Interleukin 6 (IL-6) and IL-10 Promoter Region Polymorphisms Are Associated with Risk of Lumbar Disc Herniation in a Northern Chinese Han Population

Abstract

Aim: This study assessed the association of single-nucleotide polymorphisms (SNPs) in the proinflammatory cytokines interleukin 6 (IL-6) and IL-10 with the risk of lumbar disc herniation in a Chinese Han population. Methods: We collected blood samples from 267 patients with lumbar disc herniation (case group) and 300 normals (control group) and performed analyses of the IL-6 572C/G and 174G/C SNPs as well as the IL-10 592A/C and 1082G/A SNPs using TaqMan technology. Results: The frequencies of the IL-6-572 GG, GC, and CC genotypes were 5.99%, 42.3%, and 51.6%, respectively, in the case group, and 1.6%, 24%, and 64.3%, respectively, in the control group. Thus, the relative risk of the IL-6-572 G genotype (GG plus GC) was 1.69-fold higher for developing lumbar disc herniation compared to the CC genotype (95% confidence interval: 1.16-2.39, p < 0.01). The risks associated with the IL-6-572 CG and GG genotypes were 1.55- and 4.48-fold higher, respectively, versus the CC genotype for developing lumbar disc herniation (p < 0.01). The IL-10-1082 AG genotype was significantly higher in the case group (26.22%) versus the control group (11.67%); whereas the AA genotype was lower in the case group (73.78%) versus the control group (88.33%; p < 0.05). The IL-10-1082 G allele frequency was significantly higher in the case group (13.11%) versus the control group (5.83%; p < 0.05). Conclusion: This study demonstrates that genetic variants in the promoter regions of the IL-6 and IL-10 genes are associated with lumbar disc herniation risk in this Northern Chinese Han population.

Introduction

Lumbar disc herniation frequently occurs in the clinic, especially in patients between 20 and 50 years of age, and the most common symptom is lumbocrural pain. Patients can also experience a numb or tingling feeling in one or both legs (Kraemer, 1995; Hsu, 2010). The pathogenesis of lumbar disc herniation is very complex and to date the most accepted causes for development of lumbar disc disease are physiological aging and “degeneration,” intervertebral disc injury, or spinal overload. However, tobacco smoke and others are also risk factors for developing lumbar disc disease (Martin et al., 2002; Iatridis et al., 2006; Shiri et al., 2007; Del Grande et al., 2012), and many recent studies showed that genetic factors also play an important role in the development of intervertebral disc disease (Ala-Kokko, 2002; Battie and Videman, 2006).

It has been increasingly recognized that lumbar disc pain may be pathophysiologically induced by chemical inflammation (McCarron et al., 1987; Takahashi et al., 1996; Peng et al., 2007), although the inflammatory factors that contribute to lumbar disc herniation and pain remain to be determined. With the rapid development of molecular biology and molecular immunology for studying cytokines and chemokines, increasing research attention has been given to understanding the pathogenesis of intervertebral disc degeneration. Indeed, various cytokines participate in the pathophysiological process of disc degeneration (Bachmeier et al., 2007; Li et al., 2009; Kim et al., 2011). For example, previous studies revealed that inflammatory cytokines are not only closely related to other cytokines and chemical mediators but also play an important role in lumbar disc herniation (Habtemariam et al., 1996; Kang et al., 1996). In particular, interleukin 6 (IL-6) and IL-10 were shown to be highly expressed and activated in herniated disc and surrounding tissues, which presented as important pro- and anti-inflammatory cytokines, respectively, in the human body (Sommer and Kress, 2004). Burke et al. (2002) showed that herniated intervertebral disc cells secreted a number of proinflammatory mediators and cytokines, including IL-6, that cause lumbar pain. Kang et al. (1996) demonstrated that IL-6 was expressed in both normal and herniated discs, but was significantly induced in the herniated discs. Furthermore, Ahn et al. (2002) demonstrated that IL-10 inhibited expression of IL-1, IL-6, tumor necrosis factor-α (TNF-α), and other inflammatory mediators, as well as matrix metalloproteinase (MMP), to regulate the pathogenesis of intervertebral disc degeneration. A recent study profiled inflammatory proteins in the sera of patients with discherniation to predict lumbar radicular pain within a year (Moen et al., 2016). Thus, in this study, we first assessed single-nucleotide polymorphisms (SNPs) in the promoter regions of IL-6 and IL-10 in 267 patients with lumbar disc herniation and 300 controls using TaqMan technology. We then associated these SNPs with a risk of developing lumbar disc herniation in a North Chinese Han population.

Study Population and Methods

Study population

In this study, we enrolled 267 patients with lumbar disc herniation (case group) and 300 controls (control group) who visited the Orthopedic Department of the Affiliated Hospital of Jining Medical College and The People's Hospital of Zhangqiu between January 2014 and June 2015. Patients with lumbar disc herniation showed clinical symptoms, physical signs, and had laboratory and image examinations and surgical findings. The inclusion criteria for the case group were as follows: (a) patients with typical symptoms and physical signs of lumbar disc herniation (Hu, 2005; Xu et al., 2005) and lumbar disc herniation confirmed by magnetic resonance imaging (MRI); (b) patients between 18 and 60 years of age; and (c) patients with ethnic Han Chinese background tracing back at least three generations. The exclusion criteria for the case group were as follows: (a) patients with complicated liver, kidney, cardiovascular, and cerebrovascular diseases, respiratory diseases, or psychosis; (b) patients with complicated blood diseases, diabetes, autoimmune diseases, and tumors; (c) patients with body mass index (BMI) <18.5 kg/m² or >28.0 kg/m²; and (d) patients with a smoking index (SI, average number of cigarettes per day multiplied by smoking years) >300. However, the inclusion criteria for the control group were as follows: (a) individuals of Han ethnic Chinese with similar age and geographical location; (b) individuals without any history of lumbocrural pain; (c) individuals without family history of lumbocrural pain; (d) individuals without osteoarthritis, rheumatism, rheumatoid arthritis, lupus, and other connective tissue diseases; and (e) individuals without any traumatic fracture and tendon vascular injuries.

This study was approved by The Ethics Committee of our hospitals (The Affiliated Hospital of Jining Medical College and The People's Hospital of Zhangqiu) and all studied subjects were informed regarding the study scheme and risk and signed the informed consent before participation in this study. This study was conducted according to the State Council's Regulations on Medical Institutions Management (China) [1994].

Blood sample collection and genomic DNA extraction

Peripheral blood (2 mL) from each individual was collected after the subjects underwent fasting more than 10 h before hemospasia. The blood sample was then placed in a vacuum tube containing heparin anticoagulant and mixed for immediate genomic DNA extraction or stored at 4°C overnight. Genomic DNA was extracted from these peripheral blood samples using a whole blood DNA extraction kit (QIAamp Blood Kit; Qiagen, Hilden, Germany) following the manufacturer's instructions and quantified using an ultraviolet spectrophotometer (Shanghai Furi Science & Technology Co., Ltd., Shanghai, China). The extracted DNA was then stored at −80°C until use.

SNP selection and genotyping

In this study, we selected the IL-6 gene −572C/G and −174G/C promoter region and the IL-10 gene −592A/C and −1082G/A promoter region according to previous studies (Turner et al., 1997; Muller-Steinhardt et al., 2004). For SNP genotyping, polymerase chain reaction (PCR) primers and probes were designed using the Primer Express 3.0 random software in the 7500 real-time fluorescence quantitative PCR instrument (ABI, Foster City, CA). Two probes were designed for each gene site and labeled with VIC and FAM, respectively. These primers were synthesized by Invitrogen (Shanghai, China). We then performed PCR amplification using a 10 μL experimental reaction system containing 5 μL of 2× TaqMan allelic discrimination premixture (Takara, Dalian, China), 0.25 μL (2.5 nM) of 40× primer probe mixture, 2 μL (50 ng) of genomic DNA samples, and 2.5 μL of ddH₂O₂ with PCR conditions of an initial 95°C for 10 min, 40 cycles of 95°C for 10 s, and 60°C for 1 min. The instrument automatically collected and analyzed the fluorescence signals using the instrument software to yield SNP genotyping results.

Linkage disequilibrium analyses

Pairwise linkage disequilibrium (LD) between the SNPs was calculated using the ARLEQUIN 2.0 program (http://lgb.unige.ch/arlequin) according to a previous study (Schneider et al., 2000). We used the D′ value to describe the magnitude of LD (Lewontin, 1988) and the chi-square test to calculate allelic association using the Arlequin program for statistical significance (if the null hypothesis of no LD could be rejected).

Statistical analysis

The IL-6 and IL-10 SNP representation of the population between case and control groups was analyzed using the Hardy-Weinberg equilibrium with the χ² goodness-of-fit test. IL-6 and IL-10 genotypes and allele frequencies were calculated using frequency count and statistically analyzed twice. The mean comparison of the two groups was determined by Student's t-test. The odds ratio was calculated using a logistic regression test. All data were statistically analyzed using SPSS statistical software version 17.0 (SPSS, Chicago, IL). A p value <0.05 was considered statistically significant.

Results

Characteristics of study population

In this study, we collected and analyzed 267 cases of lumbar disc herniation and 300 controls. The clinical characteristics of these subjects are shown in Table 1. There were no statistically significant differences in age, sex, BMI, and SI between the case and control groups (p > 0.05).

Table 1.

Characteristics of Study Population

Variables	Case	Control	p
Age (year), mean ± SD	44.05 ± 9.02	41.85 ± 11.02	0.761^a
Sex	267	300
Male	151	193
Female	116	107	0.234^b
BMI (kg/m²), mean ± SD	23.7 ± 0.9	23.1 1.1	0.172^a
SI ≥100, n (%)	116 (38.67)	125 (41.67)	0.327^b

Data expressed as mean ± SD unless otherwise indicated.

Student^'s t test; ^bχ² test.

BMI, body mass index; SD, standard deviation; SI, smoking index (average number of cigarettes per day multiplied by smoking years >300 branches/[day × year]).

Association of IL-6 gene polymorphism with risk of lumbar disc herniation

Distribution of the IL-6-572C/G and −174G/C genotypes and allele frequencies between the case and control groups complied with the law of Hardy-Weinberg equilibrium. The results showed that the gene frequency reached genetic equilibrium and represented the population (p > 0.05). We then compared IL-6 genotype and allele frequencies between case and control groups (Tables 2 and 3). Specifically, difference in distribution of the IL-6-572C/G genotype and allele frequency between the case and control groups was statistically significant. IL-6 GG genotype and GC allele frequency were higher in the case group compared to the control group. Frequency of the CC genotype was lower in the case group compared to the control group (p < 0.01). The relative risk of this G (GG and GC) genotype frequency was 1.69 times higher compared with the CC genotype for the development of lumbar disc herniation (95% confidence interval [CI]: 1.16-2.39, p < 0.01), while the risks of the CG and GG genotypes were 1.55- and 4.48-fold higher, respectively, than the CC genotype for developing lumbar disc herniation (Table 2). The IL-6-572 G allele frequency was also significantly associated with risk of developing lumbar disc herniation (χ² = 11.23, p < 0.01). The IL-6-1742 GG was the main genotype, although there was no statistically significant difference between case and control groups in terms of distribution of GG or GC genotypes and G or C allele frequency (Table 3).

Table 2.

Association of the IL-6-572C/G Genotype and Allele Frequency Between Case and Control Groups

		Genotype frequency, n (%)^a			Allele frequency, n (%)^b
Site −572C/G	n	CC	CG	GG	C	G
Case	267	138 (51.69)	113 (42.32)	16 (5.99)	389 (72.85)	145 (27.15)
Control	300	193 (64.33)	102 (34.0)	5 (1.67)	488 (81.33)	112 (18.67)
OR		1.00	1.55	4.48	1.00	1.62
95% CI		1.09-2.33, 1.61-15.32			1.18-2.97
p		0.039, 0.006			0.007

χ² = 12.15, p < 0.01; ^bχ² = 10.17, p < 0.01.

95% CI, 95% confidence interval; OR, odds ratio.

Table 3.

Association of the IL-6-174G/C Genotype and Allele Frequency Between Case and Control Groups

		Genotype frequency, n (%)		Allele frequency, n (%)
Site −174 G/C	N	GG	CG	G	C
Case	267	264 (98.9)	3 (1.1)	531 (99.44)	3 (0.56)
Control	300	297 (99.0)	3 (1.0)	597 (99.5)	3 (0.5)
OR		1.13		1.12
95% CI		0.62-2.07		0.61-2.03
p		0.493		0.356

Association of IL-10 polymorphism with risk of lumbar disc herniation

Distribution of the IL-10-592A/C and −1082G/A genotypes and allele frequencies between the case and control groups complied with the law of Hardy-Weinberg equilibrium (p > 0.05). The comparison of these IL-10 genotypes and allele frequencies are shown in Tables 4 and 5, respectively. Specifically, the IL-10-592A/C genotype showed no statistically significant difference between case and control groups in terms of distribution of the three genotypes and A or C allele frequency (p > 0.05; Table 4). Moreover, the IL-10-1082A/G showed no statistically significant difference between case and control groups in terms of distribution of the genotypes and A or G allele frequency (p < 0.05). However, the IL-10-1082 AG genotype was significantly higher in the case group (26.22%) compared to the control group (11.67%). In contrast, the IL-10-1082 AA genotype was lower in the case group (73.78%) versus the control group (88.33%). The IL-10-1082 G (GG and GA) allele frequency was significantly higher in the case group (13.11%) compared to the control group (5.83%). The relative risk of the IL-10-1082 genotype was 2.69 times higher than the AA genotype for developing lumbar disc herniation (95% CI: 1.42-6.33, p < 0.05). The risk of developing lumbar disc herniation was also significantly higher in individuals carrying the IL-10-1082 G allele compared to those carrying the IL-10-1082 AA allele (χ² = 4.236, p < 0.01; Table 5).

Table 4.

Association of the IL-10-592A/C Genotype and Allele Frequency Between Case and Control Groups

		Genotype frequency, n (%)			Allele frequency, n (%)
Site −592A/C	n	AA	AC	CC	A	C
Case	267	91 (34.08)	143 (53.56)	33 (12.36)	325 (60.86)	209 (39.14)
Control	300	119 (39.67)	153 (51.0)	28 (9.33)	391 (65.17)	209 (34.83)
OR		1.00	1.22	1.54	1.00	1.20
95% CI		0.69-2.15, 0.73-2.87			0.79-1.76
p		0.872, 0.374			0.413

Table 5.

Association of the IL-10-1082A/G Genotype and Allele Frequency Between Case and Control Groups

		Genotype frequency, n (%)		Allele frequency, n (%)
Site −1082A/G	n	AA	AG	A	G
Case	267	197 (73.78)	70 (26.22)	464 (86.89)	70 (13.11)
Control	300	265 (88.33)	35 (11.67)	565 (94.17)	35 (5.83)
OR		2.69		2.44
95% CI		1.42-6.33		1.39-5.94
p		0.029		0.031

LD and haplotype value between SNPs

The standardized measure of LD denoted as D′' and the corresponding p value was calculated for paired SNPs in both case and control groups. The LD pattern is fairly identical in both samples and nearly completely homogeneous. Most of the SNPs were in tight and highly significant LD with each other. Our haplotype analysis of LD1 showed that there was no statistical significance between case and control groups (p > 0.05).

Discussion

Lumbar disc herniation usually causes lumbocrural pain, which is difficult to control or alleviate (Hahne et al., 2010). Pathophysiologically, various inflammatory factors play a role in inducing lumbar disc degeneration and nervous radical pain (Bachmeier et al., 2007), and further accelerating inflammation and intervertebral disc generation (Kang et al., 1997; Li et al., 2004); thus, this vicious circle worsens lumbar disc degeneration and pain. The inflammatory reaction affects the activity of the MMP family to accelerate degradation of the extracellular matrix in the intervertebral disc, resulting in disc degeneration and chronic inflammation (Qian and Liu, 2009). This study assessed association of IL-6 and IL-10 genetic variants with risk of developing lumbar disc herniation in a North China Han population. We found that the IL-6 GG genotype and G allele frequency were higher in the case group compared to the control group, whereas the CC genotype frequency was lower in the case group compared to the control group. The relative risk of the IL-6 GG/GC genotype was 1.69 times higher than the CC genotype for developing lumbar disc herniation, while the risk of the CG/GG genotype was 1.55 and 4.48 times higher, respectively, than the CC genotype. Moreover, the IL-10-1082 AG genotype was significantly higher in the case group compared to the control group, whereas the IL-10-1082 AA genotype was lower in the case group than the control group. The relative risk of the IL-10-1082 GG/GA genotype was 2.69 times higher than the AA genotype for developing lumbar disc herniation. The risk of lumbar disc herniation was also significantly increased in individuals carrying the IL-10-1082 G allele. This study is the first to associate IL-6 and IL-10 SNPs with an increased risk of developing lumbar disc herniation. Future studies will investigate the underlying molecular mechanism by which these two proinflammatory cytokines participate in lumbar disc degeneration and inflammation.

IL-6 is a 184-amino acid-long glycoprotein and an important proinflammatory cytokine produced by activated inflammatory cells, including lymphocytes and macrophages. Burke et al. (2002) showed that herniated intervertebral disc cells were able to secrete a number of proinflammatory mediators and cytokines, including IL-6, and this was associated with lumbar pain, although Kanemoto et al. (1996) argued that IL-6 might cause the loss of proteoglycan in the extracellular matrix of the nucleus pulposus, thereby inhibiting fibroblast synthesis of collagen. Moreover, Hadjipavlou et al. (2008) reported that other inflammatory factors or MMPs might also participate in spinal disc degeneration and pain, while Jacobsen et al. (2013) further confirmed that the MMP-1 rs1799750 2G allele was associated with increased low back pain, sciatica, and disability after lumbar disk herniation. In this study, we found an association of IL-6 SNPs with an increased risk of developing lumbar disc herniation. Genetic variations in the IL-6 promoter region could result in aberrant IL-6 transcription and expression (Fishman et al., 1998), thereby affecting individual susceptibility to various diseases (Curti et al., 2012). For example, the IL-6-572C/G polymorphism leads to replacement of cytidine by guanosine in the IL-6 promoter (Kitamura et al., 2002; Muller-Steinhardt et al., 2004; Hamid et al., 2005; Lei et al., 2005), affecting IL-6 transcription and, in turn, changing IL-6 expression (Terry et al., 2000). This polymorphism was reported to affect bone mineral density and metabolic characteristics in healthy individuals and survival rate of renal transplant patients, as well as the severity of diabetic nephropathy (Kitamura et al., 2002; Muller-Steinhardt et al., 2004; Hamid et al., 2005; Lei et al., 2005). This study demonstrated that the IL-6-572GG genotype and G allele frequency were significantly higher in the case group than in the control group, whereas frequency of the CC genotype was lower in the case group than in the control group. This suggests that the IL-6-572C/G polymorphism modified the susceptibility of lumbar disc herniation in this Chinese Han population and the G allele may be the genetic risk and susceptible factor in the development of lumbar disc herniation. Indeed, a previous study showed that the IL-6-174 G/C SNP directly affected IL-6 transcription efficiency (Fishman et al., 1998). Comprehensive analysis of the IL-6 polymorphism data in other regions and populations also demonstrated that the IL-6 genotype and allele frequency varied significantly; for example, the allele frequency of the IL-6-174C site was 0.005 in our studied population, which is significantly lower than 0.353 in an American population, 0.162 in an Italian population, and 0.430 in a British population (Cox et al., 2001; Humphries et al., 2001; Margaglione et al., 2001). However, our current data were similar to that of a Korean population with the allele frequency of IL-6-174C at 0.006 (Lim et al., 2002). Indeed, the allele frequency of IL-6-174C in Asia was similar (Lim et al., 2002), but was different between Chinese Han and European populations (p < 0.001). Thus, the IL-6-174 SNP was very low and is not likely to be the main risk factor for lumbar disc herniation in Chinese.

IL-10 is an anti-inflammatory cytokine produced by T helper Th2 cells to inhibit proinflammatory production by Th1 cells (Saraiva and O'Garra, 2010). Normally, the IL-10 level is minimal in nonstimulated tissues because IL-10 expression is tightly regulated at the transcriptional and posttranscriptional level (Chen et al., 2007). IL-10 is a glycoprotein and can produce a strong immune and inflammatory inhibition after binding to the IL-10 receptor on the cell membrane. Moreover, IL-10 can affect proinflammatory cytokine gene transcription and stability, resulting in reduced protein translation (Ahn et al., 2002). The IL-10 gene promoter is located in the upstream transcription start site within the 5 kb region and contains at least 23 SNP sites, three of which have been widely studied, including 1082 (A/G), 819 (C/T), and 592 (C/A). A previous study reported that specific SNPs in the IL-10 promoter region could influence IL-10 transcription and secretion of IL-10 protein (Turner et al., 1997). The IL-10-1082A allele was associated with the downregulation of IL-10 transcription, whereas the IL-10-1082G allele was associated with high IL-10 expression (Turner et al., 1997; Rad et al., 2004). An IL-10 polymorphism was associated with susceptibility and severity of other human diseases (Nemec et al., 2009). This study showed that the IL-10-1082AG genotype and G allele frequency were significantly higher in the case group compared to the control group, whereas the AA genotype was lower in the case group than the control group. Our data indicate that the IL-10-1082A/G polymorphism can influence susceptibility of lumbar disc herniation and the G allele might be the genetic risk and susceptibility factor for developing lumbar disc herniation in Han Chinese.

Indeed, lumbar disc herniation is a multifactor-induced disease. To date, increasing evidence shows that population aging, gender, being overweight, obesity, lifestyle, and race also contribute to risk of developing lumbar disc herniation (Iatridis et al., 2006; Shiri et al., 2007; Hadjipavlou et al., 2008; Del Grande et al., 2012; Pearson, 2016). Although, how these factors modify the risk for developing lumbar disc herniation or gene expression remains undetermined. In this study, we analyzed and associated SNPs of IL-6 and IL-10 with lumbar disc herniation risk. A previous study associated SNPs of other genes, like aggrecan gene VNTR polymorphism, and obesity with symptomatic lumbar disc herniation incidence (Cong et al., 2014) and demonstrated an interaction between VNTR SNPs and obesity in symptomatic lumbar disc herniation. A recent Italian case study reported that vitamin D receptor gene SNPs were associated with lumbar spine pathologies (Colombini et al., 2016), while another Chinese study associated SNPs of apoptosis-related genes, like Fas, FasL, and caspase 9 with lumbar discherniation risk (Zhang et al., 2013). Zhang et al. (2015) demonstrated that SNPs of an apoptosis-related gene TRAIL (TNF-related apoptosis-inducing ligand) were also associated with risk and severity of lumbar disc degeneration. Furthermore, Moen et al. (2014) reported that the IL1A T/IL1RNA genotype was associated with an increased risk of a chronic outcome in patients following discherniation. Thus, further study is needed to better understand the risk factors and pathogenesis of lumbar discherniation to prevent and treat lumbar discherniation clinically.

This study does have some limitations. We did not analyze expression levels of IL-6 and IL-10 in patients with lumbar disc herniation and controls before determining association of these SNPs with expression level of these two cytokines. Also, this study did not have the ability to associate these SNPs with clinicopathological data, such as pain level, although our data demonstrate that IL-6-572C/G and IL-10-1082A/G polymorphisms could be genetic risk factors for developing lumbar disc herniation in this Han Chinese population. Our current data provide an experimental basis to further develop etiological and genetic study of lumbar disc herniation. However, lumbar disc herniation and chronic inflammation are complex pathological processes due to the combined effects of multiple genes and factors. Thus, a larger, more prospective, and more multiracial case-control study is necessary to obtain further evidence and verify the above findings.

Footnotes

Acknowledgment

This study was supported, in part, by a grant from Projects of Medical and Health Technology Development Program of Shandong Province (No. 2014WS0205).

Author Disclosure Statement

No competing financial interests exist.

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