Samir Mohindra ORCID iD: 0000-0001-8521-9295

Polymorphism in IFNL3/IL28B gene and risk to non-

cirrhotic chronic hepatitis C genotype 3 virus infection and
its effect on the response to combined daclatasvir and
sofosbuvir therapy
Accepted Article
Anwar Jamal Khan1, Vivek Aanand Saraswat1, Prabhat Ranjan1, Devendra

Parmar2, Tajwar Singh Negi1 and Samir Mohindra1

1
Department of Gastroenterology, Sanjay Gandhi Post Graduate Institute of

Medical Science, Raebareli Road, Lucknow-226014, U.P, India.

2
Developmental Toxicology Division, CSIR-Indian Institute of Toxicology

Research, P.O. Box 80, M.G. Marg, Lucknow-226 001, U.P, India.

Address for correspondence:

Dr. Samir Mohindra

Additional Professor

Department of Gastroenterology

Sanjay Gandhi Post Graduate Institute of Medical Science

Raebareli Road

This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process,
which may lead to differences between this version and the Version of Record. Please
cite this article as doi: 10.1002/jmv.25359.

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 Lucknow-226014, U.P, India

Tel.: +91-0522-2495013

FAX: +91-0522-2668561/2668017

E-mail: samir@sgpgi.ac.in
Accepted Article

Short Title: IFNL3/IL28B polymorphism & HCV infection risk

ABSTRACT

Hepatitis C virus infection is a considerable public-health problem and

an important cause of liver disease with about 71 million people infected

worldwide and more than 399,000 people die every year from hepatitis C

related liver diseases. The present study was therefore, initiated to investigate

the association of polymorphism in interferon lamda-3 (IFNL3) also known as

Interleukin-28B (IL28B) gene with chronic HCV infection and association of

these polymorphic variants with the combination daclatasvir and sofosbuvir

HCV therapy response. Genotypes were determined by the polymerase chain

reaction-restriction fragment length polymorphism (PCR-RFLP) assay in a

total of 250 chronic HCV genotype 3 patients and 500 number of healthy

controls. Our data revealed that the TT (minor) genotype of IFNL3

(rs12979860) and GG (minor) genotype of IFNL3 (rs8099917) exhibited

significant association with chronic HCV genotype 3 infection when compared

with controls. The results of treatment response showed that CC (major)

genotype of IFNL3 (rs12979860) and TT (major) genotype of IFNL3

(rs8099917) are associated with likelihood of achieving a higher sustained

virological response (SVR), to combined daclatasvir and sofosbuvir therapy,

in genotype 3 infected HCV patients, whereas the individuals with TT (minor)

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 genotype of IFNL3 (rs12979860) and GG (minor) genotype of IFNL3

(rs8099917) are more susceptible to chronic HCV infection and treatment

relapse, suggesting a role of IFNL3 (rs12979860) and (rs8099917) in the

treatment outcome of combined daclatasvir and sofosbuvir therapy in chronic

HCV genotype 3 infection.

Accepted Article

Key words: Anti-hepatitis C virus DAA, Genetic variation, Hepatitis C Virus,

Interleukin.

1. INTRODUCTION

Hepatitis C virus infection is a considerable public-health problem and

an important cause of liver disease. Globally, an estimated 71 million people

have chronic hepatitis C virus infection and about 399,000 people die every

year from hepatitis C related liver diseases.1,2 According to some studies, 55

to 85% of the individuals infected by the hepatitis C virus becomes chronic

carriers3 and in these patients, delayed diagnosis and the fact that the

infection might have remained asymptomatic over a long period of time result

in advanced stages of liver cirrhosis and even hepatocellular carcinoma in

their lifetime.4

Recently, many antiviral drugs have been developed for treatment of

HCV infection. One of the recently approved direct acting antiviral (DDA)

drugs are daclatasvir (DCV) in combined with sofosbuvir (SOF) with or without

ribavirin for the treatment of HCV genotype 3 infection. Daclatasvir is a potent,

pan-genotypic inhibitor of the HCV NS5A protein and sofosbuvir is a pan-

genotypic nucleotide analogue inhibitor of the HCV NS5B RNA polymerase.5,6

In one of the phase III studies, the 12-week, once-daily oral combination of

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 DCV and SOF, with or without ribavirin (DCV+SOF ±RBV), was well tolerated

and achieved SVR12 rates exceeding 90% in non-cirrhotic patients with

genotype 3, as well as in HIV/HCV coinfection with genotype 3 infection.7,8

These findings led to widespread approval of DCV and SOF for the treatment

of non-cirrhotic HCV genotype 3 infection.9,10

Accepted Article

The pathogenesis of HCV infection remains unclear but it is possible

that not only the virus but also the interaction between the virus and the host

immune system is important in determining the course of infection and the

response to therapy.11 Recently, functional gene polymorphisms have been

identified in cytokines such as IFN-λ which shows possible relationships

between these genotypes and the clinical outcome of HCV-related liver

disease.12

Interleukin-28B (IL28B) is a cytokine gene which encodes a protein

interferon lamda-312 and IL28B gene has recently been renamed as interferon

lamda-3 (IFNL3). Cytokines are antiviral effectors, which induce inflammatory

response as a predominant mechanism of host defence against infections.13,14

Inappropriate secretions of the cytokines in HCV infections may lead to

chronicity or the resistance to interferon treatment.15 SNPs in IFNL3/IL28B

gene has been identified to be associated with HCV treatment

response.16,17,18 Ge et al. (2009) in a GWAS study found that a SNP at IFNL3

(rs12979860) is the strongest host genetic predictor of sustained virological

response (SVR) in hepatitis C genotype 1. Among the three genotypes of

IFNL3, CC genotype is associated with two to three fold increases in SVR as

compared with either CT or TT genotype.16 The Caucasians, African–

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 American, and Hispanic patients having IFNL3 rs12979860 CC genotype

were strongly associated with SVR after peg-interferon and ribavirin

combination therapy as compared to CT or TT genotypes.19 Another GWAS

study in 2009 by Suppiah et al. (2009) and Tanaka et al. (2009) found that an

additional polymorphism of IFNL3 rs80999917 is a strong genetic determinant

Accepted Article

of SVR in HCV genotype 1 infected patients.17,18

In contrast to association of functionally important polymorphism in

IFNL3/IL28B gene with chronic HCV genotype 1 and 4 infection, data is

scarce on HCV genotype 3, which is more prevalent in Indian population, the

present study was therefore initiated to investigate the association of

functionally important polymorphism in IFNL3 gene with chronic HCV

genotype 3 infection in North Indian population. Attempts were also made to

investigate the association of these polymorphic variants with the recently

approved combined daclatasvir and sofosbuvir therapy response.

2. METHODS

A case control study was initiated to investigate the association of

functionally important polymorphisms in IFNL3 gene (rs12979860 and

rs8099917) with hepatitis C virus (HCV) infection. The study groups consist of

250 patients suffering from genotype 3 HCV infection visiting the outpatient

department (OPD) facility of Gastroenterology Department of Sanjay Gandhi

Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow, India. All

patients were positive for HCV antibodies and HCV RNA in serum for more

than 6 months and displayed an elevated serum alanine aminotransferase

(ALT). Patients were excluded if they were known to have injected drugs or

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 abused alcohol within the last 6 months, had poorly controlled psychiatric

illness, cirrhotic, or were hepatitis B surface antigen (HBsAg) or human

immunodeficiency virus (HIV) antibodies or renal insufficiency suspected

infections and autoimmune liver disease. They were also excluded if they

suffered from other significant concurrent medical conditions including chronic

Accepted Article

liver diseases of etiologies other than HCV infection. Patients were included in

the current analysis if they were of North-Indian origin, infected with HCV

genotype 3, had been treated per protocol (Oral intake of prescribed dose of

recently approved therapy of sofosbuvir and daclatasvir daily for 12 week and

follow-up sample was available to assess SVR).

Control group consist of 500 healthy individuals having no evidence of

any liver disease as judged by physical examination and normal liver function

test. HCV patients and controls were age and sex matched. Another control

group was also included which consist of 62 individuals who had

spontaneously cleared HCV infection and were positive for HCV antibodies

and negative for HCV RNA. All the patients, spontaneously cleared individuals

and controls included in the study belonged to the same geographical location

(Northern India) and same ethnicity. The clinical data of the patients,

spontaneous clearance individuals and the normal controls are given in Table

1.

The protocol for research work was approved by the Institutional

Bioethics Committee (IBC) of Sanjay Gandhi Post Graduate Institute of

Medical Sciences, Lucknow and it conforms to the provisions of the

declaration of Helsinki in October 2013. Informed consent was obtained from

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 the study subjects for inclusion in the study and before the collection of blood

samples it was ensured that subject anonymity was preserved. Information

concerning dietary habits, family history of disease, smoking, tobacco

chewing, and alcohol consumption was obtained from the questionnaire filled

in by the cases and controls.

Accepted Article

2.1 Genomic DNA isolation: One ml of blood was drawn into citrate

containing tubes from all patients as well as controls. DNA was isolated from

whole blood with the Flexi Gene DNA kit (Qiagen, CA) following the

manufacturer’s protocol. Isolated DNA was subsequently used for genotyping

studies.

2.2 Interferon lamda-3 (IFNL3/IL28B) Genotyping: The rs12979860 and

rs8099917 SNPs genotyping of IFNL3 gene was carried out by polymerase

chain reaction and restriction fragment length polymorphism as previously

described by Venegas et al. (2011).20 For rs12979860, oligonucleotide

primers were: 5’-AGGGCCCCTAACCTCTGCACAGTCT-3’ (sense), and 5’-

GCTGAGGGACCGCTACGTAAGTCACC-3’ (antisense). For rs8099917,

oligonucleotide primers were: 5’-TTCACCATCCTCCTCTCATCCCTCAT-3’

(sense) and 5’-TCCTAAATTGACGGGCCATCTGTTTC-3’ (antisense). PCR

reaction conditions (30 μl) were: initial denaturation at 94°C for 10 min,

followed by 40 cycles of: denaturation at 94°C for 1 min, annealing at 58°C for

40 sec, and extension at 72°C for 1 min. The PCR product for rs12979860

and rs8099917 were of 403 and 401 base pairs, respectively.

In order to perform RFLP assay for the rs12979860 genotype, 20 μl of

amplicons were digested with 5U of BstU I restriction endonuclease (New

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 England Biolabs, MA, United States) at 60°C for 2 h. BstU I digestion of allele

CC yields fragments of 184, 105, 89 and 25 base pairs, DNA containing the

allele TT polymorphism yields fragments of 184, 130 and 89 base pairs,

whereas DNA containing the CT polymorphism yields fragments of 184, 130,

105, 89 and 25 base pairs . For the RFLP assay for the rs8099917 genotype,
Accepted Article

20 μl of amplicons were digested with 1U of Mae III restriction endonuclease

(Roche Molecular Systems, Branchburg, NJ, United States) at 55°C for 2 h.

Mae III digestion of allele TT yields fragments of 186, 110 and 105 base pairs,

DNA containing the allele GG polymorphism yields fragments of 147, 110,

105 and 39 base pairs, whereas DNA containing the TG polymorphism yields

fragments of 186, 147, 110, 105 and 39 base pairs (supplementary figure).

Restriction digestion products for each were separated on agarose gels

stained with ethidium bromide for visualization on a UV trans-illuminator.

2.3 Treatment and treatment response: All the HCV patients included in the

study were naïve (not treated before with peg-interferon and ribavirin therapy).

These patients were only treated with the newly approved therapy, which

consist of oral intake of daclatasvir 60mg and sofosbuvir 400mg daily for 12

week. After discontinuation of treatment, they were followed for another 24

weeks. To determine the response to therapy, serum was tested for HCV

RNA by using a quantitative test at the end of therapy. HCV RNA levels were

determined using Cobas Taqman HCV test v2. (Roche Molecular system,

South Branchburg, NJ, USA), according to the manufacturer’s protocol.

Relapsers (REL) were patients which cleared HCV RNA from serum at the

end of treatment, but HCV returns in serum during the follow-up period.

Sustained virological response (SVR) patients, were patients which cleared

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 HCV RNA from serum at the end of treatment and for 24 weeks after the end

of treatment. Patients who were neither REL nor SVR were defined as non-

responders (NR). Non-responders (NR) were patients with persistent HCV

infection treated with new therapy and never lost HCV RNA during treatment.

Baseline characteristics of SVR patients, relapsers and non-responders

Accepted Article

chronic HCV patients are given in Table 2.

2.4 Hepatitis C virus genotyping: HCV genotyping was done by Hepatitis C

Virus Genotype Diagnostic Kit (PCR-Fluorescence Probing) (Sansure

Biotech, Inc, Changsha, Hunan Province, P.R. China), according to the

manufacturers protocol. Magnetic bead technology was used to extract HCV-

RNA from human serum. One-step RT-PCR technology was used with

several specific pairs of HCV primers to target conserved regions of different

HCV genotypes. TaqMan fluorescence probes were used to achieve

genotype detection of HCV RNA through fluorescent signal changes.

2.5 Statistical Analysis: The statistical data are reported as the mean ± SD

of original values. A comparison of numerical variables between the study

groups was performed using Student’s t-test to compare independent

samples from two groups when the samples were normally distributed and the

Mann–Whitney U-test to compare independent samples when the samples

were not normally distributed. The Pearson Chi-square-goodness-of-fit test

was used to test the distribution of genotypes and allele frequencies for

deviations from Hardy-Weinberg equilibrium between the patients group and

controls. Odds ratios (ORs) and 95%confidence intervals (CI) were calculated

to measure the risk associated with variant genotypes by unconditional

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 logistic regression method. Multiple logistic regression analysis was

performed to assess the impact that genotypes and other clinical variables

had on the treatment response. The differences were considered significant if

the p-value was less than 0.05. All statistical analysis was performed using

the software Statistical Package for the Social Sciences (SPSS Version 13.0,
Accepted Article

Chicago, IL) for windows. The Power of the study was found to be >80% as

analyzed by power genetic association analysis software

(http://dceg.cancer.gov/bb/tools/pga) at the level of significance α=0.05 with

sample size of 500 in controls and 250 in HCV patients.

3. RESULTS

The distribution of demographic and biochemical variables of healthy

controls, spontaneous clearance individuals and chronic HCV patients

genotype 3 are described in Table 1. The biochemical profiles including the

serum ALT, AST, ALP, total bilirubin, HCV RNA in the patients with chronic

hepatitis C were significantly higher than those in the healthy control (Table

1).

The distribution of genotype and allele frequency of IFNL3 gene among

controls and HCV patients is summarized in table 3. Both the genotype and

allele frequency of IFNL3 gene in controls and HCV patients were found to be

in Hardy-Weinberg equilibrium. The frequency of TT genotypes of IFNL3

(rs12979860) was found to be higher (13%) in HCV patients when compared

with controls (5%) (OR: 3.3; 95%CI: 1.86-5.78; p: 0.00) (Table 3). Likewise,

the frequency of CT genotypes (heterozygous) of IFNL3 (rs12979860) was

also found to be higher (51%) in HCV patients when compared with controls

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 (45%) (OR: 1.6; 95%CI: 1.14-2.18; p: 0.00) (Table 3). As evident from table 3,

the frequency of the GG genotype of IFNL3 (rs8099917) in HCV patients was

found to be higher (5%) when compared with controls (2%) (OR: 2.9; 95%CI:

1.27-6.62; p: 0.00) (Table 3). Similarly, the frequency of TG genotypes

(heterozygous) of IFNL3 (rs8099917) was also found to be higher (40%) in

Accepted Article

HCV patients when compared with controls (30%) (OR: 1.6; 95%CI: 1.16-

2.21; p: 0.00) (Table 3).

The frequency of the TT genotype of IFNL3 (rs12979860) was found to

be higher in HCV patients (13%) when compared with spontaneously clear

individuals (6.5%). The increase in frequency of the TT genotypes of IFNL3

(rs12979860) resulted in a significant increase in relative risk (OR: 3.1;

95%CI: 1.02-9.44; p: 0.03) in HCV patients when compared with

spontaneously clear individuals (Table 4).

The frequency of relapsers for TT genotype (37%) of IFNL3

(rs12979860) were found to be higher when compared with SVR patients

(9%) (OR: 15; 95%CI: 2.90-77.98; p: 0.00) (Table 5). The frequency of

relapsers for GG genotype (26.5%) of IFNL3 (rs8099917) were also found to

be higher when compared with SVR patients (3.5%) (OR: 20.5; 95%CI: 4.58-

91.37; p: 0.00) (Table 5). Similarly, the frequency of non-responder for TT

genotype of IFNL3 (rs12979860) and TG genotypes of IFNL3 (rs8099917)

were also found to be higher when compared with SVR patients (Table 5).

The frequency of TT genotype of IFNL3 (rs12979860) and GG

genotypes of IFNL3 (rs8099917) were found to be higher in non-SVR patients

when compared with SVR patients (Table 6).

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 4. DISCUSSION

Consistent with earlier studies,12,21,22,23 our data have shown that

polymorphisms exist in the IFNL3/IL28B gene in North Indian population. The

frequency of T allele (28%) of IFNL3 (rs12979860) was found to be similar

Accepted Article
that reported in Southern Indian (24%) population,24 Caucasians (30-32%)

population,12 Moroccan (32%),21 while the Oriental population carry much

lower frequency (5-8%) of the T allele.25 Similarly, the G allele frequency

(17%) of IFNL3 (rs8099917) was quite similar to that reported earlier in the

Eastern Indian (15%) population,22 Caucasians (16%) population,12 while the

same for IFNL3 (rs8099917) is relatively lower in Oriental (5-9%) population.26

The significant increase in the frequency of TT (minor) genotype of

IFNL3 (rs12979860) in chronic HCV patients when compared with controls,

suggest that polymorphism in IFNL3 (rs12979860) may modify the

susceptibility of an individual to chronic HCV infection. Explanation for the

association of TT genotype of IFNL3 (rs12979860) with chronic HCV patients

was suggested by Par et al. (2011) that the frequency of IFNL3 (rs12979860)

CC (major) genotype was lower in HCV patients than in controls, it may be

regarded as being protective against the chronic HCV infection, and that is, it

may confer protection against the disease.27 In a study on 219 Pakistani HCV

patients, rs12979860 CC genotype has been associated with protection

against HCV infection.28 Ge et al. (2009) suggested that IL28B (rs12979860)

polymorphism was associated with decreased IL28B expression because

IL28B encodes interferon lambda-3, which may be involved in interfering with

viral replication.16 Significant association of TT genotype of IFNL3

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 (rs12979860) with chronic HCV infection was also shown in the Caucasian,

Oriental, Moroccan, Pakistani and Egyptians population.12,21,23,29,30 However,

El-Awady et al. (2012) and Sharafi et al. (2012) did not find significant

association of TT genotype of IFNL3 (rs12979860) with chronic HCV

infection31,32.
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Consistent with the earlier studies in Caucasians and Oriental

population,20,30,33 our study also shows the association of IFNL3 (rs8099917)

GG (minor) genotype with chronic HCV infection when compared with healthy

controls. The high prevalence of the IFNL3 (rs8099917) G (minor) allele in

HCV genotype 3 infected patients shows that the IFNL3 (rs8099917) GG

genotype may have a causative role in HCV infection.

Our study also showed that, CC (major) genotype of IFNL3

(rs12979860) is associated with spontaneous clearance of HCV infection.

Highest clearance rate was found among individuals having CC (56.5%)

genotype and the lowest clearance rate was detected in the individuals having

TT (6.5%) genotype of IFNL3 (rs12979860). This findings were supported by

earlier studies in Caucasians, African Americans and in Moroccan

population.16,17,19,34 Thomas et al. (2009) studied 1008 subjects and found that

rs12979860 CC genotype was predictive of spontaneous HCV clearance,

among individuals of both European and African ancestry.19 An association

between rs12979860 CC genotype and spontaneous HCV clearance was also

shown by Tillmann et al. (2010) who studied 190 German women and by

Montes-Cano et al. (2010) in a study of 353 Spanish subjects.35,36 Ezzikouri et

al. (2013) and Kurbanov et al. (2011) also reported that polymorphism in

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 IFNL3 (rs12979860) plays a role in spontaneous clearance of HCV

infection.21,37

Our data further revealed that, combined DCV and SOF therapy in 250

HCV genotype 3 patients, 219 (87.6%) HCV patients achieved SVR, while 19
Accepted Article
(7.6%) were relapsers and the remaining 12 (4.8%) failed to respond the

therapy. These results are consistent with those from previous study of HCV

genotype 3 infection in which SVR12 was achieved by more than 90% of

patients without cirrhosis after 12 weeks of treatment with DCV+SOF

therapy.7,38 Nelson et al., (2015) reported a 96% SVR12 rate in genotype 3

HCV patients without cirrhosis (96%; 105 of 109) than in those with cirrhosis

(63%; 20 of 32).7 Similarly, Hezode et al., (2017) also reported higher (98%)

SVR12 rates in genotype 3 HCV patients without cirrhosis (98%; 43 of 44).39

In our study, the SVR (87.6%) rate in genotype 3 HCV patients without

cirrhosis is slightly lower compared to the above study. This could be

explained due to the ethnic differences and small sample size of genotype 3

non-cirrhotic HCV patients in the previous study compared to our study

(n=250). Further, the lower SVR rate in our study compared to the previous

study may also be due to the preexisting NS5A resistance-associated variants

or sub-genotypes. DAAs are vulnerable to drug resistance and resistance-

associated variants (RAVs) may occur naturally prior to DAA therapy or may

emerge following drug exposure.40 Our data further showed that TT (minor)

genotype of IFNL3 (rs12979860) may influence the outcome of combined

DCV and SOF therapy. The TT genotype of IFNL3 (rs12979860) was found to

present in much higher frequency in relapsers (37%), non-responders (41.5%)

and non-SVR patients (39%) when compared to SVR patients (9%) which

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 results in a significant increase in OR in relapsers, non-responders and non-

SVR patients when compared with SVR patients. This increase in TT

genotype frequency relapser, non-responders and non-SVR patients indicates

that TT genotype may be responsible for failure of combined DCV and SOF

for maintaining the SVR in chronic HCV patients. Our data also indicate that,
Accepted Article

CC (major) genotype of IFNL3 (rs12979860) was identified in much higher

frequency in SVR patients (39.5%) when compared to relapsers (10.5%), non-

responders (17%) and non-SVR patients (13%), indicating the association of

CC (major) genotype with likelihood of achieving SVR in comparison with non-

CC variants. This finding was further supported by previous studies that the

CC variant of rs12979860 was associated with increased likelihood of

achieving SVR in comparison with non-CC variants.41,42 Asselah et al. (2011)

showed a better treatment response rate of the C Allele of the IFNL3

(rs12979860). The response rates were 81.8%, 46.5% and 29.4% for

genotype CC, CT and TT respectively.43 However, da Silva Conda et al.

(2014) did not find a significant association of SNP rs1297960 with SVR.33

Despite the strong association between IFNL3 and DAAs treatment

response, the underlying mechanism of IFNL3 in determining treatment

response remains unknown. It is postulated that DAAs treatment response

mechanism is similar to the interferon treatment response mechanism. IFNL3

signals through the IFN-λ receptor complex, induces expression of IFN-

stimulated genes (ISGs) via JAK-STAT signaling and has antiviral properties.

In chronically infected individuals carrying the favourable allele of IFNL3,

basal levels of IFN-λ and ISGs will be relatively low and treatment with DAAs

can induce a potent antiviral state leading to viral clearance or SVR. In

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 individuals carrying the unfavourable allele of IFNL3, basal levels of IFN-λ and

ISGs will be high which can lead to a refractory state and unresponsiveness

to the DAAs treatment and failure to respond to treatment.44,45,46 Par et al.

(2011) reported that, the high IFNL3 cytokine producing CC genotype was

associated with a two to three fold greater rate of SVR to anti-HCV therapy
Accepted Article

compared with the low cytokine producing TT genotype.27 They added that

the frequency of IFNL3 CC genotype was lower in HCV patients than in

controls, it may be regarded as being protective against the chronic HCV

infection, and that is, it may confer protection against the disease. Thomas et

al. (2009), reported that the IFNL3 CC genotype enhanced the spontaneous

resolution of HCV infection and patients who harbour the C allele at

rs12979860 are more prone to respond to treatment and clear HCV than

patients who do not possess this genetic polymorphism, which suggests a

primary role for IFNL3 in the resolution of HCV infection.19

A statistically significant risk for GG (minor) genotype of IFNL3

(rs8099917) was also found in relapsers and non-SVR patients when

compared to SVR patients. Similar result were also obtained by Suppiah et al,

(2009) and Tanaka et al, (2009), they founded a strong association of IFNL3

(rs8099917) polymorphism with response to combination treatment with

interferon and ribavirin in Australian and Japanese patients, infected with viral

genotype 1.17,18 Rauch et al, (2010) conducted a GWAS including all viral

genotypes 1-4, and found that the rs8099917 minor allele (G) was associated

with both progression to chronic hepatitis C and failure to respond to

combined Peg-interferon and ribavirin therapy, with the strongest effects in

patients infected with genotypes 1 or 4.34 Moreover, the TT (major) genotype

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 of IFNL3 (rs8099917) who achieved SVR was 60%, which is significantly

higher compared to TT genotypes in relapsers (21%), non-responders (25%)

and non-SVR patients (22.5%). This finding was supported by previous

studies on Caucasians, Oriental and Chilean patients, which showed high

proportion of TT genotypes in SVR in HCV genotype 1, 2a, and

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2b.13,16,17,18,20,32 However, da Silva Conda et al. (2014) and Lyoo et al. (2011)

did not find any significant association of SVR with anti viral treatment in

patients with TT genotypes of IFNL3 rs8099917.33,47 Despite the strong

association between IFNL3 (rs8099917) genotype and SVR, the underlying

mechanism of IFNL3 in determining SVR remains unknown. IL28B

(rs8099917) polymorphism has been reported with decreased IL28B

expression and are in strong linkage disequilibrium with IL28B

(rs12979860).16,18 Therefore, it is predicted that rs8099917 may share similar

mechanism as rs12979860 on the association with treatment response. The

study in Switzerland tested more than 500000 SNPs. Analysis of data from

1213 individuals with available data at both rs12979860 and rs8099917 found

that rs12979860 genotype was highly associated with chronic HCV infection

and was in strong linkage disequilibrium with rs8099917.34 Further, minor

genotype of both locus of IFNL3 (rs12979860, rs8099917) were found to be

present much higher frequency in non-SVR patients when compared to SVR

patients which results in almost similar significant increase in OR of

rs12979860 (13) and rs8099917 (11.5) in non-SVR patients when compared

with SVR patients therefore both the locus of IFNL3 (rs12979860 and

rs8099917) can be used for predicating non-SVR in HCV patients when

treated with combined DCV and SOF therapy.

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 In conclusion, our data suggests that TT (minor) genotype of IFNL3

(rs12979860) and GG (minor) genotype of IFNL3 (rs8099917) is associated

with an increased susceptibility to chronic HCV genotype 3 infection. Results

of treatment response indicate that combined DCV and SOF therapy achieved

much high SVR and was well tolerated in chronic HCV genotype 3 patients. It
Accepted Article

further indicate that CC (major) genotype of IFNL3 (rs12979860) and TT

(major) genotype of IFNL3 (rs8099917) is associated with likelihood of

achieving SVR to combined DCV and SOF therapy in genotype 3 infected

HCV patients and the individuals with TT (minor) genotype of IFNL3

(rs12979860) and GG (minor) genotype of IFNL3 (rs8099917) are more

susceptible to chronic HCV infection and treatment relapse.

ACKNOWLEDGEMENTS AND DISCLOSURES

The authors are grateful to the Director, Sanjay Gandhi Post Graduate

Institute of Medical (SGPGIMS), Lucknow, India for his keen interest and

support in carrying out the study. Dr. Anwar Jamal Khan is grateful to Indian

Council of Medical Research (ICMR), New Delhi, India for providing a

Research Associate Fellowship.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest.

REFERENCES

1) WHO, World health organization. Global hepatitis report, 2017. Available:

http://www.who.int/hepatitis/publications/global-hepatitis-report2017/en/.

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 Table 1. Distribution of demographic and biochemical variables of
healthy controls, chronic HCV patients and spontaneous clearance
individuals

Healthy HCV Spontaneous p-value

Accepted Article
controls Patients
Clearance Conts Vs
(n=500) (n=250)
(n=62) HCV

Age 49.4 ± 9.2 49.8 ± 9.5 46.5 ± 8.9 >0.05

(years, mean ±

S.D.)

Sex >0.05

Male (%) 270 (54%) 135 (54%) 28 (45%)

Female (%) 230 (46%) 115 (46%) 34 (55%)

ALT (U/L) 26.5 ± 4.4 82.6 ± 75.3 30.07 ± 12.4 <0.00*

AST (U/L) 29.2 ± 4.5 108.7 ± 31.3 ± 9.6 <0.00*

84.5

ALP (U/L) 45.4 ± 7.2 110.4 ± 47.6 ± 8.6 <0.00*

34.4

Total bilirubin 0.67 ± 0.20 1.3 ± 0.58 0.69± 0.25 <0.00*

(mg/dl)

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 Albumin (g/dl) 3.72 ± 0.24 3.82 ± 0.32 3.65 ± 0.22 >0.05

Prothrombin time 12.73 ± 13.21 ± 1.3 12.69 ± 0.86 >0.05

(Seconds) 0.87
Accepted Article
HCV RNA (IU/ml) Negative Positive Negative <0.00*

HCV genotype 3 ----- 100% -----

Data are represented as means ± standard deviation (SD); p-value represent

the comparison of data between healthy controls and HCV patients; *p<0.05 is
considered statistically significant; ALT: alanine aminotransferase; AST:
aspartate aminotransferase. ALP: alkaline phosphatase

Table 2. Baseline characteristics of sustained virological response

patients, relapsers and non-responders HCV patients

SVR REL NR p-value

(n=219) (n=19) (n=12) SVR Vs

REL

Age 47.6 ± 6.6 48.8 ± 7.3 52.3 ± 6.5 >0.05

(years, mean ± S.D.)

Sex >0.05

Male (%) 122 09 (47.5%) 04 (33.5%)

(55.5%)
Female (%) 10 (52.5%) 08 (66.5%)

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 97 (44.5%)

HCV RNA of genotype <0.00*

3
127 (58%) 08 (42%) 04 (33.5%)
Accepted Article
<600,000 IU/ml
92 (42%) 11 (58%) 08 (66.5%)

≥600,000 IU/ml

Data are represented as means ± standard deviation (SD); p-value represent the
comparison of data between SVR and REL patients; *p<0.05 is considered statistically
significant; SVR: Sustained virological response; REL: Relapsers; NR: Non-responder.

Table 3: Distribution of genotype & allele frequency of IFNL3 among controls and
HCV patients

IFNL3 Genotyp Control HCV Crud p Adjuste p Allele

e s e OR d aOR valu Frequenc
rs1297986 Patient valu (95% CI) e y
0 (n=500) s (95% e
CI) Conts
(BstUI) (%) (n=250) HCV

(%)

CC 249 90 1 1 (Ref.) 0.7 0.6

(Ref.) 2 2
(50%) (36%)

CT 224 128 1.6 0.00* 1.65 0.00*

(45%) (51%) (1.14- (1.21-

2.18) 2.29)

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 TT 27 32 3.3 0.00* 3.5 0.00* 0.2 0.3
8 8
(5%) (13%) (1.86- (1.97-
5.78) 5.96)
Accepted Article

IFNL3 TT 338 138 1 1 (Ref.) 0.8 0.7

(Ref.) 3 5
rs8099917 (68%) (55%)

(MaeIII)

TG 151 99 1.6 0.00* 1.5 0.00*

(30%) (40%) (1.16- (1.19-

2.21) 2.27)

GG 11 13 2.9 0.00* 2.5 0.00* 0.1 0.2

7 5
(2%) (5%) (1.27- (1.34-
6.62) 6.69)

Ref: reference category; OR: Odds ratio; 95%CI: 95% confidence interval; *p<0.05 is considered
statistically significant; aOR adjusted with age in multivariate logistic regression models.

Table 4: Distribution of genotype & allele frequency of IFNL3 among

spontaneous clearance individuals and HCV patients

IFNL3 Genotyp spontaneou HCV OR p Allele

e s Clearance Frequenc
rs1297986 Patient (95% valu y
0 (n=62) s CI) e
SC
(BstUI) (%) (n=250) HCV

(%)

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 CC 35 90 1 0.7 0.62
(Ref.) 5
(56.5%) (36%)
Accepted Article

CT 23 128 2.1 0.00*

(37%) (51%) (1.19

-
3.91)

TT 4 32 3.1 0.03* 0.2 0.38

5
(6.5%) (13%) (1.02
-
9.44)

IFNL3 TT 43 138 1 0.8 0.75

(Ref.) 5
rs8099917 (69.5%) (55%)

(MaeIII)

TG 19 99 1.6 0.11

(30.5%) (40%) (0.89

-
2.95)

GG 0 13 --- --- 0.1 0.25

5
(0%) (5%)

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 Ref: reference category; OR: Odds ratio; 95%CI: 95% confidence interval; *p<0.05 is considered
statistically significant.

Table 5: Comparisons between sustained virological response patients and

relapsers or non-responders HCV patients with IFNL3 genotypes
Accepted Article

Genotyp SVR REL REL Vs NR NR Vs Allele

e SVR SVR Frequency
(n=21 (n=19) (n=12)
9) OR OR SVR REL
(%) (95% CI) (%) (95% CI) NR
(%)
p value p value

IFNL3 CC 86 2 1 (Ref.) 2 1 (Ref.) 0.6 0.3 0.3

5 7 8
rs129798 (39.5 (10.5 3.8(0.81- (17%) 1.9(0.36-
60 %) %) 17.82) 10.04) 0.3 0.6 0.6
CT 5 5 3 2
(BstUI) 113 10 0.07 0.44
TT (41.5
(51.5 (52.5 15(2.90- %) 10.8(1.9
%) %) 77.98) 4-59.46)
5
20 7 0.00* 0.00*
(41.5
(9%) (37%) %)

IFNL3 TT 131 4 1 (Ref.) 3 1 (Ref.) 0.7 0.4 0.6

8 7 2
rs809991 TG (60%) (21%) 4.1(1.24- (25%) 4.9(1.29-
7 13.48) 18.68) 0.2 0.5 0.3
GG 80 10 9 2 3 8
(MaeIII) 0.01* 0.01*
(36.5 (52.5 (75%)
%) %) 20.5(4.5 ---
8-91.37) ---
8 5
0.00*
(3.5%) (26.5
%)

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 SVR: Sustained virological response; REL: Relapsers; NR: Non-responder; Ref: reference category;
OR: Odds ratio; 95%CI: 95% confidence interval; *p<0.05 is considered statistically significant.

Table 6: Comparisons between sustained virological response patients and

Non-SVR (relapsers + non-responders) HCV patients with IFNL3 genotypes
Accepted Article

Genotype SVR Non- OR p Allele

SVR value Frequency
(n=219) (95% CI)
(n=31) SVR
(%) Non- SVR
(%)

IFNL3 CC 86 4 1 (Ref.) 0.06 0.65 0.37

rs12979860 CT (39.5%) (13%) 2.8 0.00* 0.35 0.63

(BstUI) TT 113 15 (0.91-

8.90)
(51.5%) (48%)
13
20 12
(3.76-
(9%) (39%) 44.21)

IFNL3 TT 131 7 1 (Ref.) 0.00* 0.78 0.53

rs8099917 TG (60%) (22.5%) 4.5 0.00* 0.22 0.47

(MaeIII) GG 80 19 (1.79-
11.04)
(36.5%) (61.5%)
11.5
8 5
(3.03-
(3.5%) (16%) 45.18)

SVR: Sustained virological response; Non-SVR (relapsers + non-responders); Ref: reference category;
OR: Odds ratio; 95%CI: 95% confidence interval; *p<0.05 is considered statistically significant.

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