TWAS is a creative and valuable analysis method that can integrate genetic variation with gene expression to identify genes whose cis-regulated expression is associated with complex traits. It captures heterogeneous signals better than individual SNPs or cis-eQTLs and focuses prediction on the genetic component of expression that avoids confounding from environmental differences caused by the trait that may influence expression. What’s more, TWAS avoids tissue acquisition challenges that may pose the greatest hurdle for producing larger datasets. Thus, TWAS has been widely applied to yield mechanistic disease insights, yet the first time for RA in this study.
RA is a systemic disease and a variety of immunological events occur not only joints but also outside the joint at mucosal surfaces and primary lymphoid tissues, especially synovium. Thus, various types of tissues and cells will be attacked by the disease, including synovium, cartilage, bone, fibroblasts, adipocytes, macrophage, immune cells and so on. In this work, we conducted cell/tissue related TWAS for RA. TWAS identified total 674 genes with transcriptome-wide-significant associations with RA in four tissues/cells. CRIPAK, MUT, FOXRED1 and EBPL, which were collectively expressed in all the four tissues/cells, were novel genes associating with RA. Consistent with the result of TWAS, eighteen genes, ANXA5, AP4B1, ATIC, C12orf65, CMAH, PDHB, RUNX3, SBF1, SH2B3, STK38, TMEM43, XPNPEP1, KIAA1530, NUFIP2, PPP2R3C, RAB24, STX6, TLR5, have been reported differently expressed in peripheral blood mononuclear cells of RA patients.
Cysteine rich PAK1 inhibitor (CRIPAK) is an endogenous inhibitor of p21-activated protein kinase 1 (PAK1), which interacts with Pak1 through the N-terminal regulatory and inhibited enhancement of estrogen receptor transactivation. The decrease in gene expression of CRIPAK could act to promote accumulation of phosphorylated myosin light chain and its stimulation of actomyosin ATPase activity in laser-captured serotonin neurons from macaques treated with ovarian hormones [22]. There are few reports about the role and mechanism of CRIPAK in diseases, especially no reports in RA. However, PAK1 has been extensively studied. PAK1, a potential mediator of Rac1/Cdc42 signaling pathway, is involved in regulating the migration, invasion, proliferation, and inflammation of fibroblast-like synoviocytes from rheumatoid arthritis patients [23, 24]. These studies indirectly support the potential role of CRIPAK in rheumatoid arthritis.
Methylmalonyl-CoA mutase (MUT) encodes the mitochondrial enzyme methylmalonyl Coenzyme A mutase. In humans, the gene encoded enzyme catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA, while this enzyme may have different functions in other species. FAD dependent oxidoreductase domain containing 1 (FOXRED1) encoded protein that is localized to the mitochondria and whose function is involved in assembly, stability and/or correct functioning of complex I [25, 26]. Numerous processes involved in mitochondrial function are related to RA. For example, oxidative stress impairs energy metabolism in primary cells and synovial tissue of RA patients [27]; the interaction of abnormal cellular metabolism, mitochondrial dysfunction, hypoxia and the proinflammatory signaling pathways in synovial cells is contributed to synovial invasiveness of RA [28]; rare/low-frequency variants of the mitochondria respiratory chain-related proteins were aggregated RA patients [29]; etc. Meanwhile, The top three GO terms in MCODE 5 of PPI network were complex I biogenesis, NADH dehydrogenase complex assembly and mitochondrial respiratory chain complex I assembly. Therefor, we predict the two genes may play roles in the pathology of RA via affecting mitochondrial function.
EBPL is an emopamil-binding protein (EBP) like protein. EBP is a high-affinity binding protein for [H-3] emopamil and belongs to the family of so-called sigma receptors. Mutations disrupted EBP impair cholesterol biosynthesis and cause X-chromosomal dominant chondrodysplasia punctate. The EBPL mRNA was expressed ubiquitously and most abundant in liver, lung and kidney. However, EBPL has a yet-to-be-discovered function [30].
In the common RA associated gene list identified by TWAS, we should paid attention to ATIC, RUNX3 and TLR5. 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) encodes a bifunctional protein, which catalyzes the last two steps of the de novo purine biosynthetic pathway. ATIC plays crucial role in the mechanisms underlying methotrexate’s anti-inflammatory and antiproliferative effects. ATIC missense variant and gene polymorphism affects response to methotrexate treatment in RA patients [31–33]. Runt related transcription factor 3 (RUNX3) encodes a member of the runt domain-containing family of transcription factors and involves on T-cell development, T-cells polarization and T cell selection [34, 35]. RA is characterized by the presence of activated T lymphocytes. It is indicated that RUNX3 may play roles on the mechanisms of T cell activation in RA. Toll like receptor 5 (TLR5) encodes a member of TLR family that plays an essential role in pathogen recognition and innate immune responses activation. TLR5 agonist, flagellin, can promote monocyte infiltration and osteoclast maturation directly through myeloid TLR5 ligation and indirectly via TNF-alpha production from RA and mouse cells [36]. Angiogenesis in RA is fostered directly by TLR5 ligation and indirectly through interleukin-17 induction [37]. TLR5 is the bridge that interconnects formation of new blood vessels with maturation of joint osteoclasts, thereby accelerating the bone destruction process in RA [38]. There have other researches indicate that TLR5 is involved on RA inflammation, bone destruction and angiogenesis; thus, TLR5 is a critical element and target for RA mechanism.
With TWAS in this study, we found that the mRNA expression of some genes in human tissues/cells can be affected by SNPs, further associating with RA susceptibility. For example, four genes (CRIPAK, MUT, FOXRED1 and EBPL) in four distinct loci (rs3755963, rs6458697, rs602735 and rs1198329) were associated with RA susceptibility. We carried out a TWAS strategy to pinpoint RA associated genes; both genomics and transcriptomics were combined, and cis-heritable genes were explored and evaluated efficiently. The study provides a potential functional mechanism of how genetic variants on chromosome may increase RA susceptibility.