Do genetic variants in the TLR4 promoters and role of innate immune system play a role in gout susceptibility?
Gout is a form of inflammatory arthritis caused by the deposition of monosodium urate crystals in the joints as a result of hyperuricemia and subsequent innate immune response. Proteins encoded by Toll-like receptor 4 (TLR4) and Nucleotide-binding leucine-rich repeat-containing receptor protein 3 (NLRP3) play a crucial role in the initiation and progression of MSU crystals-induced gouty inflammation. Previously, TLR4 rs2149356 (T-allele), which is in high linkage disequilibrium with variants in the TLR4 promoter region showed an association with a higher risk of with gout in Han Chinese/New Zealand (NZ) Europeans but with a protective effect in NZ Polynesians. There is also evidence for a multiplicative interaction between Interleukin 1 beta (IL-1β) and caspase recruitment domain family member 8 (CARD8) in the NZ populations. Therefore, this study attempted to investigate whether TLR4 promoter variants and variations in other TLR4/NLRP3 related genes confer gout risk in European and Polynesian individuals. Five TLR4 promoter variants, 55 functional variants along with genetic loci within 100kb upstream/downstream of genes in TLR4/NLRP3 signalling cascade were tested for an association with gout in the United Kingdome (UK) Biobank dataset (2149 cases, 102709 controls). Significant signals were replicated in NZ European (1066 cases, 1128 controls) and Polynesian (1369 cases, 1502 controls). Haplotype-based association and variant- variant interaction analysis of the TLR4/NLRP3 tested variants were also investigated for an association with gout in the UK Biobank and NZ datasets. Multiple adjusted logistic regression analysis was carried out using R version 3.4.1 and PLINK2. In addition, an attempt in optimization of transfection method involving THP-1 cell line was undertaken to investigate the functional effect of TLR4 promoter variants with susceptibility to gout. The TLR4: rs10983755 and rs1927914 (A allele), rs4986790 (Asp299Gly) G allele, rs1554973 C allele, and the TLR4 promoter haplotypes AGAGC and AGGGT were found to be associated with a higher risk of gout in the UK Biobank dataset. However, in the NZ dataset, only the rs10983755 (A allele) and rs1554973 (C allele) showed a protective association with gout in Easter/all Polynesians and in the mixed Eastern/Western Polynesians, respectively. Additionaly, the minor allele of four loci in TLR4, three in NLRP3, three in CARD8, two in Interleukin I receptor Type I (IL-1R2) and four tagged loci in Receptor-interacting serine threonine-protein kinase 1 (RIPK1) displayed evidence of nominal association (P<0.05) with gout in the UK Biobank dataset and, of which only, one locus in the IL-1R2 acheived the Bonferroni corrected P-value of 0.0003. Some of these loci also showed an association with gout in the NZ datasets, but mostly with opposite direction of effect. Haplotype analysis revealed that the TLR4 haplotypes CT; CAT, AGC, GTGTCC; ATACCC, NLRP3 CCGG and a CARD8 haplotype TGC were associated with a higher risk of gout in UK individuals. While the TLR4 haplotypes AGC; GGACTT, NLRP3 TTAA and CARD8 CAT were found to be associated a lower risk of gout. The haplotype NLRP3 TTAA also presented an association with gout in the NZ datasets but with the opposite direction in the UK Biobank dataset. The variant-variant interaction analysis indicated an epistatic interaction of the TLR4 rs1155928/NLRP3 rs10925010, TLR4 rs913615/CARD8 rs10500299, TLR4 rs486790/IL- 1R2 rs4851522) and TLR4 rs913615/RIPK1 rs9392453 with gout in the UK Biobank dataset (Pinteraction = 0.01, 0.01, 0.02 and 0.02). No interaction was replicated in the NZ datasets and none achieved the Bonferroni corrected threshold. Nonetheless, the genotype combination of the TLR4 rs4846989 (TT)/CARD8 rs17294094 (GG) (in the NZ Polynesian), TLR4 rs1360094 (AG and AA)/IL-1R2 rs4851522 (CC) and TLR4 rs1554973/IL-1R2 rs4851522 (CT/CC) (in NZ European) demonstrated an association with an elevated risk of gout in the UK Biobank, also a displayed similar trend of association in at least in one of the NZ datasets. Finally, the transfection of THP-1 cell line using the NeonTM Transfection System showed the highest number of GFP positive cells under the parameter 1400v, 20ms and 1 pulse, but this method requires further optimization before it can be used for functional investigations. Collectively, this research provides an insight that variants in the TLR4 promoter region and additional loci in the TLR4/NLRP3 signalling cascades might be influencing gout pathology.
Advisor: Merriman, Tony; McKinney, Cushla
Degree Name: Master of Science
Degree Discipline: Department of Biochemistry
Publisher: University of Otago
Keywords: New Zealand
Research Type: Thesis