Allele-Specific Methylation Occurs at Genetic Variants Associated with Complex Disease

We hypothesize that the phenomenon of allele-specific methylation (ASM) may underlie the phenotypic effects of multiple variants identified by Genome-Wide Association studies (GWAS). We evaluate ASM in a human population and document its genome-wide patterns in an initial screen at up to 380,678 sites within the genome, or up to 5% of the total genomic CpGs. We show that while substantial inter-individual variation exists, 5% of assessed sites show evidence of ASM in at least six samples; the majority of these events (81%) are under genetic influence. Many of these cis-regulated ASM variants are also eQTLs in peripheral blood mononuclear cells and monocytes and/or in high linkage-disequilibrium with variants linked to complex disease. Finally, focusing on autoimmune phenotypes, we extend this initial screen to confirm the association of cis-regulated ASM with multiple complex disease-associated variants in an independent population using next-generation bisulfite sequencing. These four variants are implicated in complex phenotypes such as ulcerative colitis and AIDS progression disease (rs10491434), Celiac disease (rs2762051), Crohn's disease, IgA nephropathy and early-onset inflammatory bowel disease (rs713875) and height (rs6569648). Our results suggest cis-regulated ASM may provide a mechanistic link between the non-coding genetic changes and phenotypic variation observed in these diseases and further suggests a route to integrating DNA methylation status with GWAS results

Types of allele-specific methylation candidates.

<p>Plots showing number of different categories of ASM candidates within the microarray study sample population. Of the 242533 MPRs for which there were at least 6 heterozygotes within the population (left pie chart) we detected some level of ASM in at least 116045 (left pie chart, green), of these we detected ASM in at least 6 samples in 12032 MPRs (left pie chart, dark green). Of these 12032 ASM MPRs, we detected cis-regulated ASM in 9750 MPRs (right pie chart, solid blue or solid yellow), and random or stochastic ASM in 2282 MPRs (right pie chart, mixed blue and yellow). Representation of patterns of allelic-choice in ASM within the microarray study sample population. ASM allelic choice is shown at 28 ASM and 2 non-ASM MPRs for the 42 samples in our initial microarray sample population. Non-heterozygous samples (white), samples with biallelic methylation (grey), and samples with ASM (blue and yellow) with methylation at either Allele-A (yellow) or Allele-A (blue) are shown. MPRs are organized in columns to show those determined to have no ASM (first two columns), cis-regulated ASM (both for Allele-A (3rd to 11th columns) and Allele-B (15th to final columns) or random ASM (12th to 14th columns).</p

Candidate cis-regulated ASM variants in phenotypically implicated SNPs.

<p>Mean number of candidate cis-regulated ASM variants in ten random LD pruned (ld<0.3) sets of candidate cis-regulated ASM SNPs that are also in high LD (LD>0.8) with a Genome Wide Association Study (GWAS) derived variant from the National Human Genome Research Institute (NHGRI) database or an eQTL in monocytes or peripheral blood monocytes (PBMCs), with accompanying standard deviations of the mean (sd).</p

Microarray based detection of allele-specific methylation.

<p><b>A)</b> A simplified representation of the Methyl-Sensitive Restriction Enzyme (MSRE) based Allele-Specific Methylation (ASM) assay. DNA is MSRE treated (left panels) and MSRE sites with methylated CpGs protected from digestion (upper panels, Allele-A) while its homologous chromosomal region with unmethylated CpGs are not (lower panels, Allele-B). The DNA is digested with StyI and NspI to form 200–1200 bp fragments, linkers ligated and DNA amplified to create amplicons that are hybridized to the array. Only regions with protected MSRE sites (methylated CpG) are amplified and can hybridize to show signal on the array (final panel). <b>B)</b> Bioinformatic detection of Allele-Specific Methylation (ASM) from Affymetrix SNP 6.0 arrays signals after MSRE digestion. In the scatter plot on the left, 4 different expected states after MSRE digest at a heterozygous region are compared to the typical distribution of probe intensities observed within the HapMap samples for the same MPR (here portrayed by light grey squares): biallelic methylation (dark grey circles), monoallelic A methylation (blue circles), monoallelic B methylation (yellow circles) and finally biallelic lack of methylation (red circles). The primary calling method relies on feature extraction by way of conversion of 2-dimensional A and B probe intensity data (scatter plot) from heterozygotes to log2(A/B) values and is compared against the typical log2(A/B) distribution observed for this MPR within the HapMap samples (histogram, light grey). Simply put, MPRs diverging from this distribution after MSRE treatment are called ASM. Using this method, biallelic unmethylated states have the potential to result in false positive ASM calls as any log2(A/B) value would be based on background noise, so are filtered out by removing MPRS with low total intensities (highlighted here with a red quarter-circle, for further information on how this filter was devised, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098464#pone.0098464.s008" target="_blank">Figure S8</a>).</p

Confirmation of ASM in a subset of candidate cis-regulated ASM variants.

<p>Results from microarray and next-generation bisulfite sequencing ASM assays of ten variant-containing regions. The table shows the CpG position, whether it is found within an MSRE site and the Bonferroni adjusted chi-square based p-values of the association of methylation at this CpG with either the reference of alternate alleles. The allele with the highest number percentage of methylated reads was designated the most frequently methylated allele (REF = reference, ALT = alternate) and compared to that from the microarray data; for all CpGs that were within MSRE sites and showed significant association of methylation with an allele in the sequencing assay the methylated allele matched that of the microarray assay. For more details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098464#pone.0098464.s020" target="_blank">Table S3</a>.</p

Genomic context of cis-regulated allele-specific methylation events.

<p>Illustrations showing genomic context and individual CpG methylation levels at three separate MPRs (rs10491434 (left), rs6569648 (middle) and rs943049 (right)). The chromosomal location of each amplicon is demonstrated with an ideogram. and the RefSeq genes (orange) surrounding the amplicon (red line) shown below. A section (grey box) contracts to the amplicon region itself to show the relative positions of the SNPs (black lines) and CpGs (blue lines) within the amplicons themselves (red bars); methylation levels of the alternate (grey circles) and reference (yellow circles) alleles within samples heterozygous for the SNP are graphed below each CpG. Asterixes (*) mark the CpGs illustrated within <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098464#pone-0098464-g003" target="_blank">Figure 3</a>.</p