Interactions between the MMP‐3 gene rs591058 polymorphism and occupational risk factors contribute to the increased risk for lumbar disk herniation: A case‐control study

Abstract Objective Lumbar disk herniation (LDH) is a complex condition based on lumbar disk degeneration (LDD). Previous studies have shown that genetic factors are highly associated with the severity and risk for LDH. This case‐control study was aimed to evaluate the association between the matrix metalloproteinase (MMP)‐3 gene rs591058 C/T polymorphism and LDH risk in a southern Chinese population. Methods A total of 231 LDH patients and 312 healthy controls were recruited in this study. Genotyping was analyzed using a standard polymerase chain reaction and restriction fragment length polymorphism (PCR‐RFLP). Results It was observed that TT genotype or T allele carriers of the MMP‐3 gene rs591058 C/T polymorphism was more likely associated with an increased risk for LDH. Subgroup analyses showed the following characteristics increased the risk for LDH: female sex; cigarette smoking; and alcohol consumption. Furthermore, individuals with high whole body vibration, bending/twisting, and lifting were associated with an increased risk for LDH. Conclusion Taken together, these data indicated that the MMP‐3 gene rs591058 C/T polymorphism was associated with an increased risk for LDH. The MMP‐3 gene rs591058 C/T polymorphism might serve as a clinical indicator and marker for LDH risk in the Chinese population.

gender, cigarette smoking, occupation, trauma, and obesity, may also play a role in the etiology of LDH. 4,5 Previous studies have demonstrated that various genetic modifications are involved in LDH progression. The involvement of genetic factors in the etiology LDH has become evident in past decades and likely accounts for up to 70% of an individual's risk. Ala-Kokko 6 identified various genes, including collagen IX, vitamin D receptor, and matrix metalloproteinase (MMP)-3, that are associated with LDH.
The intervertebral disk has a unique outer structure that consists of annulus fibrosus and an elastic inner nucleus pulposus, endowing the intervertebral disk with the capacity to distribute power and resist compression. 7 One of the major steps of intervertebral disk degeneration is matrix deterioration. 8 Currently, the mechanism underlying extracellular matrix deterioration, especially the role of gene expression, has become the emphasis of most studies. MMP-3 is remarkably upregulated and mediates disk degeneration by degrading matrix proteoglycans and collagens during disk degeneration, which indicates the influence on structural modifications of the disk. 9,10 MMP-3 gene is located on chromosome 11:102840607.
Rs591058 C/T polymorphism is a variant in intron region of MMP-3 gene. The data from 1000 Genomes show the minor allele frequency of rs591058 C/T is 0.355232. The relationship between MMP-3 gene rs591058 C/T polymorphism and some disease risk was explored before. Xu et al 11 found that rs591058 C/T polymorphism had no statistically significant association with frozen shoulder risk.
Foster et al 12 showed that rs591058 C/T polymorphism was associated with the risk of tendon pathology. Raleigh et al 13 observed that MMP-3 gene rs591058 C/T polymorphism was associated with Achilles tendinopathy, which possibly interacted with the COL5A1 gene. Recently, an Indian study showed that the MMP-3 gene rs591058 C/T polymorphism is associated with deceased risk for LDH. 14 No Chinese studies have addressed this issue up to date.
Thus, we conducted this hospital-based case-control study to assess the relationship between the MMP-3 gene rs591058 polymorphism and LDH risk in a Chinese Han population.

| Subjects
This case-control study enrolled 231 LDH patients and 312 sex-and age-matched controls from the First Affiliated Hospital of Nanjing Medical University between 2012 and 2018. All MRI images were evaluated by experienced radiologists and orthopedic spine surgeons. Patients with an extruded herniated disk were included in this study. Five potential risk factors for degeneration, including prolonged sitting, bending/twisting, whole body vibration, lifting, and heavy work load, were recorded. Individuals were invited to estimate the number of hours spent per day working affected by each of the five risk factors. The risk factors were classified into the following three levels, which were based on the exposure frequency and years of employment: low; moderate; and high (Table S1). The controls were selected from individuals undergoing health examinations during the same period in the hospital. The individuals with other intervertebral disk diseases were excluded.
Other demographic and risk factor information was extracted from the medical records. This study was approved by the Ethics

Committees of the First Affiliated Hospital of Nanjing Medical
University and met the standards of the Declaration of Helsinki.
Written informed consent was obtained from all subjects.

| Blood sampling and genotyping
Blood samples (2 mL) were obtained from the cubital vein of the cases and controls. Genomic DNA was extracted using a TIANamp Blood DNA kit (Tiangen Biotech). The quality and concentration of the extracted DNA were measured at optical density (OD) wavelengths (260 and 280 nm) using NanoDrop (Thermo Scientific). A

| Statistical analysis
Demographic data from the case and control groups were analyzed using a two-tailed Student's t test (continuous variables) and a chi-square test (categorical variables), as appropriate. The Hardy-Weinberg equilibrium (HWE) was also assessed in the control group through a goodness-of-fit chi-square test. Using logistic regression analysis, the odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Stratified analyses were conducted according to gender, age, alcohol consumption, and cigarette smoking. In addition, the association between the MMP-3 gene rs591058 C/T polymorphism and factors to which the LDH patients were exposed was explored. All statistical analyses were performed on SPSS 22.0 (SPSS, Inc). The significance level was set at a P < .05.

| Characteristics of the study population
The demographic data of cases and controls are summarized in

| Relationship between the MMP-3 gene rs591058 C/T polymorphism and the risk for LDH
The HWE test was not significantly different with respect to the genotype frequency of the MMP-3 gene rs591058 C/T polymorphism in the controls. The TT genotype or TT + CT genotype was associated with an increased risk for LDH (TT vs CC: OR, 1.91; 95% CI, 1.09-3.37; P = .024; TT + CT vs CC: OR, 1.46; 95% CI, 1.03-2.06; P = .034) ( Table 2). In addition, the T allele of the rs591058 C/T polymorphism was also associated with an increased risk for LDH (Table 2). Furthermore, the conclusions were still significant after adjusting for age and sex.
Stratified analyses were performed according to sex, age, alcohol consumption, and cigarette smoking (

| Correlation between the MMP-3 gene rs591058 C/T polymorphism and factors to which LDH patients are exposed
Last, we investigated the association between the MMP-3 gene rs591058 C/T polymorphism and the factors to which the LDH patients are exposed ( Table 4). The individuals with high whole body vibration, bending/twisting, and lifting were prone to LDH; however, the prolonged sitting and heavy workload had no effects on the incidence of LDH. To summarize, exposure to high

TA B L E 4
The associations between MMP-3 gene rs591058 polymorphism and clinical characteristics of lumbar disk herniation whole body vibration, bending/twisting, and lifting, individuals with the rs591058 C/T polymorphism had an increased risk for developing LDH.

| D ISCUSS I ON
In this study, we found that MMP-3 gene rs591058 C/T polymorphism was related to increased risk for LDH. Subgroup analyses showed increased risk for LDH patients was shown in the female, smokers, and drinkers. In addition, there were synergistic effects between high whole body vibration, bending/twisting, and lifting and the genotypes of rs591058 C/T polymorphism, contributing to increased risk for LDH patients. allele carriers exposing to hard labor were more vulnerable to LDH.
It is of note that a meta-analysis showed no association between the MMP-3 5A/6A polymorphism and interbody disk degeneration susceptibility. 23 Another SNP (rs632478 polymorphism) was evaluated in an Iranian study, 24 which suggested the homozygote CC of the MMP-3 rs632478 polymorphism may lead to an increased risk for IDD compared with the AA genotype. 24 For this study, we only investigated the MMP-3 gene rs591058 C/T polymorphism. An Indian study 14 involving 200 LDH patients and 200 healthy controls showed that the TT genotype of the MMP-3 gene rs591058 C/T polymorphism is a protective factor for LDH in females, which was not consistent with our findings. In this study, we found that the MMP-3 gene rs591058 C/T polymorphism was associated with an increased risk for LDH. Further analyses observed a significantly increased risk for LDH patients in females, cigarette smokers, and alcohol drinkers. The following factors may account for the conflicting findings of the current study and the Indian study. First, exposure factors were different for these two studies. Second, race differences may also be a potential factor. Third, the clinical heterogeneity of LDH was also different. Fourth, the sample sizes of these studies were important factors that could not be ignored, which may affect the final conclusions. In addition, we investigated the association between the MMP-3 gene rs591058 C/T polymorphism and the factors affecting LDH patients. The data showed that the individuals with high whole body vibration, bending/twisting, and lifting were prone to LDH.
Cautious consideration for some potential limitations in this present study is in order. First, the correlation between the MMP-3 gene rs591058 C/T polymorphism and LDH risk could be clearly clarified by a single case-control study. Second, the sample size was limited, which may underpower the facticity. Third, the lack of population representativeness could not be overlooked because of the ethnic differences. Fourth, we only investigated one SNP of MMP-3 gene in this study. Furthermore, the occupation, age, and sex discrepancies of the subjects were not taken into consideration, which may cause bias.
Collectively, the MMP-3 gene rs591058 C/T polymorphism was associated with an increased risk for LDH, suggesting an essential role of this SNP in the progression of LDH; however, the specific mechanism by which the MMP-3 gene rs591058 C/T polymorphism mediates the occurrence of LDH is unknown, which warrants further study.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.