DNA methylation of TOMM40-APOE-APOC2 in Alzheimer's disease

Abstract

The apolipoprotein E (APOE) ε4 allele is the major genetic risk factor for Alzheimer's disease (AD). Multiple regulatory elements, spanning the extended TOMM40-APOE-APOC2 region, regulate gene expression at this locus. Regulatory element DNA methylation changes occur under different environmental conditions, such as disease. Our group and others have described an APOE CpG island as hypomethylated in AD, compared to cognitively normal controls. However, little is known about methylation of the larger TOMM40-APOE-APOC2 region. The hypothesis of this investigation was that regulatory element methylation levels of the larger TOMM40-APOE-APOC2 region are associated with AD. The aim was to determine whether DNA methylation of the TOMM40-APOE-APOC2 region differs in AD compared to cognitively normal controls in post-mortem brain and peripheral blood. DNA was extracted from human brain (n = 12) and peripheral blood (n = 67). A methylation array was used for this analysis. Percent methylation within the TOMM40-APOE-APOC2 region was evaluated for differences according to tissue type, disease state, AD-related biomarkers, and gene expression. Results from this exploratory analysis suggest that regulatory element methylation levels within the larger TOMM40-APOE-APOC2 gene region correlate with AD-related biomarkers and TOMM40 or APOE gene expression in AD.

Fig. 1

Genomic map of APOE locus CpG sites. All genes are transcribed in the same direction as shown here from left to right. CpG islands are black bars. Gene exons are gray bars. Approximate CpG locations and cg ID are noted as cg number from the Infinium HumanMethylation450 BeadChip Kit (Illumina). Genes are located at chr19:45,392,813–45,456,635 UCSC Genome Browser Human Feb. 2009 (GRCh37/hg19) Assembly. Approximate 64,000 base pair region is not to scale

Fig. 2

Percent DNA methylation averages (means) across APOE locus. Control cerebellum (CB: n = 6), AD CB (n = 6), control hippocampus (HP; n = 6), AD HP (n = 6), control peripheral blood (PB: n = 24), and AD PB (n = 26) mean beta-values (percent DNA methylation) vary by genomic region, tissue type, and disease status. White dotted line is set to beta-value 0.50 for reference. Asterisk (*) denotes significantly different CpG mean for AD compared to controls (p < 0.05) using a multivariate analysis where CpG is the dependent variable and the independent variable (fixed factor) is disease status or tissue type. There was not a significant difference between control and AD in PB. The pound sign (#) represents a significant difference between control HP compared to control PB or AD HP compared to AD PB

Fig. 3

TOMM40 promoter CpG methylation is correlated with RNA expression TOMM40 promoter cg22024783 methylation in all subjects (AD and controls) is significantly positively correlated with APOE mRNA expression and non-significantly negatively correlated with TOMM40 mRNA expression in HP (a). TOMM40 promoter cg12266551 methylation is significantly negatively correlated with TOMM40 mRNA expression in controls and non-significantly positively correlated with TOMM40 mRNA expression in AD in HP (b)

Fig. 4

APOE locus CpG methylation is correlated with AD-related biomarkers. TOMM40 promoter cg22024783 methylation is significantly positively correlated with CSF Aβ₄₂ in controls, not correlated in MCI, and significantly negatively correlated in AD (a). TOMM40 promoter cg22024783 methylation is significantly positively correlated with CSF T-tau in controls, not in MCI or AD (b). TOMM40 promoter cg22024783 methylation is significantly positively correlated with CSF P-tau in controls, in MCI or AD (c). APOE CpG island cg05501958 methylation is not significantly correlated with CSF Aβ₄₂ in controls, MCI or AD (d). APOE CpG island cg05501958 methylation not correlated with CSF T-tau in controls or MCI, but is significantly positively correlated in AD (e). APOE CpG island cg05501958 methylation is not correlated CSF P-tau in controls or MCI, but is marginally positively correlated with CSF P-tau in AD (f)