SLC11A1 promoter polymorphisms, gene expression and association with autoimmune and infectious diseases
- Publication Type:
- Thesis
- Issue Date:
- 2012
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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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