Methylomic profiling implicates cortical deregulation of ANK1 in Alzheimer's disease

Abstract

Alzheimer's disease (AD) is a chronic neurodegenerative disorder that is characterized by progressive neuropathology and cognitive decline. We performed a cross-tissue analysis of methylomic variation in AD using samples from four independent human post-mortem brain cohorts. We identified a differentially methylated region in the ankyrin 1 (ANK1) gene that was associated with neuropathology in the entorhinal cortex, a primary site of AD manifestation. This region was confirmed as being substantially hypermethylated in two other cortical regions (superior temporal gyrus and prefrontal cortex), but not in the cerebellum, a region largely protected from neurodegeneration in AD, or whole blood obtained pre-mortem from the same individuals. Neuropathology-associated ANK1 hypermethylation was subsequently confirmed in cortical samples from three independent brain cohorts. This study represents, to the best of our knowledge, the first epigenome-wide association study of AD employing a sequential replication design across multiple tissues and highlights the power of this approach for identifying methylomic variation associated with complex disease.

FIGURE 1. Cortex-specific hypermethylation of ANK1 is correlated with AD-associated neuropathology in the brain

Linear regression models demonstrated that a) cg11823178 in ANK1 is the top-ranked neuropathology-associated differentially methylated position (DMP) in the EC in the London discovery cohort (N = 104). The adjacent probe, cg05066959, is also significantly associated with neuropathology. Green bars denote the location of annotated CpG islands. b) EC DNA methylation at both CpG sites is strongly associated with Braak score (cg11823178: r = 0.47, t(102) = 5.39, P = 4.59E–7; cg05066959: r = 0.41, t(102) = 5.37, P = 1.34E–5). c) Both probes are also associated with neuropathology in the other cortical regions assessed in the same individuals, being significantly correlated with Braak score in the STG (N = 113) (cg11823178: r = 0.37, t(111) = 4.15, P = 6.51E–5; cg05066959: r = 0.33, t(111) = 3.67, P = 3.78E–4) and the PFC (N = 110) (cg11823178: r = 0.29, t(108) = 3.12, P = 2.33E–3; cg05066959: r = 0.32, t(108) = 3.52, P = 6.48E–4). d) There is no association between DNA methylation and Braak score at either ANK1 probe in the CER (N = 108) (cg11823178: r = 0.01, t(106) = 0.082, P = 0.935; cg05066959: r = −0.08, t(106) = 0.085, P = 0.395), a region largely protected against AD-related neuropathology. e) cg11823178 is the top-ranked cross-cortex DMP (Fisher’s χ²(6) = 60.6, P = 3.42E–11), with cg05066959 also strongly associated with Braak score (Fisher’s χ²(6) = 52.9, P = 1.24E–9).

FIGURE 1. Cortex-specific hypermethylation of ANK1 is correlated with AD-associated neuropathology in the brain

Linear regression models demonstrated that a) cg11823178 in ANK1 is the top-ranked neuropathology-associated differentially methylated position (DMP) in the EC in the London discovery cohort (N = 104). The adjacent probe, cg05066959, is also significantly associated with neuropathology. Green bars denote the location of annotated CpG islands. b) EC DNA methylation at both CpG sites is strongly associated with Braak score (cg11823178: r = 0.47, t(102) = 5.39, P = 4.59E–7; cg05066959: r = 0.41, t(102) = 5.37, P = 1.34E–5). c) Both probes are also associated with neuropathology in the other cortical regions assessed in the same individuals, being significantly correlated with Braak score in the STG (N = 113) (cg11823178: r = 0.37, t(111) = 4.15, P = 6.51E–5; cg05066959: r = 0.33, t(111) = 3.67, P = 3.78E–4) and the PFC (N = 110) (cg11823178: r = 0.29, t(108) = 3.12, P = 2.33E–3; cg05066959: r = 0.32, t(108) = 3.52, P = 6.48E–4). d) There is no association between DNA methylation and Braak score at either ANK1 probe in the CER (N = 108) (cg11823178: r = 0.01, t(106) = 0.082, P = 0.935; cg05066959: r = −0.08, t(106) = 0.085, P = 0.395), a region largely protected against AD-related neuropathology. e) cg11823178 is the top-ranked cross-cortex DMP (Fisher’s χ²(6) = 60.6, P = 3.42E–11), with cg05066959 also strongly associated with Braak score (Fisher’s χ²(6) = 52.9, P = 1.24E–9).

FIGURE 1. Cortex-specific hypermethylation of ANK1 is correlated with AD-associated neuropathology in the brain

Linear regression models demonstrated that a) cg11823178 in ANK1 is the top-ranked neuropathology-associated differentially methylated position (DMP) in the EC in the London discovery cohort (N = 104). The adjacent probe, cg05066959, is also significantly associated with neuropathology. Green bars denote the location of annotated CpG islands. b) EC DNA methylation at both CpG sites is strongly associated with Braak score (cg11823178: r = 0.47, t(102) = 5.39, P = 4.59E–7; cg05066959: r = 0.41, t(102) = 5.37, P = 1.34E–5). c) Both probes are also associated with neuropathology in the other cortical regions assessed in the same individuals, being significantly correlated with Braak score in the STG (N = 113) (cg11823178: r = 0.37, t(111) = 4.15, P = 6.51E–5; cg05066959: r = 0.33, t(111) = 3.67, P = 3.78E–4) and the PFC (N = 110) (cg11823178: r = 0.29, t(108) = 3.12, P = 2.33E–3; cg05066959: r = 0.32, t(108) = 3.52, P = 6.48E–4). d) There is no association between DNA methylation and Braak score at either ANK1 probe in the CER (N = 108) (cg11823178: r = 0.01, t(106) = 0.082, P = 0.935; cg05066959: r = −0.08, t(106) = 0.085, P = 0.395), a region largely protected against AD-related neuropathology. e) cg11823178 is the top-ranked cross-cortex DMP (Fisher’s χ²(6) = 60.6, P = 3.42E–11), with cg05066959 also strongly associated with Braak score (Fisher’s χ²(6) = 52.9, P = 1.24E–9).

FIGURE 1. Cortex-specific hypermethylation of ANK1 is correlated with AD-associated neuropathology in the brain

Linear regression models demonstrated that a) cg11823178 in ANK1 is the top-ranked neuropathology-associated differentially methylated position (DMP) in the EC in the London discovery cohort (N = 104). The adjacent probe, cg05066959, is also significantly associated with neuropathology. Green bars denote the location of annotated CpG islands. b) EC DNA methylation at both CpG sites is strongly associated with Braak score (cg11823178: r = 0.47, t(102) = 5.39, P = 4.59E–7; cg05066959: r = 0.41, t(102) = 5.37, P = 1.34E–5). c) Both probes are also associated with neuropathology in the other cortical regions assessed in the same individuals, being significantly correlated with Braak score in the STG (N = 113) (cg11823178: r = 0.37, t(111) = 4.15, P = 6.51E–5; cg05066959: r = 0.33, t(111) = 3.67, P = 3.78E–4) and the PFC (N = 110) (cg11823178: r = 0.29, t(108) = 3.12, P = 2.33E–3; cg05066959: r = 0.32, t(108) = 3.52, P = 6.48E–4). d) There is no association between DNA methylation and Braak score at either ANK1 probe in the CER (N = 108) (cg11823178: r = 0.01, t(106) = 0.082, P = 0.935; cg05066959: r = −0.08, t(106) = 0.085, P = 0.395), a region largely protected against AD-related neuropathology. e) cg11823178 is the top-ranked cross-cortex DMP (Fisher’s χ²(6) = 60.6, P = 3.42E–11), with cg05066959 also strongly associated with Braak score (Fisher’s χ²(6) = 52.9, P = 1.24E–9).

FIGURE 1. Cortex-specific hypermethylation of ANK1 is correlated with AD-associated neuropathology in the brain

Linear regression models demonstrated that a) cg11823178 in ANK1 is the top-ranked neuropathology-associated differentially methylated position (DMP) in the EC in the London discovery cohort (N = 104). The adjacent probe, cg05066959, is also significantly associated with neuropathology. Green bars denote the location of annotated CpG islands. b) EC DNA methylation at both CpG sites is strongly associated with Braak score (cg11823178: r = 0.47, t(102) = 5.39, P = 4.59E–7; cg05066959: r = 0.41, t(102) = 5.37, P = 1.34E–5). c) Both probes are also associated with neuropathology in the other cortical regions assessed in the same individuals, being significantly correlated with Braak score in the STG (N = 113) (cg11823178: r = 0.37, t(111) = 4.15, P = 6.51E–5; cg05066959: r = 0.33, t(111) = 3.67, P = 3.78E–4) and the PFC (N = 110) (cg11823178: r = 0.29, t(108) = 3.12, P = 2.33E–3; cg05066959: r = 0.32, t(108) = 3.52, P = 6.48E–4). d) There is no association between DNA methylation and Braak score at either ANK1 probe in the CER (N = 108) (cg11823178: r = 0.01, t(106) = 0.082, P = 0.935; cg05066959: r = −0.08, t(106) = 0.085, P = 0.395), a region largely protected against AD-related neuropathology. e) cg11823178 is the top-ranked cross-cortex DMP (Fisher’s χ²(6) = 60.6, P = 3.42E–11), with cg05066959 also strongly associated with Braak score (Fisher’s χ²(6) = 52.9, P = 1.24E–9).

FIGURE 2. Neuropathology-associated DMPs are consistent across sample cohorts, with replicated evidence for ANK1 hypermethylation

a) Braak-associated DNA methylation scores for the top-ranked cross-cortex DMPs identified using linear regression models in the London discovery cohort (listed in Supplementary Table S9) are significantly correlated with neuropathology-associated differences at the same probes in both cortical regions profiled in the Mount Sinai replication cohort using linear regression models (PFC (N =142) Braak score: r = 0.64, P = 6.03E–13; STG (N = 144) Braak score: r = 0.63, P = 2.66E–12; PFC amyloid burden: r = 0.65, P = 2.87E–13; STG amyloid burden: r = 0.46, P = 1.09E–6). Shown is data for Mount Sinai PFC Braak score analysis, with the two ANK1 probes (cg11823178 and cg05066959) highlighted in red. cg11823178 and cg05066959 are significantly associated with b) Braak score in the STG (cg11823178: r = 0.28, t(142) = 3.62, P = 1.63E–04; cg05066959: r = 0.25, t(142) = 3.29, P = 5.78E–04) and PFC (cg11823178: r = 0.24, t(140) = 3.14, P = 1.07E–03; cg05066959: r = 0.21, t(140) = 2.75, P = 4.00E–03) and also c) amyloid pathology in the STG (cg11823178: r = 0.21, t(142) = 2.81, P = 4.99E–04; cg05066959: r = 0.27, t(142) = 3.47, P =5.65E–04) and PFC (cg11823178: r = 0.29, t(140) = 3.69, P = 2.35E–04; cg05066959: r = 0.19, t(140) = 2.56, P = 9.93E–03). In the Oxford replication cohort, bisulfite–pyrosequencing was used to quantify DNA methylation across eight CpG sites spanning an extended ANK1 region. Linear models, adjusting for age and gender, confirmed significant neuropathology-associated hypermethylation in all three cortical regions assessed (see Supplementary Fig. S1), d) most notably in the EC (N=51), where six of the eight CpG sites showed a significant (amplicon average P = 0.0004) neuropathology-associated increase in DNA methylation (data is represented as mean +/− SEM, with *=p<0.05, **=p<0.01, and ***=p<0.005). Meta-analyses across the three sample cohorts (London, Mount Sinai and Oxford) confirms Braak-associated cortex-specific hypermethylation for both e) cg11823178 and f) cg05066959. Finally, there is striking consistency in neuropathology-associated DMPs identified in our discovery cohort and those identified in the co-submitted study by De Jager and colleagues. g) Braak-associated DNA methylation scores for the 100 top-ranked cross-cortex DMPs identified in the London discovery cohort are significantly correlated with neuropathology-associated differences (neuritic-plaque load) at the same probes in the dorsolateral prefrontal cortex (DLPFC) identified by De Jager and colleagues in 708 individuals (r = 0.57, P = 1.55E–9). The two ANK1 probes (cg11823178 and cg05066959) are highlighted in red.

FIGURE 2. Neuropathology-associated DMPs are consistent across sample cohorts, with replicated evidence for ANK1 hypermethylation

a) Braak-associated DNA methylation scores for the top-ranked cross-cortex DMPs identified using linear regression models in the London discovery cohort (listed in Supplementary Table S9) are significantly correlated with neuropathology-associated differences at the same probes in both cortical regions profiled in the Mount Sinai replication cohort using linear regression models (PFC (N =142) Braak score: r = 0.64, P = 6.03E–13; STG (N = 144) Braak score: r = 0.63, P = 2.66E–12; PFC amyloid burden: r = 0.65, P = 2.87E–13; STG amyloid burden: r = 0.46, P = 1.09E–6). Shown is data for Mount Sinai PFC Braak score analysis, with the two ANK1 probes (cg11823178 and cg05066959) highlighted in red. cg11823178 and cg05066959 are significantly associated with b) Braak score in the STG (cg11823178: r = 0.28, t(142) = 3.62, P = 1.63E–04; cg05066959: r = 0.25, t(142) = 3.29, P = 5.78E–04) and PFC (cg11823178: r = 0.24, t(140) = 3.14, P = 1.07E–03; cg05066959: r = 0.21, t(140) = 2.75, P = 4.00E–03) and also c) amyloid pathology in the STG (cg11823178: r = 0.21, t(142) = 2.81, P = 4.99E–04; cg05066959: r = 0.27, t(142) = 3.47, P =5.65E–04) and PFC (cg11823178: r = 0.29, t(140) = 3.69, P = 2.35E–04; cg05066959: r = 0.19, t(140) = 2.56, P = 9.93E–03). In the Oxford replication cohort, bisulfite–pyrosequencing was used to quantify DNA methylation across eight CpG sites spanning an extended ANK1 region. Linear models, adjusting for age and gender, confirmed significant neuropathology-associated hypermethylation in all three cortical regions assessed (see Supplementary Fig. S1), d) most notably in the EC (N=51), where six of the eight CpG sites showed a significant (amplicon average P = 0.0004) neuropathology-associated increase in DNA methylation (data is represented as mean +/− SEM, with *=p<0.05, **=p<0.01, and ***=p<0.005). Meta-analyses across the three sample cohorts (London, Mount Sinai and Oxford) confirms Braak-associated cortex-specific hypermethylation for both e) cg11823178 and f) cg05066959. Finally, there is striking consistency in neuropathology-associated DMPs identified in our discovery cohort and those identified in the co-submitted study by De Jager and colleagues. g) Braak-associated DNA methylation scores for the 100 top-ranked cross-cortex DMPs identified in the London discovery cohort are significantly correlated with neuropathology-associated differences (neuritic-plaque load) at the same probes in the dorsolateral prefrontal cortex (DLPFC) identified by De Jager and colleagues in 708 individuals (r = 0.57, P = 1.55E–9). The two ANK1 probes (cg11823178 and cg05066959) are highlighted in red.

FIGURE 2. Neuropathology-associated DMPs are consistent across sample cohorts, with replicated evidence for ANK1 hypermethylation

a) Braak-associated DNA methylation scores for the top-ranked cross-cortex DMPs identified using linear regression models in the London discovery cohort (listed in Supplementary Table S9) are significantly correlated with neuropathology-associated differences at the same probes in both cortical regions profiled in the Mount Sinai replication cohort using linear regression models (PFC (N =142) Braak score: r = 0.64, P = 6.03E–13; STG (N = 144) Braak score: r = 0.63, P = 2.66E–12; PFC amyloid burden: r = 0.65, P = 2.87E–13; STG amyloid burden: r = 0.46, P = 1.09E–6). Shown is data for Mount Sinai PFC Braak score analysis, with the two ANK1 probes (cg11823178 and cg05066959) highlighted in red. cg11823178 and cg05066959 are significantly associated with b) Braak score in the STG (cg11823178: r = 0.28, t(142) = 3.62, P = 1.63E–04; cg05066959: r = 0.25, t(142) = 3.29, P = 5.78E–04) and PFC (cg11823178: r = 0.24, t(140) = 3.14, P = 1.07E–03; cg05066959: r = 0.21, t(140) = 2.75, P = 4.00E–03) and also c) amyloid pathology in the STG (cg11823178: r = 0.21, t(142) = 2.81, P = 4.99E–04; cg05066959: r = 0.27, t(142) = 3.47, P =5.65E–04) and PFC (cg11823178: r = 0.29, t(140) = 3.69, P = 2.35E–04; cg05066959: r = 0.19, t(140) = 2.56, P = 9.93E–03). In the Oxford replication cohort, bisulfite–pyrosequencing was used to quantify DNA methylation across eight CpG sites spanning an extended ANK1 region. Linear models, adjusting for age and gender, confirmed significant neuropathology-associated hypermethylation in all three cortical regions assessed (see Supplementary Fig. S1), d) most notably in the EC (N=51), where six of the eight CpG sites showed a significant (amplicon average P = 0.0004) neuropathology-associated increase in DNA methylation (data is represented as mean +/− SEM, with *=p<0.05, **=p<0.01, and ***=p<0.005). Meta-analyses across the three sample cohorts (London, Mount Sinai and Oxford) confirms Braak-associated cortex-specific hypermethylation for both e) cg11823178 and f) cg05066959. Finally, there is striking consistency in neuropathology-associated DMPs identified in our discovery cohort and those identified in the co-submitted study by De Jager and colleagues. g) Braak-associated DNA methylation scores for the 100 top-ranked cross-cortex DMPs identified in the London discovery cohort are significantly correlated with neuropathology-associated differences (neuritic-plaque load) at the same probes in the dorsolateral prefrontal cortex (DLPFC) identified by De Jager and colleagues in 708 individuals (r = 0.57, P = 1.55E–9). The two ANK1 probes (cg11823178 and cg05066959) are highlighted in red.

FIGURE 2. Neuropathology-associated DMPs are consistent across sample cohorts, with replicated evidence for ANK1 hypermethylation

a) Braak-associated DNA methylation scores for the top-ranked cross-cortex DMPs identified using linear regression models in the London discovery cohort (listed in Supplementary Table S9) are significantly correlated with neuropathology-associated differences at the same probes in both cortical regions profiled in the Mount Sinai replication cohort using linear regression models (PFC (N =142) Braak score: r = 0.64, P = 6.03E–13; STG (N = 144) Braak score: r = 0.63, P = 2.66E–12; PFC amyloid burden: r = 0.65, P = 2.87E–13; STG amyloid burden: r = 0.46, P = 1.09E–6). Shown is data for Mount Sinai PFC Braak score analysis, with the two ANK1 probes (cg11823178 and cg05066959) highlighted in red. cg11823178 and cg05066959 are significantly associated with b) Braak score in the STG (cg11823178: r = 0.28, t(142) = 3.62, P = 1.63E–04; cg05066959: r = 0.25, t(142) = 3.29, P = 5.78E–04) and PFC (cg11823178: r = 0.24, t(140) = 3.14, P = 1.07E–03; cg05066959: r = 0.21, t(140) = 2.75, P = 4.00E–03) and also c) amyloid pathology in the STG (cg11823178: r = 0.21, t(142) = 2.81, P = 4.99E–04; cg05066959: r = 0.27, t(142) = 3.47, P =5.65E–04) and PFC (cg11823178: r = 0.29, t(140) = 3.69, P = 2.35E–04; cg05066959: r = 0.19, t(140) = 2.56, P = 9.93E–03). In the Oxford replication cohort, bisulfite–pyrosequencing was used to quantify DNA methylation across eight CpG sites spanning an extended ANK1 region. Linear models, adjusting for age and gender, confirmed significant neuropathology-associated hypermethylation in all three cortical regions assessed (see Supplementary Fig. S1), d) most notably in the EC (N=51), where six of the eight CpG sites showed a significant (amplicon average P = 0.0004) neuropathology-associated increase in DNA methylation (data is represented as mean +/− SEM, with *=p<0.05, **=p<0.01, and ***=p<0.005). Meta-analyses across the three sample cohorts (London, Mount Sinai and Oxford) confirms Braak-associated cortex-specific hypermethylation for both e) cg11823178 and f) cg05066959. Finally, there is striking consistency in neuropathology-associated DMPs identified in our discovery cohort and those identified in the co-submitted study by De Jager and colleagues. g) Braak-associated DNA methylation scores for the 100 top-ranked cross-cortex DMPs identified in the London discovery cohort are significantly correlated with neuropathology-associated differences (neuritic-plaque load) at the same probes in the dorsolateral prefrontal cortex (DLPFC) identified by De Jager and colleagues in 708 individuals (r = 0.57, P = 1.55E–9). The two ANK1 probes (cg11823178 and cg05066959) are highlighted in red.

FIGURE 2. Neuropathology-associated DMPs are consistent across sample cohorts, with replicated evidence for ANK1 hypermethylation

a) Braak-associated DNA methylation scores for the top-ranked cross-cortex DMPs identified using linear regression models in the London discovery cohort (listed in Supplementary Table S9) are significantly correlated with neuropathology-associated differences at the same probes in both cortical regions profiled in the Mount Sinai replication cohort using linear regression models (PFC (N =142) Braak score: r = 0.64, P = 6.03E–13; STG (N = 144) Braak score: r = 0.63, P = 2.66E–12; PFC amyloid burden: r = 0.65, P = 2.87E–13; STG amyloid burden: r = 0.46, P = 1.09E–6). Shown is data for Mount Sinai PFC Braak score analysis, with the two ANK1 probes (cg11823178 and cg05066959) highlighted in red. cg11823178 and cg05066959 are significantly associated with b) Braak score in the STG (cg11823178: r = 0.28, t(142) = 3.62, P = 1.63E–04; cg05066959: r = 0.25, t(142) = 3.29, P = 5.78E–04) and PFC (cg11823178: r = 0.24, t(140) = 3.14, P = 1.07E–03; cg05066959: r = 0.21, t(140) = 2.75, P = 4.00E–03) and also c) amyloid pathology in the STG (cg11823178: r = 0.21, t(142) = 2.81, P = 4.99E–04; cg05066959: r = 0.27, t(142) = 3.47, P =5.65E–04) and PFC (cg11823178: r = 0.29, t(140) = 3.69, P = 2.35E–04; cg05066959: r = 0.19, t(140) = 2.56, P = 9.93E–03). In the Oxford replication cohort, bisulfite–pyrosequencing was used to quantify DNA methylation across eight CpG sites spanning an extended ANK1 region. Linear models, adjusting for age and gender, confirmed significant neuropathology-associated hypermethylation in all three cortical regions assessed (see Supplementary Fig. S1), d) most notably in the EC (N=51), where six of the eight CpG sites showed a significant (amplicon average P = 0.0004) neuropathology-associated increase in DNA methylation (data is represented as mean +/− SEM, with *=p<0.05, **=p<0.01, and ***=p<0.005). Meta-analyses across the three sample cohorts (London, Mount Sinai and Oxford) confirms Braak-associated cortex-specific hypermethylation for both e) cg11823178 and f) cg05066959. Finally, there is striking consistency in neuropathology-associated DMPs identified in our discovery cohort and those identified in the co-submitted study by De Jager and colleagues. g) Braak-associated DNA methylation scores for the 100 top-ranked cross-cortex DMPs identified in the London discovery cohort are significantly correlated with neuropathology-associated differences (neuritic-plaque load) at the same probes in the dorsolateral prefrontal cortex (DLPFC) identified by De Jager and colleagues in 708 individuals (r = 0.57, P = 1.55E–9). The two ANK1 probes (cg11823178 and cg05066959) are highlighted in red.

FIGURE 2. Neuropathology-associated DMPs are consistent across sample cohorts, with replicated evidence for ANK1 hypermethylation

a) Braak-associated DNA methylation scores for the top-ranked cross-cortex DMPs identified using linear regression models in the London discovery cohort (listed in Supplementary Table S9) are significantly correlated with neuropathology-associated differences at the same probes in both cortical regions profiled in the Mount Sinai replication cohort using linear regression models (PFC (N =142) Braak score: r = 0.64, P = 6.03E–13; STG (N = 144) Braak score: r = 0.63, P = 2.66E–12; PFC amyloid burden: r = 0.65, P = 2.87E–13; STG amyloid burden: r = 0.46, P = 1.09E–6). Shown is data for Mount Sinai PFC Braak score analysis, with the two ANK1 probes (cg11823178 and cg05066959) highlighted in red. cg11823178 and cg05066959 are significantly associated with b) Braak score in the STG (cg11823178: r = 0.28, t(142) = 3.62, P = 1.63E–04; cg05066959: r = 0.25, t(142) = 3.29, P = 5.78E–04) and PFC (cg11823178: r = 0.24, t(140) = 3.14, P = 1.07E–03; cg05066959: r = 0.21, t(140) = 2.75, P = 4.00E–03) and also c) amyloid pathology in the STG (cg11823178: r = 0.21, t(142) = 2.81, P = 4.99E–04; cg05066959: r = 0.27, t(142) = 3.47, P =5.65E–04) and PFC (cg11823178: r = 0.29, t(140) = 3.69, P = 2.35E–04; cg05066959: r = 0.19, t(140) = 2.56, P = 9.93E–03). In the Oxford replication cohort, bisulfite–pyrosequencing was used to quantify DNA methylation across eight CpG sites spanning an extended ANK1 region. Linear models, adjusting for age and gender, confirmed significant neuropathology-associated hypermethylation in all three cortical regions assessed (see Supplementary Fig. S1), d) most notably in the EC (N=51), where six of the eight CpG sites showed a significant (amplicon average P = 0.0004) neuropathology-associated increase in DNA methylation (data is represented as mean +/− SEM, with *=p<0.05, **=p<0.01, and ***=p<0.005). Meta-analyses across the three sample cohorts (London, Mount Sinai and Oxford) confirms Braak-associated cortex-specific hypermethylation for both e) cg11823178 and f) cg05066959. Finally, there is striking consistency in neuropathology-associated DMPs identified in our discovery cohort and those identified in the co-submitted study by De Jager and colleagues. g) Braak-associated DNA methylation scores for the 100 top-ranked cross-cortex DMPs identified in the London discovery cohort are significantly correlated with neuropathology-associated differences (neuritic-plaque load) at the same probes in the dorsolateral prefrontal cortex (DLPFC) identified by De Jager and colleagues in 708 individuals (r = 0.57, P = 1.55E–9). The two ANK1 probes (cg11823178 and cg05066959) are highlighted in red.

FIGURE 2. Neuropathology-associated DMPs are consistent across sample cohorts, with replicated evidence for ANK1 hypermethylation

a) Braak-associated DNA methylation scores for the top-ranked cross-cortex DMPs identified using linear regression models in the London discovery cohort (listed in Supplementary Table S9) are significantly correlated with neuropathology-associated differences at the same probes in both cortical regions profiled in the Mount Sinai replication cohort using linear regression models (PFC (N =142) Braak score: r = 0.64, P = 6.03E–13; STG (N = 144) Braak score: r = 0.63, P = 2.66E–12; PFC amyloid burden: r = 0.65, P = 2.87E–13; STG amyloid burden: r = 0.46, P = 1.09E–6). Shown is data for Mount Sinai PFC Braak score analysis, with the two ANK1 probes (cg11823178 and cg05066959) highlighted in red. cg11823178 and cg05066959 are significantly associated with b) Braak score in the STG (cg11823178: r = 0.28, t(142) = 3.62, P = 1.63E–04; cg05066959: r = 0.25, t(142) = 3.29, P = 5.78E–04) and PFC (cg11823178: r = 0.24, t(140) = 3.14, P = 1.07E–03; cg05066959: r = 0.21, t(140) = 2.75, P = 4.00E–03) and also c) amyloid pathology in the STG (cg11823178: r = 0.21, t(142) = 2.81, P = 4.99E–04; cg05066959: r = 0.27, t(142) = 3.47, P =5.65E–04) and PFC (cg11823178: r = 0.29, t(140) = 3.69, P = 2.35E–04; cg05066959: r = 0.19, t(140) = 2.56, P = 9.93E–03). In the Oxford replication cohort, bisulfite–pyrosequencing was used to quantify DNA methylation across eight CpG sites spanning an extended ANK1 region. Linear models, adjusting for age and gender, confirmed significant neuropathology-associated hypermethylation in all three cortical regions assessed (see Supplementary Fig. S1), d) most notably in the EC (N=51), where six of the eight CpG sites showed a significant (amplicon average P = 0.0004) neuropathology-associated increase in DNA methylation (data is represented as mean +/− SEM, with *=p<0.05, **=p<0.01, and ***=p<0.005). Meta-analyses across the three sample cohorts (London, Mount Sinai and Oxford) confirms Braak-associated cortex-specific hypermethylation for both e) cg11823178 and f) cg05066959. Finally, there is striking consistency in neuropathology-associated DMPs identified in our discovery cohort and those identified in the co-submitted study by De Jager and colleagues. g) Braak-associated DNA methylation scores for the 100 top-ranked cross-cortex DMPs identified in the London discovery cohort are significantly correlated with neuropathology-associated differences (neuritic-plaque load) at the same probes in the dorsolateral prefrontal cortex (DLPFC) identified by De Jager and colleagues in 708 individuals (r = 0.57, P = 1.55E–9). The two ANK1 probes (cg11823178 and cg05066959) are highlighted in red.