SLC11A1 promoter polymorphisms, gene expression and association with autoimmune and infectious diseases
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Solute Carrier Family 11A Member 1 (SLC11A1) is a member of a highly conserved group of ion transporters and has restricted localisation to the phagosomal membrane of monocytes/macrophages. SLC11A1 plays an immunomodulatory role in influencing macrophage activation status and the T helper 1/T helper 2 bias. As such it modulates susceptibility to infectious/autoimmune diseases. A polymorphic (GT)n promoter microsatellite repeat is known to alter SLC11A1 promoter activity. Of the nine (GT)n alleles identified, alleles 3 and 2, which account for a combined allele frequency of greater than 95%, drive high and low SLC11A1 expression, respectively. The increased SLC11A1 expression, driven by (GT)n allele 3 is hypothesised to result in a heightened activation status of classically activated macrophages, affording resistance to infectious disease, but conferring susceptibility to pro-inflammatory autoimmune diseases. Conversely, decreased SLC11A1 expression in the presence of allele 2 would confer susceptibility to infectious disease, but resistance to autoimmune disease. A large number of studies assessing the association between the presence of specific (GT)n promoter alleles with the incidence of infectious and autoimmune disease have produced inconsistent associations. Meta-analyses are powerful analytical tools which combine individual association studies to estimate the strength of an association, therefore, meta-analyses of case control association studies (from 1991-2006) analysing the association of SLC11A1 promoter (GT)n alleles 2 and 3 with the incidence of autoimmune disease were performed. The meta-analyses found a weak predominance of disease in the absence of allele 2, with a fixed effects pooled OR of 0.80 (95% CI = 0.22), however, a random effects pooled odds ratio (OR) of 0.88 (95% CI = 0.66) for allele 3 suggested no association with the incidence of autoimmune disease. The publication of additional case control studies between 2006 and the present allowed a more comprehensive meta-analysis to be completed. This analysis, which included additional SLC11A1 polymorphisms, represents the largest study assessing the association of SLC11A1 polymorphisms with disease occurrence to date. Allele 2 of the (GT)n microsatellite was associated with increased and reduced incidence of infectious [OR=1.32 (1.20-1.46)] and autoimmune diseases [OR=0.90 (0.81-1.00)], respectively. Allele 3 was significantly associated with reduced incidence of infectious disease [OR=0.82 (0.76-0.88)], however, the association with susceptibility to autoimmune disease occurrence did not reach statistical significance [OR=1.11 (0.98-1.26)]. The findings of the meta-analysis challenges the hypothesis that allele 3 is the disease causing variant at the (GT)n microsatellite repeat. The results of these meta-analyses highlight small sample sizes as a major limitation of case control association studies. Completion of large-scale studies has been impractical because conventional SLC11A1 (GT)n genotyping methodologies are time consuming and cannot differentiate all (GT)n variants. A high resolution melt curve methodology has been designed and optimised to genotype two SLC11A1 polymorphisms, the (GT)n and (CAAA)n microsatellite repeats. Assay validation yielded a 100% success rate for genotyping of the (GT)n and (CAAA)n microsatellites. The designed methodology is the first to enable accurate, sensitive and high-throughput genotyping of these microsatellites and will enable the completion of sufficiently large association studies required to determine the association between the SLC11A1 (GT)n and (CAAA)n polymorphisms and disease occurrence. In addition to the (GT)n microsatellite, the -237C/T polymorphism has also been shown to modulate SLC11A1 expression, with the T variant driving low expression in the presence of (GT)n allele 3. Little is known about SLC11A1 transcription or the mechanism by which the (GT)n and -237C/T promoter polymorphisms modulate SLC11A1 expression. Bioinformatic studies were completed to identify putative regulatory elements involved in transcription and promoter constructs, containing different lengths of the SLC11A1 promoter, were prepared and used to assess promoter function. A 581bp promoter region (-532 to +49) that controlled SLC11A1 expression in monocytes was identified. Within this region was identified a 148bp minimal promoter region (-99 to +49) containing the core elements for the formation of the basal transcriptional complex. The greatest transcriptional enhancement was identified within a 170bp region (-532 to -362) containing a novel IRF-Ets composite sequence for the recruitment of transcription factors IRF-8 and PU.1. Additionally, the promoter constructs suggested that the SLC11A1 promoter may mediate bidirectional transcription. It was further determined that, in monocytic cells, the ability of (GT)n alleles 2 and 3 to differentially modulate SLC11A1 expression was not due to their differing abilities to form Z-DNA, but to monocyte-specific factor(s) binding to a 165bp region (-362 to -197) of the SLC11A1 promoter. Additional bioinformatic and functional assays suggested that the T variant of the -237C/T polymorphism reduced SLC11A1 promoter activity independently of the (GT)n microsatellite repeat. Infectious and autoimmune diseases are major contributors to morbidity and mortality. SLC11A1 is instrumental in regulating macrophage function and hence susceptibility to infectious and autoimmune diseases. This study has provided insight into the association of SLC11A1 with disease incidence, has developed a novel genotyping methodology to allow the completion of large association studies and has elucidated mechanisms of transcriptional regulation of SLC11A1 and the influence of polymorphisms on SLC11A1 expression.
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