Association between CILP and IL-1α polymorphisms and phenotype-dependent intervertebral disc degeneration susceptibility: A meta-analysis

Background: The relationship between CILP (1184T>C) and IL-1α(+889C/T) polymorphisms and intervertebral disc degeneration (IDD) have been explored in several studies but the results were conflicting. The aim of the study was to evaluate and synthesize the currently available data on the association between CILP (1184T>C) and IL-1α(+889C/T) polymorphisms and susceptibility of phenotype-dependent radiologic IDD (RIDD) and symptomatic intervertebral disk herniation (SIDH). Methods: A computerized literature search was in PubMed, Cochrane Library, Embase, China National Knowledge Infrastructure database, and Web of Science. The pooled results were presented as odds ratios (ORs) with 95% confidence intervals (CIs). Moreover, the false-positive report probability (FPRP) test and trial sequential analysis (TSA) were applied to estimate the significant results. Results: Our evidence demonstrated that IL-1α(+889C/T) was significant associated with RIDD (allele model: OR = 1.34, 95%CI 1.03–1.74, p = 0.029) and SIDH (allele model: OR = 1.28, 95% CI 1.03–1.60, p = 0.028). However, the results were not noteworthy under the FPRP test and TSA analysis. Additionally, CILP (1184T>C) polymorphism was significantly associated with RIDD with adequate evidence (allele model: OR = 1.27, 95% CI 1.09–1.48, p = 0.002) instead of SIDH. Conclusion: The current meta-analysis illustrated firm evidence that CILP (1184T>C) polymorphism was significantly associated with the susceptibility of RIDD. However, the significant associations between IL-1α(+889C/T) and RIDD and SIDH were less credible. Thus, more multi-center studies with diverse populations were required to verify the results.


Introduction
Low back pain (LBP), one of the most common muscleskeletal diseases, contributes to lower life quality and even disability. Over 80% of elderly people are afflicted with LBP to some extent during their lifetime (Andersson, 1999). Intervertebral disc degeneration (IDD) is regarded as a major cause of LBP which has an extremely complex pathogenesis (Chou et al., 2011). Multiple risk factors such as environmental, diet, and genetic factors were reported to accelerate the pathogenesis process of IDD (Battié and Videman, 2006).
As a chondroid structure, the nucleus pulposus (NP) contains a luxuriant extracellular matrix (ECM) such as proteoglycans and collagen. Accumulating evidence has highlighted the important role of ECM homeostasis play in the development of IDD. The imbalance of ECM homeostasis may cause diverse changes in disc including height reduction, dehydration, endplate alteration, etc. (Roberts et al., 2006). Overexpression of proinflammatory and catabolic phenotypes is the major reason for the imbalance of ECM homeostasis in the NP (Sivan et al., 2014;Molinos et al., 2015). Overexpression of IL-1 in the degenerative disk tissues inhibited extracellular matrix synthesis and improved extracellular matrix degradation (Phillips et al., 2013;Li et al., 2017). Hypersensitivity to IL-1α in disc cells is a significant motivator for degeneration (Maeda and Kokubun, 2000). Cartilage intermediate layer protein (CILP) is sufficiently distributed in intervertebral discs and has a vital function in regulating the metabolism of ECM. CILP inhibits the production of cartilage matrix protein via the TGF-β1signaling pathway (Seki et al., 2005).
Given the vital function of the IL-1α and CILP in ECM metabolism, numerous candidate polymorphism loci correlated to IDD predisposition were identified. Various studies indicated that CILP (1184T>C) and IL-1α(+889C/T) polymorphisms could be significantly correlated with the susceptibility of IDD (Solovieva et al., 2004;Seki et al., 2005). However, recent studies failed to detect the association and even obtain the opposite conclusion (Virtanen et al., 2007;Kelempisioti et al., 2011;Abdollahzade et al., 2018;. More importantly, the results of previous systematic reviews are unreliable due to overlooking the phenotypes of IDD (Wang W. et al., 2016;Wang Z. et al., 2016;Rajasekaran et al., 2016). Under the circumstances, we aim to provide more exhaustive evidence and rigorous criteria to explore the correlation of IL-1α and CILP polymorphisms with radiologic IDD (RIDD) as well as symptomatic intervertebral disk herniation (SIDH).

Search strategy for literature
We performed a literature retrieval of PubMed, China National Knowledge Infrastructure database, Web of Science, Cochrane Library database, and Embase by using the following terms ("LDD" or "IDD" or "lumbar disease" or "lumbar degeneration" or "Intervertebral disc degeneration" or "herniation") AND ("polymorphism" or "allele" or "variant") AND ("IL-1α" or "CILP" or "IL-1A"). The bibliography of associated studies was also examined for additional correlative studies.

Selection criteria
Studies included in the meta-analysis should be based on the following criteria: 1) case-control study; 2) the consequence was the investigation of the correlation between CILP (1184T>C) IL-1α(+889C/T) or rs1800587 or rs2073711and IDD or SIDH; 3) genotype frequencies were available to calculate an odds ratio (OR) with 95% confidence intervals (CIs); 4) grade criteria or phenotypes of IDD were described in detail.
The exclusion standards were listed as follows: 1) other types of study design; 2) genotype frequencies were not present in the articles; 3) studies contained overlapping data from previous research; 4) publication language was not English.

Methodological quality assessment
The quality evaluation of each study was performed independently by two investigators (JCL and XQS) following the Newcastle-Ottawa Scale (NOS). Dissension between reviewers would be discussed to reach a consensus. Based on the NOS standard, only studies with calculating scores >5 were deemed of high methodological quality.

Data extraction
Two reviewers independently extracted the following information on basis of a standard form: 1) Name of the First author 2) Publication information 3) Country 4) Ethnicity 5) Genotype frequencies 6) Numbers of cases and controls 7) Phenotypes of IDD.
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FIGURE 1
Flow diagram of the study identification and selection process.

Statistical analysis
Stata 14.0 was applied to conduct the statistical analysis. The correlation strength was assessed by ORs and corresponding 95% Cl in five genetic models, including allele, domain, recessive, heterozygote, and homozygote models. THE Cochrane Q test and the I2 statistic test were performed in each model to assess the heterogeneity between studies, and significant heterogeneity was considered when I 2 >50% or p < 0.05 (Higgins and Thompson, 2002). When significant heterogeneity existed, the pooled ORs would be estimated in the random-effects model; otherwise, the fixed-effects model (Mantel-Haenszel) was selected. Hardy-Weinberg equilibrium (HWE) was utilized to explore the genotype distribution of the population included in this research. Significant disequilibrium was defined by p < 0.05 in the chi-square test. Publication bias was quantitatively assessed through the Begg's and Egger's test and p < 0.05 represented a significant public bias. To evaluate the robustness of overall findings, sensitivity analysis was performed. Subgroup analysis was performed in RIDD to evaluate the genetic effect of IL-1α(+889C/T) and CILP (1184T>C)in different phenotypes of RIDD meanwhile stratified analysis was conducted in SIDH to test the genetic effect of CILP (1184T>C) in different ethnicity. False-positive report probability (FPRP) tests were performed to correct all significant pooled ORs. According to the calculating Excel spreadsheet (Wacholder et al., 2004), the values of FPRP were estimated at the prior probability of 0.01 to detect ORs of 1.5. FPRP >0.2 was thought to be a false significant result.

Trail sequential analysis
In order to control type I and type II errors and predict the required information size (RIS) in the research, the trial sequential analysis (TSA) was carried out in the TSA software (version 0.9; www.ctu.dk/tsa) with a 5% type I error and 20% type II error (Roshanov et al., 2017). The trial sequential monitoring boundaries were based on the O'Brien-Fleming-spending function. If the cumulative Z-curve exceeds the RIS or TSA line, it suggested the significant association is credible. Otherwise, further studies are required to confirm the result (Wetterslev et al., 2017). If the cumulative Z-curve passes the futility boundary, the association is a true negative result.

Study characteristics
The detailed literature search process was present in Figure 1. After duplicate removal and records screening, 25 articles were subjected to full-text screening. Conforming to the selection criteria, 13 studies were excluded with 12 literature remaining. Among them, six recruited studies evaluated the relationship between IL-1α rs1800587 and the risk of RIDD and SIDH in Caucasian, Mexica, and Chinese populations. Six studies consisting of Japanese, Chinese, Finland, and Indian populations were included in the analysis for CILP rs2073711. The characteristics and genotype frequencies of each study were shown in Table 1 meanwhile quality assessment was listed in Supplementary Table S1.

CILP (1184T>C) polymorphism and SIDH susceptibility
A total of 898 cases and 1111 controls (Seki et al., 2005;Virtanen et al., 2007; were recruited and the pooled results demonstrated no significant relationship in any models (Table 3) (Figure 4). All models suggested high heterogeneity. Subgroup analysis revealed a significant association between CILP (1184T>C) polymorphism and SIDH risk in the Asian population. Frontiers in Genetics frontiersin.org

Publication bias and sensitivity analysis
A ruling one out in turn strategy was adopted to estimate the effect of exclusion for each study on pooling ORs. The overall estimates did not alter under any genetic model when each individual study was omitted in turn, demonstrating the robustness of the findings (Figure 6). Begg's and Egger's tests indicated the results were free from

Significance test and TSA results
We performed the FPRP test to investigate the false-positive results.
The relationship between IL-1α(+889C/T) polymorphism and IDD and SIDH was not noteworthy (FPRP >0.2) in any model while the significant association between CILP (1184T>C) polymorphism and RIDD was considered a true positive result. The cumulative z-curve did not pass the TSA and RIS line for the association between IL-1α(+889C/T) polymorphism and IDD, indicating more studies are required (Figure 7). For IL-1α(+889C/T) polymorphism and SIDH, the cumulative z-curve crossed the futility line in the allele model and did not exceed the TSA and RIS boundary in the recessive and heterozygous model (Figure 8), which suggested the association turned out to be invalid in the allele model and more studies are required for the recessive and heterozygous model. Regarding CILP (1184T>C) polymorphism and RIDD, the cumulative z-curve reached the TSA line in the allele and recessive model (Figure 9). The evidence is adequate to confirm this significant association.

Discussion
AS a multifactorial disease, IDD is thought to be mainly caused by overloading and senescence (Rannou et al., 2004;Zhang et al., 2009). In a broad sense, IDD contains multiple potential phenotypes such as SIDH, asymptomatic disc herniation, disc bulge, disc height or signal change, and Modic change. SIDH characterized by sciatica represents a more serious phenotype of IDD because over 70% of individuals with disc herniations are asymptomatic (Boden et al., 1990). Recently, mounting evidence highlights the function of genetic factors in the initiation and progression of Frontiers in Genetics frontiersin.org IDD. TT genotype for IL-1α rs1800587 induced the overexpression of IL-1 (Dominici et al., 2002). The relationship between IL-1α rs1800587 polymorphism and IDD susceptibility has been investigated in numerous studies, but the results were inconsistent. Karppinen et al. and Chen et al. found that IL-1α polymorphism (rs1800587) was associated with the susceptibility of IDD in Finland and Chinese populations (Karppinen et al., 2009;Chen et al., 2018). However, the findings were not detected in Caucasian and Iranian populations (Paz Aparicio et al., 2011;Abdollahzade et al., 2018). CILP (1184T>C) polymorphism resulted in inhibition of the extracellular matrix proteins synthesis (Seki et al., 2005;Min et al., 2010). However, updated studies failed to detect the statistical significance in the Chinese and Finland populations (Virtanen et al., 2007). In addition, studies revealed the C allele as a protective factor in Finnish and Indian populations, which contrasts with studies in the Japanese population (Kelempisioti et al., 2011;Harshitha et al., 2018). Thus, updated research with new evidence was performed to obtain a constructive conclusion. In our meta-analysis, significant associations were detected between IL-1α rs1800587 polymorphism and IDD and SIDH but more evidence is required to confirm the conclusion. The subgroup analysis confirmed that IL-1α rs1800587 polymorphism was correlated with the risk of both RIDD (Solovieva grade) and SIDH but not RIDD (Modic change). Thus, we assumed that the change of expression due to rs1800587 polymorphism might mainly participate in the degeneration of NP instead of the endplate. Moreover, the homozygous model had the highest risk score. Compared with RIDD, a stronger association was detected between IL-1α(+889C/T) and SIDH. The results highlighted the importance of IL-1α polymorphism in the occurrence and progression of IDD.
Moreover, the pooled ORs and 95% CIs and TSA result demonstrated that CILP (1184T>C) was a credible susceptibility variant for RIDD (Pfirrmann grade) instead of SIDH, indicating the genetic effect involved in the initial stage of NP degeneration. Consistent with the results of IL-1α(+889C/T), the highest risk score was detected in the homozygous model. Furthermore, CILP rs2073711 is associated with SIDH in an ethnicity-dependent manner, which was mainly due to the difference in allele frequencies Frontiers in Genetics frontiersin.org for ethnicity. The C allele was found to be a protective factor for Caucasians but a risk factor for Asians. Additionally, the C allele is also a protective factor for knee arthritis in the European population (Valdes et al., 2004), which is consistent with this conclusion. To our best knowledge, this research is the first to comprehensively assess the genetic effect of IL-1α(+889C/T) and CILP (1184T>C) in different phenotypes of IDD. The significant associations were evaluated using the FPRP test and TSA. All eligible data was extracted to provide more convincing evidence. Our findings provided a novel perspective on the relationship between genetic polymorphisms of IL-1α and CILP and IDD phenotypedependent susceptibility and emphasized its role in IDD early screening and SIDH prevention. Population with high-risk genotypes should avoid longtime overloading and other risk factors leading to the development of IDD.
However, limitations should be claimed. First, despite a larger sample size in this study compared with previous studies, samples with other phenotypes of IDD are required to estimate the false-positive result. Second, this study did not estimate multiple confounding factors such as gender, occupation, and environmental factors, which might affect the effect estimates. Third, candidate gene studies have limitations in dealing with complex disorders and the results are often unrepeatable (Arango, 2017). A large-scale meta-analysis is a promising approach to solving the defect. Additionally, the potential value of polygenic risk score analysis for complex diseases has been highlighted (Arango, 2017). Even so, it is crucial to conduct individual studies because individual datasets are the basis of large-scale research and certain hypotheses may be better addressed in smaller studies, at least initially (Thompson et al., 2017).

Conclusion
The current meta-analysis illustrated adequate evidence that CILP (1184T>C) polymorphism was significantly Frontiers in Genetics frontiersin.org associated with the susceptibility of RIDD. However, the significant associations between IL-1α(+889C/T) and RIDD and SIDH were not noteworthy. Further research with larger sample size and various populations is required to verify the outcome.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Author contributions
JL participated in designing the experiments, data extraction, data analysis, and drafting the manuscript. YC and XS participated in the data extraction. HW designed and revised the article. All authors approved the final version.