Variants of the Inosine Triphosphate Pyrophosphatase
Gene Are Associated With Reduced Relapse Risk
Following Treatment for HCV Genotype 2/3
Karolina Rembeck,1 Jesper Waldenstr€om,1 Kristoffer Hellstrand,1 Staffan Nilsson,2 Kristina Nystr€om,1
Anna Martner,1 Magnus Lindh,1 Gunnar Norkrans,1 Johan Westin,1 Court Pedersen,3 Martti F€arkkil€a,4
Nina Langeland,5 Mads Rauning Buhl,6 Kristine Mïrch,5 Peer Brehm Christensen,3 and Martin Lagging1
The present study evaluated the impact of variations in the inosine triphosphate pyrophosphatase (ITPase) gene (ITPA) on treatment outcome in patients with hepatitis C
virus (HCV) genotype 2/3 infection receiving peginterferon-a2a and lower, conventional
800 mg daily dose of ribavirin. Previous studies using higher, weight-based ribavirin dosing report that patients carrying polymorphisms encoding reduced predicted ITPase activity show decreased risk of ribavirin-induced anemia but increased risk of
thrombocytopenia, with no impact on elimination of virus. In all, 354 treatment-na€ıve
HCV genotype 2/3-infected patients, enrolled in a phase III trial (NORDynamIC), were
genotyped for ITPA (rs1127354 and rs7270101). Homo- or heterozygosity at Ars1127354
or Crs7270101, entailing reduced ITPase activity, was observed in 37% of patients and was
associated with increased likelihood of achieving sustained virological response (SVR)
(P 5 0.0003 in univariate and P 5 0.0002 in multivariate analyses) accompanied by a
reduced risk of relapse among treatment-adherent patients. The association between ITPA
variants and SVR remained significant when patients were subdivided by the 12- and 24week treatment duration arms, HCV genotype, fibrosis stage, and IL28B genotype, and
was not secondary to improved adherence to therapy or less pronounced anemia. Gene
variants predicting reduced predicted ITPase activity were also associated with decreased
risk of anemia (P < 0.0001), increased risk of thrombocytopenia (P 5 0.007), and lower
ribavirin concentrations (P 5 0.02). Conclusion: These findings demonstrate a novel
ribavirin-like association between polymorphisms at ITPA and treatment efficacy in
chronic hepatitis C mediated by reduced relapse risk. We hypothesize that patients (63%)
being homozygous for both major alleles, leading to normal ITPase activity, may benefit
more from the addition of ribavirin to present and future treatment regimens for HCV in
spite of concomitant increased risk of anemia. (HEPATOLOGY 2014;59:2131-2139)
R
ecently, a genome-wide association study demonstrated that two genetic variants, a missense
variant in exon 2 (rs1127354, P32T) and a
splicing-altering single nucleotide polymorphism
(SNP) in intron 2 (rs7270101, IVS2) of the inosine
triphosphate pyrophosphatase (ITPase) gene (ITPA)
protect against ribavirin-induced hemolytic anemia
during therapy for hepatitis C virus (HCV) genotype
1 infection,1 and this holds true also in the setting of
telaprevir-based triple therapy.2 Prior studies that have
biochemically quantified the impact of these ITPA
haplotypes show that hetero- and homozygosity are
Abbreviations: ATP, adenosine triphosphate; CRF, case report form; HCV, hepatitis C virus; IMP, inosine monophosphate; ITPase, inosine triphosphate pyrophosphatase; Peg-IFN, pegylated interferon-alpha; PP, per-protocol; SNP, single nucleotide polymorphism; SVR, sustained virological response; XTP, xanthosine
triphosphate.
From the 1Department of Infectious Diseases/Virology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; 2Department of Mathematical Sciences, Chalmers University of Technology, Gothenburg, Sweden; 3Department of Infectious Diseases, University of Southern Denmark,
Odense, Denmark; 4Department of Gastroenterology, Helsinki University, Helsinki, Finland; 5Department of Medicine, Haukeland University Hospital and Institute of Medicine, University of Bergen, Bergen, Norway; 6Department of Infectious Diseases, Aarhus University, Aarhus, Denmark.
Received October 7, 2013; accepted January 9, 2014.
The Swedish Medical Research Council and ALF Funds at Sahlgrenska University Hospital supported this study. Unrestricted grants from Roche affiliates in the
Nordic region also supported this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article.
2131
2132
REMBECK ET AL.
Fig. 1. ITP to GTP biochemical pathway.
associated with reduced inosine triphosphatase (ITPase)
activity.3-5
The evolutionarily conserved ITPase is a cytosolic
enzyme that recycles purines by the pyrophosphohydrolysis of ITP to inosine monophosphate (IMP), and
thus protects against accumulation of noncanonical
nucleotides such as ITP and xanthosine triphosphate
(XTP) as well as their deoxy forms (dITP and dXTP),
which otherwise incorrectly may be incorporated into
RNA and DNA (Fig. 1) producing genetic instability,
anomalous proteins, and altered adenosine triphosphate (ATP)-dependent signaling.3-6 The pathophysiological consequences of ITPase deficiency are largely
unknown, but it has been associated with increased
toxicity in patients receiving purine analog drugs such
as 6-mercaptopurine and azathioprine.7,8 Studies of
human cell lines have ascribed ITPase a pivotal role in
protection against DNA damage,9 and in genetic
knockout models the absence of ITPase generates
growth retardation with cardiac myofiber disarray and
death intrauterine or within 2 weeks of birth.10 The
protection against ribavirin-induced anemia in patients
with reduced predicted ITPase activity is suggested to
occur by means of avoidance of ATP reduction with
ensuing diminution of oxidative stress and hemolysis.11
Although protective against anemia, ITPase deficiency
is reported to induce a greater decline in platelet count
HEPATOLOGY, June 2014
during interferon/ribavirin therapy for HCV genotype
1 infection.12 It has been suggested that patients with
normal ITPase activity, being more likely to experience
ribavirin-induced anemia, also are more prone to
develop a reactive thrombocytosis12 secondary to
increased stimulation of megakaryocyte-erythroid progenitor cells mediated by augmented erythropoietin
production.13
In the setting of HCV 2/3 infection treated with
1,000/1,200 mg/day14 or 800-1400 mg/day weightbased dosing of ribavirin,15 rather than the conventional 800 mg daily dosing,16 ITPA variants were also
reported to convey protection against anemia but did
not impact early reduction of HCV or the likelihood
of achieving a sustained virological response (SVR).
The aim of the present study was to retrospectively
evaluate the impact of ITPA genotypes on baseline
characteristics, hemoglobin levels, platelet count, ribavirin
concentrations,
on-treatment
virological
responses, and SVR in a phase III treatment trial
(NORDynamIC; n 5 382) in a cohort of northern
European treatment-na€ıve patients with chronic HCV
genotype 2 or 3 infection receiving peginterferon-a2a
180 lg once weekly and the standard, lower ribavirin
dose of 800 mg daily.16
Materials and Methods
Patients. In all, 382 treatment-na€ıve HCV genotype 2/3 infected patients were included in a phase III,
open label, randomized, multicenter, investigatorinitiated trial (NORDynamIC) conducted at 31 centers in Denmark, Finland, Norway, and Sweden.17 All
patients were adults with compensated liver disease
and had detectable HCV RNA. At study entry,
patients were randomized to either 12 or 24 weeks of
treatment with 180 lg of peg-interferon (Peg-IFN) a2a once weekly and 800 mg/day ribavirin. Samples
from 354 Caucasian patients were available for
rs1127354 and rs7270101 genotyping. In order to
minimize the impact of adherence to therapy on outcome, patients constituting the per-protocol (PP) population, i.e., having received at least 80% of the target
dose of Peg-INF as well as at least 80% of the target
dose of ribavirin for at least 80% of the target
Address reprint requests to: Martin Lagging, M.D., Ph.D., Dept. of Infectious Diseases/Virology, Guldhedsgatan 10B, SE-413 46 G€
oteborg, Sweden.
E-mail: [email protected]; fax: 146-31-41 12 56.
C 2014 by the American Association for the Study of Liver Diseases.
Copyright V
View this article online at wileyonlinelibrary.com.
DOI 10.1002/hep.27009
Potential conflict of interest: Dr. Lagging is on the speakers’ bureau for MSD, Roche, and Janssen/Medivir.
HEPATOLOGY, Vol. 59, No. 6, 2014
REMBECK ET AL.
Table 1. Predicted ITPase Activity According to Compound
Genotype of rs1127354 and rs7270101
rs1127354
Wild type (CC)
Wild type (CC)
Wild type (CC)
Heterozygote (CA)
Heterozygote (CA)
Homozygote (AA)
rs7270101
Predicted ITPase
Activity (%)
Wild type (AA)
Heterozygote (AC)
Homozygote (CC)
Wild type (AA)
Heterozygote (AC)
Wild type (AA)
100
60
30
25
10
<5
Distribution in the
study population
% (n)
63
21
2
12
1.7
0.3
(219)
(73)
(7)
(43)
(6)
(1)
treatment duration (n 5 285), were evaluated. Adherence was monitored both by reporting of dose reductions and missed doses in the case report forms (CRF)
and in the patient diaries in which patients recorded
each dosing.
ITPA Genotyping Methods. SNPs were determined in plasma by allelic discrimination using TaqMan SNP Assays (Life Technologies): Assay ID
C_29168507_10 for rs7270101, and C_27465000_10
for rs1127354. Both SNPs were in Hardy-Weinberg
equilibrium.
Classification of Predicted ITPase Activity. This
was achieved based on the compound genotype of
rs1127354 and rs7270101 as previously determined by
biochemical analyses3-5 (detailed in Table 1), i.e.,
100% (CCrs1127354 AArs7270101), 60% (CCrs1127354
ACrs7270101), 30% (CCrs1127354 CCrs7270101), 25%
(CArs1127354
AArs7270101),
10%
(CArs1127354
ACrs7270101), and <5% (AArs1127354 AArs7270101).
HCV RNA Quantification. Plasma HCV RNA
was determined using Cobas AmpliPrep/COBAS TaqMan HCV Test (Roche Diagnostics, Branchburg, NJ),
which quantifies HCV RNA with a limit of detection
of 15 IU/mL. Quantification was performed on days
0, 3, 7, 8, 29, week 8, week 12, week 24, and 24
weeks after completion of therapy. All samples were
frozen (270 C) and centrally analyzed. Patients were
classified as achieving SVR if plasma HCV RNA was
undetectable 24 weeks after completion of therapy.
Liver Biopsies. Liver biopsies were obtained from
all patients within 24 months prior to study entry.
The evaluation was performed in a blinded fashion
according to the Ishak protocol.18 Additionally, steatosis was graded.19
Ribavirin Concentration. Plasma trough concentrations of ribavirin were evaluated at day 29 and at
week 12 by high-performance liquid chromatography
(HPLC; Merck-Hitachi, Tokyo, Japan) followed by
UV-detection (wavelength 215 nm).
Interferon a-2a Concentration. Plasma concentration of interferon-a2a was measured at days 3, 7, and
2133
29 using high absorbent enzyme-linked immunosorbent
assay (ELISA) plates (AMS Biotechnology, Oxon, UK).
Homeostatic Model Assessment-Insulin Resistance
(HOMA-IR). Baseline fasting glucose (mmol/L) was
measured locally, whereas fasting serum insulin (mU/
L, Architect Insulin, Abbott, Abbott Park, IL) was analyzed centrally. HOMA-IR was calculated: (Glucose 3
Insulin) / 22.5.20
Statistical Methods. Spearman’s rank-order correlation test was used to analyze univariate relationships with
ITPase activity. Logistic regression was performed on the
PP patients to evaluate the relationship between SVR
and ITPase activity (considered as a numerical value)
both with and without baseline covariates: age, bodymass index (BMI), liver fibrosis stage, HCV RNA level,
IP-10 level, and IL28B genotype, as well as change in
hemoglobin on days 0-29, ribavirin concentrations day
29, and treatment duration. All statistical analyses were
performed by SN using IBM SPSS statistics v. 19.0 software package (Somers, NY). All reported P-values are
two-sided, and P < 0.05 was considered significant.
Ethical Considerations. Written informed consent
was obtained from each participating patient and the
Ethics Committees in each participating country
approved the study. The study has been registered at
the NIH trial registry (ClinicalTrials.gov Identifier:
NCT00143000).
Results
The minor Ars1127354 allele was present in 14% of
patients, and the minor Crs7270101 allele in 24%, combinations of which predict varying degrees of reduced
ITPase activity3-5 (Table 1). The majority of patients
(63%) were homozygous for both major alleles, entailing normal ITPase activity. Among the 13 baseline
characteristics evaluated (Table 2), reduced predicted
ITPase activity was weakly albeit significantly associated with HOMA-IR (P 5 0.048); however, this association should be interpreted with caution, as it may
be an effect of multiple testing. In contrast, ITPase
activity was not significantly associated with
interleukin-28B (IL28B, also known as interferon-k3)
genotype or baseline plasma levels of interferon
gamma-induced protein 10 (IP-10 or CXCL10).
Carriage of a genotype predicting reduced ITPase
activity was significantly associated with a less pronounced decline in hemoglobin and a greater reduction in platelet count during the first 4 weeks of
therapy (Fig. 2). The association with protection
against anemia was significant for both HCV genotypes 2 and 3 (P < 0.0001 for both genotypes).
2134
REMBECK ET AL.
HEPATOLOGY, June 2014
Table 2. Baseline Characteristics With Patients Grouped According to Predicted ITPase Activity Based
on Compound Genotype
Predicted ITPase Activity (% of normal) Based on Compound Genotype
Age (years)‡
Gender (Male/Female)†
BMI (kg/m2)‡
HCV-RNA level (log10 IU/mL)‡
HCV Genotype (2/3)†
IP-10 (pg/mL)*
Hemoglobin (g/L)‡
Platelet count (x 109)‡
IL28B (CC/CT/TT)†
Fibrosis Stage (Ishak 0/1/2/3/4/5/6)†
Steatosis Grade (0/1/2/3)†
Fasting glucose (mmol/L)‡
HOMA-IR*
£ 10% (n 5 7)
25-30% (n 5 50)
60% (n 5 73)
100% (n 5 219)
P
41 (13)
4/3
24.0 (3.0)
6.5 (0.3)
2/5
113 (65-290)
147 (13)
239 (69)
4/2/1
0/2/0/2/2/1/0
3/1/1/2
5.2 (0.4)
2.8 (1.2-9.2)
42 (10)
30/20
26.8 (4.4)
6.1 (0.8)
10/40
277 (119-377)
151 (13)
222 (68)
20/20/6
4/5/14/12/4/5/4
11/22/10/5
5.6 (1.9)
3.6 (1.9-7.3)
43 (12)
40/33
25.3 (4.1)
6.0 (0.8)
25/48
179 (119-297)
148 (14)
221 (64)
38/26/8
0/7/24/19/11/3/7
25/27/12/7
5.2 (2.1)
2.3 (1.5-5.0)
42 (11)
133/86
25.7 (4.4)
6.0 (0.9)
62/157
196 (107-349)
149 (13)
222 (65)
97/102/19
7/30/59/52/26/10/3
69/76/32/20
5.0 (0.9)
2.6 (1.3-5.7)
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
0.048
P values obtained using Spearman’s rank correlation test. Data presented as median (IQR),* n,† or mean (SD).‡
The mean ribavirin concentrations at day 29 were
lower for patients with a genotype predicting reduced
ITPase activity (mean ribavirin concentration 1.4 versus 1.6 lg/mL for 30% and 60%, respectively;
Fig. 2C), although by treatment week 12 no such difference was observed. No significant association was
noted between predicted ITPase activity and dose
reductions of ribavirin (percent of patients taking the
planned ribavirin dose was 100%, 95%, 95%, 94%
for ITPase activity of 10%, 30%, 60%, and 100%,
respectively). Thus, there were few dose reductions of
ribavirin in this study, with only 10% of patients taking less than 80% of the planned ribavirin dose, likely
secondary to the use of the lower conventional 800
mg/day rather than the higher weight-based dosing.
No significant association was noted between ITPase
activity and on-treatment interferon concentrations or
dose reductions of Peg-IFN. The main reason for premature termination of therapy in this study was
interferon-induced neuropsychiatric side effects, as previously reported.21 Also, there were no associations
between ITPase activity and inclusion in or exclusion
from the PP analysis.
The level of predicted ITPase activity was not associated with improved on-treatment responses such as
decline in HCV RNA level on days 0-3, HCV RNA
level day 7, decline in HCV RNA level days 7-29,
likelihood of achieving rapid virological response
(RVR), i.e. undetectable HCV RNA week 4; (43%,
65%, 61%, and 63% for ITPase activity 10%, 30%,
Fig. 2. Impact of ITPase activity on change in hemoglobin (A) and platelet count days 0-29 (B), and on ribavirin concentration day 29 (C). Boxplots displaying the 10th, 25th, 50th, 75th, and 90th percentiles among patients grouped according to predicted ITPase activity based on compound ITPA genotype, i.e., 10% (CArs1127354 ACrs7270101 or AArs1127354 AArs7270101), 25-30% (CCrs1127354 CCrs7270101 or CArs1127354 AArs7270101),
60% (CCrs1127354 ACrs7270101), and 100% (CCrs1127354 AArs7270101). P-values obtained for Spearman’s rank correlation coefficient (rs).
HEPATOLOGY, Vol. 59, No. 6, 2014
REMBECK ET AL.
2135
Fig. 3. Proportion of patients (%) achieving treatment responses grouped according to predicted ITPase activity based on compound ITPA
genotype and treatment duration 12 (A) or 24 weeks (B).
60%, and 100%, respectively), or HCV RNA level at
end-of treatment (EOT) (Fig. 3). It should be noted,
however, that an HCV genotype 2/3-infected population might be suboptimal for evaluating impact on the
second phase decline in HCV RNA, as the early
reduction in virus levels in this population is rapid,
compared to HCV genotype 1-infected patients when
treated without direct antiviral agents (DAAs), as illustrated by the finding that 30% of patients had
achieved HCV RNA <1000 IU/mL already by day 7.
In contrast to the lack of association with ontreatment responses, genotypes predicting reduced
ITPase activity were significantly associated with higher
likelihood of achieving SVR for adherent patients, i.e.,
the PP population, when analyzed together (odds
ration [OR] 5 6.4 for completely reduced activity, P
5 0.0003 in univariate analysis; Table 3), as well as
when subdivided by the 12- and 24-week treatment
duration arms, HCV genotype, fibrosis stage, and
IL28B variants. Despite seemingly large deviations, the
ORs in the different subgroups of reduced ITPase
activity did not differ significantly. The increased likelihood of SVR was explained by lower relapse rates for
patients with reduced predicted ITPase activity (relapse
rate 20%, 21%, 19%, and 39% for ITPase activity
10%, 25-30%, 60%, and 100%, respectively, following 12 weeks of treatment duration, and 0%, 0%,
13%, and 14% for corresponding ITPase activities
among patients treated for 24 weeks). As these patients
had received at least 80% of the target dose of
Table 3. Proportion of Patients Achieving SVR (Per-Protocol Analysis)
Predicted ITPase Activity (% of normal)
£ 10%
All Patients
12-week Treatment Duration
24-week Treatment Duration
HCV genotype 2*
HCV genotype 3*
Non-significant Fibrosis (Ishak 0-2)
Bridging Fibrosis (Ishak 3-4)
Cirrhosis (Ishak 5-6)
IL28B CC
IL28B CT
IL28B TT
6/7
4/5
2/2
2/2
4/5
1/2
4/4
1/1
4/4
1/2
1/1
(86%)
(80%)
(100%)
(100%)
(80%)
(50%)
(100%)
(100%)
(100%)
(50%)
(100%)
25-30%
35/39
15/19
20/20
8/9
27/30
18/19
10/12
5/6
14/17
14/14
4/5
(90%)
(79%)
(100%)
(89%)
(90%)
(95%)
(83%)
(83%)
(82%)
(100%)
(80%)
60%
50/61
23/30
27/31
19/20
31/41
25/27
18/23
5/9
27/32
17/21
5/7
(82%)
(77%)
(87%)
(95%)
(76%)
(93%)
(78%)
(56%)
(84%)
(81%)
(71%)
100%
115/173
57/102
58/71
29/51
84/120
59/77
39/64
5/17
48/73
58/84
9/16
(66%)
(56%)
(82%)
(57%)
(70%)
(77%)
(61%)
(29%)
(66%)
(69%)
(56%)
*Two patients were infected with both genotype 2 and 3 and were excluded from the genotype subanalysis.
Odds ratio (0% vs. 100% ITPase activity; confidence intervals in parentheses) and P values obtained using logistic regression.
Odds Ratio
6.4
4.9
11.9
71.0
3.5
5.3
7.5
27.4
6.3
6.6
5.9
(2.1-19.6)
(1.4-17.6)
(1.03-138)
(3.5-1454)
(1.04-11.7)
(0.76-37.0)
(1.3-43.7)
(1.8-420)
(1.2-31.8)
(1.01-44.8)
(0.35-99)
P
0.0003
0.01
0.02
0.0004
0.03
0.07
0.01
0.009
0.02
0.03
0.19
2136
REMBECK ET AL.
Fig. 4. Change in hemoglobin days 0-29 among patients achieving
or not achieving SVR. Boxplots displaying the 10th, 25th, 50th, 75th,
and 90th percentiles. P-value obtained for logistic regression.
Peg-IFN as well as at least 80% of the target dose of
ribavirin for at least 80% of the target treatment duration, improved adherence was deemed unlikely to
account for the improved rate of SVR. Additionally, in
a logistic regression analysis evaluating the impact on
SVR of ITPase activity and percent of planned ribavirin dosing actually taken, ITPase activity remained
significant (P 5 0.001), whereas percent of planned
ribavirin dosing actually taken was not (P 5 0.95).
And when restricted to patients having taken 100% of
planned ribavirin dosing, ITPase activity retained a significant impact on SVR (P 5 0.001). Similarly, protection against anemia could not explain the improved
SVR among patients with reduced ITPase activity, as
there were no significant associations between change
in blood hemoglobin from baseline to day 29, hemoglobin level day 29 or hemoglobin below 100 g/L day
29 and SVR. However, a nonsignificant trend towards
lower decline in hemoglobin on day 0-29 was noted
among patients achieving SVR (median 215 versus
219 g/L for patients achieving and not achieving
SVR, respectively, P 5 0.12; Fig. 4). It should be
noted that only 1 of 265 patients had a hemoglobin
below 100 g/L day 29, likely secondary to the use of
standard, lower 800 mg dosing of ribavirin. In a logistic regression analysis evaluating the impact of both
ITPase activity and change in hemoglobin on days 029 on SVR, ITPA remained significant (P 5 0.003),
whereas change in hemoglobin on days 0-29 did not
(P 5 0.95). A threshold level regarding ITPase activity
could not be identified, as SVR continuously improved
HEPATOLOGY, June 2014
as ITPase activity decreased (OR 2.3 for 100% versus
60% and OR 4.4 100% versus 30%).
Ribavirin levels on day 29 were significantly associated with SVR (mean concentration 131 versus 102
lg/mL for SVR and non-SVR, respectively; P 5
0.007). In a logistic regression analysis evaluating the
impact of SVR, both ITPase activity and ribavirin concentrations at day 29 remained significant (P <
0.0001 for both). Similarly, baseline ribavirin dose
adjusted for body weight was significantly associated
with SVR (mean dose 139 versus 115 mg/kg for SVR
and non-SVR, respectively; P 5 0.03). In a logistic
regression analysis evaluating the impact of SVR, both
ITPase activity (P 5 0.001) and weight-adjusted ribavirin dose (P 5 0.002) remained significant.
ITPase was independently predictive of the likelihood of achieving SVR in multivariate analysis
(adjusted OR 5 11 for completely reduced activity, P
5 0.0002; Table 4). Other significantly predictive
baseline factors were liver fibrosis stage (OR 0.6),
unfavorable IL28B variants (OR 0.46), and baseline
HCV RNA level (OR 0.26), in addition to treatment
duration, i.e., 12 or 24 weeks (OR 5.0) and ribavirin
concentrations day 29 (OR 5 2.3).
When an intention-to-treat rather than a PP analysis
was performed, ITPase activity continued to have a
significant impact on SVR (P 5 0.031 univariate and
P 5 0.007 multivariate analysis; n 5 354), although
less so than in the PP analysis, as did all other
response predictors, which is likely secondary to a
dilution effect caused by the addition of nonadherent
patients who did not achieve SVR due to premature
treatment termination primarily secondary to neuropsychiatric side effects of interferon therapy.21 It is
worth noting that to avoid a similar effect of inadequate drug exposure, the genome-wide association
study by Ge et al.22 that identified the impact of
IL28B genotype on HCV genotype 1 therapy was
restricted to patients with compliance of greater than
80% for both Peg-IFN and ribavirin and a minimum
of 12 weeks of therapy.
Discussion
The results of this study suggest an unexpected association between polymorphisms predicting reduced
ITPase activity and improved treatment efficacy in
chronic HCV infection, which was explained by a
reduced incidence of relapse. This association remained
significant in multivariate analysis, and when patients
were subdivided by the 12- and 24-week treatment
duration arms or by HCV genotype, fibrosis stage,
HEPATOLOGY, Vol. 59, No. 6, 2014
REMBECK ET AL.
2137
Table 4. Impact on SVR in Univariate and Multivariate Analysis
Univariate Analysis
Odds Ratio
Predicted ITPase Activity
Treatment Duration (12 or 24 Weeks)
Ribavirin Concentration day 29 (lg/mL)
Fibrosis Stage (Ishak 0/1/2/3/4/5/6)
IL28B (CC/CT/TT)
Baseline HCV-RNA level (log10 IU/mL)
BMI (kg/m2)
Change in hemoglobin day 0-29 (g/L)
6.4
3.4
2.6
0.68
0.83
0.35
0.90
1.02
(2.1-19.6)
(1.9-6.0)
(1.4-4.7)
(0.56-0.82)
(0.56-1.8)
(0.23-0.55)
(0.85-0.96)
(0.99-1.04)
and IL28B variants. Importantly, the higher rate of
SVR among patients with genotypically lower ITPase
activity was not secondary to increased treatment
adherence or protection against anemia, as there were
no significant associations between ITPase activity and
protocol adherence. The view that the improved rate
of SVR was unrelated to protection against anemia
was further supported by the lack of significant association between change in hemoglobin during therapy
and SVR, and by previous reports, using higher
weight-based ribavirin dosing, demonstrating that anemia during peg-IFN and ribavirin therapy for HCV
infection heralds better rather than worse outcome.23
Also, the improved outcome was not explained by ontreatment viral response, in contrast to favorable
IL28B variants24 or lower baseline plasma IP-10,25
which both primarily improve the first phase decline
in HCV RNA but only weakly impacted SVR among
the HCV genotype 2/3-infected patients in this
study.24,25 Additionally, the reduced relapse risk
occurred in spite of a significant association between
genetic variants predicting reduced ITPase activity and
lower plasma concentrations of ribavirin during
treatment.
To our knowledge, this is the first report of ITPA
polymorphisms encoding reduced ITPase activity
improving treatment efficacy in HCV infection without
linkage to improved ribavirin adherence. It was previously noted that among a subset of 81 HCV genotype
1b-infected Japanese patients with the favorable IL28B
rs8099917 TT genotype treated with 600-1,000 mg
daily weight-based dosing of ribavirin, carriage of the
minor A1127354 ITPA allele was associated with
improved SVR and less relapse.26 Although these findings support the association between ITPA genotype
and outcome, the results may have been secondary to
improved ribavirin adherence, which was not the case
in the present study. Additionally, the other major ITPA
SNP, i.e., rs7270101, was not analyzed, making assessment of the impact of ITPase activity difficult.
Multivariate Analysis
P
0.0003
<0.0001
0.001
<0.0001
0.36
<0.0001
0.001
0.12
Adjusted Odds Ratio
11
5.0
2.3
0.60
0.46
0.26
(2.8-45)
(2.3-11)
(1.1-4.7)
(0.46-0.79)
(0.27-0.78)
(0.14-0.48)
P
0.0002
<0.0001
0.02
<0.0001
0.004
<0.0001
NS
NS
It is unclear how lower ITPase activity may protect
against relapse, but the underlying mechanism of
action might differ from that suggested for protection
against ribavirin-induced anemia, i.e., avoidance of
ATP reduction in erythrocytes and thus less oxidative
stress.11 While this may also occur in hepatocytes, it is
not self-evident how the avoidance of ATP depletion
would translate into reduced relapse risk.
We propose a ribavirin-like mechanism of action for
genetic variants entailing reduced ITPase activity (biochemical pathway detailed in Fig. 1). In part, the antiviral effects of ribavirin, which also has little effect on
on-treatment HCV RNA levels27 but markedly reduces
the risk of relapse,28 are assumed to be achieved by
competitive inhibition of the inosine monophosphate
dehydrogenase (IMPDH), leading to depletion of
GTP and an indirect increase of ITP by way of induction of IMP.29 Similarly, reduced ITPase activity leads
to increased levels of ITP, as shown in genetic knockout models,10 and lower intracellular levels of IMP, the
substrate of IMPDH, which in turn results in lower
levels of GTP. Thus, it may be hypothesized that
increased intracellular concentrations of ITP and
accompanying decreased levels of GTP, resulting from
either reduced ITPase activity or ribavirin treatment,
lead to increased incorporation of this unusual nucleotide3,4 by the nonstringent HCV RNA-dependentRNA-polymerase with ensuing random mutagenesis.30
Cellular polymerases, having more rigorous proofreading functions, are less likely to incorporate rogue
nucleotides such as ITP and dITP.31 In addition,
inosine-containing RNA has been proposed to act as a
danger signal triggering the activation of innate immunity.32 Inflammatory responses are critical in the antiviral immune response, and inosine-containing RNA is
known to enhance the production of IFN-b, TNF,
and IL-6 from innate immune cells, as compared to
unmodified RNA.32 Potentially, this could have additional secondary impact on adaptive immune
responses, similar to what has been reported for
2138
REMBECK ET AL.
ribavirin.33 Needless to say, this proposed mechanism
of action is speculative, and warrants further in vitro as
well as in vivo studies.
Although the introduction of interferon-free treatment regimens for HCV entails considerable improvements in efficacy and side effects, on a global
perspective health economics likely will mandate the
continued use of interferon-based therapy as a therapeutic option for several years to come, especially for
HCV genotypes 2/3. Additionally, when using
interferon-sparing therapy with sofosbuvir and ribavirin, a high proportion of genotype 3-infected
patients experience relapse despite early viral eradication,34,35 although pending trials may overcome this
problem by prolonged treatment duration or the addition of supplementary antiviral agents, further augmenting cost. Thus, our results suggesting that 800
mg daily dosing of ribavirin might be sufficient or
superfluous for HCV genotype 2/3-infected patients
with polymorphisms encoding reduced ITPase activity
when receiving interferon-based therapy, but not for
patients homozygous for both major alleles with normal ITPase activity, likely may impact HCV treatment
guidelines, even in more affluent regions. Indeed,
some patients in this latter group might benefit from
higher, weight-based ribavirin dosing, as previously has
been suggested for HCV genotype 2/3-infected
patients with BMI 30 kg/m2.36 The lack of association between ITPA variants and SVR in previous studies might be explained by the use of higher weightbased ribavirin dosing in these studies,1,14,15 as higher
intracellular ribavirin levels may overcome the negative
impact of normal ITPase activity. Indeed, the majority
of patients, having the wild-type major ITPA variant
entailing normal ITPase activity, may also benefit more
from the addition of ribavirin to some of the pending
interferon-free treatment regimens for HCV as well as
from the use of ribavirin against other viral infections,
e.g., hepatitis E virus (HEV) and respiratory syncytial
virus (RSV), although they are also more likely to
develop ribavirin-induced anemia. Additionally, pharmacological inhibition of ITPase activity could potentially also reduce the need for or substitute ribavirin.
Reduced ITPase activity, while protective against
anemia, is also associated with augmented treatmentinduced thrombocytopenia, which hitherto has hampered its clinical utility. The reduced anemia during
therapy noted in the present study was in line with
earlier reports,1,14,15,26 as was the association with
greater decline in platelet count.12 However, this did
not affect ribavirin dosing in contrast to the results
presented by Eskesen et al.15 and Kurosaki et al.,26
HEPATOLOGY, June 2014
both using weight-based ribavirin dosing, likely due to
use of the better-tolerated conventional lower 800 mg
daily dosing in the present study.
The association between reduced predicted ITPase
activity and lower ribavirin concentrations at day 29
was unexpected, but may be a consequence of reduced
hemolytic anemia during therapy. A larger number of
circulating erythrocytes would generate a larger distribution volume for the intracellular phosphorylated
forms of ribavirin, which in turn would generate lower
extracellular concentrations of unphosphorylated ribavirin. Conversely, it might be argued that the lower
ribavirin concentrations observed may have contributed to the protection against anemia noted among
patients with reduced ITPase activity.
In the present study, classification of predicted ITPase
activity was based on the compound genotype of
rs1127354 and rs7270101 rather than by means of
direct measurement of ITPase activity, which might be
considered superior. However, because direct measurement requires freshly harvested, purified erythrocytes,3-5
this is neither possible in retrospective studies nor plausible in most multicenter prospective trials. Additionally,
in light of marked variations in ITPase activity depending on the origin and age of the cells studied,37,38 other
technical difficulties including the potential presence of
inhibitory IDP in the ITP used as substrate,6 in addition to relatively high cost of the direct ITPase assay,
analysis based on the compound genotype has been suggested to be preferential, especially for population-based
screening of ITPase activity.5
In conclusion, following therapy with standard dosing of ribavirin for HCV genotype 2/3, our results
imply that ITPA polymorphisms entailing reduced
ITPase activity are associated with a higher likelihood
of achieving SVR resulting from reduced risk of
relapse unrelated to ribavirin adherence or protection
against anemia.
Acknowledgment: We thank Marie-Louise Landelius and Anne-Sofie Tyl€o for technical assistance.
References
1. Fellay J, Thompson AJ, Ge D, Gumbs CE, Urban TJ, Shianna KV,
et al. ITPA gene variants protect against anaemia in patients treated for
chronic hepatitis C. Nature 2010;464:405-408.
2. Ogawa E, Furusyo N, Nakamuta M, Kajiwara E, Nomura H, Dohmen
K, et al. Clinical milestones for the prediction of severe anemia by
chronic hepatitis C patients receiving telaprevir-based triple therapy. J
Hepatol 2013;59:667-674.
3. Sumi S, Marinaki AM, Arenas M, Fairbanks L, Shobowale-Bakre M,
Rees DC, et al. Genetic basis of inosine triphosphate pyrophosphohydrolase deficiency. Hum Genet 2002;111:360-367.
HEPATOLOGY, Vol. 59, No. 6, 2014
4. Shipkova M, Lorenz K, Oellerich M, Wieland E, von Ahsen N. Measurement of erythrocyte inosine triphosphate pyrophosphohydrolase
(ITPA) activity by HPLC and correlation of ITPA genotype-phenotype
in a Caucasian population. Clin Chem 2006;52:240-247.
5. Maeda T, Sumi S, Ueta A, Ohkubo Y, Ito T, Marinaki AM, et al.
Genetic basis of inosine triphosphate pyrophosphohydrolase deficiency
in the Japanese population. Mol Gen Metab 2005;85:271-279.
6. Simone PD, Pavlov YI, Borgstahl GE. ITPA (inosine triphosphate
pyrophosphatase): from surveillance of nucleotide pools to human disease and pharmacogenetics. Mutat Res 2013;753:131-46.
7. Stocco G, Cheok MH, Crews KR, Dervieux T, French D, Pei D, et al.
Genetic polymorphism of inosine triphosphate pyrophosphatase is a determinant of mercaptopurine metabolism and toxicity during treatment for
acute lymphoblastic leukemia. Clin Pharmacol Ther 2009;85:164-172.
8. Bierau J, Lindhout M, Bakker JA. Pharmacogenetic significance of inosine triphosphatase. Pharmacogenomics 2007;8:1221-1228.
9. Waisertreiger IS, Menezes MR, Randazzo J, Pavlov YI. Elevated levels
of DNA strand breaks induced by a base analog in the human cell line
with the P32T ITPA variant. J Nucleic Acids 2010;2010:pii 872180.
10. Behmanesh M, Sakumi K, Abolhassani N, Toyokuni S, Oka S,
Ohnishi YN, et al. ITPase-deficient mice show growth retardation and
die before weaning. Cell Death Differ 2009;16:1315-1322.
11. Hitomi Y, Cirulli ET, Fellay J, McHutchison JG, Thompson AJ,
Gumbs CE, et al. Inosine triphosphate protects against ribavirininduced adenosine triphosphate loss by adenylosuccinate synthase function. Gastroenterology 2011;140:1314-1321.
12. Thompson AJ, Clark PJ, Singh A, Ge D, Fellay J, Zhu M, et al.
Genome-wide association study of interferon-related cytopenia in
chronic hepatitis C patients. J Hepatol 2012;56:313-319.
13. Cardier JE, Erickson-Miller CL, Murphy MJ Jr. Differential effect of
erythropoietin and GM-CSF on megakaryocytopoiesis from primitive
bone marrow cells in serum-free conditions. Stem Cells 1997;15:286-290.
14. Thompson AJ, Santoro R, Piazzolla V, Clark PJ, Naggie S, Tillmann
HL, et al. Inosine triphosphatase genetic variants are protective against
anemia during antiviral therapy for HCV2/3 but do not decrease dose
reductions of RBV or increase SVR. HEPATOLOGY 2011;53:389-395.
15. Eskesen AN, Melum E, Moghaddam A, Bjoro K, Verbaan H, RingLarsen H, et al. Genetic variants at the ITPA locus protect against
ribavirin-induced hemolytic anemia and dose reduction in an HCV
G2/G3 cohort. Eur J Gastroenterol Hepatol 2012;24:890-896.
16. Hadziyannis SJ, Sette H Jr, Morgan TR, Balan V, Diago M, Marcellin
P, et al. Peginterferon-alpha2a and ribavirin combination therapy in
chronic hepatitis C: a randomized study of treatment duration and ribavirin dose. Ann Intern Med 2004;140:346-355.
17. Lagging M, Langeland N, Pedersen C, Farkkila M, Buhl MR, Morch
K, et al. Randomized comparison of 12 or 24 weeks of peginterferon
alpha-2a and ribavirin in chronic hepatitis C virus genotype 2/3 infection. HEPATOLOGY 2008;47:1837-1845.
18. Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F, et al.
Histological grading and staging of chronic hepatitis. J Hepatol 1995;
22:696-699.
19. Westin J, Nordlinder H, Lagging M, Norkrans G, Wejstal R. Steatosis
accelerates fibrosis development over time in hepatitis C virus genotype
3 infected patients. J Hepatol 2002;37:837-842.
20. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF,
Turner RC. Homeostasis model assessment: insulin resistance and betacell function from fasting plasma glucose and insulin concentrations in
man. Diabetologia 1985;28:412-419.
21. Leutscher PD, Lagging M, Buhl MR, Pedersen C, Norkrans G,
Langeland N, et al. Evaluation of depression as a risk factor for treatment failure in chronic hepatitis C. HEPATOLOGY 2010;52:430-435.
REMBECK ET AL.
2139
22. Ge D, Fellay J, Thompson AJ, Simon JS, Shianna KV, Urban TJ,
et al. Genetic variation in IL28B predicts hepatitis C treatmentinduced viral clearance. Nature 2009;461:399-401.
23. Sulkowski MS, Shiffman ML, Afdhal NH, Reddy KR, McCone J, Lee
WM, et al. Hepatitis C virus treatment-related anemia is associated
with higher sustained virologic response rate. Gastroenterology 2010;
139:1602-1611, 1611 e1601.
24. Lindh M, Lagging M, Farkkila M, Langeland N, Morch K, Nilsson S,
et al. Interleukin 28B gene variation at rs12979860 determines early
viral kinetics during treatment in patients carrying genotypes 2 or 3 of
hepatitis C virus. J Infect Dis 2011;203:1748-1752.
25. Askarieh G, Alsio A, Pugnale P, Negro F, Ferrari C, Neumann AU,
et al. Systemic and intrahepatic interferon-gamma-inducible protein 10
kDa predicts the first-phase decline in hepatitis C virus RNA and overall viral response to therapy in chronic hepatitis C. HEPATOLOGY 2010;
51:1523-1530.
26. Kurosaki M, Tanaka Y, Tanaka K, Suzuki Y, Hoshioka Y, Tamaki N,
et al. Relationship between polymorphisms of the inosine triphosphatase gene and anaemia or outcome after treatment with pegylated interferon and ribavirin. Antiviral Ther 2011;16:685-694.
27. Rotman Y, Noureddin M, Feld JJ, Guedj J, Witthaus M, Han H,
et al. Effect of ribavirin on viral kinetics and liver gene expression in
chronic hepatitis C. Gut 2014;63:161-169.
28. Hiramatsu N, Oze T, Yakushijin T, Inoue Y, Igura T, Mochizuki K,
et al. Ribavirin dose reduction raises relapse rate dose-dependently in
genotype 1 patients with hepatitis C responding to pegylated interferon
alpha-2b plus ribavirin. J Viral Hepat 2009;16:586-594.
29. Gebeyehu G, Marquez VE, Van Cott A, Cooney DA, Kelley JA,
Jayaram HN, et al. Ribavirin, tiazofurin, and selenazofurin: mononucleotides and nicotinamide adenine dinucleotide analogues. Synthesis,
structure, and interactions with IMP dehydrogenase. J Med Chem
1985;28:99-105.
30. Kuipers OP. Random mutagenesis by using mixtures of dNTP and
dITP in PCR. Methods Mol Biol 1996;57:351-356.
31. Thomas MJ, Platas AA, Hawley DK. Transcriptional fidelity and proofreading by RNA polymerase II. Cell 1998;93:627-637.
32. Liao JY, Thakur SA, Zalinger ZB, Gerrish KE, Imani F. Inosine-containing RNA is a novel innate immune recognition element and
reduces RSV infection. PLoS One 2011;6:e26463.
33. Hultgren C, Milich DR, Weiland O, Sallberg M. The antiviral compound ribavirin modulates the T helper (Th) 1/Th2 subset balance in
hepatitis B and C virus-specific immune responses. J Gen Virol 1998;
79:2381-2391.
34. Lawitz E, Mangia A, Wyles D, Rodriguez-Torres M, Hassanein T,
Gordon SC, et al. Sofosbuvir for previously untreated chronic hepatitis
C infection. N Engl J Med 2013;368:1878-1887.
35. Jacobson IM, Gordon SC, Kowdley KV, Yoshida EM, RodriguezTorres M, Sulkowski MS, et al. Sofosbuvir for hepatitis C genotype 2
or 3 in patients without treatment options. N Engl J Med 2013;368:
1867-1877.
36. Alsio A, Rembeck K, Askarieh G, Christensen PB, Farkkila M,
Langeland N, et al. Impact of obesity on the bioavailability of
peginterferon-alpha2a and ribavirin and treatment outcome for chronic
hepatitis C genotype 2 or 3. PLoS One 2012;7:e37521.
37. Holmes SL, Turner BM, Hirschhorn K. Human inosine triphosphatase:
catalytic properties and population studies. Clin Chim Acta 1979;97:
143-153.
38. Verhoef VL, Fuller SA, Morris AJ. Individual variation of nucleoside
triphosphate pyrophosphohydrolase activity in human erythrocytes,
granulocytes, lymphocytes, and platelets. Biochem Genet 1980;18:235245.