Multiomics analysis of rheumatoid arthritis yields sequence variants that have large effects on risk of the seropositive subset

Abstract

Objectives: To find causal genes for rheumatoid arthritis (RA) and its seropositive (RF and/or ACPA positive) and seronegative subsets.

Methods: We performed a genome-wide association study (GWAS) of 31 313 RA cases (68% seropositive) and ~1 million controls from Northwestern Europe. We searched for causal genes outside the HLA-locus through effect on coding, mRNA expression in several tissues and/or levels of plasma proteins (SomaScan) and did network analysis (Qiagen).

Results: We found 25 sequence variants for RA overall, 33 for seropositive and 2 for seronegative RA, altogether 37 sequence variants at 34 non-HLA loci, of which 15 are novel. Genomic, transcriptomic and proteomic analysis of these yielded 25 causal genes in seropositive RA and additional two overall. Most encode proteins in the network of interferon-alpha/beta and IL-12/23 that signal through the JAK/STAT-pathway. Highlighting those with largest effect on seropositive RA, a rare missense variant in STAT4 (rs140675301-A) that is independent of reported non-coding STAT4-variants, increases the risk of seropositive RA 2.27-fold (p=2.1×10^-9), more than the rs2476601-A missense variant in PTPN22 (OR=1.59, p=1.3×10^-160). STAT4 rs140675301-A replaces hydrophilic glutamic acid with hydrophobic valine (Glu128Val) in a conserved, surface-exposed loop. A stop-mutation (rs76428106-C) in FLT3 increases seropositive RA risk (OR=1.35, p=6.6×10^-11). Independent missense variants in TYK2 (rs34536443-C, rs12720356-C, rs35018800-A, latter two novel) associate with decreased risk of seropositive RA (ORs=0.63-0.87, p=10^-9-10^-27) and decreased plasma levels of interferon-alpha/beta receptor 1 that signals through TYK2/JAK1/STAT4.

Conclusion: Sequence variants pointing to causal genes in the JAK/STAT pathway have largest effect on seropositive RA, while associations with seronegative RA remain scarce.

Keywords: autoantibodies; polymorphism, genetic; rheumatoid arthritis.

Figure 1

Effects of the lead sequence variants associated with seropositive RA (18 019 cases) compared with RA overall (31 313 cases, left graph) and seronegative RA (8515 cases, right graph). The x-axis and the y-axis show the logarithmic estimated ORs for the associations with the three phenotypes. All effects are shown for the RA risk increasing allele based on current meta-analysis of study population from six countries in Northwestern Europe (table 1). Error bars represent 95% CIs. The red line represents slope (SD) based on a simple linear regression through the origin using MAF (1-MAF) as weights. See further results in table 2 and online supplemental tables 2; 3.

Figure 2

Identification of sequence variants that associate with seropositive RA and the multiomics approaches used to recognise candidate causal genes. (A) schematic overview of the experimental approach used to identify sequence variants that associate with seropositive RA and their systematic annotation, applying multiomics approach to identify candidate causal genes, that is, based on whether lead variants or correlated variants (R² >0.8) affect protein coding (online supplemental tables 2–4), mRNA expression (cis-eQTL (online supplemental tables 5 and 6)) or levels of proteins in plasma (pQTL (online supplemental table 7)). (B) Out of 33 lead variant associations outside the HLA-locus (online supplemental table 3), 25 candidate causal genes were identified as listed, ranked by effect (OR). All effects are shown for the risk increasing allele based on GWAS in RA study populations from Northwestern Europe (table 1). Associations that are previously unreported in RA are marked with *. Grey boxes highlight where data point to a candidate causal gene. GWAS, genome-wide association study; RA, rheumatoid arthritis.

Figure 3

STAT4 missense variant rs140675301 is associated with seropositive RA (18 019 cases), is not correlated with previously reported variants at the locus and leads to an amino acid change in a highly conserved area of the protein. (A) Locus plot for the association of variants at the STAT4 locus with seropositive RA. The upper graph illustrates that the intronic variant rs4853458, that is the lead variant at the locus, is not correlated (r² <0.2) with the missense variant rs140675301, that is coloured in purple. The missense variant rs140675301 is only highly correlated (r² >0.8) with one variant, the intronic variant rs189948717 (coloured in red), that has less effect (seropositive RA: OR=1.81, p=3.69×10⁻⁶). Neither of these variants have previously been reported in any disease. The lower graph highlights that the lead variant at the locus (rs4853458, coloured in purple) has many correlated variants, coloured by degree of correlation (r²) with rs4853458. (B) Secondary structure of STAT4 (viewed from two angles) based on a structural model with STAT1 crystal structure (PDB code: 1yvl.1.A (Mao et al, Molecular Cell 2005;17:761–71) as template. Glu128Val (red) is located in a loop connecting the N-terminal domain (blue), important for tetramer formation of STATs and nuclear translocation, and the coiled coil domain (green), which provides a carbonised hydrophilic surface that binds to regulatory factors. α-Helices are drawn as cylinders. Invariant residues are marked with asterix. (C) multiple sequence alignment of the conserved STAT4 loop between the N-terminal domain (α8) and the coiled coil (α9) domain, performed with Clustal omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). RA, rheumatoid arthritis.

Figure 4

The JAK-STAT pathway. The figure and table shows which receptors, JAK and STAT subtypes certain cytokines bind to, highlighting proteins encoded by and/or affected by causal genes in seropositive RA, based on the multiomics analysis of sequence variants associated with risk of seropositive RA (shown in bold). Binding of a cytokine to its receptor activates the associated Janus kinases (JAK). The JAK in turn phosphorylates (P) the receptor, which provides a docking for signal transducers and activators of transcription (STATs) and other signalling molecules to bind to the receptor. STATs also become phosphorylated and translocate to the nucleus, where they regulate gene expression. *Protein targeted by drugs that are registered for RA. **Proteins targeted by drugs registered or in pipeline for other diseases. RA, rheumatoid arthritis.