Epigenetic Alterations in Blood Mirror Age-Associated Dna Methylation and Gene Expression Changes in Human Liver

Figures & data

Figure 1.  Distribution of significant age-associated CpG sites in the human liver.

(A) Number of positive and negative correlations of CpG sites significantly associated with age in human liver. (B) Distribution of significant CpG sites compared with all analyzed CpG sites in relation to gene regions, in other words, TSS1500 (200–1500 bp upstream of TSS), TSS200 (200 bp immediate upstream of TSS), 5′UTR, first exon, 3′UTR and intergenic regions (not annotated to any of the regions). (C) Distribution of significant CpG sites compared with all analyzed CpG sites in relation to CpG islands, in other words, N_shore/S_shore (2 kb immediate upstream/downstream of CpG island), N_shelf/S_shelf (2 kb immediate downstream/upstream of shore regions), CpG island and open sea (not annotated to any of these regions).

*p < 0.05. p-values were corrected using Bonferroni correction.

+: Positive association; -: Negative association; N_Shelf: Northern shelf; N_Shore: Northern shore; S_Shelf: Southern shelf; S_Shore: Southern shore; TSS: Transcription start site; UTR: Untranslated region.

Figure 2.  Correlations of top ten significant CpG sites associated with age in human liver.

(A) Correlation of top most significant CpG site, cg03801902 of CCND2 and age. (B–J) Correlations of the remaining nine top CpG sites.

Figure 3.  Venn diagram presenting the number of shared CpG sites associated with age of human liver (20,396 CpG sites), blood (137,993 CpG sites), adipose tissue (31,567 CpG sites) and pancreatic islets (241 CpG sites), as presented in the Supplementary Tables 3, 5, 6 & 7.

Figure 4.  Age-associated changes in both mRNA expression and DNA methylation in human liver.

(A) Correlations between age and mRNA expression as well as DNA methylation of QTRT1. (B) Correlations between age and ZEB2 mRNA expression as well as DNA methylation of two CpG sites annotated to ZEB2.

Figure 5.  Pathway analysis.

(A) Selected significantly enriched KEGG pathways of the genes with age-associated methylation changes in human liver (Bonferroni adjusted q < 0.05); complete list is presented in Supplementary Table 9). (B) Significant KEGG pathways of the genes with age-associated methylation changes of CpG sites located in CpG islands. (C) Significant KEGG pathways of genes with age-associated methylation changes of shared CpG sites for human liver, blood and adipose tissue. (D) Significant KEGG pathways of the genes with age-associated expression and methylation changes in human liver.

ECM: Extracellular matrix.

Figure 6.  Validation of the 450K array methylation data by pyrosequencing.

(A–D) Correlations between age and DNA methylation of CpG sites cg14361627, cg08097417, cg06639320 and cg24079702 generated by 450K array for the whole cohort (n = 95). (E–H) Correlations between age and DNA methylation of CpG sites cg14361627, cg08097417, cg06639320 and cg24079702 generated by 450K array for the validation cohort (n = 20). (I–L) Correlations between age and DNA methylation of CpG sites cg14361627, cg08097417, cg06639320 and cg24079702 generated by pyrosequencing assay. The data in the figures (E–L) were analyzed by Spearman r (two tailed).

Table 1.  Characteristics of liver donors.

Characteristics, n = 95 (34 men, 61 women)	Average	Minimum–maximum
Age (years)	49.5 ± 7.6	28–65
BMI (kg/m^2)	43.0 ± 5.6	33.3–60.5

Data presented as mean ± standard deviation and range.

Table 2.  Previously reported age-associated genes by Steegenga et al. and CpG sites with DNA methylation changes (q < 0.05) in human liver based on age.

Target ID	Gene	p-value	q-value	Regression coefficient	Standard error mean	Relation to gene region	Relation to CpG island
cg18236477	ATP8A2	1.50E-06	0.0003	0.0176	0.0033	Body	Island
cg26708235	ATP8A2	4.78E-06	0.0008	0.0205	0.0042	First exon	Island
cg02057782	ATP8A2	0.000214	0.0108	0.0125	0.0031	Body	N_Shore
cg13537353	ATP8A2	0.001361	0.0363	0.0271	0.0083	Body	Island
cg18836626	ATP8A2	0.001642	0.0411	0.0165	0.0051	Body	Island
cg24799863	DLX5	5.29E-07	0.0001	0.0167	0.0029	5′UTR; first exon	Island
cg20080624	DLX5	3.73E-06	0.0006	0.0180	0.0036	Body	Island
cg18544329	DLX5	2.41E-05	0.0025	0.0173	0.0038	TSS200	Island
cg00503840	DLX5	3.92E-05	0.0034	0.0129	0.0028	Body	Island
cg27032146	DLX5	0.000132	0.0078	0.0169	0.0042	Body	Island
cg00400832	DLX5	0.000229	0.0113	0.0180	0.0047	Body	Island
cg27016494	DLX5	0.000295	0.0134	0.0148	0.0039	Body	N_Shore
cg18873386	DLX5	0.000463	0.0180	0.0123	0.0033	Body	N_Shore
cg15732768	DLX5	0.000498	0.0189	0.0169	0.0047	Body	Island
cg13344740	DLX5	0.000517	0.0193	0.0116	0.0031	5′UTR; first exon	Island
cg24724428	ELOVL2	6.16E-14	4.01E-09	0.0390	0.0044	TSS1500	Island
cg16867657	ELOVL2	1.66E-10	9.72E-07	0.0268	0.0036	TSS1500	Island
cg21572722	ELOVL2	2.80E-08	2.72E-05	0.0190	0.0030	TSS1500	Island
cg22796507	FOXE3	2.60E-08	2.58E-05	0.0317	0.0052	First exon	Island
cg19809499	FOXE3	4.35E-08	3.58E-05	0.0538	0.0092	First exon	Island
cg01281911	FOXE3	5.54E-08	4.19E-05	0.0337	0.0057	First exon	Island
cg23115387	FOXE3	2.13E-07	0.0001	0.0177	0.0030	First exon	Island
cg18815943	FOXE3	5.20E-07	0.0001	0.0374	0.0070	First exon	Island
cg15457058	FOXE3	3.49E-05	0.0032	0.0386	0.0090	First exon	Island
cg00806138	FOXE3	0.000366	0.0154	0.0159	0.0042	TSS1500	N_Shore
cg13017614	FOXE3	0.000377	0.0158	0.0205	0.0056	TSS200	N_Shore
cg27175287	FOXE3	0.001275	0.0348	0.0153	0.0046	3′UTR; first exon	S_Shore
cg22454769	FHL2	1.46E-06	0.0003	0.0185	0.0035	TSS200; TSS200; 5′UTR; TSS200	Island
cg24079702	FHL2	8.83E-06	0.0012	0.0171	0.0035	TSS200; TSS200; 5′UTR; TSS200	Island
cg066 39320	FHL2	0.000393	0.0162	0.0154	0.0041	TSS200; TSS200; 5′UTR; TSS200	Island
cg06907053	FHL2	0.000416	0.0167	0.0179	0.0049	TSS200; 5′UTR; TSS200; TSS1500	Island
cg02054792	FHL2	0.000829	0.0263	0.0222	0.0065	5′UTR; 5′UTR; 5′UTR; 5′UTR	Island
cg17129821	FHL2	0.001427	0.0375	0.0166	0.0050	Body; body; body; body	Open sea
cg02445447	FHL2	0.001162	0.0328	0.0139	0.0041	TSS200; 5′UTR; first exon; 5′UTR; 5′UTR; first exon	Island
cg17291435	FHL2	0.001185	0.0332	0.0360	0.0109	First exon; 5′UTR; 5′UTR; first exon; 5′UTR; 5′UTR; first exon	Island
cg17795240	GATA4	2.03E-05	0.0022	0.0176	0.0038	Body	S_Shore
cg00090147	GATA4	0.000926	0.0283	0.0144	0.0041	5′UTR	Island
cg21073927	GATA4	0.001324	0.0357	0.0221	0.0067	5′UTR	Island
cg10866709	GATA4	0.001456	0.0380	0.0126	0.0037	TSS1500	Island
cg27100236	GATA4	0.001941	0.0457	0.0324	0.0103	TSS1500	Island
cg08316825	GLRA1	2.14E-08	2.22E-05	0.0172	0.0026	TSS200; TSS200	Island
cg02071447	GLRA1	8.85E-07	0.0002	0.0186	0.0034	TSS200; TSS200	Island
cg27088197	GLRA1	1.24E-05	0.0015	0.0174	0.0037	Body; Body	Open sea
cg14319409	GLRA1	0.000116	0.0071	0.0281	0.0070	TSS200; TSS200	Island
cg02257793	GLRA1	0.000936	0.0286	0.0161	0.0047	Body; Body	N_Shore
cg00059225	GLRA1	4.34E-12	9.87E-08	0.0219	0.0026	First exon; first exon; 5′UTR; 5′UTR	Island
cg08097417	KLF14	9.99E-17	2.27E-11	0.0327	0.0031	TSS1500	Island
cg14361627	KLF14	7.37E-15	8.39E-10	0.0334	0.0035	TSS1500	Island
cg00094518	KLF14	5.41E-09	8.65E-06	0.0342	0.0053	First exon	Island
cg22285878	KLF14	3.23E-07	0.0001	0.0314	0.0057	TSS1500	Island
cg09499629	KLF14	1.60E-06	0.0003	0.0312	0.0061	TSS1500	Island
cg04528819	KLF14	1.85E-05	0.0020	0.0232	0.0051	First exon	Island
cg20426994	KLF14	6.14E-05	0.0046	0.0254	0.0061	First exon	Island
cg18751682	KLF14	0.000114	0.0070	0.0398	0.0100	TSS200	Island
cg08719712	KLF14	0.00043	0.0171	0.0187	0.0051	TSS1500	S_Shore
cg21449170	KLF14	0.000703	0.0237	0.0283	0.0082	TSS200	Island
cg07955995	KLF14	0.001076	0.0312	0.0481	0.0145	TSS1500	Island
cg25109431	KLF14	0.001263	0.0346	0.0244	0.0074	TSS200	Island
cg09823095	KLF14	0.001654	0.0413	0.0179	0.0055	TSS200	Island
cg26330518	NEFM	0.000142	0.0082	0.0162	0.0040	TSS1500	N_Shore
cg18898125	NEFM	0.000407	0.0165	0.0157	0.0042	TSS1500	N_Shore
cg22562942	NEFM	3.14E-09	6.17E-06	0.0232	0.0034	TSS200; first exon	Island
cg07552803	NEFM	1.73E-07	9.34E-05	0.0250	0.0044	TSS1500; first exon	Island
cg17078116	NEFM	4.18E-07	0.0001	0.0241	0.0044	TSS200; first exon	Island
cg09234518	NEFM	6.99E-07	0.0002	0.0308	0.0058	TSS200; TSS1500	Island
cg20585869	NEFM	3.00E-06	0.0005	0.0281	0.0057	TSS200; first exon	Island
cg02106941	NEFM	5.85E-06	0.0009	0.0222	0.0046	TSS1500; first exon	Island
cg04118306	NEFM	6.43E-06	0.0010	0.0229	0.0048	TSS200; first exon	Island
cg07502389	NEFM	1.36E-05	0.0016	0.0188	0.0040	TSS200; TSS1500	Island
cg02761376	NEFM	2.58E-05	0.0026	0.0196	0.0044	TSS1500; first exon	Island
cg16459364	NEFM	2.71E-05	0.0027	0.0211	0.0047	TSS200; TSS1500	Island
cg23290344	NEFM	0.000909	0.0280	0.0571	0.0170	TSS1500; first exon	Island
cg26980244	NEFM	5.40E-08	4.11E-05	0.0205	0.0033	5′UTR; first exon; body	Island
cg18267374	NEFM	4.80E-05	0.0039	0.0171	0.0039	TSS1500; 5′UTR; first exon	Island
cg16219603	PENK	9.11E-08	5.81E-05	0.0206	0.0034	TSS1500	Island
cg16419235	PENK	2.96E-05	0.0028	0.0183	0.0041	TSS1500	Island
cg11610346	PENK	0.000905	0.0280	0.0179	0.0052	TSS1500	Island
cg16072688	PENK	0.00167	0.0415	0.0104	0.0031	TSS1500	Island
cg19414741	PENK	3.48E-05	0.0032	0.0222	0.0051	Body; body	Island
cg22848150	PENK	8.45E-05	0.0058	0.0247	0.0060	Body; body	Island
cg04598121	PENK	9.32E-05	0.0061	0.0195	0.0047	Body; first exon	Island
cg04127342	PENK	0.000235	0.0115	0.0208	0.0054	Body; body	Island
cg19149681	PENK	0.001058	0.0309	0.0177	0.0052	TSS1500; TSS200	Island
cg12877723	PENK	0.001475	0.0383	0.0264	0.0082	Body; body	Island
cg21694941	PENK	0.001601	0.0404	0.0167	0.0051	5′UTR; 5′UTR; first exon	Island
cg25478614	SST	3.15E-06	0.0006	0.0196	0.0039	Body	N_Shore
cg05121480	SST	1.74E-05	0.0019	0.0275	0.0061	Body	N_Shore
cg15347189	SST	2.52E-05	0.0025	0.0175	0.0039	First exon	Island
cg00457403	SST	0.000519	0.0194	0.0220	0.0062	Body	N_Shore
cg02164046	SST	3.17E-06	0.0006	0.0229	0.0046	First exon; 5′UTR	Island
cg09805010	THRB	3.91E-05	0.0034	0.0404	0.0095	5′UTR; 5′UTR; 5′UTR	Island
cg20985755	THRB	0.000143	0.0082	0.0334	0.0085	TSS200; TSS200; TSS1500	Island
cg13790603	THRB	0.000867	0.0272	0.0358	0.0106	TSS200; TSS200; TSS1500	Island
cg22842189	THRB	0.000922	0.0283	0.0187	0.0055	5′UTR; 5′UTR; 5′UTR	Open sea
cg12258785	THRB	0.001165	0.0328	0.0148	0.0044	TSS1500; TSS1500; TSS1500	Island
cg15385623	THRB	0.000995	0.0297	0.0128	0.0037	First exon; TSS200; 5′UTR; first exon; 5′UTR	Island
cg02333852	THRB	0.000487	0.0186	0.0238	0.0066	First exon; first exon; 5′UTR; 5′UTR; first exon; 5′UTR	Island
cg00107187	TMEM179	3.16E-05	0.0030	0.0133	0.0029	First exon	Island
cg22943329	TMEM179	0.000123	0.0074	0.0186	0.0046	TSS1500	S_Shore
cg10281977	TMEM179	0.000185	0.0098	0.0155	0.0039	First exon	Island
cg03734874	TMEM179	0.000222	0.0111	0.0179	0.0046	TSS1500	Island
cg17471939	TP73	1.61E-06	0.0003	0.0230	0.0044	Body	Open sea
cg17163168	TP73	2.46E-05	0.0025	0.0179	0.0039	Body	Open sea
cg26055950	TP73	4.89E-05	0.0040	0.0131	0.0029	Body	Open sea
cg14159342	TP73	0.001625	0.0408	0.0114	0.0034	Body; body; body; body	S_Shore
cg25731359	TP73	1.55E-07	8.60E-05	0.0213	0.0037	TSS1500; TSS1500	Island
cg12475507	TP73	3.41E-07	0.0001	0.0156	0.0027	TSS1500; TSS1500	Island
cg01434649	TP73	7.24E-06	0.0010	0.0160	0.0032	TSS1500; TSS1500	Island
cg24073122	TP73	1.49E-05	0.0017	0.0182	0.0039	TSS1500; TSS1500	Island
cg07382920	TP73	4.10E-05	0.0035	0.0239	0.0056	TSS1500; TSS1500	Island
cg20611911	TP73	6.10E-05	0.0046	0.0230	0.0055	TSS1500; TSS1500	Island
cg24678611	TP73	0.000117	0.0072	0.0134	0.0032	TSS1500; TSS1500	Island
cg04391111	TP73	0.000533	0.0197	0.0174	0.0048	TSS1500; TSS1500	Island

Bold CpG sites showed age-associated DNA methylation changes both in Steegenga et al. [28] study and in liver (this study).

N_Shore: Northern shore; S_Shore: Southern shore; TSS: Transcription start site; UTR: Untranslated region.

Steegenga W , BoekschotenM , LuteCet al. Genome-wide age-related changes in DNA methylation and gene expression in human PBMCs . Age (Dordr).36 ( 3 ), 1523 – 1540 ( 2014 ).

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