Epigenome-wide analysis of long-term air pollution exposure and DNA methylation in monocytes: results from the Multi-Ethnic Study of Atherosclerosis

Figures & data

Table 1. Descriptive characteristics of 1,207 MESA participants.

Characteristic	n (%) or mean (SD)
	or median [IQR]
N	1,207
Age, years
Mean (SD)	69.6 (9.4)
Age category, years
<65	421 (35%)
65–74	378 (31%)
75–84	331 (27%)
≥85	77 (6%)
Sex
Women	623 (52%)
Men	584 (48%)
Race/ethnicity
White	570 (47%)
Hispanic	381 (32%)
Black	256 (21%)
Site
New York	398 (33%)
Maryland	301 (25%)
Minnesota	457 (38%)
North Carolina	51 (4%)
Body mass index, kg/m^2*
Mean (SD)	29.7 (5.5)
Body mass index category (WHO categories), kg/m^2*
Normal or underweight (<25)	237 (20%)
Grade 1 overweight (25–29.9)	454 (38%)
Grade 2 overweight (30–39.9)	458 (38%)
Grade 3 overweight (≥40.0)	57 (5%)
Physical activity, MET-min/wk m-su)*
Mean (SD)	5,696.2 (7,205.6)
Smoking status*
Never	484 (40%)
Former	604 (50%)
Current	112 (9%)
Second-Hand smoke (hours per week)*	3.7 (20.5)
Current alcohol use*
No	660 (55%)
Yes	543 (45%)
Education*
Less than high school	176 (15%)
High school	236 (20%)
Some college but no degree	212 (18%)
Bachelor’s/Associate/Technical	373 (31%)
Advanced degree	208 (17%)
Income*
<$24,999	297 (25%)
$25,000 – $49,999	371 (31%)
$50,000 – $99,999	333 (28%)
≥$100,000	163 (14%)
Neighbourhood socioeconomic status score*
Mean (SD)	−0.41 (1.1)
PM2.5, µg/m^3
Mean (SD)	10.7 (1.7)
Median [IQR]	10.4 [2.2]
NOX, ppb
Mean (SD)	28.7 (18.5)
Median [IQR]	19.1 [31.9]
SD, standard deviation; IQR; interquartile range; MET, metabolic equivalent of task; PM2.5, fine particulate matter; NOX, oxides of nitrogen; WHO, World Health Organization
* Contains missing values

Table 2. Differentially methylated regions for PM_2.5 and NO_X with FDR<0.05.

Chr	Start	End	Nearest Gene (hg19)	Location Relative to Gene (hg19)	CD14+ Chromatin State ^a	Average Fold Change in Methylation	P-value	FDR Cut-Off
PM2.5
5	1,594,282	1,594,863	SDHAP3	Overlaps 5' UTR	Weak promoter	1.15^b	1.4x10^−6	1.3x10^−5
6	29,648,379	29,649,024	ZFP57	Upstream	Weak promoter	1.11^b	2.1x10^−5	2.6x10^−5
16	11,374,865	11,374,865	PRM1	Inside exon	Heterochromatin/low	1.27^b	3.2x10^−5	3.9x10^−5
7	27,183,274	27,184,109	HOXA5	Overlaps 5' UTR	Weak promoter and Heterochromatin/low	1.04^b	4.6x10^−5	5.3x10^−5
NOX
5	1,594,282	1,595,048	SDHAP3	Overlaps 5' UTR	Weak promoter	1.26^c	7.7x10^−6	1.5x10^−5
6	29,648,379	29,649,024	ZFP57	Upstream	Weak promoter	1.25^c	9.8x10^−6	3.0x10^−5

PM_2.5, fine particulate matter; NO_X, oxides of nitrogen; FDR, false discovery rate; chr, chromosome; UTR, untranslated region.

^aPrediction based on histone modifications in monocyte samples from the BLUEPRINT (H3K27ac, H3K4me1, H3K4me3) and ENCODE (H3K36me3) projects

^bAverage fold change in methylation for every 2.5 µg/m³ higher exposure to PM_2.5.

^cAverage fold change in methylation for every 30 ppb higher exposure to NO_X.

Figure 1. Genome-wide significant differentially methylated region associated with PM_2.5 located at chromosome 5 (location: 1,594,282–1,594,863). The top portion of this plot is a karyogram of chromosome 5, with a red vertical bar indicating the location of interest. Below that is a graphic showing the exact chromosomal location in Mb, followed by a pictogram of the SDHAP3 gene (hg19). Tan boxes represent exons (vertically narrow portions are untranslated regions), and grey lines with arrows represent introns, as well as the direction of transcription (arrows pointing to the left indicate that this gene is transcribed from right to left, implying it is on the minus strand). The bottom section presents evidence for differential methylation of a DMR (blue line), based on a cluster of 12 CpGs (blue dots). The vertical axis for this section reflects the PM_2.5 coefficient from the linear model. Plots were generated by using the function makeMethPlot from the package methylation.

Figure 2. Genome-wide significant differentially methylated region associated with PM_2.5 located at chromosome 6 (location: 29,648,379–29,649,024). The top portion of this plot is a karyogram of chromosome 6, with a red vertical bar indicating the location of interest. Below that is a graphic showing the exact chromosomal location in Mb (hg19). The bottom section presents evidence for differential methylation of a DMR (blue line), based on a cluster of 19 CpGs (blue dots). The vertical axis for this section reflects the PM_2.5 coefficient from the linear model. Plots were generated by using the function makeMethPlot from the package methylation.

Figure 3. Genome-wide significant differentially methylated region associated with PM_2.5 located at chromosome 16 (location: 11,374,865–11,374,865). The top portion of this plot is a karyogram of chromosome 16, with a red vertical bar indicating the location of interest. Below that is a graphic showing the exact chromosomal location in Mb, followed by a pictogram of the PRM1 gene (hg19). Tan boxes represent exons (vertically narrow portions are untranslated regions), and grey lines with arrows represent introns, as well as the direction of transcription (arrows pointing to the left indicate that this gene is transcribed from right to left, implying it is on the minus strand). The bottom section presents evidence for differential methylation of a DMR (blue line), based on a cluster of 5 CpGs (blue dots). The vertical axis for this section reflects the PM_2.5 coefficient from the linear model. Plots were generated by using the function makeMethPlot from the package methylation.

Figure 4. Genome-wide significant differentially methylated region associated with PM_2.5 located at chromosome 7 (location: 27,183,274–27,184,109). The top portion of this plot is a karyogram of chromosome 7, with a red vertical bar indicating the location of interest. Below that is a graphic showing the exact chromosomal location in Mb, followed by pictograms of the HOXA5, HOXA6, and HOXA-AS3 genes (hg19). Tan boxes represent exons (vertically narrow portions are untranslated regions), and grey lines with arrows represent introns, as well as the direction of transcription (arrows pointing to the left indicate that this gene is transcribed from right to left, implying it is on the minus strand, and vice versa for arrows point to the right). The bottom section presents evidence for differential methylation of a DMR (blue line), based on a cluster of 47 CpGs (blue dots). The vertical axis for this section reflects the PM_2.5 coefficient from the linear model. Plots were generated by using the function makeMethPlot from the package methylation.

Table 3. Significant (FDR<0.05) associations between methylation of PM_2.5-associated DMRs and mRNA expression of nearby genes.

Chr	Start	End	Illumina Transcript	Gene (hg19)	β (95% CI)	P-value	FDR Cut-Off
7	27,183,274	27,184,109	ILMN_1753613	HOXA5	−0.288 (−0.332, −0.243)	1.5x10^−34	2.8x10^−3
7	27,183,274	27,184,109	ILMN_1739582	HOXA9	0.168 (0.118, 0.218)	8.1x10^−11	5.6x10^−3
7	27,183,274	27,184,109	ILMN_1689336	HOXA10	0.079 (0.047, 0.112)	2.1x10^−6	8.3x10^−3
16	11,374,865	11,374,865	ILMN_1738866	DEXI	0.012 (0.004, 0.019)	2.8x10^−3	1.1x10^−2
5	1,594,282	1,594,863	ILMN_1800197	MRPL36	0.014 (0.005, 0.024)	3.7x10^−3	1.4x10^−2

DMR, differentially methylated region; FDR, false discovery rate; CI, confidence interval.

Table 4. Differentially methylated sites for PM_2.5 and NO_X with FDR<0.05.

CpG	Chr	Nearest Gene	Distance	Location Relative to Gene (hg19)	CD14+ Chromatin State ^a	DNase HS CD14 + ^b	TFBS Any Cell ^c	β (95% CI)	P-value	FDR Cut-Off
PM2.5
cg05926640	1	TOMM20	181,469	Downstream	Strong enhancer	Yes	Yes	0.049 (0.032, 0.067)	5.6x10^−8	1.8x10^−7
cg04310517	16	KREMEN2	3,954	Upstream	Heterochromatin/low	No	No	0.052 (0.033, 0.072)	2.2x10^−7	3.5x10^−7
cg09509909	10	FGFBP3	3,489	Upstream	Heterochromatin/low	Yes	Yes	0.079 (0.049, 0.110)	4.6x10^−7	5.3x10^−7
NOX
cg11756214	19	ZNF347	0	5' UTR	Weak promoter	Yes	Yes	0.078 (0.050, 0.106)	5.6x10^−8	1.8x10^−7

PM_2.5, fine particulate matter; NO_X, oxides of nitrogen; FDR, false discovery rate; chr, chromosome; DNAse HS, DNase hypersensitivity; TFBS, transcription factor binding site; ENCODE, The Encyclopedia of DNA Elements; CI, confidence interval.

^aPrediction based on histone modifications in monocyte samples from the BLUEPRINT (H3K27ac, H3K4me1, H3K4me3) and ENCODE (H3K36me3) projects

^bDNase hypersensitivity reported in a CD14+ monocyte sample (ENCODE).

^cTFBS reported in any cell type available from the UCSC Genome Browser.

Table 5. Significant (FDR<0.05) associations between cg05926640 methylation and mRNA expression of nearby genes.

CpG	Illumina Transcript	Gene (hg19)	β (95% CI)	P-value	FDR Cut-Off
cg05926640	ILMN_2269564	ARID4B	−0.323 (−0.453, −0.192)	1.3x10^−6	9.3x10^−4
cg05926640	ILMN_2362982	ARID4B	−0.227 (−0.333, −0.120)	3.1x10^−5	1.9x10^−3
cg05926640	ILMN_1761334	ARID4B	−0.274 (−0.406, −0.143)	4.8x10^−5	2.8x10^−3
cg05926640	ILMN_1671005	IRF2BP2	−0.213 (−0.318, −0.108)	7.3x10^−5	3.7x10^−3
cg05926640	ILMN_1679796	TOMM20	0.118 (0.044, 0.191)	1.7x10^−3	4.6x10^−3

FDR, false discovery rate; CI, confidence interval.