Promoter polymorphisms in IL-6 gene influence pro-inflammatory cytokines for the risk of osteoarthritis

Background
Interleukin-6 (IL-6) gene regulates IL-6 levels, interplay of which has been found to influence pathophysiology of osteoarthritis (OA). Polymorphism within promoter region of IL-6 gene and its association with plasma levels of pro-inflammatory cytokines; IL-6, interleukin 1-beta (IL-1β) and tumor necrosis factor–alpha (TNF-α) remained to be investigated in Punjab region of India, where OA is highly prevalent.

Methods
Six single nucleotide polymorphisms (SNPs) in the promoter region of IL-6 gene; rs1800795 (−174G/C), rs1800796 (−572G/C), rs1800797 (−597G/A), rs2069827 (−1363G/T), rs12700386 (−2954G/C) and rs10499563 (−6331G/T) were investigated by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 279 confirmed osteoarthritis patients and 287 controls. Plasma levels of pro-inflammatory cytokines; IL-6, IL-1β and TNF-α were measured by sandwich Enzyme Linked Immunosorbent Assay (ELISA).

Results
Allele frequency spectrum after adjusting the effect of systolic blood pressure (SBP), diastolic blood pressure (DBP), total cholesterol (TC), low density lipoprotein (LDL), triglycerides (TG) and body mass index (BMI) revealed that major allele G of rs1800795 and T of rs10499563 were significantly associated with increased risk of OA (P < 0.01) in all the three genetic models; co-dominant (OR 4.08 & 4.12, P < 0.001), recessive (OR 3.00 & 2.51, P < 0.001) and dominant (OR 2.56 & 3.09, P < 0.05). Major allele G of rs1800796 and rs1800797 was observed to enhance OA risk in recessive mode (OR 1.75, P < 0.001 & 1.62, P = 0.01 respectively). Disease risk analysis after adjusting the effect of confounders exposed a susceptibility haplotype GGGGCT, which increased the OA risk by 2.27 times (OR 2.27, 95%CI: 1.26–4.10, P = 0.009) and a protective haplotype CGAGGC which significantly reduced the OA risk (OR 0.47, 95%CI 0.27–0.92, P = 0.031). Both of these haplotypes manifested in the recessive mode of inheritance. Subjects who had one copy of the susceptibility haplotype had lower values of IL-6 (3.6 pg/ml) and IL-1β levels (3.2 pg/ml) than those who had 2 copies of it (4.4 pg/ml & 4.2 pg/ml respectively). IL-6 and IL-1β levels were observed to be negatively associated with protective haplotype CGAGGC (P < 0.05). Carriers of 1 copy of this haplotype showed decreased IL-1β levels than those who had none (1.00 pg/ml vs. 1.3 pg/ml respectively) which further decreased to 0.9 pg/ml in those subjects who carried two copies of protective haplotype.

Conclusion
The present study discovered susceptibility (GGGGCT) and protective (CGAGGC) haplotypes within promoter region of IL-6 gene which influenced the plasma levels of IL-6 and IL-1β for the risk of osteoarthritis in the population of Punjab, India.