Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease

Hirofumi Chiba; Yoichi Kakuta; Yoshitaka Kinouchi; Yosuke Kawai; Kazuhiro Watanabe; Munenori Nagao; Takeo Naito; Motoyuki Onodera; Rintaro Moroi; Masatake Kuroha; Yoshitake Kanazawa; Tomoya Kimura; Hisashi Shiga; Katsuya Endo; Kenichi Negoro; Masao Nagasaki; Michiaki Unno; Tooru Shimosegawa

doi:10.1371/journal.pone.0194036

Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease

PLoS One. 2018 Mar 16;13(3):e0194036. doi: 10.1371/journal.pone.0194036. eCollection 2018.

Authors

Hirofumi Chiba¹, Yoichi Kakuta¹, Yoshitaka Kinouchi², Yosuke Kawai³, Kazuhiro Watanabe⁴, Munenori Nagao⁴, Takeo Naito¹, Motoyuki Onodera¹, Rintaro Moroi¹, Masatake Kuroha¹, Yoshitake Kanazawa¹, Tomoya Kimura¹, Hisashi Shiga¹, Katsuya Endo⁵, Kenichi Negoro¹, Masao Nagasaki², Michiaki Unno⁴, Tooru Shimosegawa¹

Affiliations

¹ Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan.
² Institute for Excellence in Higher Education, Tohoku University, Sendai, Japan.
³ Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan.
⁴ Department of Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan.
⁵ Division of Gastroenterology, Tohoku Medical and Pharmaceutical University, Sendai, Japan.

Abstract

Background: Inflammatory bowel disease (IBD) has an unknown etiology; however, accumulating evidence suggests that IBD is a multifactorial disease influenced by a combination of genetic and environmental factors. The influence of genetic variants on DNA methylation in cis and cis effects on expression have been demonstrated. We hypothesized that IBD susceptibility single-nucleotide polymorphisms (SNPs) regulate susceptibility gene expressions in cis by regulating DNA methylation around SNPs. For this, we determined cis-regulated allele-specific DNA methylation (ASM) around IBD susceptibility genes in CD4+ effector/memory T cells (Tem) in lamina propria mononuclear cells (LPMCs) in patients with IBD and examined the association between the ASM SNP genotype and neighboring susceptibility gene expressions.

Methods: CD4+ effector/memory T cells (Tem) were isolated from LPMCs in 15 Japanese IBD patients (ten Crohn's disease [CD] and five ulcerative colitis [UC] patients). ASM analysis was performed by methylation-sensitive SNP array analysis. We defined ASM as a changing average relative allele score ([Formula: see text]) >0.1 after digestion by methylation-sensitive restriction enzymes. Among SNPs showing [Formula: see text] >0.1, we extracted the probes located on tag-SNPs of 200 IBD susceptibility loci and around IBD susceptibility genes as candidate ASM SNPs. To validate ASM, bisulfite-pyrosequencing was performed. Transcriptome analysis was examined in 11 IBD patients (seven CD and four UC patients). The relation between rs36221701 genotype and neighboring gene expressions were analyzed.

Results: We extracted six candidate ASM SNPs around IBD susceptibility genes. The top of [Formula: see text] (0.23) was rs1130368 located on HLA-DQB1. ASM around rs36221701 ([Formula: see text] = 0.14) located near SMAD3 was validated using bisulfite pyrosequencing. The SMAD3 expression was significantly associated with the rs36221701 genotype (p = 0.016).

Conclusions: We confirmed the existence of cis-regulated ASM around IBD susceptibility genes and the association between ASM SNP (rs36221701) genotype and SMAD3 expression, a susceptibility gene for IBD. These results give us supporting evidence that DNA methylation mediates genetic effects on disease susceptibility.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adolescent
Adult
Aged
Alleles
Asian People / genetics*
CD4-Positive T-Lymphocytes / metabolism
DNA Methylation / genetics*
Female
Genetic Predisposition to Disease / genetics*
Genotype
Humans
Immunologic Memory / genetics
Inflammatory Bowel Diseases / genetics*
Male
Middle Aged
Polymorphism, Single Nucleotide / genetics
Young Adult

Grants and funding

This work was supported by Japan Society for the Promotion of Science (KAKENHI Grant Numbers 26460959, 15H04805 and 15K19311). Toshiba Corporation awarded research grants to M. Nagasaki during the conduct of the study. The specific role of this author is articulated in the "author contributions" section. The funders had not role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.