Abstract
Purpose
To determine whether polymorphisms (SNPs) in the genes encoding cytokines and nitric oxide synthase (NOS) might play some role in lumbar disc herniation (LDH).
Patients and methods
Case–control study in which 179 patients were retrospectively reviewed. The case group was made of 50 patients with symptomatic LDH diagnosed by MRI while the control group was made of 129 individuals undergoing routine hip or knee arthroplasty with a lifetime lack of low back pain. SNPs in the cytokine genes of IL-1 [IL-1α (−889 C/T), IL-1β (+3953 T/C)], TNF-α (−308 G/A and −238 G/A) and NOS genes [eNOS (r 27 bp, intron 4 and −786 T/C) and iNOS (22 G/A)].
Results
The CC genotype and C allele of the IL-1β (+3953 T/C) SNP were significantly more frequent among LDH patients compared to controls. On the other hand, eNOS (−768 T/C) and iNOS (22 G/A) SNPs were significantly more common in the control group.
Conclusions
Carriers of the CC genotype of the IL-1β (+3953 T/C) SNP were more frequent among LDH patients suggesting some potential role of the IL-1β SNP on LDH pathogenesis. The eNOS (−786 T/C) and iNOS (22 G/A) SNPs were more frequent among the control subjects, suggesting their possible protective role against LDH. Genotyping these SNPs could be useful to identify persons with an increased lifetime risk of disc herniation in whom measures to avoid LDH could be implemented.
Keywords: Lumbar disc herniation, Polymorphism, Cytokine, Nitric oxide synthase
Introduction
The prevalence of lumbar disc degeneration is variable in the general population and depends on the individuals studied and their racial background. Schmorl nodes of lumbar spine have been found in 16.4% of asymptomatic Southern Chinese in the fourth decade of life by MRI [1]. Lumbar disc bulge was observed in 84.8% and disc herniation in 18.2% of asymptomatic Americans older than 55 while less than 10% of Spanish patients with low back pain showed lumbar end plate erosions and spondylolisthesis by MRI [2, 3]. Older reports estimated the incidence of lumbar disc herniation (LDH) detected by CT in Spain at around 2% [4]. LDH occurs when the soft central nucleus pulposus of a lumbar intervertebral disc is displaced from its usual anatomical position, possibly causing nerve root compression and symptoms of low back pain and sciatica. Most frequently affected are the intervertebral spaces L4–L5 and L5–S1. LDH is a common motive for sick leave and a significant medical, social and economic burden.
Epidemiological studies in twins have shown that the most determining individual factor for suffering intervertebral disc degeneration is a family history of this condition [5, 6]. Environmental factors such as mechanical variables, lifestyle factors such as body weight or even smoking play an important role in LDH as well [7, 8]. The patient’s genetic makeup could, therefore, be the factor that under similar environmental conditions determines that some individuals develop this condition while others do not. Among these genetic factors that lead to variation in the response of an organism to a similar environmental stimulus, we find gene polymorphisms (GP).
A GP is an allelic variation of a gene that exists in stable form in a population at a frequency of at least 1%. Two types of GP have been described: variable number of tandem repeat polymorphisms (VNTR) and single nucleotide polymorphisms (SNP), which appear more frequently. Its phenotypic expression has no specific clinical effect but a GP determines, e.g., that a person is responsive or not to a given drug or susceptible to suffer a given disease. Polymorphisms differ from mutations in that the latter are much less common and are generally associated with diseases.
It is well established that cytokines play a role in intervertebral disc degeneration and, specifically, interleukin (IL)-1 has been incriminated in this process among other factors that mediate inflammation. Hence, a predisposition to disc degeneration could be related to the expression of GP in the genes that code for such mediators, particularly IL-1 [9, 10].
Nitric oxide (NO) is a small molecule of short half-life that is generated by the enzyme nitric oxide synthase (NOS). The role of NO as a physiological mediator has been the topic of intense investigation. It is known that NO regulates arterial pressure, platelet aggregation, wound repair and apoptosis, and is involved in the response to an allograft, ulcerative colitis and gastrointestinal disorders. It also participates in the processes of interest in Orthopaedic Surgery such as inflammation, arthritis, osteoporosis, sepsis, ligament repair and aseptic loosening of joint prostheses [11, 12]. It was reported that NO mediates the change of proteoglycan synthesis in the human lumbar intervertebral disc in response to hydrostatic pressure [13]. As for cytokines, the expression of NO may vary according to the GP carried by an individual.
The aim of this work was to determine a potential association between LDH and different SNPs of cytokines [IL-1α (−889 C/T); IL-1β (+3953 T/C); tumor necrosis factor (TNF) [(−308 G/A and −238 G/A)], endothelial nitric oxide synthase (eNOS) [(27 bp repeat in intron 4) and (−786 T/C)]; and inducible nitric oxide synthase (iNOS) (iNOS 22 G/A). To prove this hypothesis, a retrospective case–control study was designed in which 50 patients with MRI-confirmed symptomatic LDH and 129 individuals undergoing routine hip or knee arthroplasty at the Hospital Universitario Central de Asturias with a lifetime lack of low back pain were genotyped for the above SNPs.
Patients and methods
Patients
One hundred and seventy nine patients were recruited from the Orthopaedic Surgery and Traumatology Department of the Hospital Universitario Central de Asturias (HUCA) between July 2007 and December 2008.
The case group was made of 50 patients, 21 women (43%) and 29 men (58%) with several clinical symptoms suggestive of LDH and the condition confirmed by Magnetic Resonance Imaging (MRI). LDH symptoms were those described by the Spine Center of the University of California San Francisco Medical Center as follows: (1) lower back pain, (2) pain, weakness or tingling in the legs, buttocks and feet, (3) difficulty in moving the lower back, (4) problems with bowel, bladder or erectile function in severe cases [14]. LDH MRI findings were extension of the disk beyond margins of adjacent vertebral bodies. The mean age of the case group was 43.9 ± 11.9 years [range 23–77]. Fifteen of them (30%) underwent surgery and the LDH diagnosis was confirmed at the operation room while the 35 remaining LDH patients were on non-surgical therapy.
The control group was made of 129 patients who had been admitted to the Orthopaedic Surgery and Traumatology Department for primary hip or knee arthroplasty. This control group was made of 44 men (34.1%) and 85 women (65.9%) who has a lifetime lack of symptoms suggesting LDH. Their mean age was 68.7 ± 9.2 years [range 25–85].
Both cases and controls were from a homogeneous Caucasian population in Hardy–Weinberg equilibrium and lived in the same region (Asturias). Each participant was required to sign an informed consent form, which had been approved by the Medical Ethics Committee of the HUCA.
Genotypes
Blood samples (10 mL) were withdrawn from each subject into a vacuum tube containing K3-ethylenediaminetetraacetic acid (EDTA) as anticoagulant by puncturing a peripheral vein in the arm.
The next part of the study was performed at the Biochemistry and Molecular Biology laboratory, Oviedo University Medical School, where the necessary DNA was extracted and isolated to genotype the following SNPs: IL-1α (−889 C/T); IL-1β (+3953 T/C); TNF-α [(−308 G/A and −238 G/A); eNOS [(27 bp repeat in intron 4) and (−786 T/C)]; and iNOS (iNOS 22 G/A).
DNA was first isolated from the blood sample leucocytes by the saline precipitation method described by Miller and Polesky [15]. Next, the DNA fragment containing the polymorphic sequence was amplified by PCR. The products of amplification were visualized by 2% w/v agarose gel electrophoresis. Based on prior knowledge of the size of the possible resultant alleles and their electrophoretic patterns, the corresponding genotype for each SNP was determined. The primers used in each case are provided in Table 1 along with the annealing temperatures and restriction enzymes used.
Table 1.
Genetic polymorphisms examined in the present study and primers used with their corresponding annealing temperatures, restriction enzymes and amplification product sizes
| Polymorphism | Primers (sense/antisense) | Annealing temperature (°C) | Restriction enzyme | PCR product size | Fragment sizes |
|---|---|---|---|---|---|
| IL-1α (−889 C/T) | 5′-ATCACACCTAGTTCATTTCCTCTATTTA-3′ 5′-GATTTTTACATATGAGCCTTCCATG-3′ |
58 | NcoI | 195 bp | 195 bp (T) 166 + 29 bp (C) |
| IL-1β (+3953 T/C) | 5′-CTCAGGTGTCCTCCAAGAAATCAAA-3′ 5′-GCTTTTTTGCTGTGAGTCCCG-3′ |
60 | TaqI | 194 bp | 194 bp (C) 108 + 86 bp (T) |
| TNF-α (−308 G/A) | 5′-GCAATAGGTTTTGAGGGCCAT-3′ 5′-GGGACACACAAGCATCAAG-3′ |
58 | NcoI | 147 bp | 122 + 25 bp (G) 147 bp (A) |
| TNF-α (−238 G/A) | 5′-AAACAGACCACAGACCTGGTC-3′ 5′-CTCACACTCCCCATCCTCCCGGATC-3′ |
58 | BamHI | 154 bp | 110 + 44 (G) 154 bp (A) |
| eNOS (27 bp, intron 4) | 5′-CTATGGATGTGCCTTGGCTGGAGG-3′ 5′-TCGCCTCAAGGGACCCGCCA-3′ |
63 | – | 22 bp (6rep) 195 bp (5rep) 168 (4rep) |
– |
| eNOS (−786 T/C) | 5′-TGGAGAGTGCTGGTGACCCCA-3′ 5′-CTGTCCCACCCCCACCTCCG-3′ |
62 | MspI | 180 bp | 140 + 40 bp (T) 90 + 50 + 40 bp (C) |
| iNOS (22 G/A) | 5′-GCTGAATCTGAGTTGATGAACAGATC-3′ 5′-CTCCCGGGATCACACGCCCA-3′ |
60 | NcoI | 140 bp | 120 + 20 bp (G) 140 bp (A) |
Statistical analysis
Data analysis was performed using the programs SPSS Software, version 15.0 (Chicago, IL, USA) and BMD Statistical Software Ltd. (Cork, Ireland). Genotype frequencies in the two groups were compared using the Chi-squared test. Odds ratios (OR) and their 95% confidence intervals (CI) were also calculated. The level of significance was set at p < 0.05. Yates’ correction and Mantel–Haenszel tests were used when necessary.
Results
The genotypic and allelic frequencies observed for the different SNPs in LDH patients and controls are shown in Tables 2, 3, 4 and 5.
Table 2.
Polymorphisms in the cytokines-encoding genes IL-1α (−889C/T), IL-1β (+3953 T/C), TNF-α (−308 G/A) and (−238 G/A) in patients with lumbar disc herniation (LDH) and control patients
| Gene (%) | Genotype frequency | LDH (n = 50) | Controls (n = 129) | Pearson χ2 | Odds ratio (95% CI) | p |
|---|---|---|---|---|---|---|
| IL-1-α (−889 T/C) | TT | 3 (6) | 5 (3.9) | 0.38 | 1.58 (0.29–8.02) | 0.54 |
| CT | 25 (50) | 61 (47.3) | NS | |||
| CC | 22 (44) | 63 (48.8) | NS | |||
| IL-1-β (+3953 T/C) | CC | 4 (8) | 3 (2.3) | 3.10 | 3.65 (0.66–21.54) | 0.008* |
| CT | 16 (32) | 50 (38.8) | NS | |||
| TT | 30 (603) | 76 (58.9) | NS | |||
| TNF-α (−308 G/A) | AA | 1 (2) | 4 (3.1) | 0.16 | 0.64 (0.03–6.3) | 0.69 |
| AG | 16 (32) | 42 (32.6) | NS | |||
| GG | 33 (66) | 83 (64.3) | NS | |||
| (n = 49) | (n = 122) | |||||
| TNF-α (−238 G/A) | AA | 0 | 0 | – | – | – |
| AG | 5 (10) | 9 (7.38) | 0.33 | 1.4 (0.39–4.90) | 0.57 | |
| GG | 45 (90) | 113 (92.62) | ||||
* p value was calculated by the two-tailed Fisher’s exact test
Table 3.
Allele frequencies of polymorphisms of the cytokines-encoding genes IL-1α (−889 C/T), IL-1β (+3953 T/C), TNF-α (−308 G/A and 238 G/A) in patients with lumbar disc herniation (LDH) and controls
| Gene (%) | Allele frequency | LDH (n = 50) | Controls (n = 129) | Pearson χ2 | Odds ratio (95% CI) | p |
|---|---|---|---|---|---|---|
| IL-1-α (−889 T/C) | C | 69/100 (0.69) | 187/258 (0.73) | 0.43 | 1.2 (0.69–2.02) | 0.51 |
| T | 31/100 (0.31) | 71/258 (0.27) | ||||
| IL-1-β (+3953 T/C) | C | 32/100 (0.32) | 56/258 (0.22) | 4.12 | 1.7 (0.98–2.93) | 0.042* |
| T | 68/100 (0.68) | 202/258 (0.78) | ||||
| TNF-α (−308 G/A) | A | 18/100 (0.18) | 50/258 (0.19) | 0.09 | 0.91 (0.48–1.72) | 0.77 |
| G | 82/100 (0.82) | 208/258 (0.81) | ||||
| (n = 50) | (n = 122) | |||||
| TNF-α (−238G/A) | A | 5/100 (0.05) | 9/244 (0.03) | 0.31 | 1.37 (0.39–4.63) | 0.58 |
| G | 95/100 (0.95) | 235/244 (0.97) | ||||
* p calculated by the Mantel–Haenszel test
Table 4.
Polymorphisms in eNOS (27 bp repeat, intron 4), and (−786 T/C) and iNOS (22 G/A)-encoding genes in patients with lumbar disc herniation (LDH) and controls
| Gene (%) | Genotype | LDH (n = 50) | Controls (n = 129) | Pearson χ2 | Odds ratio (95% CI) | p |
|---|---|---|---|---|---|---|
| NOS3 (27 bp, intron 4) | 44 | 0 (0) | 3 (2.3) | 1.18 | ND | 0.28 |
| 45 | 10 (20) | 28 (21.7) | NS | |||
| 55 | 40 (80) | 97 (75.2) | NS | |||
| 65 | 0 (0) | 1 (0.8) | ||||
| NOS3 (−786 T/C) | CC | 3 (6) | 45 (34.9) | 13.88 | 0.12 (0.03–0.43) | 0.0002* |
| CT | 30 (60) | 55 (42.69 | NS | |||
| TT | 17 (34) | 29 (22.5) | NS | |||
| NOS2 (exon 22) | AA | 0 (0) | 19 (14.7) | 6.76 | ND | 0.0009* |
| GA | 35 (70) | 63 (48.8) | ||||
| GG | 15 (30) | 47 (36.4) |
Confidence intervals could not be calculated in this case and are only provided for the relative risk as: RR (95% CI) = 0.87 (0.16–4.81)
* p value was calculated by the Yates correction test
Table 5.
Allele frequencies of polymorphisms in eNOS (27 bp repeat, intron 4), and (786 T/C) and iNOS (22 G/A)-encoding genes in patients with lumbar disc herniation (LDH) and controls
| Gene (%) | Allele frequency | LDH (n = 50) | Controls (n = 129) | Pearson χ2 | Odds ratio (95% CI) | p |
|---|---|---|---|---|---|---|
| NOS3 (27 bp, intron 4) | 4 | 10/100 (0.1) | 34/258 (0.14) | 0.68 | 0.73 (0.32–1.62) | 0.41 |
| 5 | 90/100 (0.9) | 222/258 (0.86) | ||||
| 6 | 0/100 (0) | 2/258 (0.0) | ||||
| NOS (−786 T/C) | C | 36/87 (0.41) | 145/258 (0.66) | 4.1 | 0.6 (0.35–1.01) | 0.042* |
| T | 47/87 (0.59) | 113/258 (0.44) | ||||
| NOS2 (exon 22) | A | 35/100 (0.35) | 101/258 (0.39) | 0.53 | 0.84 (0.5 –1.39) | 0.47 |
| G | 65/100 (0.65) | 157/258 (0.61) |
* p calculated by the Mantel–Haenszel test
Interleukin-1β SNP: IL-1β (+3953 T/C)
Carriers of the homozygous polymorphic CC genotype were threefold more frequent among the LDH cases than in controls (χ2 = 3.1; OR = 3.65 [0.66–21.54]; p = 0.008) (Table 2). In addition, the C allele was detected more frequently in LDH patients than controls (χ2 = 4.12; OR = 1.7 [0.98–2.93]; p = 0.042) (Table 3).
Endothelial nitric oxide synthase SNP: eNOS (−786 T/C)
The polymorphic CC genotype was fivefold more frequent among controls than in the LDH patients (χ2 = 13.88; OR (95% CI) = 0.12 [0.03–0.43]; p = 0.0002) (Table 4). The C allele of the SNP NOS3 (−786 T/C) was also significantly more frequent among the controls compared to the LDH patients (χ2 = 4.1; OR (95% CI) = 0.6 [0.35–1.01]; p = 0.042) (Table 5).
Inducible nitric oxide synthase SNP: iNOS (22 G/A)
The polymorphic AA genotype was observed in 14.7% of the control subjects and in none of the patients with a herniated disc, the comparison being significant (χ2 = 6.76; p = 0.0009) (Table 4). No significant differences were detected when allele frequencies of this SNP between the two groups were compared (p = 0.47; Table 5).
Other SNPs
No significant differences were observed when the genotypic and allelic frequencies of the remaining SNPs genotyped in LDH cases and controls were compared.
Discussion
The present study shows an association between the carriage of the IL-1β (+3953 T/C) SNP and symptomatic LDH Spanish patients. Several reports have found an association between different SNPs and intervertebral disc disease so far [6, 9, 17–27]. Due to their proinflammatory role, variations in genes codifying cytokines were studied first. Of them, only the IL-1α (−889 C/T), and IL-1β (+3953 T/C)] were clearly associated with intervertebral disc disease [9, 17, 18, 21]. Our results agree with those of Solovieva et al. observed in a Finnish cohort in which the carriage of the IL-1β (+3953) T allele correlated not only with intervertebral disc degeneration, but also with the number of days of low back pain [9, 18]. However, we could not find an association between the IL-1α (−889 C/T) SNP and LDH in our Spanish cohort that others have reported not only in Finnish, but also in Chinese patients [9, 19, 21]. There is a linkage disequilibrium between IL-1α and IL-1β genes, both located in the long arm of chromosome 2, so it is difficult to be sure whether these reported associations are specific for one or the other gene.
The link between cytokines and LDH is still poorly understood. IL-1β SNP associates with increased “in vitro” expression of IL-1β [22]. IL-1 induces the expression of other proinflamatory enzymes such as NOS, matrix metalloproteases (MMPs) and cyclooxigenases (COX) that might destroy proteoglycans causing disc degeneration. This hypothesis is supported by the fact that Takahashi et al. [16] detected the presence of IL-1α, IL-1β, IL-6, TNF-α and prostaglandin E2 in the disc tissue of almost one hundred patients with a herniated disc. MMPs and COX SNPs have been associated with lumbar disc degeneration as well [23, 24].
The eNOS (−786 T/C) and iNOS (22 G/A) SNPs were more frequent among the control subjects with a lifetime lack of LDH symptoms, suggesting a potential protective role against LDH development. Ours is the first report of an association between a NOS SNP and protection against LDH in the world literature.
The IL-1β SNP carried by only 8% of LDH patients in our cohort cannot explain but a minor part of the intervertebral disc disease pathogenetic mechanisms. The genetic component associated with intervertebral disc disease is of a highly complex nature. A large number of biological mediators are involved in this pathologic process not only interleukins, but also NOS, MMPs and COX. Genes encoding one of the three alpha chains of type IX collagen, the major collagen component of hyaline cartilage or the vitamin D receptor seems to play a role as well. The multiple SNPs existing for each of the genes that encode these molecules may contribute to the disease in varying measure of protect against its development. Thus, the final outcome is determined by cumulative expression of the cluster of polymorphic genes, leading to variation in the effects observed in the subset of individuals with disc herniation. Environmental and occupational factors play an additive or role in association to the genetic background in vertebral disc degeneration, such as the effect of weight lifting or whole-body vibration on the carriage of the IL-1α (−889 T/C) allele or the synergistic effect of obesity on the carriage of the COLA9A3 SNP [17, 19, 27].
Caution is needed in the interpretation of our data. Ours is a case–control, relatively small, only Caucasian, retrospective and non-aleatorized cohort. Larger populations of other non-Caucasian ethnic backgrounds must be genotyped for this IL-1β SNP. In addition case and control groups were not matched for age or gender with a predominance of older female in the control group, made of individuals undergoing orthopaedic prosthesis surgery. Control individuals did not have a spine MRI done ruling out LDH. However, they reported a lifetime lack of low back pain. The above differences could bias our findings. However, the mean old age of the control individuals (68.7 years), with their active laboral life finished, made unlikely the further development of LDH.
Conclusions
The clinical meaning of the association between the IL-1β SNP and LDH, confirming similar findings in other international cohorts, requires further investigation including the determination of serum levels of IL-1β in carriers of this IL-1β SNP, in both LDH cases and controls and its correlation with other already reported SNPs with intervertebral disc involvement in patients with active or sedentary life-styles. If the association observed here between the IL-1β SNP and LDH is confirmed, then all subjects whose work or other activity might increase their risk of suffering disc pathology should be genetically screened for this SNP. Preventive measures could be taken in carriers of this genotype such as redesigning their sports or work life. If used as a tool for early diagnosis and prevention, the analysis of SNPs associated with skeletal pathologies could be a promising approach.
Acknowledgments
This study was funded by a Research Project grant awarded by the Fundación S.E.C.O.T. (Spanish Society of Orthopaedic and Trauma Surgery) in 2007.
Conflicts of interest The authors declare no conflicts of interest.
References
- 1.Mok FPS, Samartzis D, Karppinen J, Luk KDK, Fong DYT, Cheung KMC. Prevalence, determinants, and association of Schmorl nodes of the lumbar spine with disc degeneration. A population-based study of 2449 individuals. Spine. 2010;35:1944–1952. doi: 10.1097/BRS.0b013e3181d534f3. [DOI] [PubMed] [Google Scholar]
- 2.Arana E, Royuela A, Kovacs FM, Estremera A, Sarasibar H, Amengual G, Galarraga I, Martinez C, Muriel A, Abraira V, Gil Del Real MT, Zamora J, Campillo C. Lumbar spine: agreement in the interpretation of 1.5-T MR images by using the Nordic Modic Consensus Group classification form. Radiology. 2010;254:809–817. doi: 10.1148/radiol.09090706. [DOI] [PubMed] [Google Scholar]
- 3.Tong HC, Carson JT, Haig AJ, Quint DJ, Phalke VR, Yamakawa KSJ, Miner JA. Magnetic resonance imaging of the lumbar spine in asymptomatic older adults. J Back Musculoskeletal Rehabil. 2006;19:67–72. [Google Scholar]
- 4.Azpeitia AJ, Martinez SM, Céspedes MM, Rebolledo VM, Millan JJM. Tomografía computarizada de las hernias discales foraminales y extraforaminales. Radiología. 1992;34:345–349. [Google Scholar]
- 5.McGregor AJ, Andrew T, Sambrook PN, Spector TD. Structural, psychological and genetic influences on low back and neck pain: a study of adult female twins. Arthritis Rheum. 2004;51:160–167. doi: 10.1002/art.20236. [DOI] [PubMed] [Google Scholar]
- 6.Patel AA, Spiker WR, Daubs M, Brodke D, Cannon-Albright LA. Evidence for an inherited predisposition to lumbar disc disease. J Bone Surg Am. 2011;93:225–229. doi: 10.2106/JBJS.J.00276. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Schumann B, Bolm.-Audorff U, Bergmann A, Elegast R, Elsner G, Grifka J, Hearting J, Jäger M, Michaelis M, Seidler A. Lifestyle factors and lumbar disc disease: results of a German multi-center case-control study (EPILIFT) Arthritis Res Ther. 2011;12:R193. doi: 10.1186/ar3164. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Kim KS, Yoon ST, Park JS, Li J, Park MS, Hutton WC. Inhibition of proteoglycan and type II collagen synthesis of disc nucleus cells by nicotine. J Neurosurg Spine. 2003;99:291–297. doi: 10.3171/spi.2003.99.3.0291. [DOI] [PubMed] [Google Scholar]
- 9.Solavieva S, Kouhia S, Leino-Arjas P, Ala-Kokko L, Luoma K, Raininko R, Saarela J, Riihimäki H. Interleukin-1 polymorphisms and intervertebral disc degeneration. Epidemiology. 2004;15:626–633. doi: 10.1097/01.ede.0000135179.04563.35. [DOI] [PubMed] [Google Scholar]
- 10.Le Maitre CL, Freemont AJ, Hoyland JA. The role of interleukin-1 in the pathogenesis of human intervertebral disc degeneration. Arthritis Res Ther. 2005;7:R732–R745. doi: 10.1186/ar1732. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Evans CH, Stefanovic-Racic M, Lancaster J. Nitric oxide and its role in orthopaedic disease. Clin Orthop Rel Res. 1995;312:275–294. [PubMed] [Google Scholar]
- 12.Stea S, Visentin M, Donati ME, Granchi D, Ciapetti G, Sudanese A, Toni A. Nitric oxide synthase in tissues around failed hip prostheses. Biomaterials. 2002;23:4833–4838. doi: 10.1016/S0142-9612(02)00236-3. [DOI] [PubMed] [Google Scholar]
- 13.Liu GZ, Ishihara H, Osada R, Kimura T, Tsuji H. Nitric oxide mediates the change of proteoglycan synthesis in the human lumbar intervertebral disc in response to hydrostatic pressure. Spine. 2001;26:134–141. doi: 10.1097/00007632-200101150-00005. [DOI] [PubMed] [Google Scholar]
- 14.UCSF Medical Center. Spine Center. Lumbar disc herniation. Available at: http://www.ucsfhealth.org/conditions/lumbar_disc_herniation/
- 15.Miller SA, Dykes DD, Polesky HF. A simple salting-out procedure for extracting DNA from human nucleated cells. Nucleic Acid Res. 1988;16:1215. doi: 10.1093/nar/16.3.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Takahashi H, Suguro T, Okazima Y, Motegi M, Okada Y, Kakiuchi T. Inflammatory cytokines in the herniated disc of the lumbar spine. Spine. 1996;21:218–224. doi: 10.1097/00007632-199601150-00011. [DOI] [PubMed] [Google Scholar]
- 17.Solavieva S, Leino-Arjas P, Saarela J, Luoma K, Raininko R, Riihimäki H. Possible association of interleukin 1 gene locus polymorphisms with low back pain. Pain. 2004;109:8–19. doi: 10.1016/j.pain.2003.10.020. [DOI] [PubMed] [Google Scholar]
- 18.Solovieva S, Lohiniva J, Leino-Arjas P, Raininko R, Luoma K, Ala-Kokko L, Riihimäki H. Intervertebral disc degeneration in realtion to the COLA9A3 and the IL-1ss gene polymorphism. Eur Spine J. 2006;15:613–619. doi: 10.1007/s00586-005-0988-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Virtanen IM, Karppinen J, Taimela S, Ott J, Barral S, Kaikkonen K, Heikkilä O, Mutanen P, Noponen N, Männikkö M, Tervonen O, Natri A, Ala-Kokko L. Occupational and genetic risk factors associated with intervertebral disc disease. Spine. 2007;32:1129–1134. doi: 10.1097/01.brs.0000261473.03274.5c. [DOI] [PubMed] [Google Scholar]
- 20.Solovieva S, Noponen N, Männikkö M, Leino-Arjas P, Luoma K, Raininko R, Ala-Kokko L, Riihimäki H. Association between the aggrecan gene variable number of tandem repeats polymorphism and intervertebral disc degeneration. Spine. 2007;32:1700–1705. doi: 10.1097/BRS.0b013e3180b9ed51. [DOI] [PubMed] [Google Scholar]
- 21.Ye W, Ma RF, Su PQ, Huang DS, Liu SL, Chen WJ, Wang XG. Association of single nucleotide polymorphisms of IL-1β with lumbar disc disease. Yi Chuan. 2007;29:923–928. doi: 10.1360/yc-007-0923. [DOI] [PubMed] [Google Scholar]
- 22.Pociot F, Molvig J, Wogensen L, Worsaae H, Nerup J. A TaqI polymorphism in the human interleukin-1β (IL-1β) gene correlates withIL-1β secretion in vitro. Eur J Clin Invest. 1992;22:396–402. doi: 10.1111/j.1365-2362.1992.tb01480.x. [DOI] [PubMed] [Google Scholar]
- 23.Valdes AM, Hassett G, Hart DJ, Spector TD. Radiographic progression of lumbar spine disc degeneration is influenced by variation at inflammatory genes: a candidate SNP association study in the Chingford cohort. Spine. 2005;30:2445–2451. doi: 10.1097/01.brs.0000184369.79744.a5. [DOI] [PubMed] [Google Scholar]
- 24.Yuan HY, Tang Y, Liang YX, Lei L, Xiao GB, Wang S, Xia ZL. Matrix metalloproteinase-3 and vitamin D receptor genetic polymorphisms, and their interactions with occupational exposure in lumbar disc degeneration. J Occup Health. 2010;52:23–30. doi: 10.1539/joh.L8149. [DOI] [PubMed] [Google Scholar]
- 25.Videman T, Saarela J, Kaprio J, Näkki A, Levälahti E, Gill K, Peltonen L, Battié MC. Associations of 25 structural, degradative, and inflammatory candidate genes with lumbar disc desiccation, bulging, and height narrowing. Arthritis Rheum. 2009;60:470–481. doi: 10.1002/art.24268. [DOI] [PubMed] [Google Scholar]
- 26.Karasugi T, Semba K, Hirose Y, Kelempisioti A, Nakajima M, Miyake A, Furuichi T, Kawaguchi Y, Mikami Y, Chiba K, Kamata M, Ozaki K, Takahashi A, Mäkelä P, Karppinen J, Kimura T, Kubo T, Toyama Y, Yamamura K, Männikkö M, Mizuta H, Ikegawa S. Association of the tag SNPs in the human SKT gene (KIAA1217) with lumbar disc herniation. J Bone Miner Res. 2009;24:1537–1543. doi: 10.1359/jbmr.090314. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Solovieva S, Lohiniva J, Leino-Arjas P, Raininko R, Luoma K, Ala-Kokko L, Riihimäki H. COL9A3 gene polymorphism and obesity in intervertebral disc degeneration of the lumbar spine: evidence of gene-environment interaction. Spine. 2002;27:2691–2696. doi: 10.1097/00007632-200212010-00008. [DOI] [PubMed] [Google Scholar]