Role of nucleoside transporters SLC28A2/3 and SLC29A1/2
genetics in ribavirin therapy: protection against anemia
in patients with chronic hepatitis C
Alexandra Doehringa, Wolf Peter Hofmannb, Christina Schleckera,
Stefan Zeuzemb, Christoph Sarrazinb, Thomas Bergd, Tobias Mu ¨llere,
Eva Herrmannc, Gerd Geisslingeraand Jo ¨rn Lo ¨tscha
Background and aim The standard of hepatitis C antiviral
therapy combines pegylated interferon-a with ribavirin. This
polar guanosine analog improves the sustained virological
response (SVR) rates, but may induce hemolytic anemia.
As its pharmacokinetics depend on facilitated
transmembrane transport, we assessed whether variants
in genes that code for concentrative (concentrative
nucleoside transporters 2 and 3 coded by SLC28A2 and
SLC28A3, respectively) and equilibrative nucleoside
transporters (equilibrative nucleoside transporters 1 and 2
coded by SLC29A1 and SLC29A2, respectively) are
associated with the therapy response and side effects.
Methods Patients (n=169) chronically infected with the
hepatitis C virus genotype 1, treated with standard doses
of pegylated interferon-a and weight-based doses of
ribavirin for up to 48 weeks, were genotyped for 21 variants
in nucleoside transporter genes SLC28A2, SLC28A3,
SLC29A1, and SLC29A2, selected to include reported
functional variants and to span the complete gene loci.
The presence or absence of a SVR (n=169) and a relevant
decrease (>3g/dl, n=115) in blood hemoglobin were
associated with the genotypes.
Results The variant SLC28A3 haplotype rs10868138G/
rs56350726T (allelic frequency 0.074) was associated
with a lower incidence (35.5%) of relevant decreases
(>3g/dl) in blood hemoglobin than in noncarriers
(64.3%; P=0.024, n=115). This protection against
hemolytic anemia was not associated with decreased
SVR rates (n=169).
Conclusion A genetic variant in SCL28A3 coding for the
concentrative nucleoside transporter 3 protects patients
with chronic hepatitis C against hemolytic anemia
without affecting SVR in hepatitis C virus genotype 1.
Pharmacogenetics and Genomics 00:000–000? c 2011
Wolters Kluwer Health | Lippincott Williams & Wilkins.
Pharmacogenetics and Genomics 2011, 00:000–000
Keywords: antiviral agents, chemically induced anemia, European
population, hepatitis C drug therapy, nucleoside analog
apharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology,
Departments ofbInternal Medicine 1,cBiostatistics and Mathematical Modeling,
Johann Wolfgang Goethe-University Hospital, Frankfurt anddDepartment of
Hepatology, Clinic for Gastroenterology and Rheumatology, University Hospital
Leipzig,eDepartment of Gastroenterology and Hepatology, Charite ´ University
Hospital, Campus Virchow Klinikum, Berlin, Germany
Correspondence to Jo ¨rn Lo ¨tsch MD, pharmazentrum frankfurt/ZAFES, Institute
of Clinical Pharmacology, Johann Wolfgang Goethe-University Hospital, Theodor
Stern Kai 7, Frankfurt am Main 60590, Germany
Tel: +49 69 6301 4589; fax: +49 69 6301 7636;
Alexandra Doehring and Wolf Peter Hofmann contributed equally to this study
Received 8 June 2010 Accepted 23 December 2010
Infection with the hepatitis C virus (HCV) affects 170
million individuals worldwide and is an important cause
of liver-related morbidity and mortality . The current
standard of care consists of pegylated interferon-a
combined with the nucleoside analog ribavirin [1,2].
Orally administered ribavirin is rapidly absorbed and
distributed, and maximum plasma concentrations are
reached within 2h followed by rapid distribution to
nonplasma compartments and long terminal phases .
The main route of elimination of ribavirin is by the
kidney. The combination therapy of pegylated interferon-
a and ribavirin leads to a sustained virological response
(SVR), that is, nondetectability of HCV RNA in serum
6 months after a treatment course, in half of the patients
[2,4]. A major side effect of ribavirin is a severe reversible
hemolytic anemia , which develops in approximately
10% of treated patients  and is the most common
reason for ribavirin dose reduction and treatment discon-
tinuation [5,7,8]. Ribavirin exerts its toxicity through an
inhibition of intracellular energy metabolism and oxida-
tive membrane damage, leading to an accelerated extra-
vascular hemolysis by the reticuloendothelial system .
Concentration-dependent toxicity and improvement of
anemia on dose reduction point toward the importance of
pharmacokinetic factors. Among the factors associated
with ribavirin-induced hemolysis are so far sex, ribavirin
dose per kg, baseline hemoglobin concentration, age,
baseline creatinine clearance [9,10], and variants in the
inosine triphosphatase (ITPA) gene .
Supplemental digital content is available for this article. Direct URL citations
appear in the printed text and are provided in the HTML and PDF versions of this
article on the journal’s Website (www.pharmacogeneticsandgenomics.com).
1744-6872 ? c 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins
Personalized chronic hepatitis C (CHC) therapy strategies
gain increasing importance for the improvement of SVR
rates and the reduction of side effects. Currently, this
includes variable therapy duration from 12 to 72 weeks
depending on the HCV genotype and additional host or
viral factors such as liver fibrosis stage and baseline HCV
RNA concentrations , and recently IL28B genotypes
[13–15]. Variants in nucleoside transporters are further
primary candidates for personalized therapy, as the polar
molecule ribavirin, requires facilitated transport over
biological membranes [16–18]. Ribavirin transporters
include sodium-dependent, high-affinity concentrative
nucleoside transporters (CNT) 2 and 3, coded by SLC28A2
and SLC28A3 (solute carrier transporter), and sodium-
independent, low-affinity equilibrative nucleoside trans-
porters (ENT) 1 and 2, coded by SLC29A1 and SLC29A2.
The purine-preferring CNT2 mediates gastrointestinal
ribavirin uptake into resorptive epithelia  and subse-
quently, ENT1, located in the basolateral membrane of
epithelial cells, presumably transports it into the capillary
lumen [18,20]. In hepatocytes, ENT1 is the most im-
portant transporter, accounting for 88% of hepatic ribavirin
uptake whereas approximately 6% are mediated by ENT2
and 4.5% by CNT2 .
A role of changes in the net function of these transporters
in ribavirin therapy is suggested by the report of restored
viral sensitivity to ribavirin in vitro when ENT1 and
CNT3 were overexpressed . Therefore, the role of
nucleoside transporter genetics in the antiviral response
and ribavirin-induced hemolytic anemia was assessed in
169 patients with CHC.
Patients and methods
The study followed the Declaration of Helsinki on
Biomedical Research involving humans, was approved
by the local Ethics Committee, and all patients provided
written informed consent before enrollment. Patients
(n=169, 84 men and 85 women) chronically infected
with HCV genotype 1 [HCV RNA (mean±standard
deviation) 960563±1123402IU/ml] were treated with
standard doses of pegylated interferon-a and weight-
based doses of ribavirin for up to 48 weeks. All patients
(aged 53±12 years) were of Caucasian ethnicity by self-
assignment and were followed-up in the Hepatology
Outpatient Clinic of the Goethe-University Hospital in
Frankfurt, Germany. From 115 patients (aged 53.9±11.3
years), the blood hemoglobin concentrations were avail-
able at the baseline and after 12 weeks of therapy.
Selection of variants
For human SLC28A2-283, SLC28A3-1066, SLC29A1-172,
(SNPs), respectively, are currently listed in the National
Center for Biotechnology Information SNP database
(www.ncbi.nlm.nih.gov/SNP/; accessed at 23 February 2010).
SLC29A1/A2 are nevertheless regarded as evolutionary
conserved, with values of nucleotide diversity in coding
regions, pcoding of less than 1, compared with other
transporters, such as SLC22A1/A2, which have values of
pcodingof more than 6 [17,23]. Among SNPs described in
these genes, rs731780, rs70914, and rs747199 in the
promoter region of SLC29A1 have been associated with
higher mRNA expression . A two-nucleotide deletion
(845delA-846delC, rs8187656, and rs8187655) in SLC29A2
results in a frame shift causing inability of the gene product
to transport nucleosides . Except for rs747199, their
minor allelic frequencies (MAF) in Caucasians are less than
5%. SLC28A2 rs2413775 has been associated with reduced
CNT2 expression after reduced binding of the transcrip-
tion factor hepatic nuclear factor . Functional SNPs
were included in this choice of variants when MAFis more
than 5% in Caucasians. HapMap (http://hapmap.ncbi.nlm.nih.
gov/cgi-perl/gbrowse/hapmap27_B36/) tagging SNPs with a
reported MAF in Caucasians of more than 10% along the
gene locus were added to cover the whole gene ranges from
the 50UTR to the 30UTR. This resulted in 21 genetic
variants (Table 1).
DNA was extracted from blood on a BioRobot EZ1
workstation using the blood and body fluid spin protocol
provided in the EZ1 DNA Blood Kit (200ml; Qiagen,
PCR primers for amplification of SLC28A2, SLC28A3,
SLC29A1, and SLC29A2 gene segments of interest and
sequencing primers were designed using the PyroMark
Assay Design (version 18.104.22.168; Qiagen; Table S1
Supplemental digital content 1 http://links.lww.com/FPC/
A242). Specificity of the primers was verified by gene
alignment (http://www.ncbi.nlm.nih.gov/Blast/). PCR reac-
tions were performed in a 50-ml assay volume on a
Mastercycler ep gradient S instrument (Eppendorf,
Hamburg, Germany), using 5-ml genomic DNA (20–
30mg/ml), mixed with 0.25ml HotStarTaq plus DNA
Polymerase (5U/ml; Qiagen), 5ml of 10? PCR buffer,
10ml of 5? Q-solution, 1ml of dNTP mix (10mmol/l
each; Qiagen), 0.1ml of one biotinylated and one non-
biotinylated PCR primer (each 100mmol/l), and 28.55ml
of water purified with high-performance liquid chroma-
tography. The PCR was done with an initial denaturation
step for 5min at 951C, 40–45 cycles with a 30-s
denaturation step at 951C, an annealing step at primer-
specific temperatures (Table S) for 30s, and an elonga-
tion step at 721C for 30s with final elongation at 721C for
5min. PCR products were evaluated by electrophoresis
on ethidium bromide-stained agarose gels.
Genotyping was done by means of pyrosequencing, a real-
time pyrophosphate detection method . PCR tem-
plate (25ml; biotinylated and nonbiotinylated strands)
Pharmacogenetics and Genomics
2011, Vol 00 No 00
allelic frequencies and functional effects
Overview of all included variants with their observed minor allelic frequencies in this cohort and, if available, their reported minor
GeneDNA mutationPosition in the gene Reported
Observed and 95%
CI; n=169 Reported effects References
Asians (n=30): 55.2%
Malays (n=96): 77.1%
Chinese (n=96): 88%
Asians (n=30): 81.7%
0.35 (0.30–0.40)Enhanced transcription rate
and expression levels
rs11854484T>C Exon 1 (c.65T>C)
0.34 (0.29–0.40)No significant difference in
affinity to ribavirin
rs1060896A>C Exon 4 (c.225A>C)
0.31 (0.26–0.36)No significant difference in
affinity to ribavirin
0.22 (0.17–0.26) 
Intron 9 (c.862-196A>G)
Intron 2 (c.157-1617G>C)
Exon 5 (c.338A>G)
Exon 14 (c.1538A>T)
Intron 17 (c.1950-54T>C)
0.20 (0.16–0.25)Increased expression for
composed of this variant
and two other
(-1050G>A); both not
present in Caucasians]
Intron 3 (c.30-549C>T)
Intron 13 (c.1260-
0.39 (0.34–0.44)Tagging SNP according to
rs6458375C>T30UTR (c.*1087C>T)0.24 (0.20–0.29)
Intron 6 (c.648+64A>G)
rs2279862A>G0.29 (0.24–0.34)Tagging SNP according to
Intron 7 (c.734-50G>A)
CI, confidence interval; SNP, single nucleotide polymorphism.
Nucleoside transporters and ribavirin Doehring et al.
was pipetted into a well containing 3ml of streptavidin-
coated sepharose beads (streptavidin–sepharose high
performance, GE Healthcare Bio-Sciences AB, Uppsala,
Sweden), 37ml of binding buffer [Tris(hydroxylmethyl)-
(1mmol/l), 0.1% of polyoxyethylenesorbitan monolaure-
ate (Tween 20); pH 7.6], and 15ml of water purified with
high-performance liquid chromatography . This mixture
was incubated for 10min at room temperature (shaker
speed of 800/min) to form specific complexes between
streptavidin-coated sepharose beads and biotinylated
strands. The complexes were purified and separated
from the nonbiotinylated strands on a PyroMark Vacuum
Prep Worktable (Biotage, Uppsala, Sweden). After
removal of all liquid by suction, the specific complexes
were captured on PrepTool filters, purified in 70%
ethanol for 5s, denatured in 0.2mol/l NaOH for 5s, and
washed with Tris(hydroxymethyl)-aminomethan (10mmol/l
in water) for 10s. The complexes were transferred to a PSQ
96 Plate Low (Biotage) prefilled with 0.16ml of 100-mmol/l
sequencing primer and 39.84ml of annealing buffer
[Tris(hydroxymethyl)-aminomethan (20mmol/l) and mag-
nesium acetate tetrahydrate (2mmol/l; pH 7.6)]. The plate
was heated at 801C for exactly 2min in a PSQ 96 Sample
Prep Thermoplate Low (Biotage) and cooled down to room
temperature. Sequencing analysis took place on a Biotage
PSQ 96MA System with Biotage enzyme mix, substrate
mix, and nucleotides (Pyro Gold Reagents, Biotage, set for
SNP genotyping and mutation analysis). For assay valida-
tion, two DNA samples of each genotype and variant
were conventionally sequenced (AGOWA GmbH, Berlin,
Germany) and implemented as positive controls into all
local sequencing runs.
The correspondence between observed numbers of
homozygous and heterozygous patients with those
expected from the Hardy–Weinberg equilibrium was
checked by means of the w2goodness-of-fit tests. Linkage
disequilibrium was analyzed by calculating parameters
D0and r2. SNPs with a MAF of more than 0.05 were
included in in-silico haploblock detection using 95%
confidence bounds on D0 and subsequent haplotype
analysis (SVS 7.2.3 for Linux, Golden Helix, Bozeman,
Associations of genetic variants with SVR were analyzed
by stepwise inclusion. The specified variables were
tested for entry into the model one by one, based on
the significance level of the score statistic. The variable
with the smallest P value was entered into the model.
After each entry, variables that were already in the model
were tested for possible removal, based on the signifi-
cance of the likelihood ratio criterion. The variable with
the largest P value was removed, and the model was re-
estimated. When no more variables satisfied the removal
criterion, covariates that were not in the model were
evaluated for entry. Model building stopped when no
more variables met entry or removal criteria or when the
current model was the same as a previous model (IBM
SPSS Statistics, Chicago, Illinois, USA) of candidate
factors (21 SNPs) in binary logistic regression analysis
(PASW 18.0.1 for Linux, IBM SPSS Statistics). This was
repeated with an additional inclusion of IL28B rs12979860
as a positive genetic control marker [13,14,32]. The
association of haplotypes of the transporter genes (eight
haplotypes with observed MAF >0.05; for detailed
haplotypes, see Table 2) was tested similarly. Associations
of genetic variants with significant anemic effects, which
were defined as either a decline in hemoglobin of more
than 3g/dl or hemoglobin levels of less than 10g/dl 
being the threshold at which ribavirin dose reduction is
recommended, were analyzed analogously. In addition,
the absolute reduction in blood hemoglobin (Dhemoglo-
bin) after 12 weeks of therapy was submitted to linear
regression analysis with the same candidate factors.
Results are reported at the uncorrected a level and using
a false discovery rate correction  following the in-
structions of Verhoeven et al.  to account for multiple
testing. Finally, to control for confounding of a protective
effect of transporter genetics against hemolytic anemia by
co-existence of ITPA variants, rs1127354C>A and
rs7270101A>C, recently shown to produce that clinical
effect , the latter w2statistics were repeated with a
subgroup of patients not carrying any of the two variant
The distributions of homozygous, heterozygous, and
noncarriers of the minor alleles agreed with the expecta-
tions from the Hardy–Weinberg law (w2goodness-of-fit
tests: P>0.05; observed allelic frequencies: Table 1).
Linkage between the investigated DNA positions and the
haploblock structures of the four genes are shown in
Fig. 1. Specifically, one, two, zero and one haploblocks,
each comprising two neighbored variant gene positions,
were found in SLC28A2, SLC28A3, SLC29A1, and
SLC29A2 genes, respectively (Table 2). As in each block,
the major haplotypes (allelic frequency >0.05) were
those composed of either only the wild-type allele or only
the variant alleles at all haploblock positions, two, four,
zero and two haplotypes in the respective genes were
entered into the association analysis, in addition to all the
SNPs from Table 1.
Among the patients with CHC, 63 (37.3%) achieved an
SVR, that is, no HCV RNA detectable 24 weeks after the
completion of the treatment and the remaining 106 were
classified as having a non-SVR. Logistic regression did not
identify any genetic variant in nucleoside transport genes
(neither SNPs nor haplotypes) that discriminated be-
tween responders and nonresponders to anti-HCV
therapy. In contrast, IL28B rs12979860 (allelic frequency:
0.45) was identified as the only genetic marker associated
Pharmacogenetics and Genomics
2011, Vol 00 No 00
with therapy response (P=0.012) when included to
replicate the earlier observed association of the IL28B
SNP with sustained response [15,32]. The results did not
change when repeating the analyses with the 115 patients
from whom blood hemoglobin concentrations during
therapy were available.
Blood hemoglobin concentrations decreased by more than
3g/dl in 69 (60%) of the 115 patients with available
hemoglobin data. The two separate binary logistic
regressions identified the SLC28A3 rs10868138 A>G
SNP and the variant SLC28A3 haplotype rs10868138G/
rs56350726T as predictive for this hemoglobin change
(P=0.008 and 0.007, respectively, at uncorrected a level,
which retained its significances when applying false
discovery rate correction but not with the most con-
servative Bonferroni correction). The haplotype provided
a better prediction of Dhemoglobin of more than 3g/dl
than the SNP. That is, with only heterozygous carriers of
the haplotype (n=17 of 115; allelic frequency 0.074)
being observed, only six (35.3%) of them but 63 (64.3%)
of the 98 noncarriers had a hemoglobin decrease of more
than 3g/dl (verificatory w2test: P=0.024 uncorrected,
Fig. 2). In contrast, significance was missed with the SNP
(P=0.088). The effect was not explained by the
presence of functional ITPA minor alleles (allelic
frequencies: rs1127354A: 0.07, rs7270101C: 0.12) as the
repetition of the w2tests after excluding all patients who
carried ITPA variant alleles resulted in an even more
(P=0.002), and also the SNP reached significance
(P=0.02). The effect of the SLC28A3 haplotype was
even larger than that of ITPA genetics in which 43.8% of
carriers of any ITPA variant had a hemoglobin decrease of
more than 3g/dl. The blood hemoglobin concentrations
during 12 weeks of therapy changed by –3.4±1.4g/dl,
from 15.1±1.3g/dl at baseline to 11.7±1.4g/dl at
therapy week 12 (t-test: P=1.53?10–48). Linear
regression identified the SLC28A3 rs56350726 A>T
SNP as predictive for the numerical change in hemoglo-
bin (P=0.033). While in carriers of this haplotype blood
hemoglobin decreased by 3.1±1.7g/dl, it decreased in
noncarriers by 3.4±1.4g/dl.
When added to pegylated interferon-a, ribavirin increases
SVR rates in patients treated for CHC but is associated
with many side effects including hemolytic anemia.
Accumulation of phosphorylated ribavirin in erythrocytes
is a likely prerequisite of its hemolytic side effects . In
erythrocytes, ribavirin is metabolically phosphorylated to
its monophosphate, diphosphate, and triphosphate by the
adenosine kinase. As these ribavirin phosphates are
unable to cross the erythrocyte membrane and as no
ribavirin phosphate-hydrolyzing enzymes are present in
erythrocytes, ribavirin accumulates and is only eliminated
slowly (half-life of 40 days compared with elimination
from plasma with a half-life of 1 day) . The emerging
60-fold higher ribavirin concentration in erythrocytes
compared with plasma concentrations is thought to cause
an oxidative damage of the cell membrane, which
promotes premature erythrocyte senescence and its
phagocytic removal . As ribavirin requires facilitated
transport across cells membranes, nucleoside transporter
gene variants are primary candidates for an individual
modulation of its clinical effects. This is supported by
evidence of a role of these transporters in ribavirin
pharmacokinetics [16–21]. In this study, we identified a
mutant SLC28A3 haplotype associated with a lower
incidence of severe hemolytic anemia in patients with
CHC receiving pegylated interferon and ribavirin. The
size of the effects was, in this patient cohort, comparable
with those of the recently reported protective effects of
ITPA gene variants causing ITPA deficiency and leading
to accumulation of active forms of ribavirin in red blood
cells . However, in the original publication of the
ITPA variants associated with protective effects against
anemia, the effect was larger than in this cohort and
therefore, comparative judgment of their protective
rs10868138G/rs56350726T haplotype is as frequent
as the ITPA rs1127354A SNP (allelic frequency app-
roximately 0.07), but less frequent than the ITPA
rs7270101C SNP (allelic frequency of 0.12). The
probability to carry any of the two functional ITPA SNPs,
calculated as 1-the probability of not carrying any of the
two, that is, 1–[(1–0.07)?(1–0.12)]=0.1816 is larger
than that of carrying the SLC28A3 haplotype, which
places the likely clinical importance of this finding after
that with ITPA.
The concentrative nucleoside transporters 3, coded by
SLC28A3, are inward transporters. The straightforward
explanation of the effect is possibly a decreased trans-
porter function due to exchanges of tyrosine at protein
positions 113 and 513 with cysteine and phenylalanine,
respectively. This may lead to lower concentrations of
ribavirin in red blood cells and subsequently to decreased
lytic effects of ribavirin triphosphate on erythrocytes;
a similar effect as caused by ITPA rs7270101  al-
though probably involving different mechanisms. However,
SLC29A2 genes that were found at an allelic frequency of more
than or equal to 5%. SLC29A1 contained no haploblock and
therefore, no haplotype was identified (see Fig. 1)
Overview of the haplotypes in the SLC28A2, SLC28A3, and
Observed allelic frequency
(and 95% CI; n=169)
CI, confidence interval.
Nucleoside transporters and ribavirin Doehring et al.
Distribution of selected single nucleotide polymorphisms (SNPs) along the SLC28A2, SLC28A3, SLC29A1, and SLC29A2 genes (top to bottom).
The SNPs are given in the notation suggested in (http://www.hgvs.org/mutnomen ): nucleotide 1 is the A of the ATG translation initiation codon,
nucleotides toward 50of the ATG translation initiation have negative numbers, the nucleotide 30of the translation stop codon is *1, the next *2, etc.
Intronic nucleotides for a coding DNA reference sequence are named as follows: beginning of the intron; the number of the last nucleotide of the
preceding exon, a plus sign and the position in the intron, such as c.1566+135A>C, and end of the intron; the number of the first nucleotide of the
following exon, a minus sign and the position upstream in the intron, such as c.862-196A>C. Below each gene, results of the in-silico haploblock
detection  and subsequent haplotype analysis are given. Linkage disequilibrium is quantified by values r2indicated in each box within the
triangular plot whereas complementary values of D0are indicated as color code. The SLC28A3 haplotype associated with less hemolytic anemia is
marked with a dotted frame. CNT, concentrative nucleoside transporter; ENT, equilibrative nucleoside transporter.
Pharmacogenetics and Genomics
2011, Vol 00 No 00
in-vitro experiments did not show differences of the
variant Tyr113Cys CNT3 transporters, coded by SLC28A3
rs10868138A>G, with adenosine, cladribine, and fludar-
abine, whereas ribavirin was not tested yet  and
therefore, the molecular consequences of the rs10868138G
allele or the rs10868138G/rs56350726T haplotype have to
be tested after this assessment provided their clinical
As ribavirin plasma concentrations have been shown to
correlate with favorable SVR rates [39–44] and consider-
ing the role of nucleoside transporters in ribavirin
pharmacokinetics [16–21], SLC28A2, SLC28A3, SLC29A1,
and SLC29A2 had also been candidates for an individual
modulation of anti-HCV therapy success. However, a
modulation of the therapy success was not seen, in con-
trast to the replication of the earlier observed association
of the IL28B rs12979860 SNP with sustained response
[15,32]. Ribavirin concentrations were neither assessed in
plasma nor intracellularly. Therefore, a modulation of
these concentrations by nucleoside transporter genetics
cannot be excluded but this would be restricted to
pharmacokinetics not translating into therapy. The
sample sizes to observe differences in allelic frequencies
between therapy response groups were large (4236 and
3676 for rs10868138 and rs56350726, respectively, and
ranging from 433 to 2052646 for all 21 SNPs). As the
selection of SNPs covered the gene ranges and included
reportedly functional or tagging SNPs, the results suggest
that the nucleoside transporter variants do not create
strong population-level differences as IL28B variants on
SVR. Nonassociation of nucleoside transporter genotypes
with SVR is so far restricted to HCV genotype 1, which
is less sensitive to the current pegylated interferon
and ribavirin combination treatment than HCV of
genotypes 2 or 3. Possible changes in ribavirin concentra-
tions might play a role in eradication of more sensitive
Finally, because possible alteration in CNT3 function
seems to be a benign condition and not to affect the SVR
rate, it may be possible to protect a patient from ribavirin-
induced anemia by pharmacological intervention against
this nucleoside transporter. The identification of a further
protective factor against ribavirin-induced hemolytic
anemia may provide a valuable pharmacogenetic diagnosis
of patients likely to tolerate ribavirin. Given the small
number of carriers of this haplotype, replication in a larger
sample is advised.
This work was supported by the Deutsche Forschungs-
gemeinschaft, Klinische Forschergruppe KFO 129 to Wolf
Peter Hofmann, Stefan Zeuzem, Christoph Sarrazin, Eva
Herrmann, Gerd Geisslinger, and Jo ¨rn Lo ¨tsch. This work
was supported by the German Competence Network for
Viral Hepatitis (Hep-Net), funded by the German Ministry
of Education and Research (BMBF, Grant No. 01 KI 0437,
Project No. 10.1.3 Core Project No. 10.1 Genetic host
factors in viral hepatitis and Genetic Epidemiology Group
in viral hepatitis), and by the EU-Vigilanz network of
excellence combating viral resistance (VIRGIL, Projekt
No. LSHM-CT-2004-503359 and the BMBF Project:
Host and viral determinants for susceptibility and
resistance to hepatitis C virus infection (Grant No.
01KI0787). The authors declare no conflict of interest.
1National Institutes of Health Consensus Development Conference
Statement. Management of hepatitis C: 2002–June 10-12, 2002.
Hepatology 2002; 36 (5 Suppl 1):S3–S20.
2Strader DB, Wright T, Thomas DL, Seeff LB. Diagnosis, management, and
treatment of hepatitis C. Hepatology 2004; 39:1147–1171.
3Khakoo S, Glue P, Grellier L, Wells B, Bell A, Dash C, et al. Ribavirin and
interferon alfa-2b in chronic hepatitis C: assessment of possible
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4National Institutes of Health Consensus Development Conference
Statement. Management of hepatitis C 2002 (June 10-12, 2002).
Gastroenterology 2002; 123:2082–2099.
Δ Hemoglobin>3 g/dl
Percentages of ribavirin-treated patients with a hemoglobin decline
of more than 3g/dl (light grey, P<0.01) or hemoglobin concentrations
of less than 10g/dl (grey, not significant) at week 12, stratified for the
SLC28A3 rs10868138G/rs56350726T haplotype (top; compare with
the figure 3 from ). Top, light grey columns: among the 98
noncarriers, 64.3% had a hemoglobin decrease of more than 3g/dl
(column ‘0’) whereas among the 17 carriers of this haplotype (column
‘1’) only 35.3% had a hemoglobin decrease of more than 3g/dl. Top:
grey columns: a similar yet nonsignificant tendency was found when
looking at absolute hemoglobin concentrations of less than 10g/dl.
Again, relatively more noncarriers than carriers of the haplotype had this
Nucleoside transporters and ribavirin Doehring et al.
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