The discovery that a signature of coordinately overexpressed IFN-inducible genes is prominent in a substantial fraction of lupus patients has fueled interest in the IFN pathway as a potential target for therapeutic intervention. This molecular signature is correlated with increased disease activity and specific clinical manifestations such as low complement levels, high levels of anti-dsDNA autoantibodies, higher sedimentation rates, and increased renal complications (35
). The goal of this study was to identify the genetic variation that leads to the dysregulation of the IFN-related pathways and genes, including IFN-inducible genes and their direct regulators. This essentially Bayesian approach of selecting candidate genes based on prior knowledge serves to increase the reliability and likelihood of finding genes truly associated with disease (36
). To our knowledge, this study represents the most comprehensive IFN pathway-based genetic analysis to date. Using independent cohorts for discovery and replication, we have evaluated a total of 1,754 genes. Eight genes were confirmed as associated with SLE.
The overall most significant associations of SLE with IFN-related genes were observed with SNPs located in regions that have been previously reported and firmly established as risk factors (HLA
, STAT4, and TNFAIP3
). Two additional genes with previous evidence for association include the ubiquitin enzyme UBE2L3
) and IRF8
). The majority of additional loci we report represent novel genetic associations with SLE, underscoring the power of this candidate pathway approach.
The strongest novel genetic effect locates in the CD44 gene. CD44 is an integral cell membrane glycoprotein important for cell-cell interactions. As a key regulator of many molecules, including IFNG and LCK, CD44 has important roles in lymphocyte activation, recirculation and homing, hematopoiesis, tumor metastasis and inflammation. It has also been shown that T cells from SLE patients display an increased and abnormal distribution of CD44 (39
). Kaufman et al. (40
) genotyped 4 SNPs within CD44 in 13 African-American families and found no evidence of association. However, given the number of SNPs and families tested, this study was likely underpowered to detect the effect that we report.
Another significant association signal was identified in the heparin-binding pleiotrophin (PTN) gene. PTN is a developmentally regulated cytokine with fibrinolytic, anti-apoptotic, mitogenic, transforming, angiogenic, and chemotactic activities (41
). PTN has recently been shown to induce the expression of inflammatory cytokines including TNF-α, IL-1β and IL-6 in quiescent human peripheral blood mononuclear cells (PBMCs) (42
). Other than being a regulator of several of the IFN-signature genes, no link between PTN and SLE has been established to date. Nevertheless, its expression is upregulated in experimental autoimmune encephalomyelitis (EAE) (43
We have also confirmed an association with DNAJ
(also known as heat shock protein
(Hsp) 40) homolog, subfamily A, member 1
gene. DnaJ is a heat shock protein that assists the chaperone Hsp70 in protein translation, folding, unfolding, translocation, and degradation. As a stress response protein, DnaJ is involved in repair and removal of damaged proteins, and is therefore important for maintaining cell viability. Hsps are potential targets of an autoimmune response and have been implicated in the induction and propagation of autoimmunity in several diseases, including rheumatoid arthritis and type 1 diabetes (44
). Experimental evidence suggests that improper protein folding may promote autoimmunity (45
). Thus, DnaJ and related proteins have potentially important but poorly understood roles in autoimmune diseases.
Karyopherin alpha 1 (KPNA1) binds RAG1, a lymphoid-specific recombinase essential for V(D)J recombination, and influences its sub-nuclear localization, hence controlling the generation of immunoglobulins and T cell receptors (46
). It has also been shown to bind activated STAT1 and IRF5 proteins and transport them to the nucleus (47
Even though our objective was to perform a comprehensive analysis of all IFN related genes, we cannot exclude the possibility that strong associations were missed due to the genomic coverage of the genotyping arrays or the a priori selection of specific genes found in the literature to be IFN-related or interactors. We, therefore, could have missed some unknown interactions. In addition, the dysregulation of IFN pathway genes is not a uniform feature across all lupus patients, and as such we would expect to detect moderate genetic effects that affect probably half of our cases. Nonetheless, replication of the novel effects we have identified in a second cohort, correction for multiple comparisons, and confirmation in a third cohort increase our confidence in the robustness of these associations.
In addition to the conventional statistical approaches, we used a data mining approach, Alternating Decision Trees (ADTrees), to try to corroborate the association results and identify novel variants that best discriminate cases vs. controls, as well as confirm and unveil potential interactions between genetic variants. The ADTrees validate the association results, and replication analyses are underway to confirm the uncovered two-locus interactions.
In summary, we have identified multiple IFN pathway-related genes that show confirmatory evidence for association with SLE. For the majority of loci, this is the first report of a genetic association with SLE with confirmation. Taken together, these new data expand the growing list of genes that show association with SLE, and emphasize the genetic contribution of dysregulated IFN pathways. Understanding how these genetic factors might contribute to pathogenesis should ultimately lead to important opportunities for developing therapeutic targets to control the active IFN signature seen in SLE patients.