Genetically predicted C-reactive protein mediates the association between rheumatoid arthritis and atlantoaxial subluxation
基因预测的 C 反应蛋白介导类风湿性关节炎与寰枢椎脱位之间的关系

Abstract 摘要

Introduction 导言

Rheumatoid arthritis (RA) is a chronic inflammatory immune system disease characterized by synovitis and cartilage destruction, which mainly affects the synovial membrane, tendon sheaths and synovial bursae of the joints (1). It mainly manifests as clinical symptoms such as joint pain, stiffness, swelling, deformity, and dysfunction (2). Its global prevalence is approximately 1% and ranks 42nd among disabling diseases worldwide (3). As the global population ages, the prevalence continues to increase. Due to RA’s high mortality and morbidity rates, patients’ quality of life is lower, and the economic burden on society is greater. The National Audit Office (NAO) reports that RA costs the UK approximately £560 million a year in health care costs, not including the cost of sick leave and work-related disability (4).
类风湿性关节炎（RA）是一种以滑膜炎和软骨破坏为特征的慢性免疫系统炎症性疾病，主要影响关节滑膜、腱鞘和滑液囊（1）。它主要表现为关节疼痛、僵硬、肿胀、变形和功能障碍等临床症状（2）。其全球发病率约为 1%，在全球致残性疾病中排名第 42 位 ( 3)。随着全球人口的老龄化，发病率也在不断上升。由于 RA 的死亡率和发病率较高，患者的生活质量较低，对社会造成的经济负担也较大。英国国家审计署（NAO）报告称，RA 每年给英国带来约 5.6 亿英镑的医疗费用，这还不包括病假和与工作相关的残疾费用 ( 4)。

The active segment of the cervical vertebra is the basic functional unit of the cervical spine. It consists of two adjacent cervical vertebrae and their attached soft tissues and is the smallest functional unit of the cervical vertebra. Cervical instability refers to excessive or abnormal cervical spine movement that cannot maintain the normal position between the vertebral bodies under physiological loads (5). Atlantoaxial subluxation (AAS) in RA patients mostly involves the atlantoaxial joint, which may be caused by head and neck trauma, congenital diseases (bone dysplasia), autoimmune diseases (rheumatoid arthritis), etc. However, the exact reason is not yet clear. Observational studies have shown that upper cervical instability occurs in 29.6% of RA patients, of which atlantoaxial subluxation accounts for 24.6% (6). However, epidemiological studies may suffer from measurement error, uncontrolled confounding factors, and reverse causality. Ultimately, the results may be subject to various biases. Therefore, a design is needed to avoid or reduce some biases further to demonstrate the causal relationship between RA and AAS.
颈椎活动节段是颈椎的基本功能单元。它由两个相邻的颈椎及其附着的软组织组成，是颈椎最小的功能单位。颈椎不稳是指在生理负荷下，椎体之间不能保持正常位置的过度或异常的颈椎运动 ( 5)。RA 患者的寰枢关节脱位（AAS）大多涉及寰枢关节，可能由头颈部外伤、先天性疾病（骨发育不良）、自身免疫性疾病（类风湿性关节炎）等引起。然而，确切的原因尚不清楚。观察性研究显示，29.6% 的 RA 患者存在上颈椎不稳，其中寰枢椎脱位占 24.6% ( 6)。然而，流行病学研究可能存在测量误差、未控制的混杂因素和反向因果关系。最终，研究结果可能会出现各种偏差。因此，需要在设计上进一步避免或减少一些偏差，以证明 RA 与 AAS 之间的因果关系。

Moreover, potential pathways related to RA and AAS have not been investigated. Previous studies have provided evidence that C-reactive protein (CRP) is elevated in both RA and AAS (7, 8). Consequently, CRP might be a potential mediator between RA and AAS.
此外，与 RA 和 AAS 相关的潜在途径尚未得到研究。先前的研究已经提供了证据，证明C反应蛋白（CRP）在RA和AAS中都会升高（7，8）。因此，CRP可能是RA和AAS之间的潜在媒介。

Mendelian randomization (MR) is a potential causal inference method that uses genetic variation as an instrumental variable to obtain the effect of exposure factors on outcomes from observational data (9). MR can reduce the effects of nonmeasurement errors or confounding factors while avoiding reverse causality through Mendelian inheritance laws (9). Therefore, we aimed to (i) determine whether RA is causally related to AAS and (ii) assess the extent to which CRP mediates the effects of RA on AAS.
孟德尔随机化（Mendelian randomization，MR）是一种潜在的因果推断方法，它利用遗传变异作为工具变量，从观察数据中获取暴露因素对结果的影响（9）。MR 可以减少非测量误差或混杂因素的影响，同时通过孟德尔遗传规律避免反向因果关系（9）。因此，我们的目的是：(i) 确定 RA 是否与 AAS 存在因果关系；(ii) 评估 CRP 在多大程度上介导了 RA 对 AAS 的影响。

Methods 方法

Primary analysis 初步分析

Figure 1 shows a schematic summary of the analysis. We conducted a two-sample bidirectional MR to evaluate the mutual causality between RA and AAS ( Figure 1A ), which was designated as the total effect.
图 1 显示了分析概要示意图。我们进行了双样本双向 MR，以评估 RA 与 AAS 之间的相互因果关系（图 1A），并将其定为总效应。

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Figure 1 图 1

Diagrams illustrating associations examined in this study. (A) The total effect between rheumatoid arthritis(RA) and atlantoaxial subluxation(AAS). c is the total effect using genetically predicted RA as exposure and AAS as outcome. d is the total effect using genetically predicted AAS as exposure and RA as outcome. (B) The total effect was decomposed into: (i) indirect effect using a two-step approach (where a is the total effect of RA on CRP, and b is the effect of CRP on AAS) and the product method (a × b) and (ii) direct effect (c′ = c – a × b). Proportion mediated was the indirect effect divided by the total effect.
本研究中的关联示意图。(A) 类风湿性关节炎（RA）与寰枢关节脱位（AAS）之间的总效应。c 是以基因预测的 RA 为暴露，AAS 为结果的总效应。(B) 总效应分解为：(i) 采用两步法的间接效应（a 是 RA 对 CRP 的总效应，b 是 CRP 对 AAS 的效应）和乘积法（a×b）；(ii) 直接效应（c′=c-a×b）。中介比例是间接效应除以总效应。

Inverse variance weighting (IVW) uses meta-analysis to combine the Wald ratios of causal effects for each SNP (15, 19). Then, MR-Egger (20) and weighted-median (21) methods were used as a complement to IVW. Different methods adapted to different validity assumptions were applied to obtain MR estimates. The application of IVW is based on the premise that all SNPs are valid instrumental variables. Therefore, this method can obtain accurate estimation results. MR-Egger assesses directional pleiotropy for instrumental variables, where the intercept can be interpreted as an estimate of the average pleiotropy of genetic variation. The weighted median has the advantage of maintaining higher precision (smaller standard deviation) compared to the MR-Egger analysis. In the presence of horizontal pleiotropy, the weighted median provides a consistent estimate even if 50% of the genetic variants are invalid IVs (22).
逆方差加权法（IVW）利用荟萃分析将每个 SNP 的因果效应 Wald 比率结合起来 ( 15, 19)。随后，MR-Egger（20）和加权中值（21）方法被用作 IVW 的补充。不同的方法适用于不同的有效性假设，以获得 MR 估计值。应用 IVW 的前提是所有 SNP 都是有效的工具变量。因此，这种方法可以获得准确的估计结果。MR-Egger评估的是工具变量的方向性褶积，截距可解释为遗传变异平均褶积的估计值。与 MR-Egger 分析法相比，加权中位数法的优点是精度更高（标准偏差更小）。在存在水平多向性的情况下，即使 50%的遗传变异是无效的 IV，加权中值也能提供一致的估计值 ( 22)。

Results 成果

Association of RA with AAS
RA 与 AAS 的关系

After removing palindromic and ambiguous SNPs, SNPs without proxy and SNPs with wrong causal directions identified by MR Steiger filtering, there were 80 SNPs in RA and 4 SNPs in AAS as instrumental variables ( Supplementary Tables S3, S4 ). Since AAS did not reach the level of gene-wide significance for SNPs, SNPs with less than genome-wide significance (P < 5 × 10^-6) were used as instrumental variables. The variance explained by and the F-statistic for SNPs instrumenting RA exposure were 5.8% and 45, respectively. Our study provides 100% power to detect the causal effect of RA on AAS risk.
在剔除了宫位和模糊 SNP、无代理的 SNP 和 MR Steiger 过滤发现的因果方向错误的 SNP 后，RA 中有 80 个 SNP，AAS 中有 4 个 SNP 作为工具变量（补充表 S3、S4）。由于 AAS 中的 SNPs 未达到全基因组显著性水平，因此将小于全基因组显著性（P < 5 × 10 ^-6 ）的 SNPs 作为工具变量。RA暴露SNPs的解释方差和F统计量分别为5.8%和45。我们的研究提供了100%的功率来检测RA对AAS风险的因果效应。

IVW, MR-Egger, and weighted median regression were used to estimate the causal relationship between genetically predicted RA and AAS ( Figures 2 , 3 ). Across all three MR methods, there was broad and consistent support for the positive association of RA with AAS (IVW odds ratio [OR] per SD increase in RA = 1.61 [95% CI, 1.36-1.90], P < 0.0001; MR-Egger OR per SD increase in RA = 1.66 [95% CI, 1.30-2.13], P < 0.001; weighted median OR per SD increase in RA = 1.80 [95% CI, 1.39-2.34], P < 0.0001). However, the results of our MR analysis showed no reverse causality for genetically predicted RA on AAS (i.e., no causality for genetically predicted AAS on RA.). The OR was 1.001 [95% CI, 0.97-1.03; p = 0.87] by using the IVW method. The results are shown in Figure 3 .
采用IVW、MR-Egger和加权中位数回归估算遗传预测RA与AAS之间的因果关系（图2、图3）。在所有三种MR方法中，RA与AAS的正相关性得到了广泛而一致的支持（RA每增加一个SD的IVW比值比[OR]=1.61 [95% CI, 1.36-1.90]，P < 0.0001；RA每增加一个SD的MR-Egger比值比[OR]=1.66 [95% CI, 1.30-2.13]，P < 0.001；RA每增加一个SD的加权中位数比值比[OR]=1.80 [95% CI, 1.39-2.34]，P < 0.0001）。然而，我们的 MR 分析结果显示，遗传预测的 RA 与 AAS 没有反向因果关系（即遗传预测的 AAS 与 RA 没有因果关系）。使用 IVW 方法得出的 OR 值为 1.001 [95% CI, 0.97-1.03; p = 0.87]。结果如图 3 所示。

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Figure 2 图 2

Forest plot to visualize causal effect of each single SNP on total AAS risk.
森林图显示每个单一 SNP 对 AAS 总风险的因果效应。

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Figure 3 图 3

Forest plot to visualize the causal effects of CRP with RA and AAS.
显示 CRP 与 RA 和 AAS 因果关系的森林图。

Proportion of the association between RA and AAS mediated by CRP
由 CRP 介导的 RA 与 AAS 之间关联的比例

We analyzed CRP as a mediator of the pathway from RA to AAS. We found that RA was associated with increased CRP, which in turn was associated with an increased risk of AAS. As shown in Figure 4 , our study showed that CRP accounted for 3.7% of the increased risk of AAS associated with RA (proportion mediated: 3.7%; 95% CI = 0.1%−7.3%).
我们分析了 CRP 作为从 RA 到 AAS 途径的介导因素。我们发现，RA 与 CRP 升高有关，而 CRP 升高又与 AAS 风险升高有关。如图 4 所示，我们的研究表明，CRP 占与 RA 相关的 AAS 风险增加的 3.7%（介导比例：3.7%；95% CI = 0.1%-7.3%）。

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Figure 4 图 4

Schematic diagram of the CRP mediation effect.
CRP 中介效应示意图。

Discussion 讨论

Recent studies (6, 29, 30) have examined the relationship between RA and AAS. However, the current evidence is limited to observational studies, and the results may be influenced by confounding factors. Our study aimed to illustrate the causal effects between RA and AAS. We used MR analysis to investigate the association between RA and AAS based on existing GWAS and to demonstrate whether the causal relationship between them is mediated through CRP. Our results suggested that genetically predicted RA was associated with an increased risk of AAS (61% increased risk of AAS for every 1 SD increase in RA), and 3.7% of this effect was mediated through CRP.
最近的研究（6、29、30）探讨了 RA 与 AAS 之间的关系。然而，目前的证据仅限于观察性研究，研究结果可能会受到干扰因素的影响。我们的研究旨在说明 RA 与 AAS 之间的因果关系。我们基于现有的全球基因组研究，使用磁共振分析来研究 RA 和 AAS 之间的关联，并证明它们之间的因果关系是否通过 CRP 介导。我们的研究结果表明，遗传预测的 RA 与 AAS 风险的增加有关（RA 每增加 1 SD，AAS 风险增加 61%），而这种效应的 3.7% 是通过 CRP 介导的。

To date, we are the first to investigate the causal relationship between RA and the risk of instability in the upper cervical spine by MR methods, while also demonstrating CRP as their mediator. Our findings are consistent with previous findings from traditional observational designs. Yurube et al. (31) showed that atlantoaxial instability occurred in 43.6% of RA patients, with atlantoaxial subluxation being the most common (at least 32.1%) through a prospective follow-up study of RA patients with no initial cervical involvement for at least 5 years. Similarly, in a retrospective study of 240 RA patients, Kotecki et al. (32) showed that the incidence of cervical spine involvement in RA patients was 75%, the most common lesion was anterior atlantoaxial subluxation (approximately 58%), and C-reactive protein levels increased (OR, 19.0; 95% CI, 7.0–32.0; P = 0.016). However, both studies were of observational design. First, they had low response rates between the two groups, and second, their results were more influenced by reverse causality or other potential mixed effects than MR analyses.
迄今为止，我们首次通过磁共振方法研究了 RA 与上颈椎不稳定风险之间的因果关系，同时还证明 CRP 是其中介因素。我们的研究结果与之前传统观察设计的结果一致。Yurube 等人（31）对至少 5 年未受累颈椎的 RA 患者进行了前瞻性随访研究，结果显示 43.6% 的 RA 患者存在寰枢椎不稳，其中寰枢椎脱位最为常见（至少 32.1%）。同样，Kotecki 等人 ( 32) 对 240 名 RA 患者进行的回顾性研究显示，RA 患者颈椎受累的发生率为 75%，最常见的病变是寰枢关节前脱位（约 58%），且 C 反应蛋白水平升高（OR，19.0；95% CI，7.0-32.0；P = 0.016）。然而，这两项研究都是观察性设计。首先，两组之间的应答率较低，其次，与 MR 分析相比，其结果受反向因果关系或其他潜在混合效应的影响更大。

Atlantoaxial instability refers to the structural changes and dysfunction of the atlanto-occipital joint and atlantoaxial joint between the atlas, the axis and the base of the skull due to various reasons (e.g., deformity, trauma, degeneration, tumor and infection), which lead to excessive or abnormal activity or abnormal position under physiological load. Atlantoaxial joint instability and dislocation are rare in normal adults and are mostly secondary to trauma and disease. Cervical instability can be further divided into atlantoaxial subluxation (AAS), vertical subluxation (VS) and subaxial subluxation (SAS). AAS is the most common, followed by VS, and SAS is less common (6). VS usually occurs after AAS. VS is considered a serious condition in RA patients because it can lead to sudden death (33). Synovitis is the initial link of rheumatoid arthritis, and it is also the basic pathological change. The characteristics of multiple synovial sacs of the atlantoaxial joint provide conditions for its involvement. At the same time, synovial tissue macrophages produce tumor necrosis factor to promote the inflammatory response of the atlantoaxial joint (34). Sorimachi et al. (35) have suggested that synovitis invades the atlantoaxial joint in three stages. First, the medial and lateral atlantoaxial joints are invaded, the joint capsule is destroyed, and the joint capsule is swollen and exuded; then, the synovium begins to proliferate, and the ligaments are edematous and destroyed, after which finally, it erodes hyaline cartilage and penetrates into subchondral bone to produce bone tissue destruction. In addition, the stability of the atlantoaxial joint mainly depends on the maintenance of the transverse ligament and other ligaments, which are characterized by high stiffness and insufficient willfulness. Another characteristic product of RA is pannus, which not only blocks the bone from obtaining nutrition through the synovium but also grows to the cartilage surface in the joint cavity, produces adhesions, and locally releases more inflammatory factors, proteolytic enzymes, etc. (36). When inflammation involves the transverse ligament, it not only destroys the fibrous structure and relaxes the ligament but also erodes the odontoid process and causes erosion and rupture near the attachment point, which finally leads to the instability of the atlantoaxial joint (37).
寰枢关节不稳定是指由于各种原因（如畸形、外伤、退行性变、肿瘤和感染等），导致寰枢关节和寰枢轴与颅底之间的寰枢关节在生理负荷下出现过度或异常活动或位置异常的结构变化和功能障碍。寰枢关节失稳和脱位在正常成人中很少见，大多是继发于创伤和疾病。颈椎不稳可进一步分为寰枢关节脱位（AAS）、垂直脱位（VS）和轴下脱位（SAS）。寰枢椎脱位最常见，垂直脱位次之，轴下脱位较少见 ( 6)。VS通常发生在AAS之后。VS在RA患者中被认为是一种严重的疾病，因为它可能导致猝死（33）。滑膜炎是类风湿性关节炎的最初环节，也是基本病理变化。寰枢关节多滑膜囊的特点为其受累提供了条件。同时，滑膜组织巨噬细胞产生肿瘤坏死因子，促进寰枢关节的炎症反应（34）。Sorimachi 等人（35）认为滑膜炎入侵寰枢关节分为三个阶段。首先，内侧和外侧寰枢关节受到侵袭，关节囊受到破坏，关节囊肿胀并渗出；然后，滑膜开始增生，韧带水肿并受到破坏；最后，滑膜炎侵蚀透明软骨并渗入软骨下骨，产生骨组织破坏。此外，寰枢关节的稳定性主要依赖于横韧带和其他韧带的维持，而这些韧带的特点是僵硬度高，韧性不足。RA 的另一个特征性产物是骨赘，骨赘不仅阻碍骨通过滑膜获得营养，还会向关节腔内的软骨表面生长，产生粘连，并在局部释放更多的炎症因子、蛋白水解酶等（36）。当炎症累及横韧带时，不仅会破坏纤维结构，使韧带松弛，还会侵蚀骨突，造成附着点附近的侵蚀和断裂，最终导致寰枢关节失稳（37）。

CRP, a member of the pentraxin family of proteins, consists of five 23 kDa subunits that can be increased 1,000-fold or more during infection, inflammation and tissue damage. Although hepatocytes are the main source of CRP, other cells, such as monocytes and lymphocytes, also produce CRP (38). Fang et al. (39) suggested that synovial tissue from RA patients also produces CRP. Therefore, one of the reasons for the increased CRP concentrations in synovial fluid and serum CRP levels in RA patients may be the local production of CRP in inflammatory synovial tissue (40). On the one hand, the interaction of CRP with Fcγ receptor I and FcγRIIA promotes the production of proinflammatory cytokines, leading to an amplification loop of the inflammatory response; on the other hand, CRP initiates bone destruction by inducing the receptor activator of nuclear factor-κB ligand protein and directly stimulating osteoclast generation, thus causing a vicious cycle between inflammation and bone destruction in RA (41). Therefore, CRP contributes to atlantoaxial joint instability by mediating synovial inflammation and bone destruction in RA.
CRP 是五联蛋白家族的一员，由五个 23 kDa 的亚基组成，在感染、炎症和组织损伤时可增加 1000 倍或更多。虽然肝细胞是 CRP 的主要来源，但单核细胞和淋巴细胞等其他细胞也会产生 CRP ( 38)。Fang 等人（39）认为，RA 患者的滑膜组织也会产生 CRP。因此，RA 患者滑液中 CRP 浓度和血清 CRP 水平升高的原因之一可能是炎性滑膜组织局部产生了 CRP（40）。一方面，CRP 与 Fcγ 受体 I 和 FcγRIIA 相互作用，促进促炎细胞因子的产生，导致炎症反应的放大循环；另一方面，CRP 通过诱导核因子-κB 配体蛋白受体激活剂，直接刺激破骨细胞的生成，启动骨质破坏，从而造成 RA 炎症与骨质破坏之间的恶性循环（41）。因此，CRP 通过介导 RA 的滑膜炎症和骨质破坏，导致寰枢关节不稳定。

Our findings also suggest that RA may increase the risk of atlantoaxial subluxation through other important mediators. Zhang et al. (6) showed that low hemoglobin levels may be associated with atlantoaxial instability in RA. This may be because low hemoglobin levels are partly a chronic inflammatory manifestation of the disease and are thought to be associated with joint damage in RA (42), thus showing a correlation with cervical instability. In addition, CD5⁺ B cells in RA patients can produce IgG with the help of T lymphocytes, and rheumatoid factor and IgG form immune complexes deposited in the synovium, which are blocked during the clearance process, resulting in bone destruction and fusion.
我们的研究结果还表明，RA 可能会通过其他重要介质增加寰枢关节脱位的风险。Zhang等人（6）的研究表明，低血红蛋白水平可能与RA患者的寰枢椎不稳有关。这可能是因为低血红蛋白水平部分是该病的慢性炎症表现，被认为与 RA 的关节损伤有关（42），从而显示出与颈椎不稳的相关性。此外，CD5 ⁺ RA患者的B细胞可在T淋巴细胞的帮助下产生IgG，类风湿因子和IgG形成免疫复合物沉积在滑膜中，在清除过程中被阻断，导致骨质破坏和融合。

This study has several limitations. First, our analysis was performed using the European population, which limits its prevalence (43). Second, the smaller number of cases in AAS is in the GWAS dataset of AAS, and it is hoped that larger GWAS data will be available for validation in the future. Third, even if we took steps to identify and eliminate outlier variants, we cannot exclude the possibility that horizontal pleiotropy influenced our results. Fourth, we used summary-level statistics in our study, not individual-level data. Therefore, we cannot further explore causal links between subgroups such as females and males. Fifth, our study demonstrates that genetic prediction of rheumatoid arthritis mediated by C-reactive protein is 3.7%, which is very low. Thus, more studies are needed to quantify other mediators.
这项研究有几个局限性。首先，我们的分析是利用欧洲人群进行的，这限制了其流行率( 43)。其次，AAS 的 GWAS 数据集中的病例较少，希望将来能有更大的 GWAS 数据进行验证。第三，即使我们采取了措施来识别和剔除离群变异，我们也不能排除水平褶积影响我们结果的可能性。第四，我们在研究中使用的是汇总层面的统计数据，而不是个体层面的数据。因此，我们无法进一步探讨女性和男性等亚群之间的因果联系。第五，我们的研究表明，C 反应蛋白介导的类风湿关节炎遗传预测率为 3.7%，这是非常低的。因此，还需要更多的研究来量化其他介导因素。

Conclusion 结论

In conclusion, our study identified a causal relationship between RA and atlantoaxial subluxation, with a small proportion of the effect mediated by CRP, but a majority of the effect of RA on atlantoaxial subluxation remains unclear. Further research is needed on additional risk factors as potential mediators. In clinical practice, lesions of the upper cervical spine in RA patients need to be given more attention.
总之，我们的研究确定了 RA 与寰枢关节脱位之间的因果关系，其中小部分影响是由 CRP 介导的，但 RA 对寰枢关节脱位的大部分影响仍不清楚。需要进一步研究作为潜在介导因素的其他风险因素。在临床实践中，RA 患者上颈椎的病变需要得到更多关注。

Data availability statement
数据可用性声明

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.
该研究中的原始贡献载于文章/补充材料中。如需进一步咨询，请联系通讯作者。

Author contributions 作者供稿

All authors designed this study. JT, WW, and XX performed the catalog and literature search and data extraction with suggestions and help from JY. JY, JZ, and XX performed the statistical analyses. All authors contributed to the data interpretation and manuscript writing. All authors contributed to the article and approved the submitted version.
所有作者均为本研究的设计者。JT、WW和XX在JY的建议和帮助下进行了目录和文献检索以及数据提取。JY、JZ和XX进行了统计分析。所有作者都参与了数据解读和稿件撰写。所有作者都参与了文章的撰写，并批准了提交的版本。

Acknowledgments 致谢

References 参考资料