Impact of tobacco, alcohol, and marijuana on genome-wide DNA methylation and its relationship with hypertension

Figures & data

Table 1. Characteristics of participants in the TruDiagnostic DNA Biobank.

	N = 3424
Sex, male	2010 (58.7%)
Age in years, mean (range)	52.9 (13.3–97.8)
Ethnicity
European	2584 (75.5%)
African American or Black	70 (2.0%)
Asian	41 (1.2%)
Latino or Hispanic	276 (8.1%)
Middle Eastern or North African	76 (2.2%)
Native American or Alaska Native	26 (0.8%)
Pacific Islander or Oceanian	23 (0.7%)
Sub-Saharan African	7 (0.2%)
Other	321 (9.4%)
BMI (kg/m^2), median (range)	25.4 (10.1–58.2)
Tobacco consumption
None	3275 (95.6%)
Less than 1 cigarette per week	48 (1.4%)
Less than 1 cigarette per day	25 (0.7%)
1–5 cigarettes per day	27 (0.8%)
6–10 cigarettes per day	21 (0.6%)
11–20 cigarettes per day	20 (0.6%)
More than 20 cigarettes per day	8 (0.2%)
Alcohol consumption
Never	634 (18.5%)
On special occasions	976 (28.5%)
Once per week	578 (16.9%)
3–5 times per week	794 (23.2%)
Regularly	442 (12.9%)
Marijuana consumption
Missing	149
Never	2908 (88.8%)
On special occasions	180 (5.5%)
Once per week	46 (1.4%)
3–5 times per week	73 (2.2%)
Regularly	68 (2.1%)

Note: All the continuous variables are shown as mean (range) and the categorical variables as n (%).

BMI: body mass index.

Figure 1. Manhattan plots of the epigenome-wide association study (EWAS) of tobacco (a), alcohol (b), and marijuana (c) consumption. The Y-axis represents the -log10(p) values and the X-axis the position of the CpG sites within the chromosome. The blue line is the suggestive nominal p-value threshold (0.0001) and the red line is the p-value adjusted threshold lower than 0.05.

Figure 2. Boxplots showing the association between CpG methylation and substance consumption. (a) Association between cg05575921 methylation (AHRR) and tobacco consumption. (b) Association between cg06690548 methylation (SLC7A11) and alcohol consumption. The Y-axis represents the residuals for beta values after adjusting by covariates. The X-axis represents the number of cigarettes smoked and the frequency of drinking, respectively. Methylation means for each tobacco consumption level are represented with their 95% confidence intervals.

Table 2. Top 15 differentially methylated CpG sites by tobacco consumption.

CpG	chr	position	Gene Symbol	Gene Group	Less than 1 per week	Less than 1 per day	1–5 per day	6–10 per day	11–20 per day	More than 20 per day	F	P-Value	Adjusted P-Value	EWAS catalogue (same direction/total studies)
cg05575921	chr5	373378	AHRR	Body	−0.028	−0.054	−0.097	−0.216	−0.183	−0.217	208.2	1.3E–226	9.7E–221	26/26
cg21566642	chr2	233284661			−0.030	−0.058	−0.066	−0.109	−0.111	−0.160	59.7	1.6E–70	5.9E–65	22/22
cg01940273	chr2	233284934			−0.016	−0.034	−0.045	−0.066	−0.069	−0.109	53.6	2.4E–63	5.9E–58	22/22
cg03636183	chr19	17000585	F2RL3	Body	−0.007	−0.017	−0.029	−0.064	−0.053	−0.094	50.9	2.8E–60	5.2E–55	21/21
ch.6.2768623F	chr6	144037285	PHACTR2	Body	−0.002	−0.001	0.000	0.009	−0.005	0.350	49.1	4.7E–58	7.0E–53	Not reported
cg21161138	chr5	399360	AHRR	Body	−0.008	−0.016	−0.015	−0.071	−0.043	−0.069	40.6	5.7E–48	7.1E–43	16/16
cg17739917	chr17	38477572	RARA	5‘UTR	−0.008	−0.053	−0.034	−0.069	−0.082	−0.116	38.2	4.5E–45	4.8E–40	7/7
cg15928392	chr2	81694446			0.000	0.000	0.001	−0.001	−0.002	−0.106	34.1	3.4E–40	3.2E–35	Not reported
cg16774290	chr7	4824628	AP5Z1	Body	0.000	0.000	0.000	−0.001	0.000	−0.100	33.2	4.1E–39	3.4E–34	Not reported
cg11173636	chr10	65632259			−0.008	0.017	0.024	0.018	0.339	−0.020	30.5	8.5E–36	6.3E–31	Not reported
cg09935388	chr1	92947588	GFI1	Body	−0.018	−0.030	−0.029	−0.100	−0.069	−0.134	30.0	3.5E–35	2.4E–30	20/20
cg26703534	chr5	377358	AHRR	Body	−0.004	−0.013	−0.016	−0.052	−0.034	−0.049	29.9	4.0E–35	2.4E–30	14/14
cg14990808	chr6	28493651	GPX5	TSS200	−0.002	−0.022	0.005	0.002	0.001	0.246	26.3	1.1E–30	6.2E–26	Not reported
cg25648203	chr5	395444	AHRR	Body	−0.006	−0.005	−0.011	−0.047	−0.037	−0.057	24.8	6.2E–29	3.3E–24	18/18
cg04176674	chr14	21121564			−0.004	−0.002	0.001	0.005	0.000	0.343	24.7	7.8E–29	3.9E–24	Not reported

Note: The CpG sites are annotated based on the chromosome (chr), the position (pos), the gene symbol from HGNC, and the gene group (based on the position of the CpG regarding the nearest gene). Each CpG site has a beta value for each consumer group vs non-consumers, a F-statistic (F), a nominal p-value, and an adjusted p-value by Bonferroni. The last column represents the number of studies in the EWAS catalogue that reported the same effect direction compared to the total studies that reported the beta value for the specific CpG site.

Table 3. Top 15 differentially methylated CpG sites by alcohol consumption.

CpG	chr	position	Gene Symbol	Gene Group	On special occasions	Once per week	3–5 times per week	Regularly	F	P-Value	Adjusted P-Value	Lohof et al (direction)
cg06690548	chr4	139162808	SLC7A11	Body	−0.003	−0.005	−0.015	−0.031	103.1	5.9E–83	4.4E–77	–
cg11376147	chr11	57261198	SLC43A1	Body	−0.001	−0.002	−0.006	−0.010	49.3	2.4E–40	8.9E–35	–
cg26457483	chr1	120256112	PHGDH	Body	−0.001	−0.004	−0.017	−0.028	39.7	1.5E–32	3.8E–27	–
cg14476101	chr1	120255992	PHGDH	Body	−0.002	−0.005	−0.016	−0.031	36.2	1.1E–29	2.0E–24	–
cg18120259	chr6	43894639	LINC01512	Body	−0.002	−0.004	−0.009	−0.019	35.0	1.1E–28	1.6E–23	–
cg06088069	chr14	75895604	JDP2	5‘UTR	−0.002	−0.003	−0.007	−0.013	34.2	5.0E–28	6.1E–23	–
cg21998542	chr10	33605101	NRP1	Body	−0.002	−0.005	−0.009	−0.019	29.0	9.2E–24	9.8E–19	–
cg15837522	chr8	117892654			−0.003	−0.011	−0.022	−0.031	28.7	1.7E–23	1.6E–18	–
cg06644515	chr1	173834831	SNORD47;GAS5; SNORD81;SNORD80; SNORD78;SNORD79	TSS1500;Body; TSS1500;TSS1500; TSS200;TSS1500	−0.001	−0.001	−0.007	−0.013	27.6	1.3E–22	1.1E–17	–
cg12116137	chr17	1576449	PRPF8	Body	0.001	0.001	0.008	0.018	25.5	7.5E–21	5.5E–16	++
cg19693031	chr1	145441552	TXNIP	3‘UTR	−0.004	−0.003	−0.011	−0.026	25.1	1.8E–20	1.2E–15	–
cg12825509	chr3	185648568	TRA2B	Body	0.000	−0.004	−0.010	−0.016	24.6	4.5E–20	2.8E–15	–
cg14346162	chr21	44490229	CBS	Body	0.001	0.004	0.009	0.014	23.5	3.1E–19	1.8E–14	Not reported
cg12973487	chr19	1623075	TCF3	Body	0.004	0.005	0.007	0.014	22.6	2.0E–18	1.0E–13	++
cg05713943	chr13	97912352	MBNL2	5‘UTR	0.001	0.000	−0.005	−0.008	21.8	8.7E–18	4.2E–13	–

Note: The CpG sites are annotated based on the chromosome (chr), the position (pos), the gene symbol from HGNC, and the gene group (based on the position of the CpG regarding the nearest gene). Each CpG site has a beta value for each consumer group vs non-consumers, a F-statistic (F), a nominal p-value, and an adjusted p-value by Bonferroni. We also included one column with the direction of the effect in a previous study (Lohoff et al (31)) for those that were significant in their analysis.

Table 4. Top 15 differentially methylated CpG sites by marijuana consumption.

CpG	chr	position	Gene Symbol	Gene Group	On special occasions	Once per week	3–5 times per week	Regularly	F	P-Value	Adjusted P-Value
cg05107281	chr11	47072710	CSTPP1	Body	−0.004	−0.010	−0.014	0.001	9.4	1.6E–07	0.12
cg06499565	chr20	35374747	NDRG3	TSS1500	0.001	0.006	0.000	−0.001	8.7	5.7E–07	0.17
cg10054857	chr18	61816543	LINC00305	TSS1500	−0.004	−0.007	−0.002	−0.002	8.4	1.0E–06	0.17
cg24344693	chr10	133273964			−0.004	0.003	−0.001	−0.002	8.2	1.5E–06	0.17
cg20226924	chr5	133985272	SEC24A	Body	0.001	0.003	0.003	0.001	8.2	1.5E–06	0.17
cg05575921	chr5	373378	AHRR	Body	−0.003	−0.011	−0.012	−0.020	8.1	1.7E–06	0.17
cg21161138	chr5	399360	AHRR	Body	−0.004	−0.011	−0.009	−0.012	8.1	1.7E–06	0.17
cg19730404	chr9	139361517	SEC16A; SEC16A	ExonBnd;Body	−0.003	−0.001	0.001	−0.004	7.8	2.8E–06	0.25
cg19308363	chr12	116290566			0.006	0.005	−0.005	−0.015	7.7	3.4E–06	0.25
cg08258765	chr5	24841586	LOC340107	TSS1500	0.005	0.002	0.004	−0.002	7.7	3.8E–06	0.25
cg03838168	chrX	48433876	RBM3	Body	−0.003	−0.004	0.015	0.005	7.7	3.9E–06	0.25
cg16822035	chr13	113633379	MCF2L;MCF2L	Body;TSS1500	0.001	0.004	0.009	0.012	7.6	4.0E–06	0.25
cg11756734	chr12	9028945	A2ML1	3‘UTR	−0.005	0.002	−0.007	0.003	7.5	4.8E–06	0.26
cg08415592	chr22	36648973	APOL1	TSS200	0.008	0.016	−0.004	0.007	7.4	5.8E–06	0.26
cg17325792	chr1	77042560	ST6GALNAC3; ST6GALNAC3	3‘UTR;Body	0.003	0.001	0.003	−0.011	7.4	6.6E–06	0.26

Note: The CpG sites are annotated based on the chromosome (chr), the position (pos), the gene symbol from HGNC, and the gene group (based on the position of the CpG regarding the nearest gene). Each CpG site has a beta value for each consumer group vs non-consumers, a F-statistic (F), a nominal p-value, and an adjusted p-value by Bonferroni.

Figure 3. Venn diagrams comparing CpG sites and genes differentially methylated for tobacco and alcohol use. (a) Comparison between differentially methylated CpG sites with a P-value adjusted lower than 0.05. (b) Comparison between genes where the CpG sites differentially methylated are annotated.

Figure 4. Forest plot of the association between tobacco (a) and alcohol (b) consumption with hypertension. OR: Odds Ratio.

Data availability statement

The data that support the findings of this study are available upon reasonable request due to ensure the privacy of the participants. Please email [email protected] for data requests. Any custom code or software used in our analysis is available at DOI: 10.5281/zenodo.6417926 (URL: https://zenodo.org/badge/latestdoi/296552532).