DCS is a common musculoskeletal disease that is caused by a combination of multiple risk factors, including genetic, psychological, biological and social factors. Although recent GWASs have successfully identified multiple DCS risk loci, the biological interpretations and functional understanding of these associations remain poorly understood. TWAS is a powerful approach to identify associated genes by combining the GWAS results and expression data. TWAS can identify genes whose cis-regulated expression is associated with complex traits. TWAS has been widely applied to multiple degenerative diseases, yet the first time for DCS in this study.
By integrating the TWAS analysis and RNA expression profiles of DCS, we identified apelin receptor (APLNR), the receptor for apelin receptor early endogenous ligand (APELA) and apelin (APLN) hormones coupled to G proteins that inhibit adenylate cyclase activity [17]. The Apelin/APLNR system participates in many basic activities of multiple cells with autocrine or paracrine and Apelin promotes cell proliferation, maturation, and induces mitochondrial autophagy [18]. Liu W et al. demonstrated Apelin/APLNR system plays a key role in intervertebral disc degeneration by reducing the degradation of the extramedullary matrix of nucleus pulposus, promoting the proliferation of nucleus pulposus cells, reducing the level of apoptosis and inflammation [18]. This may provide a new direction for the pathogenesis of the DCS.
Nerve growth factor (NGF) also identified by both TWAS analysis and RNA expression profiles of DCS. NGF has been shown to play an important role in many degenerative diseases such as osteoarthritis and Alzheimer's Disease [19, 20]. By perturbing NGF-TrkA signaling could strongly enhance human chondrocyte matrix calcification, Yangzi J et al. found a novel NGF-mediated chondrocyte calcification, which may provide new insights regarding the pathologic mechanism in early OA [19]. In summary, our results demonstrated the potential roles of NGF in DCS by TWAS analysis and RNA expression profiles. Although more experiments are needed to further confirm the biological mechanism of NGF.
RPS15A was identified in the weight of muscle-skeleton by TWAS analysis. Ribosomal protein S15A (RPS15A) is a member of the RPS family, maps to human chromosome 16p12.3 locus and encodes a highly conserved 40S ribosomal protein [21]. RPS15A has also exhibited various extra-ribosomal functions, such as cell division, tumorigenesis, and progression. For example, RPS15A encodes Ribosomal protein S15a. It is noteworthy that the NF-κB pathway is activated in the process of intervertebral disc degeneration [22]. To sum up, RPS15A could provide new clues for the pathogenesis of DCS by regulating the NF-κB pathway.
SELL (L-selectin) was identified in the weight of whole blood by TWAS analysis. L-selectin is a cell adhesion molecule consisting of a large, highly glycosylated, extracellular domain, a single spanning transmembrane domain and a small cytoplasmic tail [23]. L-selectin can express on most circulating leukocytes and contribute to adhesion, migration, and signal transduction in various diseases. In recent years some studies found L-selectin can regulate the expression of ICAM1(CD54) and the activation of ICAM1 is part of specific pathophysiology in intervertebral degeneration [24]. We have already introduced the pathogenesis of DCS is the degeneration of the intervertebral discs, so L-selectin may provide novel clues for understanding the genetic mechanism of DCS.
We used FUMA and Metascape tools to perform gene functional enrichment and annotation analysis. Several GO terms and KEGG pathways were detected to explore the functions of candidate genes and how they are distributed in DCS. Degradation of the extracellular matrix (R-HSA-1474228) was identified by both TWAS analysis and RNA expression profiles. The degeneration of intervertebral disc can cause the breakdown of the extracellular matrix [25]. By using the protein gel electrophoresis analysis, another study found gradually increased extracellular matrix fragmentation in the model of degenerative intervertebral discs, which also confirms our result [26]. In all, the degradation of the extracellular matrix pathway may be a powerful therapeutic prospect for DCS.
Mast cell degranulation (GO:0043303), mast cell activation involved in immune response (GO:0002279), and mast cell mediated immunity (GO:0002448) were identified as enriched GO terms for TWAS analysis. A study shows mast cells play a significant role in degenerative musculoskeletal diseases especially as they secrete several pro-inflammatory, neurovascular, and catabolic factors [27]. During aging and degeneration, mast cells are recruited into the intervertebral disc by up-regulation of the mast cell chemoattractant stem cell factor, and then mast cells can be activated through several different mechanisms, one of them likely being cellular interactions with intervertebral disc cells [28]. Once activated, inflammatory cytokines are released into the immediate microenvironment, inducing a catabolic/pro-inflammatory phenotype in the disc cells which then secrete factors that further promote mast cell activation [28]. This degenerative cycle can lead to a state of chronic inflammation in the intervertebral disc and result in DCS. Here we demonstrated that mast cells are present in the intervertebral disc, and they play potential roles in the pathogenesis of DCS.
Sphingolipid signaling pathway (ko04071) is significantly identified for DCS. Sphingolipid is an important part of the plasma membrane and implicated in a multitude of cellular processes [29]. Its function has been investigated in the muscle skeleton system. For example, sphingomyelin phosphodiesterase 3 (SMPD3), an important regulator of sphingolipid metabolism in the skeleton. SMPD3 cleaves sphingomyelin to generate ceramide and phosphocholine and the deficiency of SMPD3 can impair the mineralization in both cartilage and bone extracellular matrices leading to severe skeletal deformities [30]. In addition, Navone SE et al found sphingosine-1-phosphate is a metabolic product of cell membrane sphingolipids which are involved as a microenvironmental signal in the intervertebral disc degeneration process by inducing chemotaxis, migration, and secretion of pro-inflammatory cytokines [31]. We demonstrated sphingolipid plays potential roles in the pathogenesis of muscle skeleton diseases, further experiments are needed to prove our results.
The strength of our study is that we conducted TWAS analysis by using the latest GWAS summary data of DCS. On the one hand, TWAS analysis is a creative method that can predict the gene expression in DCS and avoid confounding from environmental differences caused by the trait that may influence expression. On the other hand, the large sample size of GWAS summary data ensures the accuracy of our results. In addition, we verified the candidate genes by comparing them with the RNA expression profiles. This study also has some limitations. The GWAS summary data are based on European ancestry and may not apply to other ancestry studies. Therefore, it should be cautious to apply our results to other populations. Further TWAS analysis on other populations are needed to prove our results. In addition, our results lacked sufficient mechanism-based experiments. More experiments are warranted to further confirm the biological rationality and clarify the biological mechanism of our study results.