Figures and data in Effects of smoking on genome-wide DNA methylation profiles: A study of discordant and concordant monozygotic twin pairs

Version of Record: September 14, 2023
Accepted Manuscript: August 10, 2023

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Jenny van Dongen

Gonneke Willemsen

BIOS Consortium

Eco JC de Geus

Dorret I Boomsma

Michael C Neale

(2023)
Effects of smoking on genome-wide DNA methylation profiles: A study of discordant and concordant monozygotic twin pairs

eLife 12:e83286.

https://doi.org/10.7554/eLife.83286
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Figures
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Additional files

4 figures, 5 tables and 12 additional files

Figures

Figure 1

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DNA methylation analysis in smoking discordant and smoking concordant monozygotic twin pairs.

Blood DNA methylation profiles (Illumina 450k array) from six groups of monozygotic twin pairs were analysed.

Figure 2

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Figure 3 with 1 supplement

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Top differentially methylated loci identified in monozygotic twin pairs discordant for current smoking.

(a) Manhattan plot of the epigenome-wide association study (EWAS) in 53 smoking discordant monozygotic twin pairs (current versus never). The red horizontal line denotes the epigenome-wide …

Figure 3—figure supplement 1

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DNA methylation levels in current/never smoking discordant monozygotic twin pairs.

Figures show the DNA methylation β-values (unadjusted for any covariates) in 53 discordant monozygotic twin pairs for the 13 significant CpGs.

Figure 4

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DNA methylation differences in smoking discordant and smoking concordant pairs.

(a) Distributions of the mean absolute within-pair differences in discordant and concordant pairs at the top 1000 CpGs with the lowest p-value from the epigenome-wide association study (EWAS) in …

Tables

Table 1

Descriptive statistics for smoking discordant and concordant monozygotic twin pairs.

	Discordant current/never(53 pairs)				Discordant former/never(72 pairs)				Discordant current/former(66 pairs)				Concordant current(83 pairs)				Concordant never(406 pairs)				Concordant former(88 pairs)
	Current smoker	Never-smoker	Mean diff	p-value	Former smoker	Never-smoker	Mean diff	p-value	Current smoker	Former smoker	Mean diff	p-value	Twin 1	Twin 2	Mean diff	p-value	Twin 1	Twin 2	Mean diff	p-value	Twin 1	Twin 2	Mean diff	p-value
% Female pairs	60.4%	60.4%	n.a.	n.a.	77.80%	77.80%	n.a.	n.a.	69.7%	69.7%	n.a.	n.a.	61.4%	61.4%	n.a.	n.a.	73.6%	73.6%	n.a.	n.a.	64.8%	64.8%	n.a.	n.a.
Age at blood sampling, mean (SD)	33.1 (8.0)	33.0 (7.9)	0.10	0.34	41.4 (13.2)	41.4 (13.1)	0.02	0.83	42.2 (12.6)	42.2 (12.5)	−0.06	0.45	33.8 (10.3)	33.9 (10.5)	−0.12	0.10	33.1 (11.3)	33.0 (11.2)	0.06	0.08	45.2 (13.4)	45.2 (13.4)	0.09	0.29
Cigarettes per day at blood sampling, mean (SD), N missings	8.9 (6.4), 6	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	11.9 (7.2), 9	n.a.	n.a.	n.a.	11.1 (7.0), 2	10.9 (6.9), 1	0.00	1.00	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.
Packyears, mean (SD), N missings	6.8 (7.0), 13	n.a.	n.a.	n.a.	5.9 (11.1), 15	n.a.	n.a.	n.a.	13.6 (13.2), 9	9.3 (8.7), 10	3.9	0.05	9.7 (9.3), 10	8.3 (7.6), 9	0.22	0.82	n.a.	n.a.	n.a.	n.a.	10.6 (11.5), 7	9.8 (10.4), 11	0.78	0.55
Years since quitting smoking, mean (SD), N missings	n.a.	n.a.	n.a.	n.a.	13.5 (11.4), 9	n.a.	n.a.	n.a.	n.a.	9.0 (10.2), 2	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	11.9 (9.1), 8	13.6 (11.8), 7	−1.62	0.20
Plasma cotinine level, mean (SD), N missings*	222 (197.5), 1	1.8 (2.6), 28	261.1	1.6 × 10⁻⁵	1.4 (1.8), 49	0.9 (1.0), 52	0.62	0.43	286.7 (330.5), 2	19.2 (70.0), 28	293.8	2.5 × 10⁻⁶	267 (290.9), 3	279 (308.1), 3	−8.7	0.78	1.3 (9.7), 274	0.5 (0.8), 260	0.07	0.50	55.8 (222.2), 61	6 (16.2), 64	83.3	0.26
Educational Attainment, N (%)				0.04				0.97				0.34				0.60				0.76				0.83
N missing	18	11			13	13			14	14			28	34			105	108			14	20
1. Primary school only	0 (0%)	1 (2.3%)			0 (0%)	1 (1.7%)			2 (3.8%)	5 (9.6%)			1 (1.8%)	2 (4.1%)			3 (1.0%)	4 (1.3%)			6 (8.1%)	4 (5.9%)
2. Lower vocational schooling	1 (2.9%)	0 (0%)			7 (11.9%)	6 (10.2%)			10 (19.2%)	4 (7.7%)			4 (7.3%)	1 (2.0%)			8 (2.7%)	10 (3.4%)			2 (2.7%)	10 (14.7%)
3. Lower secondary schooling (general)	2 (5.7%)	2 (4.8%)			11 (18.6%)	8 (13.6%)			7 (13.5%)	4 (7.7%)			5 (9.1%)	7 (14.3%)			11 (3.7%)	11 (3.7%)			10 (13.5%)	6 (8.8%)
4. Intermediate vocational schooling	12 (34.3%)	15 (35.7%)			13 (23.7%)	19 (32.2%)			8 (15.4%)	13 (25.0%)			16 (29.1%)	16 (32.7%)			81 (26.9%)	85 (28.5%)			26 (35.1%)	17 (25.0%)
5. Intermediate/higher secondary schooling (general)	2 (5.7%)	1 (2.4%)			3 (6.8%)	3 (5.1%)			2 (3.8%)	2 (3.8%)			4 (7.3%)	6 (12.2%)			15 (5.0%)	17 (5.7%)			3 (4.1%)	2 (2.9%)
6. Higher vocational schooling	14 (40.0%)	14 (33.3%)			14 (22.0%)	11 (18.6%)			15 (28.8%)	16 (30.8%)			19 (34.5%)	11 (22.4%)			97 (32.2%)	81 (27.2%)			17 (23.0%)	17 (25.0%)
7. University	4 (11.4%)	9 (21.4%)			10 (16.9%)	11 (18.6%)			8 (15.4%)	8 (15.4%)			6 (10.9%)	6 (12.2%)			86 (28.6%)	90 (30.2%)			10 (13.5%)	12 (17.6%)
BMI, mean (SD), N missings	23.8 (3.8), n.a.	24.0 (3.4), n.a.	−0.17	0.74	25.0 (3.6), n.a.	25.1 (4.2), n.a.	−0.13	0.75	23.7 (3.1)	25.2 (4.2)	−1.47	3.4 × 10⁻⁴	23.6 (3.5), n.a.	23.1 (3.2), n.a.	0.47	0.06	23.8 (3.8),4	23.6 (3.4), 2	0.27	0.03	25.6 (4.0), n.a.	24.9 (3.7), n.a.	0.63	0.02
Percentage monocytes, mean (SD), N missings	8.0 (2.3), 0	8.5 (2.4), 0	−0.44	0.19	8.6 (2.0), 0	8.3 (1.8), 0	0.29	0.19	8.6 (1.9),0	9.2 (3.1), 0	−0.57	0.14	8.3 (2.1), 0	8.1 (1.9), 0	0.20	0.36	8.5 (2.0), 0	8.5 (2.2), 0	0.03	0.75	8.4 (2.4), 0	8.5 (2.4), 0	−0.06	0.75
Percentage lymphocytes, mean (SD), N missings	35.0 (8.9), 0	35.9 (10.0)	−0.94	0.50	33.6 (8.5), 0	34.0 (8.7)	−0.37	0.77	35.8 (8.2),0	35.6 (8.5), 0	0.25	0.84	33.7 (8.3), 0	34.1 (8.3), 0	−0.44	0.67	36.3 (8.4), 0	36.2 (8.4), 0	0.04	0.92	35.0 (7.7), 0	34.1 (8.4), 0	0.95	0.26
Percentage neutrophils, mean (SD), N missings	53.4 (9.5), 0	52.1 (9.8), 0	1.34	0.38	54.4 (9.1), 0	54.5 (9.1), 0	−0.08	0.95	51.6 (9.0),0	51.7 (8.9), 0	0–0.06	0.96	53.7 (8.9), 0	53.7 (9.3), 0	0.03	0.98	51.8, (8.7), 0	51.9 (9.3), 0	−0.08	0.86	52.8 (8.2), 0	53.7 (8.4), 0	−0.84	0.35
Percentage eosinophils, mean (SD), N missings	3.1 (2.5), 0	3.1 (2.1), 0	0.05	0.91	3.1 (1.9), 0	2.9 (2.0), 0	0.15	0.53	3.3 (1.9),0	3.1 (1.7), 0	0.21	0.33	3.4 (2.2), 0	3.4 (1.8), 0	−0.03	0.91	2.9 (1.8), 0	2.9 (1.9), 0	−0.04	0.66	3.1 (1.9), 0	3.2 (2.4), 0	−0.06	0.77
Percentage basophils, mean (SD), N missings	0.5 (0.7), 0	0.5 (0.7), 0	−0.01	0.96	0.3 (0.3), 0	0.4 (0.5), 0	−0.02	0.76	0.6 (0.9),0	0.4 (0.4), 0	0.18	0.12	0.9 (3.1), 0	0.6 (1.1), 0	0.25	0.49	0.5 (0.7), 0	0.4 (0.7), 0	0.06	0.21	0.6 (1.1), 0	0.6 (0.9), 0	−0.01	0.97

*

Missing values include values that are below the detection limit. BMI = body mass Index.

Table 2

Epigenome-wide significant differentially methylated CpGs in monozygotic pairs discordant for current smoking status.

						Current smoking discordant pairs				Former smoking discordant pairs (former/never)
IlmnID	CHR	MAPINFO	Gene*	Nearest gene	Mean diff	p-value	95conf_L	95conf_H	T-Statistic	Mean diff	p-value	95conf_L	95conf_H	T-Statistic
cg05575921	5	373378	AHRR	AHRR	0.132	4.9 × 10⁻¹¹	0.100	0.165	8.265	0.027	3.3 × 10⁻⁴	0.013	0.041	3.778
cg21566642	2	233284661		ALPPL2	0.092	1.5 × 10⁻¹⁰	0.069	0.115	7.960	0.033	3.2 × 10⁻⁶	0.020	0.046	5.067
cg05951221	2	233284402		ALPPL2	0.066	1.8 × 10⁻⁹	0.048	0.084	7.270	0.026	4.6 × 10⁻⁶	0.016	0.037	4.964
cg01940273	2	233284934		ALPPL2	0.060	2.1 × 10⁻⁹	0.044	0.077	7.240	0.018	1.3 × 10⁻⁴	0.009	0.027	4.052
cg13411554	3	53700276	CACNA1D	CACNA1D	−0.038	6.0 × 10⁻⁹	−0.049	−0.027	−6.947	−0.007	0.10	−0.016	0.002	−1.655
cg01901332	11	75031054	ARRB1	ARRB1	0.025	8.0 × 10⁻⁹	0.018	0.033	6.868	0.006	0.16	−0.002	0.013	1.425
cg21161138	5	399360	AHRR	AHRR	0.046	1.9 × 10⁻⁸	0.032	0.059	6.642	0.002	0.64	−0.006	0.009	0.466
cg00336149	3	53700195	CACNA1D	CACNA1D	−0.027	2.0 × 10⁻⁸	−0.035	−0.019	−6.615	−0.002	0.60	−0.008	0.005	−0.524
cg22132788	7	45002486	MYO1G	MYO1G	−0.056	2.4 × 10⁻⁸	−0.073	−0.039	−6.596	−0.011	4.5 × 10⁻³	−0.019	−0.004	−2.930
cg21188533	3	53700263	CACNA1D	CACNA1D	−0.036	3.9 × 10⁻⁸	−0.047	−0.025	−6.437	−0.006	0.24	−0.015	0.004	−1.196
cg09935388	1	92947588	GFI1	GFI1	0.052	4.1 × 10⁻⁸	0.035	0.068	6.423	0.002	0.61	−0.006	0.010	0.519
cg25648203	5	395444	AHRR	AHRR	0.035	5.3 × 10⁻⁸	0.024	0.046	6.353	0.002	0.48	−0.004	0.008	0.710
cg19089201	7	45002287	MYO1G	MYO1G	−0.040	7.5 × 10⁻⁸	−0.053	−0.028	−6.260	−0.007	0.13	−0.017	0.002	−1.529

Coordinates are given based on genome build 37. Mean differences represent non-smoking twin minus smoking-twin (hence positive values indicate a higher methylation level in non-smoking twins). The table shows the 13 epigenome-wide significant CpGs from the within-pair EWAS in 53 discordant monozygotic twin pairs (current versus never smokers). Results from the comparison within 72 monozygotic pairs discordant for former smoking are also shown.
*

CpGs without a gene name are located in intergenic regions. 95conf_L = 95% confidence interval lower bound, 95conf_H = 95% confidence interval upper bound.

Table 3

Correlations of within-pair differences in DNA methylation with within-pair differences in cigarettes per day and packyears in 83 concordant current smoking monozygotic pairs.

cgid	CHR	Position	Gene	Nearest gene	Cigarettes per day			Packyears		p-value
cgid	CHR	Position	Gene	Nearest gene	r	p-value	r		r	p-value
cg05575921	5	373378	AHRR	AHRR	−0.52	5.9 × 10⁻⁷	−0.55	1.2 × 10⁻⁶	−0.08	0.50
cg21566642	2	233284661		ALPPL2	−0.49	4.7 × 10⁻⁶	−0.56	8.1 × 10⁻⁷	−0.19	0.10
cg05951221	2	233284402		ALPPL2	−0.44	4.7 × 10⁻⁵	−0.56	9.0 × 10⁻⁷	−0.18	0.12
cg01940273	2	233284934		ALPPL2	−0.56	7.0 × 10⁻⁸	−0.65	2.0 × 10⁻⁹	−0.23	0.04
cg13411554	3	53700276	CACNA1D	CACNA1D	0.27	1.4 × 10⁻²	0.32	7.4 × 10⁻³	0.20	0.08
cg01901332	11	75031054	ARRB1	ARRB1	−0.23	3.8 × 10⁻²	−0.34	5.5 × 10⁻³	−0.04	0.71
cg21161138	5	399360	AHRR	AHRR	−0.52	8.4 × 10⁻⁷	−0.52	7.4 × 10⁻⁶	−0.05	0.65
cg00336149	3	53700195	CACNA1D	CACNA1D	0.36	1.0 × 10⁻³	0.42	3.5 × 10⁻⁴	0.16	0.16
cg22132788	7	45002486	MYO1G	MYO1G	0.41	2.1 × 10⁻⁴	0.41	8.4 × 10⁻⁴	0.14	0.23
cg21188533	3	53700263	CACNA1D	CACNA1D	0.32	4.1 × 10⁻³	0.38	1.4 × 10⁻³	0.27	0.01
cg09935388	1	92947588	GFI1	GFI1	−0.42	9.1 × 10⁻⁵	−0.59	1.4 × 10⁻⁷	−0.03	0.80
cg25648203	5	395444	AHRR	AHRR	−0.28	1.2 × 10⁻²	−0.42	4.3 × 10⁻⁴	−0.06	0.61
cg19089201	7	45002287	MYO1G	MYO1G	0.15	1.8 × 10⁻¹	0.27	2.6 × 10⁻²	0.23	0.05

Table 4

Significantly associated transcripts in cis for CpGs that are differentially methylated in smoking discordant monozygotic twin pairs.

CpG	Gene	Z score	p-value	FDR
cg25648203	EXOC3	−7.34	2.11e−13	0
cg19089201	RP4-647J21.1	5.55	2.84e−8	0
cg05575921	EXOC3	−4.86	0.00000119	0.00039
cg21161138	EXOC3	−3.82	0.000133	0.0254

Author response table 1

check
	Frequency	Percent	Valid Percent	Cumulative Percent
Valid	1,00 no changes in original status	9349	97.1	97.1	97.1
	2,00 original status were adjusted based on data checks	33	.3	.3	97.4
	3,00 original status was missing, added from survey data	192	2.0	2.0	99.4
	4,00 made missing based on data checks	11	.1	.1	99.6
	5,00 original status missing and insufficient data to add status	43	.4	.4	100.0
	Total	9628	100.0	100.0

Additional files

Supplementary file 1 Questions on smoking that were asked at blood draw and quality control of longitudinal smoking data.: https://cdn.elifesciences.org/articles/83286/elife-83286-supp1-v2.docx
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Supplementary file 2 Genomic inflation factor of each epigenome-wide association study (EWAS) analysis.: https://cdn.elifesciences.org/articles/83286/elife-83286-supp2-v2.xlsx
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Supplementary file 3 Within-pair analysis results for 13 epigenome-wide significant CpGs in current/never discordant pairs.: https://cdn.elifesciences.org/articles/83286/elife-83286-supp3-v2.xlsx
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Supplementary file 4 Epigenome-wide significant CpGs in MZ twin pairs discordant for current/former smoking.: https://cdn.elifesciences.org/articles/83286/elife-83286-supp4-v2.xlsx
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Supplementary file 5 Correlations between the within-pair difference in DNA methylation level and time since quitting smoking in MZ pairs discordant for former smoking.: https://cdn.elifesciences.org/articles/83286/elife-83286-supp5-v2.xlsx
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Supplementary file 6 Epigenome-wide association study (EWAS) atlas trait enrichment analysis results for top CpGs identified in current/never smoking discordant twin pairs.: https://cdn.elifesciences.org/articles/83286/elife-83286-supp6-v2.xlsx
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Supplementary file 7 Kegg pathway enrichment analysis results for top CpGs identified in current/never smoking discordant twin pairs.: https://cdn.elifesciences.org/articles/83286/elife-83286-supp7-v2.xlsx
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Supplementary file 8 GO pathway enrichment analysis results for top CpGs identified in current/never smoking discordant twin pairs.: https://cdn.elifesciences.org/articles/83286/elife-83286-supp8-v2.xlsx
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Supplementary file 9 Biobank-based Integrative Omics Studies (BIOS) consortium investigators.: https://cdn.elifesciences.org/articles/83286/elife-83286-supp9-v2.docx
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MDAR checklist: https://cdn.elifesciences.org/articles/83286/elife-83286-mdarchecklist1-v2.docx
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Source code 1 R-script for within-MZ pair epigenome-wide association study (EWAS analysis).: https://cdn.elifesciences.org/articles/83286/elife-83286-code1-v2.zip
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Reporting standard 1 This manuscript follows the guidelines of STROBE (Strengthening the reporting of observational studies in epidemiology). The STROBE checklist has been included in the submission.: https://cdn.elifesciences.org/articles/83286/elife-83286-repstand1-v2.docx
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Figures

DNA methylation analysis in smoking discordant and smoking concordant monozygotic twin pairs.

Study flowchart.

Top differentially methylated loci identified in monozygotic twin pairs discordant for current smoking.

DNA methylation levels in current/never smoking discordant monozygotic twin pairs.

DNA methylation differences in smoking discordant and smoking concordant pairs.

Tables

Descriptive statistics for smoking discordant and concordant monozygotic twin pairs.

Epigenome-wide significant differentially methylated CpGs in monozygotic pairs discordant for current smoking status.

Correlations of within-pair differences in DNA methylation with within-pair differences in cigarettes per day and packyears in 83 concordant current smoking monozygotic pairs.

Significantly associated transcripts in cis for CpGs that are differentially methylated in smoking discordant monozygotic twin pairs.

Additional files

Supplementary file 1

Supplementary file 2

Supplementary file 3

Supplementary file 4

Supplementary file 5

Supplementary file 6

Supplementary file 7

Supplementary file 8

Supplementary file 9

MDAR checklist

Source code 1

Reporting standard 1

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DNA methylation analysis in smoking discordant and smoking concordant monozygotic twin pairs.

Study flowchart.

Top differentially methylated loci identified in monozygotic twin pairs discordant for current smoking.

DNA methylation levels in current/never smoking discordant monozygotic twin pairs.

DNA methylation differences in smoking discordant and smoking concordant pairs.

Descriptive statistics for smoking discordant and concordant monozygotic twin pairs.

Epigenome-wide significant differentially methylated CpGs in monozygotic pairs discordant for current smoking status.

Correlations of within-pair differences in DNA methylation with within-pair differences in cigarettes per day and packyears in 83 concordant current smoking monozygotic pairs.

Significantly associated transcripts in cis for CpGs that are differentially methylated in smoking discordant monozygotic twin pairs.

Supplementary file 1

Supplementary file 2

Supplementary file 3

Supplementary file 4

Supplementary file 5

Supplementary file 6

Supplementary file 7

Supplementary file 8

Supplementary file 9

MDAR checklist

Source code 1

Reporting standard 1