1. Epidemiology and Global Health
  2. Genetics and Genomics

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

  1. Jenny van Dongen  Is a corresponding author
  2. Gonneke Willemsen
  3. BIOS Consortium
  4. Eco JC de Geus
  5. Dorret I Boomsma
  6. Michael C Neale
  1. Department of Biological Psychology, Vrije Universiteit Amsterdam, Netherlands
  2. Amsterdam Public Health Research Institute, Netherlands
  3. Amsterdam Reproduction and Development (AR&D) Research Institute, Netherlands
  4. Virginia Institute for Psychiatric and Behavioral Genetics, Virginia Commonwealth University, United States
https://doi.org/10.7554/eLife.83286
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Cite this article
  1. Jenny van Dongen
  2. Gonneke Willemsen
  3. BIOS Consortium
  4. Eco JC de Geus
  5. Dorret I Boomsma
  6. 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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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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Study flowchart.
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 significance threshold (Bonferroni correction) and 13 CpGs with significant differences are highlighted. (b) Mean within-pair differences in monozygotic twin pairs at the 13 CpGs that were epigenome-wide significant in smoking discordant monozygotic pairs. Mean within-pair differences of the residuals obtained after correction of methylation β-values for covariates are shown for 53 monozygotic pairs discordant for current/never smoking, 66 monozygotic pairs discordant for current/former smoking, 72 monozygotic pairs discordant for former/never smoking, 83 concordant current smoking monozygotic pairs, 88 concordant former smoking monozygotic pairs, and 406 concordant never smoking monozygotic pairs. (c) QQ-plot showing p-values from the EWAS in 53 smoking discordant monozygotic twin pairs (current versus never). P-values for CpGs located nearby significant SNPs from the genome-wide association study (GWAS) of smoking initiation are plotted in blue and all other genome-wide CpGs are plotted in orange.

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 discordant monozygotic pairs (current versus never smokers). (b) Scatterplot of cigarettes smoked per day in 80 concordant current smoking monozygotic pairs with complete data. (c) Scatterplot of within-pair differences in cigarettes smoked per day versus DNA methylation at cg05575921 (AHRR) in 80 concordant current smoking monozygotic pairs with complete data. (d) Scatterplot of within-pair differences in DNA methylation at cg05575921 (AHRR) versus time since quitting smoking (years) in 63 pairs discordant for former smoking.

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 smokerNever-smokerMean diffp-valueFormer smokerNever-smokerMean diffp-valueCurrent smokerFormer smokerMean diffp-valueTwin 1Twin 2Mean diffp-valueTwin 1Twin 2Mean diffp-valueTwin 1Twin 2Mean diffp-value
% Female pairs60.4%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)0.100.3441.4 (13.2)41.4 (13.1)0.020.8342.2
(12.6)		42.2
(12.5)		−0.060.4533.8
(10.3)		33.9
(10.5)		−0.120.1033.1
(11.3)		33.0
(11.2)		0.060.0845.2
(13.4)		45.2
(13.4)		0.090.29
Cigarettes per day at blood sampling, mean (SD), N missings8.9 (6.4), 6n.a.n.a.n.a.n.a.n.a.n.a.n.a.11.9 (7.2), 9n.a.n.a.n.a.11.1
(7.0), 2		10.9
(6.9), 1		0.001.00n.a.n.a.n.a.n.a.n.a.n.a.n.a.n.a.
Packyears, mean (SD), N missings6.8 (7.0), 13n.a.n.a.n.a.5.9 (11.1), 15n.a.n.a.n.a.13.6
(13.2), 9		9.3
(8.7), 10		3.90.059.7 (9.3), 108.3 (7.6), 90.220.82n.a.n.a.n.a.n.a.10.6
(11.5), 7		9.8
(10.4), 11		0.780.55
Years since quitting smoking, mean (SD), N missingsn.a.n.a.n.a.n.a.13.5 (11.4), 9n.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		−1.620.20
Plasma cotinine level, mean (SD), N missings*222 (197.5), 11.8 (2.6), 28261.11.6 × 10−51.4 (1.8), 490.9 (1.0), 520.620.43286.7 (330.5), 219.2 (70.0), 28293.82.5 × 10−6267 (290.9), 3279 (308.1), 3−8.70.781.3 (9.7), 2740.5 (0.8), 2600.070.5055.8 (222.2), 616 (16.2), 6483.30.26
Educational Attainment, N (%)   0.04   0.97   0.34   0.60   0.76   0.83
N missing1811  1313  1414  2834  105108  1420  
1.  Primary school only0 (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 schooling1 (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 schooling12 (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 schooling14 (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. University4 (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 missings23.8 (3.8), n.a.24.0 (3.4), n.a.−0.170.7425.0 (3.6), n.a.25.1 (4.2), n.a.−0.130.7523.7 (3.1)25.2 (4.2)−1.473.4 × 10−423.6 (3.5), n.a.23.1 (3.2), n.a.0.470.0623.8 (3.8),423.6 (3.4), 20.270.0325.6 (4.0), n.a.24.9 (3.7), n.a.0.630.02
Percentage monocytes, mean (SD), N missings8.0 (2.3), 08.5 (2.4), 0−0.440.198.6 (2.0), 08.3 (1.8), 00.290.198.6
(1.9),0		9.2
(3.1), 0		−0.570.148.3 (2.1), 08.1 (1.9), 00.200.368.5 (2.0), 08.5
(2.2), 0		0.030.758.4
(2.4), 0		8.5
(2.4), 0		−0.060.75
Percentage lymphocytes, mean (SD), N missings35.0 (8.9), 035.9 (10.0)−0.940.5033.6 (8.5), 034.0 (8.7)−0.370.7735.8
(8.2),0		35.6
(8.5), 0		0.250.8433.7 (8.3), 034.1 (8.3), 0−0.440.6736.3 (8.4), 036.2 (8.4), 00.040.9235.0
(7.7), 0		34.1
(8.4), 0		0.950.26
Percentage neutrophils, mean (SD), N missings53.4 (9.5), 052.1 (9.8), 01.340.3854.4 (9.1), 054.5 (9.1), 0−0.080.9551.6
(9.0),0		51.7
(8.9), 0		0–0.060.9653.7 (8.9), 053.7 (9.3), 00.030.9851.8, (8.7), 051.9 (9.3), 0−0.080.8652.8
(8.2), 0		53.7
(8.4), 0		−0.840.35
Percentage eosinophils, mean (SD), N missings3.1 (2.5), 03.1 (2.1), 00.050.913.1 (1.9), 02.9 (2.0), 00.150.533.3
(1.9),0		3.1
(1.7), 0		0.210.333.4 (2.2), 03.4 (1.8), 0−0.030.912.9 (1.8), 02.9
(1.9), 0		−0.040.663.1
(1.9), 0		3.2
(2.4), 0		−0.060.77
Percentage basophils, mean (SD), N missings0.5 (0.7), 00.5 (0.7), 0−0.010.960.3 (0.3), 00.4 (0.5), 0−0.020.760.6
(0.9),0		0.4
(0.4), 0		0.180.120.9 (3.1), 00.6 (1.1), 00.250.490.5 (0.7), 00.4
(0.7), 0		0.060.210.6
(1.1), 0		0.6
(0.9), 0		−0.010.97
                        
  1. *

    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 pairsFormer smoking discordant pairs (former/never)
IlmnIDCHRMAPINFOGene*Nearest geneMean diffp-value95conf_L95conf_HT-StatisticMean diffp-value95conf_L95conf_HT-Statistic
cg055759215373378AHRRAHRR0.1324.9 × 10−110.1000.1658.2650.0273.3 × 10−40.0130.0413.778
cg215666422233284661ALPPL20.0921.5 × 10−100.0690.1157.9600.0333.2 × 10−60.0200.0465.067
cg059512212233284402ALPPL20.0661.8 × 10−90.0480.0847.2700.0264.6 × 10−60.0160.0374.964
cg019402732233284934ALPPL20.0602.1 × 10−90.0440.0777.2400.0181.3 × 10−40.0090.0274.052
cg13411554353700276CACNA1DCACNA1D−0.0386.0 × 10−9−0.049−0.027−6.947−0.0070.10−0.0160.002−1.655
cg019013321175031054ARRB1ARRB10.0258.0 × 10−90.0180.0336.8680.0060.16−0.0020.0131.425
cg211611385399360AHRRAHRR0.0461.9 × 10−80.0320.0596.6420.0020.64−0.0060.0090.466
cg00336149353700195CACNA1DCACNA1D−0.0272.0 × 10−8−0.035−0.019−6.615−0.0020.60−0.0080.005−0.524
cg22132788745002486MYO1GMYO1G−0.0562.4 × 10−8−0.073−0.039−6.596−0.0114.5 × 10−3−0.019−0.004−2.930
cg21188533353700263CACNA1DCACNA1D−0.0363.9 × 10−8−0.047−0.025−6.437−0.0060.24−0.0150.004−1.196
cg09935388192947588GFI1GFI10.0524.1 × 10−80.0350.0686.4230.0020.61−0.0060.0100.519
cg256482035395444AHRRAHRR0.0355.3 × 10−80.0240.0466.3530.0020.48−0.0040.0080.710
cg19089201745002287MYO1GMYO1G−0.0407.5 × 10−8−0.053−0.028−6.260−0.0070.13−0.0170.002−1.529

  1. 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.

  2. *

    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.
cgidCHRPositionGeneNearest geneCigarettes per dayPackyearsp-value
rp-valuerr
cg055759215373378AHRRAHRR−0.525.9 × 10−7−0.551.2 × 10−6−0.080.50
cg215666422233284661ALPPL2−0.494.7 × 10−6−0.568.1 × 10−7−0.190.10
cg059512212233284402ALPPL2−0.444.7 × 10−5−0.569.0 × 10−7−0.180.12
cg019402732233284934ALPPL2−0.567.0 × 10−8−0.652.0 × 10−9−0.230.04
cg13411554353700276CACNA1DCACNA1D0.271.4 × 10−20.327.4 × 10−30.200.08
cg019013321175031054ARRB1ARRB1−0.233.8 × 10−2−0.345.5 × 10−3−0.040.71
cg211611385399360AHRRAHRR−0.528.4 × 10−7−0.527.4 × 10−6−0.050.65
cg00336149353700195CACNA1DCACNA1D0.361.0 × 10−30.423.5 × 10−40.160.16
cg22132788745002486MYO1GMYO1G0.412.1 × 10−40.418.4 × 10−40.140.23
cg21188533353700263CACNA1DCACNA1D0.324.1 × 10−30.381.4 × 10−30.270.01
cg09935388192947588GFI1GFI1−0.429.1 × 10−5−0.591.4 × 10−7−0.030.80
cg256482035395444AHRRAHRR−0.281.2 × 10−2−0.424.3 × 10−4−0.060.61
cg19089201745002287MYO1GMYO1G0.151.8 × 10−10.272.6 × 10−20.230.05
Table 4
Significantly associated transcripts in cis for CpGs that are differentially methylated in smoking discordant monozygotic twin pairs.
CpGGeneZ scorep-valueFDR
cg25648203EXOC3−7.342.11e−130
cg19089201RP4-647J21.15.552.84e−80
cg05575921EXOC3−4.860.000001190.00039
cg21161138EXOC3−3.820.0001330.0254
Author response table 1
check
FrequencyPercentValid PercentCumulative Percent
Valid1,00 no changes in original status934997.197.197.1
2,00 original status were adjusted based on data checks33.3.397.4
3,00 original status was missing, added from survey data1922.02.099.4
4,00 made missing based on data checks11.1.199.6
5,00 original status missing and insufficient data to add status43.4.4100.0
Total9628100.0100.0

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
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
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
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
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
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
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
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
Supplementary file 9

Biobank-based Integrative Omics Studies (BIOS) consortium investigators.

https://cdn.elifesciences.org/articles/83286/elife-83286-supp9-v2.docx
MDAR checklist
https://cdn.elifesciences.org/articles/83286/elife-83286-mdarchecklist1-v2.docx
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
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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  1. Jenny van Dongen
  2. Gonneke Willemsen
  3. BIOS Consortium
  4. Eco JC de Geus
  5. Dorret I Boomsma
  6. 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