The Association Between PNPLA2 Methylation in Peripheral Blood and Early-Stage Lung Cancer in a Case–Control Study

Figures & data

Table 1 Clinical Characteristics of LC Cases

Sample Characteristics	Type	N	%
Age	≤ 55 years	64	38.1
	> 55 years	104	61.9
Gender	Female	60	35.7
	Male	108	64.3
Tumor subtype	Adenocarcinoma in situ	37	22.0
	Microinvasive adenocarcinoma	34	20.2
	Invasive adenocarcinoma	97	57.8
Tumor size	T1	147	87.5
	T2 and T3	21	12.5
Tumor length	≤ 1 cm	68	40.5
	> 1 cm	100	59.5
Lymph node involvement	0	164	97.6
	1 and 2	4	2.4
Tumor stage	Stage I	158	94.0
	Stage II	10	6.0

Abbreviations: LC, lung cancer; N, number.

Table 2 The Association Between PNPLA2 Methylation and LC

CpG Sites	Controls (N = 187) Median (IQR)	LC Cases (N = 168) Median (IQR)	OR (95% CI)^# per −10% Methylation	P-value^#
PNPLA2_CpG_1	0.28 (0.17–0.40)	0.29 (0.19–0.43)	1.06 (0.87–1.28)	0.566
PNPLA2_CpG_2,3	0.17 (0.12–0.37)	0.18 (0.10–0.41)	0.96 (0.84–1.09)	0.495
PNPLA2_CpG_4	0.02 (0.02–0.04)	0.03 (0.02–0.04)	0.71 (0.27–1.88)	0.490
PNPLA2_CpG_5	0.04 (0.03–0.05)	0.04 (0.03–0.05)	0.87 (0.63–1.21)	0.414
PNPLA2_CpG_6	0.37 (0.32–0.43)	0.36 (0.27–0.48)	0.89 (0.77–1.03)	0.130
PNPLA2_CpG_7	0.23 (0.17–0.27)	0.22 (0.17–0.28)	1.08 (0.89–1.31)	0.453
PNPLA2_CpG_8,10	0.35 (0.31–0.38)	0.33 (0.28–0.38)	1.44 (1.06–1.96)	0.018
PNPLA2_CpG_9	0.28 (0.24–0.33)	0.28 (0.23–0.34)	0.82 (0.69–0.98)	0.029
PNPLA2_CpG_11,12	0.46 (0.38–0.53)	0.45 (0.35–0.51)	1.17 (0.97–1.42)	0.100
PNPLA2_CpG_13	0.15 (0.13–0.18)	0.15 (0.11–0.19)	0.85 (0.64–1.14)	0.288
PNPLA2_CpG_14,15,16,17	0.11 (0.08–0.13)	0.10 (0.07–0.14)	1.07 (0.62–1.85)	0.809
PNPLA2_CpG_18	0.12 (0.09–0.14)	0.11 (0.08–0.15)	0.94 (0.69–1.29)	0.718
PNPLA2_CpG_20,21	0.40 (0.34–0.45)	0.38 (0.30–0.46)	1.04 (0.85–1.28)	0.675

Notes: ^#Logistic regression, adjusted for age, gender and experimental batches. Significant P-values are in bold.

Abbreviations: LC, lung cancer; N, number; IQR, interquartile range; OR, odds ratio; CI, confidence interval.

Table 3 The Association Between PNPLA2 Methylation and LC at Very Early Stage (Stage I)

CpG Sites	Controls (N = 187) Median (IQR)	LC Cases (N = 158) Median (IQR)	OR (95% CI)^# per −10% Methylation	P-value^#
PNPLA2_CpG_1	0.28 (0.17–0.40)	0.28 (0.18–0.40)	1.06 (0.88–1.29)	0.525
PNPLA2_CpG_2,3	0.17 (0.12–0.37)	0.18 (0.10–0.40)	0.96 (0.84–1.09)	0.507
PNPLA2_CpG_4	0.02 (0.02–0.04)	0.03 (0.02–0.04)	0.62 (0.23–1.67)	0.342
PNPLA2_CpG_5	0.04 (0.03–0.05)	0.04 (0.03–0.05)	0.87 (0.63–1.20)	0.394
PNPLA2_CpG_6	0.37 (0.32–0.43)	0.36 (0.27–0.48)	0.89 (0.76–1.03)	0.112
PNPLA2_CpG_7	0.23 (0.17–0.27)	0.22 (0.16–0.27)	1.08 (0.89–1.32)	0.432
PNPLA2_CpG_8,10	0.35 (0.31–0.38)	0.33 (0.28–0.38)	1.42 (1.05–1.93)	0.025
PNPLA2_CpG_9	0.28 (0.24–0.33)	0.29 (0.23–0.35)	0.80 (0.66–0.96)	0.017
PNPLA2_CpG_11,12	0.46 (0.38–0.53)	0.45 (0.35–0.51)	1.17 (0.97–1.42)	0.101
PNPLA2_CpG_13	0.15 (0.13–0.18)	0.15 (0.11–0.19)	0.84 (0.62–1.12)	0.238
PNPLA2_CpG_14,15,16,17	0.11 (0.08–0.13)	0.10 (0.07–0.14)	0.99 (0.57–1.73)	0.982
PNPLA2_CpG_18	0.12 (0.09–0.14)	0.11 (0.09–0.16)	0.91 (0.66–1.26)	0.578
PNPLA2_CpG_20,21	0.40 (0.34–0.45)	0.39 (0.30–0.47)	1.00 (0.81–1.23)	0.980

Notes: ^#Logistic regression, adjusted for age, gender and experimental batches. Significant P-values are in bold.

Abbreviations: LC, lung cancer; N, number; IQR, interquartile range; OR, odds ratio; CI, confidence interval.

Table 4 Gender-Stratified Association Between PNPLA2 Methylation and LC

CpG Sites	Controls Median (IQR)	LC Cases Median (IQR)	OR (95% CI)^# per −10% Methylation	P-value^#
Female (63 controls vs 60 LC cases)
PNPLA2_CpG_1	0.33 (0.19–0.52)	0.26 (0.15–0.48)	1.10 (0.86–1.41)	0.451
PNPLA2_CpG_2,3	0.14 (0.10–0.20)	0.17 (0.12–0.24)	0.80 (0.63–1.01)	0.063
PNPLA2_CpG_4	0.03 (0.02–0.03)	0.03 (0.02–0.05)	0.19 (0.02–1.95)	0.163
PNPLA2_CpG_5	0.03 (0.02–0.04)	0.03 (0.02–0.04)	0.95 (0.68–1.35)	0.786
PNPLA2_CpG_6	0.37 (0.33–0.43)	0.37 (0.29–0.48)	0.90 (0.71–1.13)	0.360
PNPLA2_CpG_7	0.24 (0.21–0.28)	0.24 (0.17–0.28)	1.21 (0.80–1.83)	0.375
PNPLA2_CpG_8,10	0.34 (0.32–0.37)	0.32 (0.28–0.36)	1.85 (1.07–3.22)	0.029
PNPLA2_CpG_9	0.27 (0.24–0.32)	0.26 (0.22–0.33)	0.87 (0.70–1.09)	0.235
PNPLA2_CpG_11,12	0.46 (0.39–0.53)	0.45 (0.32–0.50)	1.33 (0.99–1.78)	0.057
PNPLA2_CpG_13	0.15 (0.12–0.18)	0.14 (0.10–0.17)	0.90 (0.62–1.30)	0.566
PNPLA2_CpG_14,15,16,17	0.11 (0.09–0.13)	0.09 (0.07–0.13)	1.51 (0.57–4.01)	0.409
PNPLA2_CpG_18	0.12 (0.11–0.14)	0.11 (0.08–0.16)	0.94 (0.64–1.39)	0.761
PNPLA2_CpG_20,21	0.40 (0.35–0.47)	0.41 (0.30–0.47)	1.01 (0.72–1.43)	0.934
Male (124 controls vs 108 LC cases)
PNPLA2_CpG_1	0.25 (0.17–0.37)	0.29 (0.22–0.40)	0.99 (0.73–1.34)	0.951
PNPLA2_CpG_2,3	0.29 (0.12–0.44)	0.25 (0.10–0.47)	1.07 (0.90–1.27)	0.446
PNPLA2_CpG_4	0.02 (0.01–0.04)	0.02 (0.01–0.04)	0.94 (0.32–2.76)	0.909
PNPLA2_CpG_5	0.04 (0.03–0.05)	0.04 (0.03–0.05)	0.68 (0.36–1.30)	0.243
PNPLA2_CpG_6	0.37 (0.31–0.42)	0.35 (0.27–0.46)	0.89 (0.74–1.08)	0.228
PNPLA2_CpG_7	0.21 (0.15–0.27)	0.22 (0.16–0.28)	1.04 (0.83–1.31)	0.743
PNPLA2_CpG_8,10	0.35 (0.30–0.38)	0.34 (0.28–0.39)	1.27 (0.87–1.84)	0.214
PNPLA2_CpG_9	0.29 (0.25–0.33)	0.29 (0.24–0.35)	0.75 (0.56–1.00)	0.052
PNPLA2_CpG_11,12	0.45 (0.35–0.53)	0.44 (0.36–0.54)	1.06 (0.82–1.38)	0.633
PNPLA2_CpG_13	0.15 (0.13–0.18)	0.15 (0.11–0.20)	0.80 (0.51–1.24)	0.316
PNPLA2_CpG_14,15,16,17	0.10 (0.08–0.12)	0.10 (0.07–0.14)	0.90 (0.46–1.77)	0.769
PNPLA2_CpG_18	0.12 (0.09–0.13)	0.11 (0.09–0.14)	0.95 (0.55–1.64)	0.858
PNPLA2_CpG_20,21	0.40 (0.34–0.45)	0.37 (0.30–0.46)	1.06 (0.82–1.37)	0.673

Notes: ^#Logistic regression, adjusted for age and experimental batches. Significant P-values are in bold.

Abbreviations: LC, lung cancer; IQR, interquartile range; OR, odds ratio; CI, confidence interval.

Table 5 Age-Stratified Association Between PNPLA2 Methylation and LC

CpG Sites	Controls Median (IQR)	LC Cases Median (IQR)	OR (95% CI)^# per −10% Methylation	P-value^#
≤ 55 years old (100 controls vs 64 LC cases)
PNPLA2_CpG_1	0.32 (0.20–0.44)	0.24 (0.16–0.39)	1.19 (0.84–1.67)	0.327
PNPLA2_CpG_2,3	0.18 (0.11–0.39)	0.16 (0.08–0.40)	0.92 (0.78–1.10)	0.381
PNPLA2_CpG_4	0.02 (0.02–0.03)	0.02 (0.01–0.04)	0.82 (0.23–2.94)	0.758
PNPLA2_CpG_5	0.04 (0.02–0.05)	0.03 (0.02–0.06)	0.68 (0.38–1.25)	0.215
PNPLA2_CpG_6	0.39 (0.32–0.44)	0.39 (0.28–0.53)	0.77 (0.63–0.95)	0.012
PNPLA2_CpG_7	0.23 (0.18–0.27)	0.23 (0.16–0.28)	1.30 (0.92–1.84)	0.134
PNPLA2_CpG_8,10	0.35 (0.31–0.38)	0.33 (0.24–0.38)	2.43 (1.45–4.05)	0.001
PNPLA2_CpG_9	0.29 (0.24–0.33)	0.29 (0.23–0.39)	0.75 (0.60–0.94)	0.011
PNPLA2_CpG_11,12	0.45 (0.38–0.54)	0.45 (0.36–0.51)	1.28 (0.98–1.67)	0.073
PNPLA2_CpG_13	0.15 (0.13–0.18)	0.15 (0.11–0.21)	0.77 (0.50–1.18)	0.227
PNPLA2_CpG_14,15,16,17	0.11 (0.09–0.13)	0.11 (0.08–0.14)	1.38 (0.53–3.63)	0.512
PNPLA2_CpG_18	0.12 (0.10–0.14)	0.12 (0.09–0.16)	0.79 (0.52–1.21)	0.279
PNPLA2_CpG_20,21	0.39 (0.34–0.45)	0.39 (0.29–0.46)	1.00 (0.75–1.32)	0.983
> 55 years old (87 controls vs 104 LC cases)
PNPLA2_CpG_1	0.23 (0.14–0.39)	0.30 (0.22–0.45)	0.86 (0.66–1.14)	0.293
PNPLA2_CpG_2,3	0.17 (0.12–0.36)	0.20 (0.12–0.42)	0.95 (0.77–1.17)	0.602
PNPLA2_CpG_4	0.02 (0.01–0.04)	0.03 (0.02–0.04)	0.53 (0.11–2.60)	0.430
PNPLA2_CpG_5	0.04 (0.03–0.05)	0.04 (0.03–0.05)	1.07 (0.60–1.91)	0.812
PNPLA2_CpG_6	0.36 (0.31–0.41)	0.35 (0.27–0.44)	0.97 (0.74–1.26)	0.801
PNPLA2_CpG_7	0.22 (0.17–0.28)	0.22 (0.17–0.28)	0.92 (0.69–1.22)	0.571
PNPLA2_CpG_8,10	0.34 (0.31–0.38)	0.33 (0.29–0.38)	0.84 (0.53–1.34)	0.464
PNPLA2_CpG_9	0.28 (0.25–0.32)	0.28 (0.23–0.34)	0.82 (0.58–1.17)	0.286
PNPLA2_CpG_11,12	0.47 (0.39–0.53)	0.44 (0.34–0.52)	1.08 (0.81–1.44)	0.606
PNPLA2_CpG_13	0.14 (0.12–0.17)	0.15 (0.10–0.18)	0.95 (0.62–1.45)	0.810
PNPLA2_CpG_14,15,16,17	0.10 (0.08–0.11)	0.10 (0.07–0.14)	0.79 (0.38–1.65)	0.540
PNPLA2_CpG_18	0.12 (0.09–0.13)	0.11 (0.08–0.15)	1.30 (0.70–2.43)	0.403
PNPLA2_CpG_20,21	0.40 (0.35–0.45)	0.37 (0.30–0.46)	1.15 (0.82–1.60)	0.425

Notes: ^#Logistic regression, adjusted for age and experimental batches. Significant P-values are in bold.

Abbreviations: LC, lung cancer; IQR, interquartile range; OR, odds ratio; CI, confidence interval.

Table 6 The Correlation Between PNPLA2 Methylation and the Clinical Characteristics of LC Patients

Clinical Characteristics Group (N)	Median Methylation Level of Each CpG Site
	CpG_1	CpG_2,3	CpG_4	CpG_5	CpG_6	CpG_7	CpG_8,10	CpG_9	CpG_11,12	CpG_13	CpG_14,15,16,17	CpG_18	CpG_20,21
Tumor subtype
Adenocarcinoma in situ (37)	0.33	0.15	0.02	0.04	0.37	0.22	0.32	0.31	0.42	0.15	0.10	0.11	0.41
Microinvasive adenocarcinoma (34)	0.29	0.16	0.03	0.04	0.37	0.23	0.33	0.28	0.44	0.15	0.11	0.12	0.41
Invasive adenocarcinoma (97)	0.26	0.21	0.03	0.04	0.36	0.22	0.33	0.27	0.45	0.14	0.10	0.11	0.36
P-value*	0.377	0.193	0.912	0.892	0.781	0.976	0.895	0.284	0.990	0.833	0.500	0.752	0.275
Tumor size
T1 (147)	0.29	0.18	0.03	0.03	0.36	0.22	0.33	0.28	0.45	0.15	0.10	0.11	0.39
T2 and T3 (21)	0.25	0.28	0.02	0.04	0.36	0.22	0.31	0.27	0.42	0.13	0.09	0.09	0.35
P-value^$	0.791	0.780	0.545	0.331	0.948	0.639	0.812	0.146	0.167	0.486	0.481	0.058	0.206
Tumor length
≤ 1 cm (68)	0.31	0.15	0.02	0.03	0.36	0.23	0.32	0.28	0.46	0.14	0.10	0.12	0.41
> 1cm (100)	0.25	0.25	0.03	0.04	0.37	0.22	0.33	0.27	0.43	0.15	0.10	0.11	0.36
P-value^$	0.183	0.006	0.587	0.380	0.598	0.261	0.612	0.398	0.098	0.724	0.230	0.131	0.597
Lymph node involvement
0 (164)	0.29	0.19	0.03	0.04	0.36	0.22	0.33	0.28	0.45	0.15	0.10	0.11	0.38
1 and 2 (4)	0.24	0.14	0.02	0.04	0.30	0.25	0.32	0.23	0.43	0.16	0.08	0.08	0.32
P-value^$	0.818	0.530	0.058	0.946	0.443	0.578	0.776	0.067	0.818	0.935	0.396	0.236	0.119
Tumor stage
Stage I (158)	0.28	0.18	0.03	0.04	0.36	0.22	0.33	0.29	0.45	0.15	0.10	0.11	0.39
Stage II (10)	0.43	0.31	0.02	0.04	0.37	0.25	0.31	0.25	0.42	0.12	0.08	0.11	0.32
P-value^$	0.114	0.332	0.118	0.358	0.828	0.452	0.827	0.018	0.698	0.377	0.234	0.139	0.008

Notes: *P-value for Kruskal–Wallis test; ^$P-value for Mann–Whitney U-test; Significant P-values are in bold.

Abbreviations: LC, lung cancer; N, number; IQR, interquartile range; OR, odds ratio; CI, confidence interval.