This study aimed to offer new perspectives and methods for the treatment and prevention of dorsopathies. In our research, the identification of candidate genes and the establishment of causal inference were conducted via MR methods, while the mediation analysis was used to explore disease related gene-mediated methylation sites. As a result, our study revealed four genes as potential therapeutic targets for dorsopathies, including NLRC4, CGREF1, KHK and RNF212. Among these genes, elevated transcription levels of the NLRC4, CGREF1 and KHK were significantly associated with a decreased risk of dorsopathies, whereas a raised transcription level of RNF212 was significantly related a higher risk of dorsopathies.

In order to find the novel drug targets for dorsopathies, we employed an integrative analysis that combined colocalization with MR to assess causal genes for dorsopathies. Later, we conducted tests for horizontal pleiotropy to ensure the reliability of our MR genetics findings. In addition, TWAS analyses on NLRC4, CGREF1, KHK and RNF212 was conducted through Fusion and UTMOST software, which further illuminated the transcriptional associations of these genes with dorsopathies and bolstered the evidence supporting their potential causal role. Moreover, we conducted a GWAS analyses whose results were consistent with those from the SMR analyses, which reinforcing the evidence underpinning the potential causal association between these four genes with dorsopathies. Finally, we evaluated the influence of methylation sites on those genes expression and their links to dorsopathies. The results exhibited that the rise in cg23387401 was entirely mediated by RNF212, the elevations of cg26638505 and cg18948125 were primarily mediated by NLRC4, and the up-regulation of cg06112415 was fully mediated by CGREF1, leading to increased dorsopathies risk; while the rise of cg22740783 was totally mediated by CGREF1, resulting in decreased dorsopathies risk.

NOD-like receptors (NLRs) family caspase activation and recruitment domain-containing protein 4 (NLRC4) gene encodes NLRC4, which is mainly expressed in macrophages, neutrophils, dendritic cells and glial cells[26]. As a member of the caspase recruitment domain-containing NLR family, NLRC4 partners with NLR family of apoptosis inhibitory proteins (NAIP) to assemble inflammasome complexes, which termed NAIP/NLRC4 inflammasomes[27]. Inflammasomes function as intracellular multi-protein platforms that are activated by pathogen-associated molecular patterns or damage-associated molecular patterns, triggering innate immune reactions and inflammatory caspase-activation to defense pathogens and danger signals and maintain the homeostasis[27–29]. The activation of caspases results in the proteolytic activation of the pro-inflammatory cytokines interleukin-1β (IL-1β) and/or interleukin-18 (IL-18)[27]. Meanwhile, activated caspase-1 also cleaves gasdermin D, whose N-terminal fragments become inserted into cell membranes, thereby facilitating the release of more IL-1β and IL-18[28,29]. In particular, inflammasomes were classified into canonical inflammasomes, which activating caspase-1, and non-canonical inflammasomes, which primarily activate caspase-4 and/or caspase-5 in human cells[29].

Specifically, the NAIP/NLRC4 inflammasome serves as a as part of the innate immune response and senses a range of intracellular bacteria and bacterial components in the host cell cytosol, such as S. typhimurium, Legionella pneumophila, flagellin and components of the virulence-associated type III secretion apparatus, thereby to mediate host defense against bacterial pathogens[26,27,30]. NAIP proteins function as specific cytosolic receptors for a variety of bacterial protein ligands[31], while NLRC4 interacts directly with caspase-1 to induce cell death, such as the pyroptosis of macrophage, and to cause rapidly initiate inflammation and vascular fluid loss[26]. The localized effects of NAIP/NLRC4 inflammasome could defend against bacterial pathogens. However, the aberrant activity of this cluster also leads to multiple clinical manifestations, including macrophage activation syndrome, neonatal enterocolitis and autoimmune disorders[28,31], and promotes the process of some diseases, such as gliomas[29], premature rupture of membranes[26], ulcerative colitis[28], rheumatoid arthritis[30] and so on. Sim et al. found that non-canonical pathway molecules of NAIP/NLRC4 inflammasomes, including caspase-4, caspase-5, and N-cleaved GSDMD, were significantly increased with each glioma grade[29]. Additionally, Zhu et al. reported that NLRC4 was upregulated in the membranes of patients with premature rupture of fetal membranes and recruited more caspase-1, which promotes the process of rupture of fetal membranes by inducing apoptosis and degrading extracellular matrix[26]. The study of An et al. presented that the persistent activation of NAIP/NLRC4 inflammasome induces macrophage pyroptosis mediated by caspase1-dependent cleavage of GSDMD and releases proinflammatory cytokines including IL-1b and IL-18, facilitating the occurrence and progression of UC[28]. In addition, research conducted by Delgado-Arévalo et al. indicated that NLRC4 as an inflammasome sensor differentially upregulated in CD1c+ cDC from patients with rheumatoid arthritis, and this sensor seems to be nonredundantly involved in the detection of intracellular dsDNA[30]. Nevertheless, our research observed the significantly inhibitory effect of up-regulated NLRC4 expression on dorsopathies. Thus, further research on the roles and mechanisms of NLRC4 and inflammatsome in the progress of dorsopathies is needed.

Cell growth regulator with EF-hand domain 1 (CGREF1) gene encodes a novel secretory protein with 2 Ca2+-binding EF-hand domains which plays an important role in eukaryotic cellular signaling[32]. CGREF1 mRNA has high expressions in HCT116, H1299 and HepG2 cells while expressing at low levels in other cell lines including Raji, Jurkat, BT325, PC12[32]. Mechanistically, CGREF1 is regulated by p53[33], and the overexpression of CGREF1 significantly inhibits the transcriptional activity of AP-1, reduces the phosphorylation of ERK (extracellular signal-regulated kinases) and p38 MAPK (mitogen-activated protein kinases), and suppresses the proliferation of HEK293T and HCT116 cells[32]. Furthermore, CGREF1 can decrease the percent of G2/M and S phase and repress cell proliferation while overexpressing[32]. We found that the increased expression of CGREF1 is significantly related to the decreased risk of dorsopathies. In the research of Xiang et al., the colorectal cancer patients with higher expression of CGREF1 were found to have significantly better overall survival than patients with lower expression, and the role of CGREF1 in the prognosis of Early-onset colorectal cancer was reported[33]. Furthermore, Xie et al. observed that CGREF1 expression were high in the tissue of osteosarcoma patients while it were lowly expressed in normal tissue, and speculated that CGREF1 can predict drug resistance to osteosarcoma[34]. However, the biological function of CGREF1 is poorly explored, and further study for its mechanisms and roles in these diseases is warranted.

Ketohexokinase (KHK) is an enzyme that phosphorylates fructose to produce fructose-1-phosphate (F-1-P) at the first rate-limiting step in fructose metabolism[35,36]. The gene KHK encodes two isoforms, KHK-C and KHK-A. Considered as the primary enzyme in fructose metabolism, KHK-C is exclusively expressed in a few tissues, especially in the liver, and drives the aforementioned reaction rapidly, resulting in the accumulation of uric acid and transient depletion of intracellular phosphate and ATP[35–38]. In the contrary, KHK-A has a much weaker affinity and a higher Michaelis constant for fructose[38]. However, KHK-A can directly phosphorylate phosphoribosyl pyrophosphate synthetase 1 (PRPS1) by prevention of inhibitory nucleotide binding and facilitation of ATP binding[37]. As a result, KHK-A improves nucleic acid synthesis and promotes the G1/S phase transition in the cell cycle, accelerating cell proliferation[36,38].

Recent studies have highlighted the importance of KHK as the key mechanism stimulating the various adverse metabolic effects of fructose, such as impaired insulin sensitivity, hypertriglyceridemia, and oxidative stress[35]. In addition, fructose metabolism seems to provide cancer cells with the supplementary fuel required for proliferation and metastasis in colon cancer, and glioma[36,38]. Moreover, alternated expression of KHK is also related to several diseases. For instance, knocked down the expression of KHK dramatically reduced fructose-induced production of reactive oxidative species (ROS) in proximal tubular cells[35], indicating the association between KHK and ROS. Furthermore, KHK-A activates and upregulates PRPS1, which could lead to enhanced nucleic acid synthesis for tumourigenesis[38]. Kim et al. reported that most cancer cell lines predominantly expressed KHK-A rather than KHK-C, and KHK-A overexpression could augment cell invasion[36]. Moreover, in their study, upon fructose stimulation, KHK-A acted as a nuclear protein kinase and triggered Epithelial-mesenchymal transition in breast cancer, promoting breast cancer metastasis[36]. Besides, loss-of-function variants in KHK can cause essential fructosuria, an autosomal recessive disease characterized by intermittent appearance of fructose in the urine[39]. Yang et al. found the the overexpression of KHK-A in cell lines of oesophageal squamous cell carcinoma, which may finally promote the proliferation, tumourigenicity and motility of ESCC cells[38]. Similarly, KHK-A was considered to play an instrumental role in promoting de novo nucleic acid synthesis and hepatocellular carcinoma development[37]. In our study, the raised expression of KHK is found to be positively related to decreased risk of dorsopathies. In oder to understand the mechanism of changed expression level of KHK in dorsopathies, further research are needed.

Regulatory factor X2 (RFX2) gene is essential for maintaining normal spermatogenesis and involved in spermatogenesis impairment and male infertility in mice[40]. In particular, Ring finger protein 212 (RNF212) gene is important for crossing over and chiasma formation during meiosis[41]. Mouse Rnf212 has a central role in designating crossover sites and coupling chromosome synapsis to the formation of crossover-specific recombination complexes. In humans, RNF212 has been associated with variation in the genome-wide recombination rate[41]. The protein encoded by RNF212 has homology to two meiotic procrossover factors: Zip3 and ZHP-3[42]. It functions to couple chromosome synapsis to the formation of crossover-specific recombination complexes[43]. Moreover, with the symbolic RING-finger domains, RNF212 protein is a RING-family E3-ligase for Small ubiquitin-like protein (SUMO), and the latter plays an important role in assembly and disassembly of synaptonemal complex by regulating protein–protein inter action during meiosis[42,43,43]. In addition, RNF212 stabilizes association of a subset of MutSγ complexes with recombination sites[42,44]. MutSγ complex is a kind of meiosis-specific recombination factors, working as an attractive target for non-crossover/crossover differentiation[42]. It binds and stabilizes DNA strand-exchange intermediates to promote both homolog synapsis and crossingover[44]. In mammals, every pair of chromosomes obtains at least one crossover, while the majority of recombination sites yield non-crossovers. Non-crossovers are inferred to arise from the disassembly of D-loops and annealing of DNA double-strand breaks ends in a process termed synthesis dependent strand annealing[42]. Designation of crossovers involves the formation of metastable joint molecules and selective localization of SUMO-ligase RNF212 to a minority of recombination sites where it stabilizes pertinent factors, such as MutSγ[42,44]. Furthermore, this differential RNF212-dependent stabilization of key recombination proteins at precrossover sites is thought to be the basic feature of crossover/non-crossover differentiation[42]. It is suggested that RNF212-mediated SUMOylation may stabilizes the association of MutSγ with nascent crossover CO intermediates in a number of ways, such as promoting protein-protein interactions, altering ATP binding and hydrolysis (which modulate the binding and dissociation of MutSγ complexes) or antagonizing ubiquitin-dependent protein turnover[41,42].

Insufficient RNF212 accumulating at recombination sites can lead to crossing-over stochastically fails[43]. Fujiwara et al. reported that Rnf212 knock out (KO) in mice leads to infertility of male and female due to the loss of SPCs(spermatocytes) at post-anaphase stage. Moreover, crossing over is diminished by ≥90% in Rnf212−/− mice[43]. In particular, the Rnf212 KO spermatocytes lack chiasmata and exhibit depletion of spermatids and mature spermatozoa[41]. A nonsense mutation in the Rnf212 gene was discovered in repro57 mutant mice, and this mutant mice exhibited male infertility, arrest of spermatogenesis in meiosis, and defects in cytological markers of recombination and chiasma formation, which is similar to the Rnf212 KO phenotype[41]. In humans, the rate of crossing-over varies significantly between individuals, and higher maternal crossover rates have been associated with greater fecundity[42]. However, in the absence of RNF212, designation of crossover sites fails because no MutSγ complexes are stabilized beyond early pachynema[44]. Yu et al. detected that the frequencies of allele C and the genotype CC at the rs4045481 locus in RNF212 gene were significantly higher in patients with azoospermia in comparison with controls. Furthermore, they reported that homozygous of allele C (genotype CC) may decrease the activity of pre-mRNA due to the disappearance of the binding motifs of SRSF5, leading to the reduced expression of RNF212 and influencing normal spermatogenesis, consequently increasing ther risk of azoospermia[40]. Nevertheless, we found that the increased expression of RNF212 exhibited a positive correlation with dorsopathies. The influence of raised expression of RNF212 is poor classified, and further researches are required to illustrate the mechanism of increased RNF212 level and dorsopathies.

To summary, our study revealed the causal associations between the genetically predicted expression of four genes (NLRC4, CGREF1, KHK and RNF212) and dorsopathies risk, which offers new perspectives and strategies for the improved diagnosis, treatment and prevention of dorsopathies.

Based on our results, we hypothesized that increased expression level of NLRC4, CGREF1 and KHK are associated with decreased risk of dorsopathies, while the increased expression level of RNF212 has the opposite effect. Moreover, further study focusing on the mechanism of these expression changes in dorsopathies are needed, and future clinical studies should be conducted.