Genetic variants in interferon-λ 4 influences HCV clearance in Chinese Han population
Genetic variants in interferon-? 4 influences HCV clearance in Chinese Han population
OPEN Recent many studies indicated a novel dinucleotide variant in ss469415590 (TT vs. ?G) of interferon-? 4 (IFNL4) gene strongly associated with hepatitis C virus clearance. To evaluate the impact and clinical usefulness of IFNL4 ss469415590 genotype on predicting both spontaneous HCV clearance and response to therapy in Chinese population, we genotyped 795 chronic HCV carriers, 460 subjects with HCV natural clearance and 362 patients with pegylated interferon-? and ribavirin (PEG IFN-?/ RBV) treatment. IFNL4 ss469415590 variant genotypes significantly decreased host HCV clearance, both spontaneous (dominant model: OR = 0.50, 95% CI = 0.36-0.71) and IFN-? induced (dominant model: OR = 0.32, 95% CI = 0.18-0.56). Multivariate stepwise analysis indicated that ss469415590, rs12979860, the level of baseline HCV RNA and platelet were as independent predictors for sustained virological response (SVR). But the area under the ROC curve (AUC) was only 0.58 for ss469415590, and it was elevated to 0.71 by adding rs12979860, baseline HCV RNA and platelet in the prediction model of SVR. Therefore, these findings underscore that although genetic factors of host and pathogen were commonly important during HCV clearance, ss469415590 may be also a strongly predictive marker in the Chinese population.
HCV RNA (IU/L)
2 or 3
AFP (ng/mL) b
Participant characteristics. Participant profiles were shown in Table?1. Among 795 persistent HCV
carriers and 460 subjects with spontaneous clearance, elder, female and subject infected with viral genotype 1 were
significantly more likely to be chronic infection (P < 0.05 for both comparisons). And patients with spontaneous
HCV clearance had normal aspartate aminotransferase (AST) and alanine aminotransferase (ALT) than
persistent HCV cases (P < 0.001 for AST and P = 0.015 for ALT). In this study, 362 chronic infection patients treated
with PEG IFN-?/RBV therapy were recruited from a population of former paid-blood donors, which the viral
genotype of these CHC were all genotype 1. 65.7% patients achieved SVR. There were more patients with
abnormal AST, higher baseline HCV RNA, and lower platelet among none sustained virological response (N-SVR)
group (P = 0.038, <0.01, 0.039, respectively).
The observed genotype frequencies for the two SNPs in subjects with spontaneous HCV clearance were all in
Hardy-Weinberg equilibrium (HWE) (P > 0.05). But the allele frequencies of rs12979860 were not in accordance
with the predicted HWE in patients who attained SVR (P < 0.001). The LD was moderate between rs12979860
and ss469415590 both for spontaneous resolver (r2 = 0.62) and for patients who attained SVR (r2 = 0.79).
ss469415590 variants and HCV spontaneous clearance. IFNL4 ss469415590 genotype had a
significant impact on spontaneous HCV clearance. Clearance rate was higher in patients carrying the beneficial
TT/TT genotype (39.4%) than in patients with TT/?G (23.8%) or ?G/?G (18.7%) (P < 0.001). Meanwhile,
IL28B rs12979860 variants were also negatively related with viral clearance and clearance rate was 38.9%, 27.6%
and 17.2% for CC, CT and TT genotype (P < 0.001). After adjusting for age, gender, and viral genotype,
logistic regression analyses showed that IFNL4 ss469415590 and IL28B rs12979860 variant genotypes significantly
decreased the ability of host HCV clearance (dominant model: OR = 0.50, 95% CI = 0.36?0.71 for ss469415590;
OR = 0.59, 95% CI = 0.42?0.83 for rs12979860) (Table?2). This effect of ss469415590 and rs12979860 on HCV
clearance remained in existence after being conditioned on each other (P = 0.005 for ss469415590 and P = 0.016
for rs12979860) and therefore they was included in further combined analyses by adding up the number of
unfavorable alleles of the independent SNPs ss469415590-?G and rs12979860-T. The result showed that subjects
carrying three to four unfavorable alleles had a 90% decrease in clearance in comparison with those without
unfavorable allele (OR = 0.10, 95% CI = 0.02?0.42) (Table?3).
ss469415590 variants and response to treatment. In 361 HCV genotype 1 patients, SVR was strongly
associated with IFNL4 ss469415590 genotype: 70.0% of patients with ss469415590 TT/TT had SVR, whereas only
43.6% of patients with TT/?G and 16.7% of ?G/?G achieved SVR (P < 0.001). Subjects with IL28B rs12979860
variant TT were also fewer to achieve SVR (Table?2). Multivariate stepwise analysis indicated that these two
SNPs, the level of baseline HCV RNA and platelet were as independent predictors for SVR (Table?4). After
adjustment with age, gender, baseline HCV RNA and platelet, multivariate logistic regression showed that the OR of
ss469415590 and rs12979860 was 0.32 and 0.34 in dominant model respectively, compared with patients with
favorable genotypes (Table). The combined effect of unfavorable alleles on SVR indicated that carrying two
unfavorable alleles offered the highest risk effect (OR= 0.13, 95% CI = 0.07?0.25), as showed in Table?3.
We used decision tree ensembles in the form of a random forest classifier to quantify the relative predictive
power of ss469415590, rs12979860, selected demographic characteristics, and clinical features. As a result of this
multivariate analysis, the predictive power of a variable was expressed as the Gini. The Fig.?1A showed that the
first four stronger factors were ss469415590, baseline HCV RNA, rs12979860 and platelet. The AUC was 0.58 for
ss469415590, and was 0.64 for the set of ss469415590 and rs12979860 (Fig.?1B,C). But the AUC was elevated to
0.71 by adding baseline HCV RNA and platelet in the prediction model, suggesting that genetic factors of host
and pathogen were commonly important to resolution of virus during treatment (Fig.?1D).
Meanwhile ss469415590-?G variant (dominant model: OR = 0.26, 95% CI = 0.12?0.54 for RVR; OR = 0.11,
95% CI = 0.06?0.22 for EVR) and rs12979860-T variant (dominant model: OR = 0.37, 95% CI = 0.23?
0.62 for RVR; OR = 0.46, 95% CI = 0.26?0.81 for EVR) were also negatively related with RVR and EVR
ss469415590 variants and HCV time-dependent clearance. Patients were stratified according to
their IFNL4 ss469415590 and IL28B rs12979860 allele type, and the rate of undetectable HCV-RNA at 4, 8, 12,
24, 48 weeks after the start of therapy and 24 weeks after cessation of treatment were analyzed (Fig.?2A,B). The
rate of undetectable HCV RNA was significantly higher in patients with the ss469415590 TT and rs12979860 CC
Response to treatment
Chronic N(%) Resolver N(%)
genotype than ss469415590 TT/?G or ?G/?G and rs12979860 CT/TT genotype. Especially, the difference was
most significant when stratified by ss469415590 genotype: 44.2% vs. 18.6% for 4 weeks, 67.7% vs. 30.5% for 12
weeks and 85.9% vs. 58.3% for 48 weeks.
Meta-analysis. To further study the impact of IFNL4 ss469415590 on HCV clearance, a meta-analysis was
performed by the published data, including four data for HCV spontaneous clearance and 12 data for HCV SVR
(Supplemental?Table?3)7,8,11?17. Figure?3 indicated significant association between IFNL4 ss469415590 variant
and decreased ability of HCV clearance (pooled OR = 0.30, 95% CI = 0.23?0.39 for HCV spontaneous
clearance; pooled OR = 0.26, 95% CI = 0.22?0.32 for HCV SVR in dominant model). Subgroup analysis showed that
the estimated OR was 0.21 (95% CI = 0.17?0.27) in HCV genotype 1 and 0.36 (95% CI = 0.26?0.48) in other
genotypes. These results indicated that patients with ss469415590 variant types may have a lower ability of HCV
clearance, especially in viral genotype 1.
In this study, we investigated the associations between IFNL4 ss469415590 and risk of HCV clearance and this
is the first study that validated the observations of Prokunina-Olsson et al. and of Bibert et al. on the prediction
of the eradication of HCV, both spontaneous and IFN-? induced, in a large cohort of Chinese Han
populations7,8. The main finding was that the predictive value of IFNL4 ss469415590 was almost identical to that of
IL28B rs12979860, which were all consistent with previous studies in other races12?14. But, unexpectedly, the
linkage disequilibrium among these two SNPs in our study was only moderate, especially in spontaneous resolver
(r2 = 0.62). Previous studies have reported that ss469415590 and rs12979860 were in strong linkage
disequilibrium in chronic hepatitis C virus type 1 or 4 infection in Caucasians12,18. Both studies were conducted to predict
the treatment response of peginterferon alpha and ribavirin. However, the moderate linkage disequilibrium in
our study was performed in spontaneous resolver. Besides, the linkage disequilibrium between ss469415590 and
rs12979860 in Chinese Han population may be not as strong as in Caucasians.
We performed a mini meta-analysis with a dominant genetic model by pooling the published data to study the
effect of IFNL4 ss469415590 on HCV clearance. The pooled ORs were lower than these in our current research
for both spontaneous clearance and IFN-? induced eradication, suggesting the negative effectiveness of IFNL4
ss469415590 variant was stronger in the meta-analysis. The mainly reason may be the discrepancy of genetic
background in different races. The frequency of ss469415590-TT/?G or ?G/?G was about 12.2% in Chinese
patients with HCV spontaneous clearance of our study, while that was about 69.9% in non-Chinese patients of the
meta-analysis. This extreme variation also was found in SVR patients infected with genotype 1 (10.5% vs. 60.8%).
But this discrepancy of frequency of ss469415590-TT/?G or ?G/?G in SVR patients infected with genotype 1
was reduced among our study and three Japanese studies of the meta-analysis (10.5% vs. 24.7%).
Although GWAS defined the strong association between IL28B SNPs and viral eradication in hepatitis C,
the exact mechanism or function of IL28B has yet to be testified, and even some paradoxes existed in various
researches. On one hand, based on different IL28B alleles, the level of expression of IL28B was dissimilar between
in peripheral blood mononuclear cells (PBMCs) and in liver tissue. Many studies identified that the level of
In this study the environment factors (baseline viral load and platelet) as well as genetic factors
independently contribute to the response to treatment. The predict value of ss469415590 for SVR was only moderate
(AUC = 0.58) and almost 30% patients carrying with beneficial IFNL4 TT/TT genotype did not achieve SVR.
These results were confirmed by some recent findings, and reflected a complex genotype -environment
interaction for predicting HCV clearance.
Our study had numerous strengths. First of all, enough statistical power was guaranteed with a relatively
large sample size in this study, and it is the first study demonstrating that IFNL4 ss469415590 variants influence
HCV clearance in Chinese Han population, which validated the effect of the novel marker in non-Caucasians.
Moreover, patients with treatment came from parts of HCV persistent carriers who were all infected with HCV
by blood donation, which may have reduced potential selection bias. Finally, it is rare to study the association
between IFNL4 ss469415590 and HCV clearance, both spontaneous and IFN-? induced in one study. However,
little is known regarding the biological mechanism of the significant SNP in the clearance of HCV. Therefore,
validations with functional characterizations are warranted.
In conclusion, our study validated that IFNL4 ss469415590 was also strongly associated with HCV clearance
in Chinese Han population.
Ethical statement. This study was approved by the medical ethics committee of Nanjing Medical University.
All subjects provided informed consent to participate in the study. The experiment was carried out according to
the Declaration of Helsinki.
Participants. 1255 participants for studying spontaneous HCV clearance were recruited from the Nanjing
compulsory detoxification center (Nanjing, China) during May and Dec 2006, nine hospital hemodialysis centers
in southern China during Oct 2008 and Jan 2010, and a population of former paid-blood donors (Zhenjiang,
China) from April 2010 to January 2013. The information about these participants had been described in our
previous studies23,24. 362 treatment-na?ve CHC patients were recruited from Jurong People?s Hospital (Jurong, China)
from Jan 2011 to Oct 2013, which aimed to evaluate the factors that impacted on response to anti-viral therapy.
Eligibility criteria for therapy included age between 18 and 70 years, detectable HCV RNA in serum over a span of
more than 6 months of treatment initiation, negative for hepatitis B infection, without other types of liver diseases
such as alcoholic diseases, autoimmune liver diseases or metabolic liver diseases.
Each participant was interviewed by a structured questionnaire to collect information on demographic data
and environmental exposure history. After interview, an approximately 5 mL venous blood sample was collected
from each participant. The serum and peripheral blood mononuclear cells were separated and stored at ?70 ?C
Treatment therapy and outcome. Patients received weekly injections of pegylated IFN-?2a (180 g) and
ribavirin (RBV) was administered orally for 48 weeks. The amount of ribavirin was adjusted based on body
weight (600 mg for <60 kg, 800 mg for 60?80 kg, 1000 mg for >80 kg).
According to the manufacturers? instructions, HCV RNA was quantified in all patients at baseline and after
4, 12, 24 and 48 weeks of treatment and 24 weeks after cessation of treatment by Cobas Amplicor HCV Monitor
Test, v2.0 (Roche, Basel, Switzerland). In this study, rapid virological response (RVR) was defined as undetectable
HCVRNA at 4 weeks during therapy. Early virological response (EVR) was defined as ?2 log reduction in HCV
RNA level compared to baseline HCV RNA level or undetectable HCVRNA at 12 weeks during treatment. SVR
was defined as HCV RNA negative 24 weeks after treatment-free follow-up.
SNP genotyping. DNA extraction was with protease K digestion and phenol-chloroform purification, which
was described previously25. Genotyping was performed by the TaqMan allelic discrimination assay on ABI PRISM
7900HT Sequence Detection system (Applied Biosystems, San Diego, CA, USA). The information on primers and
probes are shown in Supporting Table?1. Two blank controls and 5 repeated samples were assigned in each
genotyping assay, and a 100% concordant was achieved. The IL28B rs12979860 allele C and IFNL4 ss469415590 alleles
TT were defined as wild-type alleles, whereas the T and ?G as mutant alleles respectively.
Statistical analysis. Differences in the general demographic characteristics were calculated by the Student t
test and the chi-square (?2) test. The associations of SNPs with HCV spontaneous clearance, RVR, EVR and SVR
were estimated by the odds ratios (ORs) and 95% confidence intervals (CIs) using multivariate logistic regression
analysis. Age, gender and viral genotype were adjusted for HCV spontaneous clearance, while, age, gender,
baseline HCV RNA and platelet were adjusted for RVR, EVR and SVR during regression analysis. A stepwise forward
procedure was used for selecting the final logistic regression model for predicting outcomes of HCV treatment.
The trend analysis was assessed with Cochran-Armitage trend test.
Random forest, an advanced tree classifier, was used to evaluate the importance of each variable and to
improve the performance of classification. The Gini or information entropy was the standard to classify the
partner node into two child nodes. In our study, the area under the curve (AUC) and the plot of variables importance
indicated the result of this method26.
All the statistical analyses were carried out by STATA 12.0 software (StataCorp LP, College Station, TX, USA),
and P < 0.05 in a two-sided test was considered as statistical significance.
We thank the students who have worked on the study, including Yuangyuan Zhang, Hui Zheng, Wenzhe Ma,
Wenjuan Zhang, Xuecheng Song. We also thank doctors and the nurses from the Jurong People?s Hospital for
helping sample collection and organization of the field investigation. This research would not have been possible
without the consent and help of the participants. The current study was supported in part by National Natural
Science Foundation of China (No.81473029 and 81473028), Priority Academic Program Development of Jiangsu
Higher Education Institutions (PAPD), Medical Reform Project of Health and Family Planning Commission of
Jiangsu Province of China (YG201413), Science and Technology Program of Zhenjiang, China (SH20141).
R.B.Y., P.H. participated in the design of the study. Y.N.Y., T.T. and M.Y. carried out the surveys and experiments.
P.H, M.Z.C performed the statistical analysis. H.B.C., Y.Z., J.W. contributed materials and analysis tools. P.H.,
Y.N.Y. wrote the paper. All authors read and approved the final manuscript.
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Huang, P. et al. Genetic variants in interferon-? 4 influences HCV clearance in Chinese
Han population. Sci. Rep. 7, 42408; doi: 10.1038/srep42408 (2017).
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