An investigation of the functional significance of 5'-flanking region polymorphisms of interleukin-6 in systemic arthritis
This thesis investigates the hypothesis that the high levels of IL-6 in SA is pathogenic and is influenced by polymorphisms in the 5'-flanking region of IL-6, resulting in altered IL-6 transcriptional regulation, susceptibility to and severity of SA. Genotype studies of SA cases and controls found the -174G/C polymorphism to be associated with SA. The -174CC genotype was under-represented in SA and this was due to under-representation of this genotype in the early disease onset group ( 5 years of age), indicating association with susceptibility and severity of SA. The -572G/C and -597G/A polymorphisms were not associated with disease despite strong allelic association between -174C -597A and weak allelic association between -174G -572C. The most common naturally occurring haplotypes were -597G-572G-174G and-597A-572G-174C. In HeLa cell transfections with luciferase reporter constructs stimulated with IL-lB or TNF , the -174G allele was associated with higher levels of expression than the -174C allele. The -597 polymorphism lacked independent function, whereas the -572G allele was associated with higher levels of expression than -572C. The AT-tract polymorphism showed functional influence, with A8T12 associated with higher levels of expression than A9T11. These effects were cell-specific, with no difference in levels of transcription between haplotypes in Huh7 cells. Deletion mutant constructs indicated that the -174G/C allelic effect was modulated by a region between -310 and -550, which includes the AT-tract. Dexamethasone inhibition of IL-1-induced IL-6 transcription was associated with significantly more inhibition of expression for the -174G allele than-174C. Electrophoretic mobility shift assays with HeLa cell nuclear extracts found a transcription factor(s) bound to the IL-6 gene between -134 and -184 more strongly with the -174C allele than -174G. This appeared to involve C/EBPB, and NF1 may have been involved. An additional novel finding was the binding of an IL-1B inducible factor between -161 and -235.