Genome-wide peripheral blood leukocyte DNA methylation microarrays identified a single association with inflammatory bowel diseases

Abstract

Background: Crohn's disease (CD) and ulcerative colitis (UC) are common forms of inflammatory bowel disease (IBD). Monozygotic (MZ) twin discordance rates and epidemiologic data implicate that environmental changes and epigenetic factors may play a pathogenic role in IBD. DNA methylation (the methylation of cytosines within CpG dinucleotides) is an epigenetic modification, which can respond to environmental influences. We investigated whether DNA methylation might be connected with IBD in peripheral blood leukocyte (PBL) DNA by utilizing genome-wide microarrays.

Methods: Two different high-throughput microarray-based methods for genome-wide DNA methylation analysis were employed. First, DNA isolated from MZ twin pairs concordant (CD: 4; UC: 3) and discordant (CD: 4; UC: 7) for IBD was interrogated by a custom-made methylation-specific amplification microarray (MSAM). Second, the recently developed Illumina Infinium HumanMethylation450 BeadChip arrays were used on 48 samples of PBL DNA from discordant MZ twin pairs (CD: 3; UC: 3) and treatment-naive pediatric cases of IBD (CD: 14; UC: 8), as well as controls (n = 14). The microarrays were validated with bisulfite pyrosequencing.

Results: The MSAMs did not yield significant IBD associations. The Methylation BeadChip approach identified a single DNA methylation association of IBD at TEPP (testis, prostate and placenta-expressed protein) when DNA isolated selectively from peripheral blood mononuclear cells was analyzed (8.6% increase in methylation between CD and control, FDR = 0.0065).

Conclusions: Microarray interrogation of IBD-dependent DNA methylation from PBLs appears to have limited ability to detect significant disease associations. More detailed and/or selective approaches may be useful for the elucidation of connections between the DNA methylome and IBD in the future.

Figure 1

Scatter plot of methylation levels obtained by Infinium HumanMethylation450 Microarrays (Microarray) versus bisulfite pyrosequencing at the SCL6A9, FAM53B, and TEPP associated CpG sites. Significant correlation was observed. r=0.963; p<<0.00001

Figure 2

Methylation levels at the TEPP associated CpG site in the select pediatric PBMC cohort of the study (see Table 2). A. Bisulfite pyrosequencing results from the PBMC samples of the Infinium HumanMethylation450 Microarrays. The significant increase in methylation was confirmed by bisulfite pyrosequencing in the CD and UC cohorts compared to controls (n=6–14). B. Extension of the measurements to larger groups of PBMC DNA from controls and treatment naïve CD patients further supported the validity of our findings (n=19–22). C. The increased level of methylation in treatment naïve (CD) patients was lost when compared to CD patients receiving therapy (CD-rx; 3 biologic monotherapy; 2 biologic+immunomodulator, 2 mesalamine alone, 1 steroid alone, 1 immunomodulator alone, 1 steroid+immunomodulator) (n=10–22).