JOURNAL OF INTERFERON & CYTOKINE RESEARCH FULL MANUSCRIPT

Volume 00, Number 00, 2017

ª Mary Ann Liebert, Inc.
DOI: 10.1089/jir.2017.0002

Confounding by Single Nucleotide Polymorphism

rs117648444 (P70S) Affects the Association of Interferon
Lambda Locus Variants with Response
to Interferon-a-Ribavirin Therapy in Patients
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with Chronic Genotype 3 Hepatitis C Virus Infection

Anand Bhushan, Sumona Ghosh, Samsiddhi Bhattacharjee, and Sreedhar Chinnaswamy

Genome-wide association studies discovered interferon lambda (IFNL or IFN-l) locus on chromosome 19 to be
involved in clearance of chronic hepatitis C virus (HCV) infection in patients following interferon-a-ribavirin
(IFN-RBV) therapy. Subsequent studies established a dinucleotide polymorphism rs368234815, as the prime
causal variant behind this association. The DG allele of this variant gives rise to a new IFNL gene, IFNL4, coding
for IFN-l4 whose activity paradoxically associates with lesser viral clearance rates. A low-frequency, non-
synonymous single nucleotide polymorphism (SNP) rs117648444 within the 2nd exon of IFNL4 changes the
70th amino acid from proline to serine resulting in lower activity of the functional IFN-l4 protein, thereby
increasing HCV clearance rates. In the present study, we used a cohort of genotype 3 HCV-infected patients,
drawn from different geographical regions of India who underwent IFN-RBV therapy, to examine the association
of several important IFNL locus SNPs/variants with sustained virological response (SVR). Intriguingly, the
causal variant rs368234815 did not show the best strength and significance of association with SVR, while
further analysis revealed that a negative confounding effect of rs117648444 was responsible for this phenom-
enon. Our results indicate that IFNL locus SNPs are subject to either a positive or a negative confounding effect
by rs117648444; the nature of confounding depends on the linkage of the IFNL SNPs with the low-activity IFN-
l4-generating minor allele of rs117648444. Thus, our work demonstrates that the linkage disequilibrium
structure of the IFNL region may confound the results of association studies. These results have implications for
the design and understanding of future case–control studies involving IFNL locus SNPs/variants.

Keywords: confounder SNPs, genotype 3 HCV, HCV, IFN-l4, IFN-RBV, IFNL SNP, rs368234815

Introduction emerged as the top hits in the initial GWAS (Ge and others
2009; Rauch and others 2010; Suppiah and others 2009;

H uman interferon lambda (IFNL) locus on chromo-

some 19 comprises 4 genes, IFNL1-4 encoding for
potent antiviral and immunomodulatory cytokines (IFN-l1-
4) belonging to the type III IFN family (Kotenko and others
2003; Sheppard and others 2003; Prokunina-Olsson and
others 2013; Chinnaswamy 2016). This locus and its genes
gained importance after several genome-wide association
studies (GWAS) in the year 2009 and showed involvement
of single-nucleotide polymorphisms (SNPs) in clearance
of chronic hepatitis C virus (HCV) infections following
interferon-a-ribavirin (IFN-RBV) therapy (Ge and others 2009;
Suppiah and others 2009; Tanaka and others 2009; Rauch
and others 2010). The SNPs rs12979860 and rs8099917
Tanaka and others 2009; Chinnaswamy 2014) (Fig. 1);
later, fine-mapping studies led to identification of more
SNPs in the region (Ge and others 2009; Rauch and others
2010; di Lulio and others 2011; Smith and others 2011).
Among them, SNPs rs28416813 and rs4803217 in 5¢ and 3¢
untranslated regions of IFNL3 gene were shown to influence
reporter gene expression thereby implicating expression dif-
ferences of IFN-l3 protein as a likely causal mechanism re-
sponsible for HCV clearance (Chinnaswamy and others 2013;
Mcfarland and others 2014). Other SNPs such as rs7248668 and
rs4803221 were identified to have best predictive values after
screening for all variants in a region spanning 100 kb at the locus
by DNA deep-sequencing methods (Smith and others 2011).

National Institute of Biomedical Genomics, Kalyani, India.

1
 2 BHUSHAN ET AL.
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FIG. 1. A schematic diagram of the genomic structure of IFNL locus highlighting the SNPs/variants that were genotyped
for this study. The SNP rs12979860 is referred to as the ‘‘tag SNP’’ due to its close correlation with the ‘‘functional
variant.’’ Position of each SNP/variant is marked by black vertical lines and approximate distance among rs8099917,
rs368234815, and rs117648444 is given by dotted lines; the alternate alleles for each SNP/variant are given in parenthesis.
Depending on the presence of the alternate alleles of the ‘‘activity’’ variant rs117648444, active IFN-l4 protein (shown as
3D structure, PDB ID: K9M1U5; visualized in UCSF CHIMERA) can be of 2 types: IFN-l4 P70 (DG-G, haplotype of
rs368234815-rs117648444) and IFN-l4 S70 (DG-A) and their properties are highlighted based on results from Terczyńska-
Dyla and others (2014). IFNL, interferon lambda; SNP, single-nucleotide polymorphism.

A combination of genetic and molecular methods identi-
fied a dinucleotide variant rs368234815 as the prime causal
variant (referred to as functional variant hereafter) at the
IFNL locus in chronic HCV infections (Prokunina-Olsson and
others 2013). The DG allele of the functional variant causes a
frameshift leading to the expression of a fully functional IFN-
l4 protein (Fig. 1). The TT allele that obliterates the expression
of a functional IFN-l4 protein seems to have been acquired
during evolution just before the out-of-Africa migration of
modern humans (Key and others 2014). Present-day human
populations harbor the DG allele in varying proportions (from
0.05 to 0.78) with the African population having the maximum
frequency (0.78) (Prokunina-Olsson and others 2013). Another
low-frequency variant rs117648444 (referred to as activity
variant hereafter) changes the amino acid from Pro to Ser at the
70th position in IFN-l4 protein (Fig. 1), altering its functional
properties (Prokunina-Olsson and others 2013; Terczyńska-
Dyla and others 2014).
In HCV infections, expression of a functional IFN-l4 pro-
tein by the DG allele of the functional variant is associated with
nonclearance of the virus from the host either spontaneously
after an acute infection (Aka and others 2014) or following
treatment with IFN-RBV in chronic cases (Prokunina-Olsson
and others 2013). This is referred to as the ‘‘IFN-l4-HCV
paradox’’ (O’Brien and others 2014), the reasons for which are
unclear so far.
Even though the SNP rs8099917 is located *4 kb away
from the functional variant rs368234815 (Fig. 1) and not in
high LD (linkage disequilibrium) with it in many world
populations (Prokunina-Olsson and others 2013; Chinnas-
wamy 2016), it has been consistently reported to be asso-
ciated with response to IFN-RBV therapy (referred to as
sustained virological response or SVR) in HCV infections; it
is also the ‘‘top hit’’ associated with SVR in 3 GWAS
(Suppiah and others 2009; Tanaka and others 2009; Rauch
and others 2010). In some reports, the association is even
stronger than the ‘‘tag SNP’’ rs12979860 (Fig. 1, referred as
tag SNP hereafter) (Doehring and others 2010; Lindh and
others 2011; Smith and others 2011; Suppiah and others
2011; Antaki and others 2013; Khudayberganova and others
2014; O’Connor and others 2016).
Some studies showed that rs8099917 is useful as a
predictor of SVR in those patients who were heterozygous
for the tag SNP (Fischer and others 2012, 2013; Palmieri
and others 2014; Ragheb and others 2014; Palenzuela
Gardón and others 2016), suggesting that there may be an
underlying and yet unidentified gene structure that links
the 2 SNPs. A recent study, on a European HCV cohort
(Terczyńska-Dyla and others 2014), showed that the minor
allele of rs8099917 is strongly correlated with the DG-G
haplotype involving the functional variant and the activity
variant rs117648444 (Fig. 1).
 LINKAGE DISEQUILIBRIUM STRUCTURE AT THE IFNL LOCUS
This discovery certainly provides a convincing explana-
tion for the association of rs8099917 with clearance of HCV
and indirectly implicates the function/activity of IFN-l4 to
be the causal link behind IFNL SNP association with HCV
clearance (Terczyńska-Dyla and others 2014). However, no
confirmation regarding this has been reported thus far, es-
pecially from other populations.
There is no consensus on which SNP should be targeted
for association studies involving the IFNL locus even
though numerous studies have been reported in this area
(Chinnaswamy 2014, 2016). There is agreement in the lit-
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erature that rs368234815 is the causal variant (Chinnaswa-
my 2016), but less is known on how the activity variant
rs117648444 influences association studies involving the
functional variant. In view of this, we undertook the current
study in a cohort of genotype 3 HCV-infected patients in an
Indian population who underwent IFN-RBV therapy.
Even though IFN-RBV therapy has become obsolete in
many parts of the world with the introduction of direct-
acting antivirals (DAAs) in 2011, they have become avail-
able only recently in India (Puri and others 2016); also,
since genotype 3 HCV is no longer an ‘‘easy-to-treat’’ ge-
notype with DAAs (Gondeau and others 2015; Kattakuzhy
and others 2016), genotyping of IFNL SNPs/variants may
still be important especially since response to DAA therapy
is also influenced by IFNL locus SNPs (Akuta and others
2010; Zeuzem and others 2013; Meissner and others 2014).
Materials and Methods
Study participants
The cohort consisted of 72 chronic HCV (genotype 3a/b)-
infected patients who had received Peg-IFN-RBV combination
Table 1. Clinical Characteristics of Hepatitis C Virus Patients Used in the Study
Parameters
Median age (IQR)
Males (%)
Mean BMI in kg/m2 (range)
Mean HCV RNA (copies/mL) at baseline (range)
Median ALT (IU/L) at baseline (range)
Median ALT (IU/L) at end of therapy (range)
Median AST (IU/L) at baseline (range)
Median AST (IU/L) at end of therapy (range)
Mean hemoglobin (g/dL) at baseline (range)
Median platelet count (per cumm) at baseline (range)
Median dose of ribavirin (mg/day) used (range)
Median dose of peginterferon-alpha used (mg) (range)
Median SAP (IU/L) at baseline (range)
Median SAP (IU/L) at end of therapy (range)
Median bilirubin total (mg/dL) at baseline (range)
Median bilirubin conjugated (mg/dL) at baseline (range)
Median fasting blood glucose level (mg/dL) at baseline (range)
Cirrhotic (%)
Diabetic (%)
Patients attaining RVR (%), n/N
Patients attaining SVR (%), n/N
Patients attaining EVR (%), n/N
End of therapy response (%), n/N
Data were missing for some patients for some parameters.
ALT, alanine transaminase; AST, aspartate transaminase; BMI, body mass index; EVR, early virological response; HCV, hepatitis C
virus; IQR, interquartile range; RVR, rapid virological response, SAP, serum alkaline phosphatase; SVR, sustained virological response.
3
therapy at 7 centers across India. Demographic characteristics,
clinical details, and viral load [including status of rapid viro-
logical response (RVR), SVR] of patients are shown in Table 1.
Forty-five healthy controls were enrolled with similar demo-
graphic characteristics. Among them, 14 controls were from an
asthma clinic (based on immunological, X-ray, and spirometry
analysis) and 31 controls were from a gastroenterology clinic
(based on liver ultrasound grade and biochemical analysis).
Blood samples were drawn for SNP genotyping from patients
and healthy controls. This study was approved by the Institu-
tional Ethics Committee: Review Committee for Protection of
Research Risks to Humans, National Institute of Biomedical
Genomics, Kalyani. Informed written consent was obtained
from all patients.
Genotyping of IFNL SNPs/variants
Samples were genotyped using PCR-RFLP (polymerase
chain reaction-restriction fragment length polymorphism) or
by direct sequencing. For patient samples, SNPs rs117648444
and rs8099917 were genotyped by PCR-RFLP and confirmed
by Sanger sequencing where 100% concordance was obtained.
The remaining SNPs/variants were genotyped by Sanger se-
quencing. Human genomic DNA was extracted from whole
blood using a QIAamp Blood DNA Midi Kit (Qiagen) as
per the manufacturer’s protocol. DNA was quantified using
NanoDrop device (Thermo Scientific). Two hundred to four
hundred nanograms of template DNA was used for each PCR.
SNPs/variants rs368234815, rs12979860, rs117648444,
and rs4803221 were sequenced from an amplicon generated
by using the primers (1) IL28B4kbxhoRev: 5¢-GATATCCTC
GAGCCCGGATTTCAGGAC-3¢ and (2) IL28B4kbKpnFor:
5¢-GATATCGGTACCGCCCTGGACGGGAAAG-3¢. SNPs
rs8099917 and rs8113007 were sequenced from an amplicon
Values
40 (15)
69.4
25.17 (20.45)
19.7 · 105 (31.99 · 105)
79.5 (394)
38 (158)
77 (273)
39 (153)
13.14 (12.8)
178,000 (349,998)
800 (1,100)
90 (130)
156 (709)
156 (319)
0.8 (10.8)
0.2 (1.9)
92 (102)
31
7
80, 54/67
71, 51/72
91, 62/68
90, 60/67
 4 BHUSHAN ET AL.
generated by the primers (1) 917For700bp: 5¢-CAAGTCAG
GCAACCACATGC-3¢ and (2) 917REV700bp: 5¢-GCTCT
GTCTGTCTCAATCAATC-3¢. Sequencing was carried out
either at the National Institute of Biomedical Genomics,
Kalyani, India (ABI-3500xL Genetic Analyzer) or at Eurofins
Genomics India Pvt. Ltd. (Bengaluru, India).
Correction for the altered activity of IFN-k4
protein by restricted analysis
We used 2 models (referred to as restricted models) to
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account for the altered activity of the IFN-l4 protein in
our association analysis of the tag SNP/functional variant
with SVR. We had a total of 8 patients who were G/A at
rs117648444; 6 were also heterozygous for rs368234815
(TT/DG), all of them belonged to the SVR group; 2 patients
were DG/DG at rs368234815, 1 belonged to the SVR group
and the other belonged to the nonresponder (NR) group. In
the first model, samples harboring TT/DG at rs368234815
and G/A genotype at rs117648444 (ie, a total of 6 patients)
were left out from the analysis (denoted as adjusted 1); and
in the second model, all samples that are heterozygous for
rs117648444 (a total of 8 patients) were left out from the
analysis (denoted as adjusted 2). The restricted models were
also applied for analysis of association of SVR with the
remaining SNPs rs8099917, rs4803221, and rs8113007.
Plasmids and site-directed mutagenesis
A 4 kb fragment of IFNL3 promoter was constructed in a
pGL3 basic vector (Invitrogen). The construct described as
‘‘p1.4kbIL28B’’ in Chinnaswamy and others (2013) was used
to clone the remaining portion of the IFNL3 promoter using
the following primers: IL28B4kbKpnFor: 5¢-GATATC
GGTACCCAGTGGAATTCAGGGCAAATTAC-3¢ and IL
28B1.1kbEcoR1Rev: 5¢-GTAATTTGCCCTGAATTCCAC
TG-3¢. The clone was confirmed by Sanger dideoxy se-
quencing of plasmid DNA. All the enzymes used for DNA
cloning were from NEB. We used a site-directed mutagen-
esis kit (NEB) to incorporate the alternate allele at rs4803221
in the promoter construct and further confirmed the mutation
by Sanger sequencing of the plasmid.
Methylation and luciferase assay
One microgram of plasmid DNA (referred as 4 kb
rs4803221 ‘‘C’’ and 4 kb rs4803221 ‘‘G’’) was methyl-
ated with CpG methyltransferase (M.SssI) and GpC me-
thyltransferase (M.CviPI) enzymes (NEB). Briefly, 4 U of
enzyme and 160 mM of S-adenosylmethionine (SAM) were
added to 1 mg of plasmid DNA to make a reaction volume
of 20 mL and incubated overnight at 37C. Reaction mix
without enzyme and substrate was used as negative controls.
The methylated plasmid DNA was transfected into
HEK293T cells as described previously (Chinnaswamy and
others 2013). Ten nanograms of the reporter construct, along
with 30 ng of each of plasmids coding for p65 (NF-kB), IRF7
(interferon regulatory factor), HCV NS5B polymerase (ge-
notype 2a), and 0.5 ng of RIG-I (retinoic acid-inducible gene I)
plasmids, was used per well. Renilla luciferase plasmid
pRLTK was used as a transfection control at 0.01 ng/well.
Twenty-four hours after transfection, cells were lysed and
luminescence was recorded by GloMax 96-well plate reader
(Promega) with the dual injection system. Dual Luciferase
Assay Kit (Promega) was used for the assay. The ratios of
firefly to Renilla luciferase values were used for analysis.
Statistical analysis
Haplotypes were inferred using PHASE software, version
2.1 (University of Washington, http://stephenslab.uchicago
.edu/software.html). For LD analysis, haplotypes were coded
as AA, AB, and BB; BB for the absence of haplotype, AB for
the presence of 1 copy of the haplotype, and AA for the
presence of 2 copies of the haplotype. LD was calculated by
‘‘R programming’’ using ‘‘LD measure’’ function. Goodness
of fit was measured by chi-square tests (with Yate’s correc-
tion) and Fisher’s exact test (2-tailed). A P value of <0.05 was
considered significant.
We tested 4 models for association of IFNL variants with
SVR: dominant, recessive, allelic, and additive models
(Supplementary Table S1; Supplementary Data are available
online at www.liebertpub.com/jir). This being a fine-mapping
study of an established associated locus rather than a dis-
covery study, multiple testing correction was deemed to be
unnecessary and, to prevent loss of power, was not carried out
for any analysis. GraphPad Prism, version 6.0, was used for
odds ratio (OR) calculation, receiver operating characteristic
(ROC) curve, and area under the curve (AUC) analysis.
Comparison of areas under the ROC for different models
were done by DeLong’s test using the function ‘‘roc.test’’
from ‘‘pROC’’ R package (R Core Team 2015).
Genotype data of IFNL variants for superpopulations (or
subpopulation) of EAS, SAS, AMR, EUR, and AFR were
extracted from the 1000 genome browser; Ensembl GRCh37
release 87—December 2016. In the 1000 Genomes data, the
genotype status of rs368234815 (TT/DG) was not available
for all populations, and hence, we used proxy SNP data
[rs74597329 (T/G)].
Logistic regression analysis was performed to test the as-
sociation of each SNP adjusting for rs117648444 as a po-
tential confounder using the R function ‘‘glm’’ with ‘‘logit’’
link (R Core Team 2015). We regressed the binary response
variable (SVR) on additively coded SNP genotype 1 at a time
with rs117648444 held as a binary covariate. Estimates of
coefficients and standard errors were used to construct 95%
confidence interval (CI) for the adjusted log odds ratio and
then transformed to corresponding CIs for adjusted odds ratio.
Log likelihood ratio test: We performed logistic regression
of SVR on additively coded SNP genotype by using R func-
tion ‘‘glm.’’ Two models were compared, 1 a marginal model
(eg, rs368234815), nested within another model (eg, both
rs368234815 and rs4803221). Log likelihood ratio test was
performed to compare the models by using the ‘‘lrtest’’
command from ‘‘lmtest’’ (R Core Team 2015) R package.
Results
LD map of IFNL SNPs/variants in an Indian
HCV (genotype 3) cohort
The HCV cohort used in this study is described previously
(Chinnaswamy and others 2014) where we showed association
of rs8099917 with response to IFN-RBV therapy in patients
infected with genotype 3 (subtype a or b) HCV. The patients
(n = 72), who underwent standard IFN-RBV therapy for 6
months between the year 2009 and 2012, were drawn from
tertiary care centers in different parts of the country except from
 LINKAGE DISEQUILIBRIUM STRUCTURE AT THE IFNL LOCUS
southern India (Chinnaswamy and others 2014). SVR (defined
as HCV serum RNA test-negativity at 6 months after the ter-
mination of IFN-RBV therapy) was achieved by 51 patients,
while 21 were NRs to therapy as determined by their serum
HCV RNA status at 6 months after completion of therapy.
The clinical characteristics of the patients are shown in Ta-
ble 1. For the present study, we genotyped 6 additional SNPs/
variants in this cohort: rs12979860, rs368234815, rs4803221,
rs8113007 (Tipu and others 2014), rs117648444, and rs7248668
(Fig. 1). All the 7 SNPs/variants in the patient cohort were
genotyped by DNA resequencing. The minor allele frequencies
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(MAFs) of the SNPs/variants are shown in Fig. 2A. Among the
7 SNPs/variants, rs7248668 was monomorphic in our study
population. LD between the remaining 6 SNPs/variants was
calculated and is shown in Fig. 2B (r2 values are depicted; for D¢
values, refer Supplementary Table S2). The LD between
rs12979860 and rs368234815 was complete (LD, r2 = 1). We
genotyped these 2 SNPs/variants in an additional control cohort
(n = 45; see the Materials and Methods section) and saw that the
LD was complete even in this control population.
In the rest of the article, we shall refer to only the func-
tional variant while we are showing results of either
rs368234815 or rs12979860.
Association of IFNL SNPs/variants
with RVR and SVR
We tested the association of the different IFNL SNPs/
variants with RVR and SVR. We had previously recorded
from the same cohort that the covariates such as age, gender,
HCV RNA copy numbers, and baseline ALT (alanine
transaminase) and AST (aspartate transaminase) levels were
not confounders in the association of an IFNL SNP with
RVR or SVR (Chinnaswamy and others 2014). RVR, de-
fined as HCV serum RNA test-negativity after 4 weeks of
IFN-RBV therapy, is known to be a predictor of subsequent
FIG. 2. LD structure at the IFNL locus in the Indian HCV-infected patient cohort. (A) IFNL SNPs/variants and their
MAFs in our study population (N = 72; SVR, n = 51 and NR, n = 21). (B) The LD plot is based on the pairwise estimates of
disequilibrium coefficient (r2). Lighter regions are with low r2 values; while darker regions show higher r2 values. HCV,
hepatitis C virus; LD, linkage disequilibrium; MAFs, minor allele frequencies; NR, nonresponder; SVR, sustained viro-
logical response.
5
SVR (Martinot-Peignoux and others 2009). On testing for
RVR using a dominant model, all the SNPs/variants except
the activity variant rs117648444 showed significant associ-
ation with RVR (Table 2).
When we tested the association of the 5 SNPs/variants with
SVR using a dominant model, only 2 SNPs rs8099917 and
rs4803221 showed significant association (Table 2, ORcr).
The allele and genotype frequencies of the different variants
in the SVR and NR group are shown in Supplementary
Table S1. Surprisingly, the IFN-l4-generating variant,
rs368234815, fell short of significance (P = 0.09). Among the
2 significantly associated SNPs, rs4803221 had the better
strength (OR = 5.9) and significance (P = 0.002) of association
with SVR. The activity variant rs117648444 failed to show
any significant association with SVR too. A previous study
reported from a northern Indian population has shown sig-
nificant association of rs12979860 with SVR in a larger co-
hort of genotype 3 HCV-infected patients (Gupta and others
2014), suggesting that our study may have lacked sufficient
power to detect an association with this SNP.
However, we can conclude from these results that SNPs
that are not in high LD with the causal variant rs368234815
(ie, rs8099917, r2 = 0.57 and rs4803221, r2 = 0.64) in our
study cohort are more strongly and significantly associated
with SVR than the ones which are in high (rs8113007,
r2 = 0.82) or complete (rs12979860, r2 = 1) LD with it (Fig. 2).
Activity variant rs117648444 is a confounder
in the association of IFNL locus
SNPs/variants with SVR
The S70 variant of IFN-l4 protein resulting from the minor
A allele of the nonsynonymous variant rs117648444 leads to
decreased activity of the protein and has been previously
reported to be crucial in association analysis with SVR
(Fig. 1) (Galmozzi and Aghemo 2014; Terczyńska-Dyla and
 6 BHUSHAN ET AL.

number of samples in either RVR (N = 67) or SVR (N = 72, for crude and regression models; N = 66 for adjusted 1 and N = 64 for adjusted 2 models, also shown in Supplementary Table S1).
3.75 (0.72–19.41)

0.31 (0.036–2.72)

All the ORs shown are from univariate analysis (except ORreg); age and gender were also included in multivariate models but did not affect association of SNPs/variants with RVR/SVR. RVR
data were available only for 67 patients (54 achieved RVR, while 13 did not). SVR data were available for all 72 patients (n, SVR = 51; NR = 21). All SNPs/variants were tested for the same
others 2014). Therefore, the altered activity of the protein has

rs117648444
to be factored into the analysis presuming that IFN-l4-
generating polymorphism is the causal variant and increased

0.248

0.49
—
—
—
—
—
—
IFN-l4-mediated ISG activity is the causal mechanism be-
hind the association of IFNL locus SNPs with treatment re-
sponse to IFN-RBV therapy in chronic HCV infections.
We observed that we had 8 individuals who were het-
erozygous at rs117648444 and among them 6 were also

CI, confidence interval; NR, nonresponder; ORadj, odds ratio adjusted; ORcr, odds ratio crude; ORreg, odds ratio regression; SNPs, single-nucleotide polymorphisms.
heterozygous at rs368234815 (all of them in the SVR group)
6.66 (1.62–27.28)

3.66 (1.23–10.89)

3.66 (1.20–11.18)
and 2 were homozygous for the DG allele at rs368234815 (1
2.44 (0.86–6.89)

3.33 (1.25–8.84)
in SVR and the other in NR, NR group). We corrected for
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rs8113007

the altered activity of the protein in our association analysis

0.033

0.039

0.015
0.01

0.14

of rs368234815, rs8099917, rs4803221, and rs8113007 with

Dominant model of inheritance for the minor alleles (eg TT/TT versus TT/DG +DG/DG for rs368234815) was used to compute OR in univariate models.
SVR, by using 2 models: adjusted 1 and adjusted 2 (refer to
the Materials and Methods section for a description of each
Association of Interferon Lambda Variants with Virological Response

model; analysis is shown in Supplementary Table S1). In

brief, in these models, we did a restricted analysis by ex-
cluding those patients who were able to express the low-
7.09 (1.87–26.92)

5.9 (1.95–17.89)

5.02 (1.65–15.28)

5.10 (1.63–15.94)

4.30 (1.56–11.83)

activity variant of the IFN-l4 protein.

The results are shown in Table 2. Intriguingly, by restricting
rs4803221

0.005

0.002

0.007

0.008

0.004

our analysis to those patients where only the high-activity IFN-

l4 could be expressed, functional variant rs368234815 now
showed significant association with SVR in both the models,
suggesting that the activity variant rs117648444 was a negative
confounder in the initial crude analysis (compare ORcr with
ORadj1, ORadj2 in Table 2). Similar results were seen for
rs8113007 (Table 2), which is in high LD (r2 = 0.82; Fig. 2) with
6.42 (1.70–24.20)

4.33 (1.47–12.75)

3.66 (1.22–10.88)

3.66 (1.20–11.17)

the functional variant. The activity variant rs117648444, unlike

3.33 (1.25–8.84)
rs8099917

its effect on the functional variant and rs8113007, does not seem
0.008

0.013

0.033

0.039

0.015

to negatively affect the association of rs8099917 and rs4803221

with SVR (Table 2). In fact, we saw evidence that the activity
variant had a positive confounding effect on rs8099917 and
rs4803221 (compare ORcr with ORadj1, ORadj2 in Table 2).
We used logistic regression analysis to test for the effect of
the activity variant on different IFNL SNPs/variants for their
association with SVR (Table 2, ORreg). After regressing out
6.14 (1.50–25.04)

4.00 (1.39–11.91)

4.00 (1.31–12.18)
2.73 (0.95–7.80)

3.68 (1.42–9.48)

the effect of the activity variant, rs368234815 showed a better

rs368234815

strength and significance of association than rs8099917, sim-

0. 015

0.098

0.022

0.025

0.006

ilar to our observations above with the restricted models. Si-

milarly, rs4803221 retained its strongest association among all
SNPs/variants with SVR even after adjusting for the effect of
the activity variant (Table 2) in the regression analysis model.
A recent study showed that rs8099917, even though is
*4 kb (Fig. 1) away from the functional variant rs368234815,
shows strong association with SVR, due to its high correlation
ORadj1 (95% CI)

ORadj2 (95% CI)

ORreg (95% CI)

(r2 = 0.74) with the DG-G haplotype giving rise to P70 variant
Table 2.

ORcr (95% CI)

OR (95% CI)

of IFN-l4 (Terczyńska-Dyla and others 2014). We did a

similar analysis by deducing the haplotypes involving
rs368234815 and rs117648444 in each of our patients by
haplotype phasing. We found 3 haplotypes to be present in
our cohort (rs368234815-rs117648444): 74.3% of TT-G;
P

20.1% DG-G; and 5.5% DG-A. We did not find any TT-A
haplotype in our cohort. We examined the correlation of the
Regression

SNPs in our study cohort with TT-G, DG-G, and DG-A hap-
Restricted
Model

lotypes (Table 3; for LD, r2 values are shown; for D¢ values

Crude

Crude

refer Supplementary Table S3). We saw that rs4803221, which

P < 0.05 is in bold.

shows the strongest association with SVR in our cohort, had

the highest correlation with DG-G haplotype (r2 = 0.87) and the
least correlation (r2 = 0.0007) with the DG-A haplotype.
To statistically validate the hypothesis that rs4803221
Endpoint

was the best predictor SNP in our cohort, we used log

RVR

SVR

likelihood ratio tests (Table 4). Comparing the different

variants for predicting SVR in nested models confirmed that
 LINKAGE DISEQUILIBRIUM STRUCTURE AT THE IFNL LOCUS 7

Table 3. Correlation of the Single-Nucleotide Polymorphisms/Variants in Our Study

Cohort with Haplotypes Formed by the Functional and Activity Variants
and Their Predictive Values of Sustained Virological Response
LD (r2)
Haplotypea rs8099917 (T/G) rs4803221 (C/G) rs368234815 (TT/DG) rs8113007 (A/T)
TT-G/non TT-G 0.537 0.636 0.998 0.810
DG-G/non-DG-G 0.748 0.873 0.727 0.531
DG-A/non-DG-A 0.013 0.0007 0.191 0.214
TT-A/non-TT-A 0 0 0 0
PPV (95% CI) 0.81 (0.69–0.90) 0.83 (0.69–0.92) 0.80 (0.63–0.90) 0.78 (0.63–0.89)
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NPV (95% CI) 0.50 (0.29–0.70) 0.54 (0.33–0.73) 0.41 (0.24–0.59) 0.40 (0.22–0.59)
AUC 0.67 0.7 0.64 0.61
95% CI 0.52–0.81 0.56–0.84 0.49–0.78 0.46–0.76
P 0.025 0.007 0.067 0.127
a
Haplotypes between rs368234815 (TT/DG) and rs117648444 (G/A).
AUC, area under the curve; LD, linkage disequilibrium; NPV, negative predictive value; PPV, positive predictive value.

rs4803221 is significantly a better predictor of SVR than Protective haplotypes TT-G and DG-A are linked
rs368234815, rs8113007, and rs8099917. We calculated the to the major alleles of rs4803221 and rs8099917
predictive values of the different SNPs/variants on SVR by
using AUC analysis under 2 cutoff levels (dominant and re- We tested the association of SVR among the 3 haplotypes
cessive models of inheritance). The best predictive SNP involving the functional and activity variants (Fig. 3A).
rs4803221 had the best AUC value of 0.7 and also had the best We saw that the DG-A haplotype showed a trend toward
positive and negative predictive values (Table 3). being protective in our cohort in line with previous stud-
However, when we statistically compared the AUC values ies (Galmozzi and Aghemo 2014; Terczyńska-Dyla and
of each of the other variants with that of the best predictor others 2014). (TT-G versus DG-G: OR = 3.33, 95% CI =
SNP rs4803221, we saw that rs4803221 was significantly 1.42–7.82, P = 0.006; DG-A versus DG-G: OR = 7.5, 95%
a better predictor of SVR than the functional variants CI = 0.81–68.93, P = 0.1 using 2-tailed Fisher’s exact test;
rs368234815 and rs8113007 but did not reach significance Fig. 3A.) By using log likelihood ratio tests, we obtained a
to be a better predictor than rs8099917 (Table 4). The significant P value (P = 0.026) by comparing the nested model
above results and those from regression models (Table 2) of TT-G versus TT-G+DG-A for association with SVR, sug-
suggested that rs4803221 may have an effect independent gesting a significant effect of the minor A allele of the activity
of both the functional and activity variants. To further test variant rs117648444 on attaining SVR in our cohort.
this hypothesis, we used the log likelihood ratio test and We sought to examine how the 2 protective haplo-
saw that the effect was not statistically significant (Table 4; types are linked to the major alleles of different IFNL SNPs.
compare rs368234815+rs117648444+rs4803221 versus First, we deduced haplotypes involving the 3 variants
rs368234815+rs117648444). rs368234815, rs4803221, and rs117648444 to examine how
the protective TT-G and DG-A haplotypes are linked to the
Table 4. Single-Nucleotide Polymorphism best predictive SNP rs4803221. Phasing at the 3 variant
rs4803221 Is a Better Predictor of Sustained positions showed that 100% of both the protective haplo-
Virological Response than rs368234815 types TT-G and DG-A were linked to the major allele C of
rs4803221 (Fig. 3B). Similar phasing by including the tag
Model 1 Model 2 Pa
SNP rs12979860, in stark contrast, showed that 100% of the
Log likelihood ratio testa protective DG-A haplotype was linked to its minor allele T
rs368234815+rs4803221 rs368234815 0. 021 (Fig. 3B). The protective DG-A haplotype was 100% linked
rs8099917+rs4803221 rs8099917 0.032 to the major allele T of rs8099917 and to the minor allele T
rs8113007+rs4803221 rs8113007 0.003 of rs8113007 (Fig. 3B) similar to the distribution at rs4803221
rs8099917+rs4803221 rs4803221 0.171 and the tag SNP, respectively.
rs368234815+rs4803221 rs4803221 0.466 However, the distribution of the DG-G and TT-G haplo-
rs8113007+rs4803221 rs4803221 0.139 types among the 2 alleles of rs8099917 was proportionally
½ 
½ 
rs368234815+ rs368234815+ different when compared to rs4803221 (86% of DG-G
rs117648444+ rs117648444 0.347
rs4803221 rs4803221 0.593 linked to the minor allele G and 99% of TT-G linked to
major allele T of rs8099917 versus 90% of DG-G linked to
Variant 1 Variant 2 P the minor allele G and 100% of TT-G linked to major allele
C of rs4803221). These results suggest that rs4803221 op-
DeLong’s test for comparing AUC values timally incorporates information from both the primary
rs368234815 rs4803221 0.015 functional variant (rs368234815) and a secondary modifying
rs8099917 0.194 functional variant (ie, the activity variant rs117648444) and
rs8113007 0.011 hence could be acting as the best ‘‘tagging-SNP’’ to report
a
Log likelihood ratio test compares model 1 versus model 2 (one on IFN-l4 function.
is nested in the other). Pa and P value <0.05 is significant and is We next sought to examine the nature of linkage of
shown in bold. IFNL SNPs with the functional and activity variant alleles
 8 BHUSHAN ET AL.
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FIG. 3. SNPs rs8099917 and rs4803221 are better predictors of SVR due to their linkage with the protective haplotypes
(of functional and activity variants) TT-G and DG-A. (A) DG-A haplotype shows a protective phenotype in our cohort (n,
SVR = 51; NR = 21) (left). Haplotypes were obtained for each patient after phasing at the 2 positions: rs368234815 and
rs117648444. Haplotypes were grouped as No IFN-l4 (TT-G), IFN-l4 P70 (DG-G), and IFN-l4 S70 (DG-A). The 3
haplotype groups were distributed into SVR and NR groups based on the status of the respective samples/patients they were
derived from and compared. ORs shown in the left panel are from univariate analysis. Logistic regression was also used to
calculate OR and 95% CI (TT-G: OR = 3.68, 95% CI = 1.42–9.48, P = 0.006; DG-A: OR = 9.82, 95% CI = 0.90–107.01,
P = 0.06). The right panel depicts the number of haplotypes in each of the 3 types in SVR and NR groups. (B) Distribution
of haplotypes involving rs368234815 and rs117648444 in linkage between the 2 alleles of rs4803221, rs12979860,
rs8099917, and rs8113007. Haplotypes were obtained for each patient after phasing at all the 6 variant positions. Proportion
of haplotypes involving rs368234815 and rs117648444 (TT-G, DG-A, and DG-G) that are linked with the 2 alleles of the
remaining SNPs are plotted. CI, confidence interval; OR, odds ratio.

in other world populations. For this purpose, we used the
genotype data of all the representative world populations
comprising around 2,500 individuals deposited in the 1000
genomes project database and deduced the haplotypes in-
volving all the SNPs/variants studied in our cohort (Sup-
plementary Table S4).
The results showed that the DG-A haplotype (rs368234815-
rs117648444) was linked to the SNPs rs12979860, rs4803221,
and rs8099917 as a single haplotype in all the populations in
the 1000 genomes database (Supplementary Table S4). Similar
to the linkage in our cohort, both the DG and A alleles (of the
functional and activity variants, respectively) were always
linked to the T and C alleles of rs8099917 and rs4803221,
respectively; and always to the T allele of rs12979860 in all
world populations, including the African population. This
shows that, due to this linkage structure, all world popu-
lations may be affected by the confounding effect of the
activity variant in genetic association studies similar to
the one observed in our study, involving SNPs/variants
rs368234815, rs12979860, rs4803221, and rs8099917.
No effect of rs4803221 variants
on IFNL3 promoter activity
Our results described above allowed us to speculate that
rs4803221 could be the best predictor of therapy response
because of the local LD structure that defines its correla-
tion with the 2 important IFNL4 variants rs368234815 and
rs117648444. However, another possible mechanism by
which IFNL SNPs can affect HCV clearance is by affecting
the expression of IFNL3 (Chinnaswamy and others 2013;
Chinnaswamy 2014; McFarland and others 2014; Lu and
others 2015). Therefore, it is important to examine alternate
mechanisms by which rs4803221 affects HCV clearance,
including that of regulation of IFNL3 promoter activity.
In fact, a previous study (Smith and others 2011), which
identified rs4803221 by deep sequencing-based fine-mapping
analysis of the IFNL locus, found that it was 1 of the 2 SNPs
that had the best predictive value of IFN-RBV therapy response
in an Australian cohort, and further speculated that rs4803221
may be affecting the DNA methylation-based transcriptional
 LINKAGE DISEQUILIBRIUM STRUCTURE AT THE IFNL LOCUS 9
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FIG. 4. SNP rs4803221 does not affect transcription activity of the IFNL3 promoter. (A) Schematic representation of the
4 kb fragment of IFNL3 promoter (upper part). The rectangular box shows the CpG Island (position: chr19q13.2:
39737689–39739288; CpG count-137, UCSC genome browser, https://genome.ucsc.edu). The horizontal arrow denotes
transcription start site. This fragment is cloned into pGL3basic vector (Invitrogen) in front of the firefly luciferase gene for
reporter assay (lower part). (B, C) The wild-type and mutant constructs were methylated by CpG methyltransferase (M.SssI)
(B) and GpC methyltransferase (M.CviPI) (C) enzymes. The constructs were transfected along with a TK promoter-driven
Renilla luciferase construct and genes encoding p65, IRF7, HCV NS5B, and RIG-I in a 96-well plate containing HEK293T
cells. The ratios of firefly to Renilla luciferase values are plotted with error bars showing standard deviation from triplicates.
The data are representative of at least 2 independent experiments. Plus (‘‘+’’) and minus (‘‘-’’) represent the presence and
absence of different components (E, enzyme; S, substrate; TFs, transcription factors). IRF, interferon regulatory factor;
RIG-I, retinoic acid-inducible gene.

regulation of IFNL3 expression. This is because the allelic
variants of rs4803221 disrupt a CpG motif in the CpG island
present upstream of the IFNL3 gene that also overlaps most of
the IFNL4 genes (Chinnaswamy 2016) (Fig. 4A).
To test this possibility, we cloned a 4 kb promoter DNA
region upstream of IFNL3 gene encompassing the CpG island
into a pGL3 basic vector (Invitrogen). DNA sequencing
showed the presence of C allele at rs4803221 in this promoter
clone; we used site-directed mutagenesis to generate the al-
ternate G allele at rs4803221 in the promoter construct. The 2
constructs were subject to in vitro methylation by the enzymes
M.Sss I and M.CviPI. Constructs subject to in vitro methyl-
ation or not were transfected into HEK293T cells as described
before (Chinnaswamy and others 2013) along with genes for
transcription factors (TFs) NF-kB and IRF7 that were also co-
overexpressed along with the HCV polymerase gene and
RIG-I gene to activate the RLR (RIG-I-like receptor) pathway
as described previously (Chinnaswamy and others 2016).
The 4 kb promoter construct showed promoter activity
only when TFs were also co-overexpressed and the RLR
pathway activated (Fig. 4B, C). Methylation of the plasmids
by both enzymes decreased the promoter activity by 3- to
4-folds. However, we did not see any major difference in
the luciferase activity arising from either of the alleles of
rs4803221 either before or after methylation by either en-
zyme. We do note a minor increase in the activity from the
construct that carried the minor G allele at rs4803221
compared to the one with the major C allele, after methyl-
ation by both enzymes (Fig. 4B, C); however, this difference
was within the margin of error. These results argue against a
major effect of rs4803221 alone on IFNL3 expression and
the possibility that the SNP singularly affects HCV clear-
ance by this mechanism is therefore less likely.
Discussion
A true causal SNP or its closely correlated SNP(s) will have
the strongest association with the phenotype in genetic asso-
ciation studies (O’Brien and others 2015). In this study, we
show that the activity variant rs117648444 has a confounding
effect on the association of IFNL locus SNPs/variants with
response to IFN-RBV therapy in chronic HCV infections. The
activity variant rs117648444 is a nonsynonymous variant
whose minor allele has been shown in vitro to decrease the
activity of IFN-l4 protein (Terczyńska-Dyla and others 2014)
thereby having a direct effect on its function. Two previous
studies have also shown the influence of rs117648444 on
genetic association of the functional variant rs368234815
 10 BHUSHAN ET AL.

(Galmozzi and Aghemo 2014; Terczyńska-Dyla and others
2014) with response to therapy in HCV infections, elucidating
the protective role of the low-activity IFN-l4 protein com-
pared to the high-activity variant.
Our study explains how the activity variant modulates the
association test results involving certain important IFNL
SNPs/variants due to the confounding effect and how the
nature of confounding is dependent on whether the minor A
allele of the activity variant is linked to the major or minor
alleles of IFNL SNPs (Fig. 3B). Even though the restricted
models were artificial and involved ‘‘neglecting’’ some sam-
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founding effect of the activity variant (Fig. 5A). Since the
minor A allele of the activity variant is always linked to the
DG risk allele of the functional variant (Fig. 3B and Supple-
mentary Table S4), the latter is always subject to the negative
confounding effect of the former; as a consequence, any SNP/
variant that has the minor A allele of the activity variant linked
to its protective allele will always be subject to a positive
confounding effect (eg, rs4803221, rs8099917) (Fig. 5A).
The most widely genotyped SNPs at the IFNL locus both
in HCV studies and others are rs12979860 and rs8099917
(Chinnaswamy 2016); since our results show the linkage of

ples from analysis, our logistic regression analysis gave results
that were consistent with our models (Table 2).
We further show that the linkage structure involving some
of the most important IFNL SNPs/variants (Chinnaswamy
2014, 2016) is highly conserved across several human
populations (Supplementary Table S4), and therefore, our
findings should be relevant in all other populations, in-
cluding the African populations or those admixed with it.
However, the extent of confounding would vary based on
(1) the relative protective effect of DG-A haplotype com-
pared to the TT-G haplotype (the higher the effect of DG-A
haplotype, the larger will be the confounding effect) and (2)
the LD (r2) values of the different IFNL SNPs with the
functional variant rs368234815.
A strong LD of any given IFNL SNP with the functional
variant also makes it more susceptible to the negative con-
the minor A allele of the activity variant being conserved
across all human populations (Supplementary Table S4),
these 2 ‘‘popular’’ SNPs should be corrected for the con-
founding effect of the activity variant in future studies to
investigate causal mechanism (s) behind IFNL SNP asso-
ciation with human diseases.
In our study, we saw that 7 out of 8 patients carrying the
DG-A haplotype attained SVR, suggesting a predominant
effect of the IFN-l4S70 variant in our cohort on viral
clearance. However, whether this can be replicated in larger
cohorts of genotype 3 HCV patients remains to be tested.
The small sample size is indeed a major limitation of our
study, and therefore, the results from this study pertaining to
the predictive abilities of various SNPs/variants and also the
large observed effect of the activity variant have to be
cautiously interpreted. However, our results offer an

FIG. 5. Activity SNP rs117648444 acts as a negative or positive confounder in association of IFNL SNPs/variants with
human diseases dictated by an underlying linkage structure. (A) Activity variant minor A allele when linked to risk allele of an
IFNL SNP (eg, rs12979860) exerts a negative confounding effect on that SNP; the same when linked to the protective allele of
an IFNL SNP (eg, rs8099917) exerts a positive confounding effect of that SNP in genetic association studies. Arrows indicate
haplotypes formed between the alleles of the activity and functional variants, which in turn are linked to any other IFNL SNP.
Dotted line denotes moderate to high LD between an IFNL SNP with the functional variant rs368234815. P, protective; R, risk;
P2P, extent of positive confounding; P2N, extent of negative confounding. (B) Confounding by the activity variant likely arises
due to early evolutionary events that shaped the nucleotide changes at the 2 key variant positions. Both the changes (at
functional and activity variant positions) seem to have happened on distinct allelic backgrounds of IFNL SNPs/variants.
Concordance denotes concurrence of the given IFNL SNP with the phenotype derived from the haplotype involving the
functional and activity variants. For example, ‘‘C’’ allele at rs4803221 is predicted to have a protective phenotype (pred.),
while the ‘‘G’’ allele is predicted to show a risk phenotype due to the nature of LD with the functional variant rs368234815 (in
dominant models of inheritance). These predicted phenotypes of rs4803221 perfectly concur with the predicted phenotypes of
3 haplotypes TT-G, DG-G, and DG-A (involving the functional and activity variants) linked to alternate alleles, and therefore,
score 3 out of 3 on the concordance score. The numbers in parenthesis show the MAFs of the 3 variants rs4803221 (0.18),
rs12979860/rs368234815 (0.25), and rs117648444 (0.05) in our HCV patient cohort.
 LINKAGE DISEQUILIBRIUM STRUCTURE AT THE IFNL LOCUS
explanation of why certain SNPs such as rs8099917 and
rs4803221, which are not in strong LD with the functional
variant unlike the tag SNP rs12979860, show stronger as-
sociation with the phenotype in several previous studies
involving different populations (Doehring and others 2010;
Lindh and others 2011; Smith and others 2011; Suppiah and
others 2011; Antaki and others 2013; Khudayberganova and
others 2014; O’Connor and others 2016). It would also mean
that the best predictive SNP would not be the same in dif-
ferent populations but would vary according to the local LD
structure at the IFNL locus.
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The DG allele is thought to be the ancient allele and the TT
allele was acquired at a later stage in human evolution (Key
and others 2014). Such information is not available for the
activity variant. We can speculate that the events leading to
both these changes affecting the expression and activity of the
IFN-l4 protein seem to have occurred on the protective allele
background of some SNPs such as rs8099917 and rs4803221
(Fig. 5B). For other SNPs such as rs12979860 (and others
in high LD with it), events leading to the generation of the
activity variant seem to have occurred on the risk allele
background; however, since this change leads to a protective
phenotype, it becomes the source of confounding that we
observed in our study (Fig. 5B). Population genetics studies
will reveal on how these events have led to the current
structure of the IFNL locus in different world populations.
In our cohort, rs4803221 even though was the best predictor
SNP for SVR based on results from some tests, other statistical
tests did not agree that it had an effect independent of both the
functional and activity variants (Tables 2 and 4). However, the
low sample size of our study may have not had the power to
unravel any independent effect of the SNP rs4803221 in at-
taining SVR. Therefore, to examine an alternate mechanism,
we carried out reporter assays to examine if rs4803221 regu-
lates IFNL3 promoter activity as speculated by Smith and
others (2011). Our reporter assays do not show evidence for
rs4803221 to modulate IFNL3 promoter activity either before
or after in vitro methylation of promoter constructs.
However, we have not tested if rs4803221 in conjunction
with other SNPs (such as rs12979860) present on the CpG
island could affect promoter activity before or after meth-
ylation of the CpG island. Since there is evidence from a
recent article (Waring and others 2016) that the minor allele
of rs12979860 associates in a dominant model of inheritance
with the methylation levels of the CpG island, such studies
need to be carried out to test if alternate mechanisms are
also behind the association of IFNL locus SNPs with HCV
infections and other human diseases.
In summary, we show that a confounding effect is manifest
due to the activity variant rs117648444 in our association
study between IFNL SNPs/variants and viral clearance in-
volving genotype 3 HCV-infected patients. The confounding
arises due to the linkage of the activity variant with different
IFNL SNPs and likely due to its direct effect on the phenotype
as a result of modulation of the activity of the IFN-l4 protein.
The nature and level of confounding vary with the level of LD
that the activity variant (or its haplotype with the functional
variant) has with the particular IFNL SNP and depend on
whether the minor allele A of the activity variant is linked to
the major or minor alleles of the concerned SNP(s). Under-
standing the confounding effect is critical to identify the best
predictive SNP in a population and to adjust for its effect in
association studies, especially since IFNL locus SNPs have
11
now been associated with several other human diseases and/or
their clinical characteristics (Gaudieri and others 2012; An-
gulo and others 2015; Eslam and others 2015; Arpaci and
others 2016; Corrales and others 2016; Dominguez-Molina
and others 2016; Morán-Auth and others 2016; Price and
others 2016). Our results should be useful in designing and
interpreting future case–control studies with IFNL SNPs and
different human diseases.
Acknowledgments
The funding for this work was provided by the Department
of Biotechnology, Government of India, as grant No. BT/
PR7035/MED/12/579/2012 to S.C. We thank Prof. Partha
Majumder, Founder Director, NIBMG (Kalyani, India) for
his support and encouragement for this work. We thank the
following clinicians who provided us the patients for the
study: Drs. Kaushik Das (IPGME&R, Kolkata, India);
Shalimar (AIIMS, New Delhi, India); Ajay Duseja (PGI,
Chandigarh, India); Deepak Amarapurkar (Bombay Hos-
pital, Mumbai, India); Rosangluaia (Civil Hospital, Ai-
zawl, India); Sujit Chowdhury (AMRI Hospital, Kolkata,
India); Ashokanand Konar (Peerless Hospital, Kolkata,
India). Our special thanks to Prof. Abhijit Chowdhury
(SDLD, IPGME&R, Kolkata, India) for coordinating with
all clinicians for this study. We thank Dr. Analabha Basu,
NIBMG, for helpful discussions. We thank Dr. Priyodarshi
Basu and Ankita Chatterjee for some of the control sam-
ples and Dr. Parthasarathi Bhattacharyya, IPCR (Kolkata,
India), for the asthma control samples. Finally, we thank
all the study participants who consented for the study.
Author Disclosure Statement
No competing financial interests exist.
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 14 BHUSHAN ET AL.

Waring JF, Davis JW, Dumas E, Cohen D, Idler K, Abel S, Address correspondence to:
Georgantas R, Podsadecki T, Dutta S. 2017. Epigenetic Dr. Sreedhar Chinnaswamy
analysis of the IFNl3 gene identifies a novel marker for re- National Institute of Biomedical Genomics
sponse to therapy in HCV-infected subjects. J Viral Hepat P.O.:N.S.S.
24(5):397–403. Kalyani
Zeuzem S, Soriano V, Asselah T, Bronowicki JP, Lohse AW, West Bengal 741251
Mullihaupt B, Schuchmann M, Bourliere M, Buti M, Ro- India
berts SK, Gane EJ, Stern JO, Vinisko R, Kukoji G, Gal-
livan JP, Bocher W, Mensa FJ. 2013. Faldaprevir and E-mail: sc2@nibmg.ac.in
deleobuvir for HCV genotype 1 infection. N Engl J Med 369:
630–639. Received 11 January 2017/Accepted 7 June 2017
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