Efficient Arsenic Metabolism — The <em>AS3MT</em> Haplotype Is Associated with DNA Methylation and Expression of Multiple Genes Around <em>AS3MT</em>

<div><p>Arsenic is a very potent toxicant. One major susceptibility factor for arsenic-related toxicity is the efficiency of arsenic metabolism. The efficiency, in turn, is associated with non-coding single nucleotide polymorphisms (SNPs) in the arsenic methyltransferase <em>AS3MT</em> on chromosome 10q24. However, the mechanism of action for these SNPs is not yet clarified. Here, we assessed the influence of genetic variation in <em>AS3MT</em> on DNA methylation and gene expression within 10q24, in people exposed to arsenic in drinking water. DNA was extracted from peripheral blood from women in the Argentinean Andes (N = 103) and from cord blood from new-borns in Bangladesh (N = 127). <em>AS3MT</em> SNPs were analyzed with Sequenom or Taqman assays. Whole genome epigenetic analysis with Infinium HumanMethylation450 BeadChip was performed on bisulphite-treated DNA. Whole genome gene expression analysis was performed with Illumina DirectHyb HumanHT-12 v4.0 on RNA from peripheral blood. Arsenic exposure was assessed by HPLC-ICPMS. In the Argentinean women, the major <em>AS3MT</em> haplotype, associated with more efficient arsenic metabolism, showed increased methylation of <em>AS3MT</em> (p = 10⁻⁶) and also differential methylation of several other genes within about 800 kilobasepairs: <em>CNNM2</em> (p<10⁻¹⁶), <em>NT5C2</em> (p<10⁻¹⁶), <em>C10orf26</em> (p = 10⁻⁸), <em>USMG5</em> (p = 10⁻⁵), <em>TRIM8</em> (p = 10⁻⁴), and <em>CALHM2</em> (p = 0.038) (adjusted for multiple comparisons). Similar, but weaker, associations between <em>AS3MT</em> haplotype and DNA methylation in 10q24 were observed in cord blood (Bangladesh). The haplotype-associated altered CpG methylation was correlated with reduced expression of <em>AS3MT</em> and <em>CNNM2</em> (r_s = −0.22 to −0.54), and with increased expression of <em>NT5C2</em> and <em>USMG5</em> (r_s = 0.25 to 0.58). Taking other possibly influential variables into account in multivariable linear models did only to a minor extent alter the strength of the associations. In conclusion, the <em>AS3MT</em> haplotype status strongly predicted DNA methylation and gene expression of <em>AS3MT</em> as well as several genes in 10q24. This raises the possibility that several genes in this region are important for arsenic metabolism.</p> </div

Correlations^* and effect estimates (β) ^† between total urinary arsenic (As), DNA methylation and gene expression in Argentina, as well as the influence of haplotype on gene expression.

<p>A. denotes CpG sites that were positively associated with the <i>AS3MT</i> haplotype and B. denotes CpG sites that were inversely associated with the haplotype.</p>*<p><i>r</i>_s and p-value from Spearman correlation.</p>†<p>β and p-value from multivariable regression.</p>‡<p>Model (the β stated in the table is β₁): Gene expression = α+β₁× total urinary arsenic (natural ln transformed)+β₂× RIN.</p>§<p>Model (the β stated in the table is β₁): Gene expression = α+β₁×copies of <i>AS3MT</i> haplotype+β₂× total urinary arsenic (natural ln transformed)+β₃×RIN.</p>¶<p>Model (the β stated in the table is β₁): Gene expression = α+β₁×methylation level+β₂× total urinary arsenic (natural ln transformed)+β₃×RIN.</p

The relationship between <i>CNNM2</i> DNA methylation and <i>AS3MT</i> gene expression, stratified for <i>AS3MT</i> haplotype.

<p>Scatterplot depicting the relationship between DNA methylation (expressed as fraction of methylated CpG) for <i>CNNM2</i> (X-axis) and gene expression for <i>AS3MT</i> (3′UTR; expression relative to the median for individuals with 2 copies of the <i>AS3MT</i> haplotype) (Y-axis), where individuals are stratified for <i>AS3MT</i> haplotype (total N  = 48). Outliers are denoted by *; the outlier to the left has a relative gene expression of 22.8 and the outlier to the right has a relative gene expression of 24.9.</p

Statistically significant correlations between <i>AS3MT</i> haplotype and DNA methylation in either Argentina or in Bangladesh, as well as descriptions of the DNA methylation sites.

*<p>According to NCBI Ref. Sequence: NC_000010.10, H sapiens chromosome 10, GRCh37.p5 Primary Assembly <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053732#pone.0053732-NCBI2" target="_blank">[42]</a>.</p>†<p>Situated in a CpG island, shore, or shelf according to Emboss CpGPlot <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053732#pone.0053732-Rice1" target="_blank">[41]</a>.</p>‡<p>Enhancer elements were defined according to Illumina’s annotations (<a href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL13534" target="_blank">http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL13534</a>).</p>§<p>According to <a href="http://www.rforge.net/IMA/snpsites.txt" target="_blank">http://www.rforge.net/IMA/snpsites.txt</a>. The numbers in parentheses indicate the distance of the SNP from the CpG site.</p>¶<p><i>r</i> = Pearson product-moment correlation coefficient.</p>∥<p>Beta (β)-values represent the fraction of methylation and hence range from 0 (unmethylated) to 1 (methylated).</p><p>Abbreviations: FDR; False Discovery Rate, SNP = Single nucleotide polymorphism.</p

Characteristics of the study subjects in Argentina and Bangladesh.

<p>For comparison, also the full study population in Argentina <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053732#pone.0053732-Engstrom1" target="_blank">[12]</a> is included.</p>*<p>Median stratified for haplotype. N = 7 (0 copies), 41 (1 copy), and 45 (2 copies) in Argentina (one individual missing haplotype data), N = 90 (0 copies), 32 (1 copy), 5 (2 copies) in Bangladesh, and N = 17 (0 copies), 71 (1 copy), and 84 (2 copies) in the full study population in Argentina.</p>†<p>P-values from non-parametric Kruskal-Wallis test, numbers of haplotype copies is the grouping variable.</p>‡<p>Adjusted for specific weight. Measured at gestational week 6–8 in Bangladesh.</p>§<p>NA =  not applicable, since no RNA was available for analysis in the Bangladeshi study population.</p

Univariable and multivariable regression analyses comprising DNA methylation sites that were significantly correlated with <i>AS3MT</i> haplotype in either Argentina or in Bangladesh.

*<p>The β–coefficient refers to the slope of the regression line (change in methylation level) with increasing numbers of copies of the haplotype.</p>†<p>β and p-value from univariable regression. The models look as follows: Methylation level = α+β₁×copies of <i>AS3MT</i> haplotype.</p>‡<p>β and p-value from multivariable regression (Argentina). The model look as follows: Methylation level = α+β₁×copies of <i>AS3MT</i> haplotype+β₂×total urinary arsenic (natural ln transformed)+β₃×age+β₄×use of coca leaves (yes/no).</p>§<p>β and p-value from multivariable regression (Bangladesh). The model look as follows: Methylation level = α+β₁×copies of <i>AS3MT</i> haplotype+β₂× total arsenic in maternal urine around gestational weeks 6–8 (natural ln transformed)+β₃×maternal age+β₄×sex of child.</p

Map of genes in chromosome band 10q24 associated with <i>AS3MT</i> haplotype-related DNA methylation.

<p>Positions of DNA methylation and gene expression assays are also shown. Black circles denote DNA methylation sites statistically significantly associated with <i>AS3MT</i> haplotype in Argentina and the white circle denotes the DNA methylation site statistically significantly associated with <i>AS3MT</i> haplotype in Bangladesh. A grey box around the DNA methylation ID denotes DNA methylation sites statistically significantly associated with <i>AS3MT</i> haplotype in both Argentina and Bangladesh.</p