Association of cigarette smoking and CRP levels with DNA methylation in α-1 antitrypsin deficiency

Figures & data

Table 1. Characteristics of 316 subjects from 162 families studied

Males	n (%)	143 (45.3)
Age in years	mean (SD)	52.6 (9.7)
Ever-smokers	n (%)	196 (62.0)
Current smokers	n (%)	13 (4.1)
Pack-years smoked**	mean (SD)	18.1 (14.4)
Age at smoking initiation**	mean (SD)	17.7 (4.7)
Duration of smoking in years**	mean (SD)	18.5 (8.4)
Time since quitting smoking in years***	mean (SD)	15.4 (10.3)
CRP level in mg/L	mean (SD)	4.8 (12.8)
ZZ AAT genotype at rs28929474	n(%)	316 (100%)

** Within 196 ever-smokers; ***within 183 former smokers; SD, standard deviation; CRP, C-Reactive Protein.

Table 2. Quality control criteria applied in the current study

Step	Number of subjects	Number of CpG sites
Starting data set	480	1,505
Remove positive/negative controls and samples that failed bisulfite conversion	398	1,505
Remove duplicates	364	1,505
Remove subjects with gender inconsistency	361	1,505
Remove subjects with who failed “First Hybridization” control	358	1,505
Remove sex chromosome markers	358	1,421
Remove CpG sites with mean detection p value ≥ 0.05	358	1,411
Normalize methylation Beta levels	358	1,411
Keep subjects with ZZ genotype at SERPINA1 locus – final data set	316	1,411

SERPINA1, serpin peptidase inhibitor; clade A (α-1 antiproteinase, antitrypsin), member 1.

Table 3. CpG sites associated with ever-smoking status with p < 10⁻⁴ in a univariate linear regression analysis and corresponding results from the linear mixed effect model and Mann-Whitney test

Gene	CG id	Methylation level in 316 subjects studied			Linear regression		LME model		Mann-Whitney test p value
		Median	IQR		B	p	B	p
TGFBI	cg07852148	0.038	0.027	0.055	-0.0148	1.55x10^−6 *	-0.0143	5.89x10^−6	1.54x10^−6
SCGB3A1	cg19421379	0.047	0.035	0.070	-0.0177	2.40 x10^−6 *	-0.0175	5.22x10^−6	1.81x10^−5
ISL1	cg16203270	0.052	0.037	0.081	-0.0180	3.35 x10^−6 *	-0.0187	2.30x10^−6	1.95x10^−5
FZD9	cg25707686	0.196	0.155	0.243	-0.0386	3.70x10^−6 *	-0.0384	7.01x10^−6	0.00013
SOX2	cg18036037	0.049	0.037	0.062	-0.0130	6.06x10^−6 *	-0.0131	8.59x10^−6	7.14x10^−5
SLIT2	cg13875122	0.075	0.051	0.108	-0.0266	6.58x10^−6 *	-0.0267	9.58X10^−6	8.91x10^−6
FAT	cg06830038	0.076	0.053	0.105	-0.0238	9.99x10^−6 *	-0.0254	4.49x10^−6	7.49x10^−5
EPHA3	cg10315231	0.046	0.034	0.072	-0.0160	1.08x10^−5	-0.0160	1.78x10^−5	4.18x10^−5
RAB32*	cg21792703	0.025	0.019	0.038	-0.0105	1.45x10^−5	-0.0108	1.19x10^−5	0.00019
DBC1	cg14445814	0.067	0.046	0.094	-0.0223	1.70x10^−5 *	-0.0216	4.39x10^−5	1.19x10^−5
NR2F6	cg24945194	0.060	0.046	0.075	-0.0113	1.79x10^−5	-0.0117	1.41x10^−5	0.00012
DLK1	cg20175079	0.067	0.048	0.096	-0.0198	1.85x10^−5	-0.0202	2.15x10^−5	0.00053
ISL1	cg14024101	0.041	0.030	0.058	-0.0130	2.90x10^−5	-0.0133	2.71x10^−5	0.001
TGFBI	cg00833799	0.236	0.179	0.300	-0.0437	3.09x10^−5	-0.0446	3.16x10^−5	6.10x10^−5
RAB32	cg01851450	0.040	0.028	0.057	-0.0136	3.10x10^−5	-0.0147	9.00x10^−6	9.35x10^−5
EYA4	cg23916819	0.087	0.062	0.118	-0.0244	3.52x10^−5	-0.0253	2.72x10^−5	4.44x10^−5
HTR1B	cg26034501	0.112	0.079	0.153	-0.0293	3.69x10^−5	-0.0299	2.78x10^−5	6.45x10^−5
FRZB	cg01872931	0.067	0.050	0.094	-0.0191	3.99x10^−5	-0.0193	4.77x10^−5	0.00014
DAPK1	cg01984172	0.029	0.021	0.040	-0.0079	5.25x10^−5 *	-0.0079	7.08x10^−5	1.40x10^−5
RARB	cg14265392	0.016	0.014	0.022	-0.0056	6.40x10^−5	-0.0056	8.11x10^−5	7.61x10^−5
DCC	cg22444505	0.083	0.061	0.110	-0.0195	7.48x10^−5	-0.0195	9.52x10^−5	0.00012
ADAMTS12	cg13887940	0.054	0.041	0.071	-0.0129	9.04x10^−5	-0.0131	9.21x10^−5	0.00018
GABRB3	cg20421990	0.041	0.031	0.053	-0.0100	9.57x10^−5	-0.0101	1.07x10^−4	1.612x10^−5

Association p values < 3.54 × 10⁻⁵ are depicted in bold. *p < 3.54 × 10⁻⁵ after Box-Cox transformation of the methylation Beta level. B, regression/LME model coefficient for the ever-smoking variable set as independent variable and coded as “1” for ever-smokers and as “0” for never-smokers (i.e., the reference group); IQR, interquartile range; LME, linear mixed effect.

Table 4. Five CpG sites most significantly associated with the age at smoking initiation, pack-years smoked, time since quitting smoking or duration of smoking in a univariate linear regression model and corresponding results from the linear mixed effect model

Phenotype	Gene	CG id	Methylation level*			Linear regression		LME model
			Median	IQR		B	p	B	p
Age at smoking initiation	MME	cg20228377	0.048	0.030	0.072	0.00378	7.62x10^−8	0.00380	4.34x10^−7
	TGFBI	cg07852148	0.034	0.025	0.049	0.00157	1.16x10^−6	0.00157	4.73x10^−6
	CSF1	cg06450700	0.026	0.020	0.035	0.00083	7.05x10^−5	0.00083	0.00016
	GABRB3	cg20421990	0.038	0.028	0.048	0.00121	7.19x10^−5	0.00121	0.00013
	TUSC3	cg10249582	0.050	0.037	0.070	0.00174	0.00012	0.00192	5.46x10^−5
Duration of smoking	RET	cg21859545	0.052	0.040	0.068	0.00071	0.00072	0.00065	0.0025
	CEBPA	cg23071874	0.035	0.027	0.047	0.00057	0.00374	0.00057	0.0046
	EGFR	cg24522859	0.037	0.030	0.046	-0.00040	0.00484	-0.00042	0.0033
	MYH11	cg15581089	0.039	0.026	0.058	-0.00068	0.00874	-0.00071	0.0075
	NOTCH1	cg26924342	0.055	0.041	0.075	0.00067	0.00932	0.00057	0.0284
Pack-years smoked	RET	cg21859545	0.052	0.040	0.068	0.00040	0.0007	0.00039	0.0014
	HLA-DOA	cg23794671	0.151	0.117	0.215	-0.00120	0.0013	-0.00113	0.0022
	GABRA5	cg17525249	0.734	0.693	0.777	0.00090	0.0018	0.00082	0.0048
	AXL	cg20384525	0.099	0.070	0.137	-0.00070	0.0078	-0.00070	0.006
	APC	cg24136097	0.206	0.166	0.248	-0.00090	0.0079	-0.00073	0.0254
Time since quitting smoking	PLSCR3	cg26114392	0.394	0.339	0.455	0.0019	0.0010	0.0018	0.0028
	ERN1	cg13884260	0.219	0.180	0.261	-0.0015	0.0010	-0.0015	0.0016
	CAV1	cg07838272	0.161	0.129	0.187	-0.0012	0.0013	-0.0012	0.0019
	EYA4	cg24842760	0.072	0.054	0.093	0.0006	0.0016	0.0006	0.0030
	BCR	cg01574123	0.797	0.751	0.840	-0.0017	0.0023	-0.0016	0.0054

Association p values < 3.54 × 10⁻⁵ are depicted in bold. *In subjects with non-missing, smoking-related phenotype studied. B, regression/LME model coefficient for the continuously defined smoking-related phenotype set as independent variable; IQR, interquartile range; LME, linear mixed effect.

Table 5. Five CpG sites most significantly associated with CRP (as a dependent or independent variable)

Level of CRP set as:	Gene	CG id	Methylation level in 316 subjects studied			Linear regression		LME model
			Median	IQR		B	p	B	p
Independent variable*	RIPK4	cg11719297	0.087	0.068	0.106	0.00042	0.0009	0.00041	0.0012
	ST6GAL1	cg23385525	0.097	0.072	0.147	0.00082	0.0025	0.00074	0.0039
	TIE1	cg19492156	0.328	0.255	0.384	-0.00130	0.0034	-0.00131	0.0034
	RUNX3	cg21368948	0.873	0.837	0.901	0.00070	0.0044	0.00071	0.0040
	KRT1	cg06030058	0.823	0.798	0.841	0.00055	0.0047	0.00051	0.0071
Dependent variable (log2-transformed)**	RUNX3	cg21368948	0.873	0.837	0.901	7.1	6.57x10^−5	7.1	6.04x10^−5
	KRT1	cg06030058	0.823	0.798	0.841	8.8	8.18x10^−5	8.1	0.00028
	JAK3	cg05244380	0.746	0.692	0.792	-4.3	0.00059	-4.1	0.0010
	CD82	cg24700286	0.248	0.186	0.316	-3.4	0.00075	-3.6	0.00024
	MMP9	cg12243192	0.049	0.036	0.064	-11.1	0.0014	-11.0	0.0013

* Tested with univariate linear regression analysis. **Regression analysis was additionally adjusted for gender. CRP, C-Reactive Protein; B, regression/LME model coefficient; IQR, interquartile range; LME, linear mixed effect.

Table 6. Association of mean methylation Beta level with smoking related phenotypes and CRP level in a univariate linear regression analysis

Phenotype	Linear regression		LME model
	B	p	B	p
Ever-smoking	-0.00452	0.007	-0.00467	0.006
Age at smoking initiation	0.000384	0.062	0.000452	0.031
Duration of smoking	0.00000877	0.939	-0.0000399	0.729
Pack-years smoked	-0.0000492	0.464	-0.0000425	0.525
Time since quitting smoking	-0.000153	0.118	-0.000137	0.171
CRP level	-0.000029	0.651*	-0.0000452	0.478**

P values < 0.05 are depicted in bold. *p = 0.84 for log₂-transformed level of CRP set as dependent variable and adjusted for gender. **p = 0.708 for log₂-transformed level of CRP set as dependent variable and adjusted for gender. CRP, C-Reactive Protein; B, regression/LME model coefficient for the continuously defined smoking-related or CRP level phenotype set as independent variable; IQR, interquartile range; LME, linear mixed effect.