Genetic Variability in Markers of HLA-C Expression in Two Diverse South African Populations

<div><p>An insertion-deletion (indel) polymorphism within the 3′ untranslated region (UTR) of <i>HLA-C</i> has been shown to be involved in the regulation of HLA-C expression. Individuals who carry a deletion at this position exhibit increased HLA-C expression, which associates with lower viral set point in HIV-1 infected individuals. This <i>263</i> indel (rs67384697) is reported to be in strong linkage disequilibrium (LD) with a single nucleotide polymorphism (SNP) 35 kilobases upstream of <i>HLA-C</i> (<i>-35T/C</i>; rs9264942) in Caucasian individuals, making this SNP a potential marker for both HLA-C expression and HIV-1 disease progression. We therefore examined genetic variation within the <i>HLA-C</i> 3′ UTR of 265 Black and Caucasian South Africans by direct sequencing and identified haplotypes encompassing the <i>263</i> indel and another indel at position 230 in both populations. Concomitant evaluation of variability at the <i>−35</i> SNP revealed this polymorphism to be an inappropriate marker for the <i>263</i> indel in these populations. These findings provide important insights into genetic variability within the regulatory regions of <i>HLA-C</i> that have potential implications for our understanding of the regulation of HLA-C expression and its impact on HIV-1 disease progression.</p></div

Linkage disequilibrium between the <i>-35</i> SNP and the <i>HLA-C</i> alleles present in the Black and Caucasian South African population groups.

1<p>the total number of individuals genotyped in each population group.</p>2<p>the observed frequency of each two-locus haplotype.</p>3<p>Lewontin's D' measure of linkage disequilibrium <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067780#pone.0067780-Lewontin1" target="_blank">[19]</a>.</p>4<p>p-values are calculated using an exact test for linkage disequilibrium <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067780#pone.0067780-Slatkin1" target="_blank">[20]</a>, and are significant at p<0.05.</p>5<p>the number of chromosomes on which the two-locus haplotype was found to occur.</p

Genetic variation within <i>HLA-C</i> 3′ UTR sequences of the <i>HLA-C</i> alleles observed in the Caucasian South African population group.

1<p>positions are given relative to the start of the HLA-C 3′ UTR.</p>2<p><i>HLA-C</i> alleles <i>C*02∶02</i> and <i>C*03∶04</i> were found to have more than one 3′ UTR sequence.</p>3<p>where more than one allele has been observed alleles are reported using standard IUB ambiguity codes.</p>4<p>I refers to an insertion and D to a deletion.</p

The haplotypes identified within the <i>HLA-C</i> 3′ UTR.

<p>The positions involved in the two haplotypes identified are indicated in colour. The haplotype encompassing the <i>263</i> indel is shown in pink, while the haplotype encompassing the <i>230</i> indel is shown in blue. The major and minor alleles at each position are also indicated. Positions were only included in the haplotypes if both D' and r² measures of pairwise LD were equal to 1. Polymorphic positions are indicated by their position relative to the start of the <i>HLA-C</i> 3′ UTR.</p

Genetic variation within <i>HLA-C</i> 3′ UTR sequences of the <i>HLA-C</i> alleles observed in the Black South African population group.

1<p>positions are given relative to the start of the HLA-C 3′ UTR.</p>2<p><i>HLA-C</i> alleles <i>C*02∶02, C*02∶10, C*03∶04, C*07∶01, C*16∶01</i> and <i>C*18∶01</i> were found to have more than one 3′ UTR sequence.</p>3<p>where more than one allele has been observed alleles are reported using standard IUB ambiguity codes.</p>4<p>I refers to an insertion and D to a deletion.</p

Correlation of FOXP3 with CD4 count and viral load.

<p><i>Panel A</i>: CD4 count was negatively correlated with day 1 FOXP3 percentage in the HIV infected group. <i>Panel B</i>: The control group showed no correlation between CD4 count and FOXP3 percentage. Panel C: FOXP3 percentage stratified by CD4 count. <i>Panel D</i>: FOXP3 percentage was positively correlated with viral load.</p

Representative plot illustrating CD25 gating strategy.

<p>Lymphocytes were gated according to forward and side scatter. CD3 positive lymphocytes were selected. CD25 expression on the x-axis was plotted against CD4 on the y-axis. A gate was set according to CD25 expression on the CD4⁻ population. This gave a percentage of CD3⁺CD4⁺CD25⁺ lymphocytes as a percentage of CD3⁺ (figure in top-right quadrant). This percentage was then used to calculate the percentage of CD3⁺CD4⁺CD25⁺ lymphocytes as a percentage of the CD3⁺CD4⁺ population (CD4 population being top-left and right quadrants added together).</p

FOXP3 expression after T cell receptor stimulation.

<p><i>Panel A</i>: FOXP3 expression as a percentage of T lymphocytes at baseline and after 4 days of cell culture, with and without T cell receptor stimulation with anti-CD3. <i>Panel B</i>: Proliferation of total CD4⁺ T cells and FOXP3⁺ expression in proliferated CD4⁺ T cells following T cell receptor stimulation with aCD3. <i>Panel C</i>: Representative plot of FOXP3⁺ expression in proliferated T cells (left-hand plot) following anti-CD3 stimulation compared with an unstimulated sample(right-hand plot). The small plots above show ancestry – lymphocytes were gated; followed by exclusion of events with high CFSE; followed by selection of CD3⁺CD4⁺ T cells. Proliferation is demonstrated by halving of CFSE fluorescence in cells that have divided (large plots below). In the anti-CD3 stimulated sample, FOXP3 expression is noted in cells which have proliferated (top-left quadrant) as well as those that have not proliferated (top-right quadrant).</p

FOXP3 expression at baseline in HIV infected patients.

<p><i>Panel A</i>: Subgroup analysis of the HIV and HIV/TB coinfected groups showed no difference in FOXP3 expression as a percentage of CD3⁺CD4⁺ T lymphocytes between the HIV and HIV/TB co-infected groups. <i>Panel B</i>: Representative plots of baseline FOXP3 expression in CD3⁺CD4⁺ lymphocytes plotted against CD25 for a control and an HIV positive sample. <i>Panel C</i>: Expression of FOXP3, CD25, CTLA-4 and GITR as a percentage of the CD3⁺CD4⁺ population. <i>Panel D</i>: Absolute numbers of total and FOXP3 expressing CD4⁺ T cells at baseline.</p

Associations between age at starting ART and cell-associated HIV-1 DNA levels after a median of 4.3 years of treatment among 146 HIV-infected children.

<p>a: Scatterplot of HIV-1 DNA log₁₀ copies/10⁶ cells (Y-axis) by age at ART start in months (x-axis). LOcally WEighted Scatter-plot Smoother (LOWESS) and linear regression prediction lines are shown in blue and red, respectively; and the grey area shows the 95% prediction elipse. b: Box and whisker plot of HIV-1 DNA log₁₀ copies/10⁶ cells by age at ART start groups; c: Histograms and estimated normal and kernel distributions of HIV-1 DNA log₁₀ copies/10⁶ cells among those who started ART <4.5 and ≥4.5 months of age.</p