Genetic association study of interleukin 6 receptor and liver X receptor in systemic lupus erythematosus

Abstract

Background: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by polyclonal B-cell activation and elevated production of pathogenic autoantibodies. The human interleukin 6 receptor (IL6R) consists of 2 membrane-bound glycoproteins interacting with IL6, which is a multifunctional cytokine essential to the regulation of the immune response and acute-phase reaction. Liver X receptor alpha (LXRA, NR1H3) and beta (LXRB, NR1H2) are established sensors of lipid and cholesterol homeostasis. The recent studies have reported that LXRs are involved in regulation of inflammation and immune responses.

Objective: We attempted to identify single nucleotide polymorphisms (SNPs) of the IL6RA, IL6RB, NR1H3 and NR1H2 genes, associated with the susceptibility to SLE in Korean populations, and to elucidate their significance in clinical phenotypes of SLE.

Materials and Methods: Blood samples were collected from Korean SLE patients diagnosed in the rheumatology clinic at the Ajou University Hospital and normal healthy controls (NC). All of the patients fulfilled the American College of Rheumatology classification criteria for SLE. Genomic DNA was extracted and IL6RA, IL6RB, NR1H3 and NR1H2 genes were amplified by polymerase chain reaction. We screened for genetic variations in the IL6RA, IL6RB, NR1H3 and NR1H2 genes using directed sequencing. SNP genotyping was performed by using the SNaPSHOT ddNTP primer extension kit. The transcriptional activity according to SNP genotype was analyzed by luciferase reporter assay in Hep3B cells and COS-7 cells. To investigate the effects of the stimulation, we used a functional assay of transcriptional activity and B cell proliferation assay with lipopolysaccharide, GW3965 and T0901317.

Results: We have identified three SNPs (-208 G>A in the promoter region, 48841 T>A C in the intron 8 region and 48864 A>C in the exon 9 region) in the IL6R？？ gene. There were no significant differences between SLE and NC in the observed genotype frequencies. The 48864 A>C polymorphism was significantly associated with rash (p=0.008). The five common haplotypes for three polymorphisms were constructed: HT1 [GTA], HT2 [ATC], HT3 [ATA], HT4 [GTC] and HT5 [GAA]. There were no significant differences between SLE and NC. However, the frequency of rash (p=0.037), leukopenia (p=0.033) and lymphopenia (p=0.027) were significantly higher in SLE patients having the haplotype HT2 [ATC].

In addition, we identified five polymorphisms (-1851 T>C and -1830 T>C in the promoter region, and -1003 G>A, -840 C>A and -115 G>A in the intron 1 region) including one novel SNPs (-1003 G>A) in the NR1H3 gene. Two SNPs in the NR1H3 gene, -840 C>A and -115 G>A, showed the complete linkage disequilibrium. Therefore, -840 C>A was excluded in genotype and haplotype analysis. There was significant difference in the -1830 T>C polymorphism (co: p=0.001, re: p=0.002), -1003 G>A polymorphism (co: p=0.002, re: p=0.002), -115 G>A polymorphism (co: p<0.001, re: p=0.001) between SLE and NC. Three common haplotypes for four polymorphisms were constructed: HT1 [TTGG], HT2 [CTGG] and HT3 [TCAA]. There was significant difference between SLE and NC in the observed haplotype HT1 [TTGG] (co: p=0.033, re: p=0.012) and HT3 [TCAA] (co: p=0.008, do: p=0.009). In the -1830 T>C polymorphism, arthritis was significantly more common in the SLE patients with the -1830 C allele (p=0.005). The -1003 G>A polymorphism was significantly associated with oral ulcer (p=0.039), arthritis (p=0.006), anti-dsDNA (p=0.041) and elevated triglyceride (p=0.007). The -115 G>A polymorphism was significantly associated with oral ulcer (p=0.024), arthritis (p=<0.001) and elevated triglyceride (p=0.011). The frequency of arthritis was significantly lower in patients who had haplotype HT1 [TTGG] (re: p=0.004), however arthritis was more common in patients with HT3 [TCGG] (do: p=0.003). The frequency of lymphopenia was significantly lower in patients who had haplotype HT2 [CTGG] (re: p=0.027). Luciferase activity of the constructs containing -1830 T and -1003 G was higher than that of the constructs containing -1830 C and -1003 A (p=0.009). The same results was observed in the GW3965 and T0901317 treated cells (p=0.034 and p<0.001, respectively). Proliferation rate of the B cells having -1830 TC type was higher than the B cells having -1830 TT type in the basal, GW3965 and T0901317 treated cells from SLE patients (p=0.011, p= 0.040 and p=0.017, respectively). Proliferation rate of the B cells having -1830 TC type was increased when compared to that of the B cells having -1830 TT type in T0901317 treated cells from NC (p=0.032).

Conclusion: These results suggest that genetic polymorphisms within IL6RA gene may be associated with disease phenotype of SLE in Koreans. Also, the NR1H3 gene genetic polymorphisms may be associated with disease susceptibility and clinical manifestations of SLE in Korean population. Specially, -1830 T>C polymorphism within NR1H3 promoter region may be involved in regulation of NR1H3 expression.