of soluble BAFF lead to higher numbers of B cells and immunoglobulins, reduced levels of monocytes, and an increased risk of autoimmunity.