Impact of PNPLA3 p.I148M and Hepatic Steatosis on Long-Term Outcomes for Hepatocellular Carcinoma and HBsAg Seroclearance in Chronic Hepatitis B

Figures & data

Figure 1 Derivation of the study population and case-cohort sample.

Table 1 Baseline Characteristics of Study Subjects and Main Outcomes

Variables	Entire Cohort (N=2385) No. of Participants (%)	Case-Cohort Study (N=961) No. of Participants (%)
Age (years)
30–39	829 (34.8)	315 (32.8)
40–49	894 (37.5)	376 (39.1)
50–59	400 (16.8)	162 (16.9)
≥60	262 (11.0)	108 (11.2)
ALT≥ 40 U/L^a	214 (9.0)	84 (8.8)
History of liver disease	169 (7.1)	70 (7.3)
First-degree family history of HCC^a	156 (6.5)	72 (7.5)
Obesity (BMI ≥25 kg/m^2)	724 (30.4)	292 (30.4)
Diabetes mellitus	51 (2.1)	23 (2.4)
PNPLA3^a
II	940 (39.5)	381 (39.8)
MI	1103 (46.3)	442 (46.2)
MM	337 (14.2)	134 (14.0)
Cigarette smoking	730 (30.6)	297 (30.9)
Alcohol consumption	473 (19.8)	205 (21.3)
Heavy alcohol consumption (≥210 gram of ethanol per week)^a	121 (5.1)	55 (5.8)
Ultrasonography
Hepatic steatosis	1092 (45.8)	419 (43.6)
Liver cirrhosis	114 (4.8)	57 (5.9)
Viral markers
HBeAg positive^a		97 (10.3)
HBV DNA ≥ 4 log copies/mL		484 (50.4)
HBV genotype^a
B or B+C		790 (83.2)
C		159 (16.8)
BCP double mutations^a		311 (33.5)
Follow-up
HBsAg seroclearance	628 (26.3)	254 (26.4)
Incident HCC	217 (9.1)	113 (11.8)

Notes: ^aData not available for all participants. Missing data in the entire cohort: ALT (n=6), HCC family history (n=1), PNPLA3 genotype (n=5), and amount of alcohol consumed (n=17); missing data in the case-cohort sample: ALT (n=3), PNPLA3 genotype (n=4), amount of alcohol consumed (n=10), HBeAg (n=23), HBV genotype (n=12), and BCP double mutations (n=34).

Abbreviations: ALT, alanine aminotransferase; BCP, basal core promoter; HCC, hepatocellular carcinoma.

Table 2 Baseline Factors Associated with HBsAg Seroclearance

Variable	Entire Cohort (N=2385)									Subcohort (N=919)
	Total No.	No. of HBsAg Seroclearance	Cumulative Incidence, %		Univariate Analysis^a			Multivariate Analysis^b		Multivariate Analysis^c
			10-y	20-y	sHR	95% CI	p-value	sHR	95% CI	sHR	95% CI
Hepatic steatosis
No	1293	300	10.0	23.6	1.0			1.0		1.0
Yes	1092	328	13.7	33.2	1.43	(1.22–1.67)	<0.0001	1.27	(1.07–1.50)	1.35	(1.03–1.78)
BMI (kg/m^2)
Normal weight/underweight (<23)	932	192	8.3	22.2	1.0			1.0		1.0
Overweight (23–24.9)	729	214	13.1	31.5	1.50	(1.23–1.82)	<0.0001	1.47	(1.20–1.80)	1.55	(1.10–2.19)
Obesity (≥25)	724	222	14.7	31.3	1.58	(1.30–1.92)	<0.0001	1.55	(1.26–1.91)	1.51	(1.09–2.09)
PNPLA3^d
II	940	221	9.2	25.7	1.0			1.0^e		1.0^f
MI	1103	310	13.2	29.5	1.19	(1.01–1.42)	0.0429	1.19	(1.00–1.41)	1.35	(1.00–1.81)
MM	337	95	13.4	28.5	1.25	(0.98–1.59)	0.0688	1.25	(0.98–1.59)	1.42	(0.95–2.13)
HBeAg
Negative										1.0
Positive										0.42	(0.21–0.85)
HBV DNA (log copies/mL) (continuous)										0.85	(0.79–0.90)
HBV genotype
B or B+C										1.0
C										2.09	(1.49–2.93)
BCP double mutations
No										1.0
Yes										1.27	(0.96–1.67)

Notes: ^aAdjusted for age at baseline. ^bAdjusted for age (continuous), ALT (continuous), history of liver disease, diabetes, cigarette smoking (non-smokers, ex-smokers, current smokers), alcohol consumption, and variables listed in the table ^cAdjusted for age (continuous), ALT (continuous), and variables listed in the table ^d5 subjects with missing data on PNPLA3 genotype. ^ep-value for trend=0.0316. ^fp-value for trend=0.0376.

Abbreviations: sHR, sub-distribution hazard ratio; BCP, basal core promoter.

Figure 2 Additive joint effect between excess BMI, PNPLA3 148M-variant, and hepatic steatosis on the probability of HBsAg seroclearance. (A) Cumulative incidence function curve of HBsAg seroclearance according to a prognostic score ranging from 0 to 5 (points: BMI [normal weight/underweight=0, overweight=1, obesity=2]; PNPLA3 [II=0, MI=1, MM=2]; steatosis [No=0, yes=1]). (B) Sub-distribution hazard ratios (sHRs) with 95% CIs estimated from competitive risk Cox model. Solid circles and lines indicated sHRs and 95% CIs. sHRs derived from the entire cohort were adjusted for age, ALT, history of liver disease, diabetes, cigarette smoking, and alcohol consumption. sHRs derived from the subcohort were further adjusted for HBeAg, viral load, HBV genotype (C vs non-C), and BCP double mutations.

Table 3 Multivariate-Adjusted sHRs of HCC Derived from the Entire Cohort and the Case-Cohort Analysis

Variable	Entire Cohort (N=2385)				Case-Cohort Analysis (N=961)
	No. of HCC	sHR^a	95% CI	p-value	HR^b	95% CI	p-value
HBsAg seroclearance
No	192	1.0			1.0
Yes	25	0.33	(0.21–0.50)	<0.0001	0.44	(0.23–0.82)	0.0107
Baseline hepatic steatosis
No	149	1.0			1.0
Yes	68	0.49	(0.36–0.66)	<0.0001	0.53	(0.33–0.86)	0.0096
Baseline BMI (kg/m^2)
Normal weight/underweight (<23)	79	1.0
Overweight (23–24.9)	69	1.34	(0.95–1.88)	0.0948	1.0^c
Obesity (≥25)	69	1.51	(1.07–2.13)	0.0193	1.85	(1.18–2.89)	0.0072
PNPLA3
II	65	1.0^d			1.0^e
MI	115	1.64	(1.20–2.25)	0.0019	1.61	(1.00–2.60)	0.0500
MM	37	1.83	(1.20–2.78)	0.0052	2.22	(1.13–4.35)	0.0206
Baseline diabetes
No	207	1.0			1.0
Yes	10	1.43	(0.73–2.80)	0.3000	3.71	(1.50–9.19)	0.0046
Baseline HBV DNA (log copies/mL) (continuous)					1.37	(1.23–1.52)	<0.0001
Baseline HBV genotype
B or B+C					1.0
C					3.16	(2.01–4.96)	<0.0001
Baseline BCP double mutations
No					1.0
Yes					2.32	(1.45–3.70)	0.0004

Notes: ^aAdjusted for age (continuous), ALT (≥40 vs <40 U/L), history of liver disease, first-degree family history of HCC, cigarette smoking (non-smokers, ex-smokers, current smokers), and alcohol consumption at baseline, and variables listed in the table ^bDerived from a weighted Cox regression model adjusted for age (continuous), ALT (≥40 vs <40 U/L), history of liver disease, and first-degree family history of HCC at baseline, and variables listed in the table ^cBMI<25 kg/m² was used as reference group. ^dp-value for trend=0.0006. ^ep-value for trend=0.0090.

Abbreviations: sHR, sub-distribution hazard ratio; BCP, basal core promoter.

Table 4 Associations of Hepatic Steatosis at Baseline and PNPLA3 p.I148M Variation with Risk of HCC by Follow-Up Time^a

Exclusion criteria	Remaining Participants Included in Analysis		Hepatic Steatosis		PNPLA3 p.I148M
					MI vs II		MM vs II
	No. of HCC	Person-years	sHR	95% CI	sHR	95% CI	sHR	95% CI
Excluding 114 subjects detected with liver cirrhosis at baseline from analysis	152^b	59700.3	0.65	(0.46–0.92)	1.64	(1.13–2.37)	1.70	(1.03–2.81)
Excluding 114 subjects detected with liver cirrhosis at baseline plus HCC cases who occurred within following time period from analysis
<3 years	150	59696.4	0.67	(0.47–0.95)	1.65	(1.13–2.39)	1.72	(1.04–2.86)
<6 years	140	59653.7	0.64	(0.45–0.91)	1.76	(1.19–2.60)	1.95	(1.16–3.27)
<9 years	124	59530.2	0.64	(0.43–0.94)	1.70	(1.12–2.58)	2.00	(1.16–3.45)
<12 years	105	59328.0	0.64	(0.42–0.98)	1.97	(1.24–3.11)	2.14	(1.17–3.91)

Notes: ^aHepatic steatosis and PNPLA3 genotype were mutually adjusted, and HBsAg seroclearance and baseline age (continuous), ALT (≥40 vs <40 U/L), history of liver disease, first-degree family history of HCC, BMI (<23, 23–24.9, ≥25 kg/m²), diabetes status, cigarette smoking (non-smokers, ex-smokers, current smokers), and alcohol consumption were also included as covariates in the multivariate analyses. ^b65 HCC cases detected with liver cirrhosis at baseline were excluded.

Abbreviation: sHR, sub-distribution hazard ratio.

Table 5 Longitudinal Cohort Analysis for Factors Associated with Episodes of Liver Enzyme Elevation and Liver Cirrhosis Detected by Imaging Modalities^{a, b}

Variable	Elevated ALT (≥40 U/L)		Elevated AST (≥40 U/L)		Liver Cirrhosis
	OR	95% CI	OR	95% CI	OR	95% CI
HBsAg seroclearance
No	1.0		1.0		1.0
Yes	0.49	(0.42–0.59)	0.43	(0.35–0.54)	0.39	(0.26–0.58)
Baseline BMI (kg/m^2)
Normal weight/underweight (<23)	1.0		1.0		1.0
Overweight (23–24.9)	1.05	(0.88–1.26)	1.04	(0.84–1.28)	1.91	(1.32–2.75)
Obesity (≥25)	1.35	(1.13–1.61)	1.24	(1.01–1.54)	2.35	(1.62–3.40)
PNPLA3
II	1.0		1.0		1.0
MI	1.22	(1.04–1.43)	1.14	(0.94–1.37)	1.16	(0.84–1.61)
MM	1.56	(1.26–1.94)	1.48	(1.14–1.91)	1.64	(1.04–2.59)
Time-varying covariate^c
Hepatic steatosis
No	1.0		1.0		1.0
Yes	1.27	(1.13–1.43)	0.76	(0.65–0.89)	0.16	(0.12–0.23)
Diabetes
No	1.0		1.0		1.0
Yes	0.73	(0.60–0.90)	1.19	(0.89–1.60)	1.68	(1.13–2.47)

Notes: ^aOf the 2304 subjects with ≥2 follow-up imaging examinations, 6 lacked AST data. ^bORs were adjusted for age and ALT (continuous) at baseline, number of visits, as well as variables listed in the table ^cStatus at each visit during follow-up were included as a time-varying covariate in the generalized linear mixed model.

Abbreviation: OR, odds ratio.