Allele-specific bisulfite pyrosequencing.

<p>To analyze the allele-specific DNA methylation, we individually measured the DNA methylation of each allele and compared methylation between the two alleles. Thus, the allele-specific polymerase chain reaction (PCR) was performed and the methylation levels of each of the two alleles, given allele-specific amplicons, were measured separately using pyrosequencing. The allele-specific PCR primers were designed such that each primer of 3′ end was a pair of base of heterozygous SNP. (a) The reference sequence of rs36221701 before and after bisulfite conversion. Rs36221701 contained two CpG sites denoted as CpG1 and CpG2 in 5′ to 3′ direction. By bisulfite conversion, unmethylated C is converted to U (U is converted to T by PCR). Rs362210701 (C/T) is converted to (T/T); it was impossible to distinguish the converted T and original T. Thus, we were unable to detect the origin of the allele for this SNP by PCR primers. (b) Allele-specific PCR. We designed PCR primer for rs13239907 (A/G), which is located 297 bp downstream of rs36221701 and was in complete linkage disequilibrium with rs36221701 (A→C, G→T: r² = 1.0) in JPT data from 1,000 genome project. The forward primer was common to both alleles; reverse primers were designed such that each primer of 3′ end was a pair of bases of genotypes (A or G: complementary strand). (c) Allele-specific pyrosequencing. The two given allele-specific amplicons were separately analyzed using pyrosequencing. Sequence primer 1 targeted rs13239907 and was used to check the accuracy of allele-specific PCR, whereas sequence primer 2 targeted CpG1 and CpG2 was used to analyze the ratio of methylation around rs36221701. (d) Primer details. The 3′ end of PCR primer R was a pair of base of heterozygous SNP. 5′ end of PCR primer R was biotinylated.</p

The relation between genotype of rs36221701 and the expression levels of genes around rs36221701.

<p>The relation between genotype of rs36221701 and the expression levels of genes around rs36221701.</p

Analysis of CpGs methylation levels around rs36221701 comparing with C and T allele in heterozygous samples.

<p>Analysis of CpGs methylation levels around rs36221701 comparing with C and T allele in heterozygous samples.</p

The relation between rs36221701 genotype and SMAD3 expression.

<p>The scatter plot shows the expression levels of the genotypes.*Simple linear regression analysis.</p

The signal cluster plot in typical allele-specific DNA methylation (ASM) by methylation-sensitive SNP array (MSNP) analysis.

<p>(a, b, c) Array data of rs1130368 located around inflammatory bowel disease (IBD) susceptibility gene showing ASM. Rs1130368 is located on the exon of <i>HLA-DQB1</i> (6p21. 32). (a) All samples, (b) Homozygous samples, (c) Heterozygous samples. Heterozygous changed to homozygous after digestion ( = 0.23 was the top score in candidates). This change was monoallelic (G/T changed only T/T); thus, ASM regulated in <i>cis</i> with genotype and not genomic imprinting. This indicated that hypermethylation existed around T allele, whereas hypomethylation existed around G allele. (d, e, f) Array data of rs36221701 located around IBD susceptibility gene showing ASM. Rs36221701 is located on the upstream of <i>SMAD3</i> (15q22.33). (d) All samples, (e) Homozygous samples, (f) Heterozygous samples. Heterozygous samples tended to change to homozygous after digestion ( = 0.14). This change was monoallelic (C/T changed only C/C); thus, ASM regulated in <i>cis</i> with genotype and not genomic imprinting. This indicated that hypermethylation existed around C allele, whereas hypomethylation existed around T allele. RAS, relative allele score.</p

Flowchart of <i>cis</i>-regulating allele-specific DNA methylation SNP candidate selection around IBD susceptibility genes.

<p>SNP, single-nucleotide polymorphism; MSRE, methylation-sensitive restriction enzyme; RAS, relative allele score; IBD, inflammatory bowel disease; (G), genomic DNA; (D), DNA digested MSREs; (U), whole-genome-amplified fully unmethylated DNA digested MSREs.</p

Six of candidate <i>cis</i>-regulating allele-specific DNA methylation SNPs around inflammatory bowel disease susceptibility genes.

<p>Six of candidate <i>cis</i>-regulating allele-specific DNA methylation SNPs around inflammatory bowel disease susceptibility genes.</p

Clinical characteristics of the patients with Crohn’s disease and ulcerative colitis.

<p>Clinical characteristics of the patients with Crohn’s disease and ulcerative colitis.</p