A meta-analysis of epigenome-wide association studies in Alzheimer’s disease highlights novel differentially methylated loci across cortex

Epigenome-wide association studies of Alzheimer’s disease have highlighted neuropathology-associated DNA methylation differences, although existing studies have been limited in sample size and utilized different brain regions. Here, we combine data from six DNA methylomic studies of Alzheimer’s disease (N = 1453 unique individuals) to identify differential methylation associated with Braak stage in different brain regions and across cortex. We identify 236 CpGs in the prefrontal cortex, 95 CpGs in the temporal gyrus and ten CpGs in the entorhinal cortex at Bonferroni significance, with none in the cerebellum. Our cross-cortex meta-analysis (N = 1408 donors) identifies 220 CpGs associated with neuropathology, annotated to 121 genes, of which 84 genes have not been previously reported at this significance threshold. We have replicated our findings using two further DNA methylomic datasets consisting of a further >600 unique donors. The meta-analysis summary statistics are available in our online data resource (www.epigenomicslab.com/ad-meta-analysis/).


Supplementary Figure 3: Forest plot of the most significant differentially methylated position (DMP) in the prefrontal cortex inverse variance fixed effects meta-analysis (cg22962123).
The methylation (beta) effect size (ES) is shown in the prefrontal cortex (red; N = 961), temporal gyrus (green; N = 608) and entorhinal cortex (blue; N = 189) for the different cohorts. The X-axis shows the beta ES, with dots representing ES and arms indicating standard error (SE). ES from the intra-tissue meta-analysis using all available individual cohorts are represented by polygons. Figure 4: Neuropathology-associated Bonferroni significant differentially methylated positions (DMPs) identified in the prefrontal cortex meta-analysis are replicated in other cortical regions. The methylation (beta) effect size (ES) of the 236 Braak-associated Bonferroni significant DMPs identified in the prefrontal cortex (N = 961) from our inverse variance fixed effect meta-analysis model (X-axis) were significantly correlated with the ES of the same probes in the temporal gyrus (green; N = 608, r = 0.94, P = 6.17 x 10 -112 ) and entorhinal cortex (blue; N = 189, r = 0.58, P = 1.80 x 10 -22 ) (Y-axis).

Supplementary Figure 5: The most significant differentially methylated region (DMR) in the prefrontal cortex inverse variance fixed effects metaanalysis (chr7:26955524-27473741) contained 20 probes and resided in the HOXA region (N = 961).
The horizontal red line denotes the Bonferroni significance level of P < 1.238 x 10 -7 . Red probes represent a positive methylation (beta) effect size (ES) ≥ 0.01, blue probes represent a negative ES ≥ 0.01. Underneath the gene tracks are shown in black with CpG islands in green.

Supplementary Figure 6: Volcano plot of differentially methylated positions (DMPs) identified in the temporal gyrus inverse variance fixed effects meta-analysis (N =608)
. The X-axis shows beta effect size (ES) and the Y-axis shows -log10(p). Gray probes indicate an ES ≥ 0.01, whilst blue probes indicate an ES ≥ 0.01 and a Bonferroni significant P-value (P < 1.238 x 10 -7 ). Exact p-values are provided in Supplementary Data 3.

Supplementary Figure 7: Forest plot of the most significant differentially methylated position (DMP) in the temporal gyrus and entorhinal cortex inverse variance fixed effects meta-analysis (cg11823178).
The methylation (beta) effect size (ES) is shown in the prefrontal cortex (red; N = 961), temporal gyrus (green; N = 608) and EC entorhinal cortex (blue; N = 189) for the different cohorts. The X-axis shows the beta ES, with dots representing ES and arms indicating standard error (SE). ES from the intra-tissue meta-analysis using all available individual cohorts are represented by polygons.

Supplementary Figure 8: Neuropathology-associated Bonferroni significant differentially methylated positions (DMPs) identified in the temporal gyrus meta-analysis are replicated in other cortical regions.
The methylation (beta) effect size (ES) of Braak-associated Bonferroni significant DMPs identified in the temporal gyrus (N = 608) from our inverse variance fixed effect meta-analysis model (X-axis) were significantly correlated with the ES of the same probes in the prefrontal cortex (red; N = 961, r = 0.91, P = 5.09 x 10 -38 ) and entorhinal cortex (blue; N = 189, r = 0.77, P = 4.02 x 10 -20 ) (Y-axis). ANK1 gene (N = 608). The horizontal red line denotes the Bonferroni significance level of P < 1.238 x 10 -7 . Red probes represent a positive methylation (beta) effect size (ES) ≥ 0.01, blue probes represent a negative ES ≥ 0.01. Underneath the gene tracks are shown in black with CpG islands in green.

Supplementary Figure 10: Volcano plot of differentially methylated positions (DMPs) identified in the entorhinal cortex inverse variance fixed effects meta-analysis (N =189)
. The X-axis shows methylation (beta) effect size (ES) and the Y-axis shows -log10(p). Gray probes indicate an ES ≥ 0.01, whilst blue probes indicate an ES ≥ 0.01 and a Bonferroni significant p-value (P < 1.238 x 10 -7 ). Exact p-values are provided in Supplementary Data 5. Figure 11: Neuropathology-associated Bonferroni significant differentially methylated positions (DMPs) identified in the entorhinal cortex meta-analysis are replicated in other cortical regions. The methylation (beta) effect size (ES) of Braak-associated Bonferroni significant DMPs identified in the entorhinal cortex (N = 189) from our inverse variance fixed effect meta-analysis model (X-axis) were significantly correlated with the ES of the same probes in the prefrontal cortex (red; N = 961, r = 0.74, P = 0.01) and temporal gyrus (green; N = 608, r = 0.85, P = 1.82 x 10 -3 ) (Y-axis).