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Single-nucleotide polymorphisms in GALNT8 are
associated with the response to interferon therapy
for chronic hepatitis C
Rikita Nakano,1,2Toshiro Maekawa,1Hiromi Abe,3Yasufumi Hayashida,1
Hidenori Ochi,1,3Tatsuhiko Tsunoda,4Hiromitsu Kumada,5
Naoyuki Kamatani,6Yusuke Nakamura7and Kazuaki Chayama1,3
Received 17 May 2012
Accepted 26 September 2012
1Laboratory for Digestive Diseases, RIKEN Center for Genomic Medicine, Hiroshima, Japan
2Pharmacology Research Laboratories, Drug Research Division, Dainippon Sumitomo Pharma Co.,
3Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical &
Health Sciences, Hiroshima University, Hiroshima, Japan
4Laboratory for Medical Informatics, RIKEN Center for Genomic Medicine, Yokohama, Japan
5Department of Hepatology, Toranomon Hospital, Tokyo, Japan
6Laboratory for International Alliance, RIKEN Center for Genomic Medicine, Yokohama, Japan
7Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, University
of Tokyo, Tokyo, Japan
New anti-hepatitis C virus (HCV) therapeutics developed recently are more effective and lead to
improvements in sustained viral response. However, interferon (IFN) monotherapy is still used to a
limited extent for fear of adverse effects. This study investigated host genetic factors affecting the
IFN response in patients with chronic hepatitis C (CHC). Using a two-step design, a large-scale
association screening including 1088 Japanese CHC patients treated with IFN was performed
employing ~70000 gene-based single-nucleotide polymorphisms (SNPs). Replication was
tested in an independent Japanese cohort of 328 patients. Fine-mapping and functional analyses
were also performed. Through two-step screening, it was found that rs2286580 in intron 6 of the
gene encoding N-acetylgalactosaminyltransferase 8 (GALNT8) on chromosome 12 was
significantly associated with a sustained viral response (combined P53.9?10”6, odds ratio 1.52,
95% confidence interval 1.29–1.82). The association was replicated in an additional cohort of
328 Japanese patients. In subgroup analysis, GALNT8 variants were associated with treatment
outcome independently of HCV genotype. By contrast, the outcome of pegylated IFN and ribavirin
combined therapy was not affected by the SNP. Fine-mapping analysis revealed that the
association peak was at rs10849138 in intron 6 of GALNT8. Allele-specific transcription analysis
demonstrated that GALNT8 expression was upregulated by an unfavourable allele of the variant. A
luciferase reporter assay demonstrated that overexpression of GALNT8 attenuated IFN-a-induced
gene transcription via the IFN-stimulated response element. These results suggest that GALNT8
variants contribute to the response to IFN therapy against CHC, providing a new insight into
antiviral mechanisms of IFN.
Type I interferons – alpha interferon (IFN-a) and beta
interferon – have been widely used as antiviral agents for
hepatitis C virus (HCV) infection. Although the improved
efficacy of pegylated (PEG)-IFN-a plus ribavirin combined
therapy is well recognized, it was shown that .50% of
patients infected with HCV genotype 1b and ~20% of
those with genotype non-1b still failed to eradicate the
virus (NIH Consensus Statement on Management of
Hepatitis C, 2002). Addition of telaprevir to therapy with
PEG-IFN and ribavirin is a new strategy that improves the
eradication rate of HCV genotype 1. However, telaprevir is
Three supplementary figures and three tables are available with the
online version of this paper.
Journal of General Virology (2013), 94, 81–89
044396G2013 SGMPrinted in Great Britain81
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associated with increased rates of adverse effects including
rash and anaemia (He ´zode et al., 2009; McHutchison
et al., 2009), which can be severe enough to necessitate
discontinuation of treatment.
Both viral (e.g. HCV genotype and serum HCV RNA
levels) and host factors (e.g. age, sex, race, liver fibrosis
and obesity) have been shown to be associated with the
outcome of IFN therapy. Several genetic factors including
cytokine-, chemokine- and IFN-stimulated genes have
been reported to influence the response to IFN therapy.
We have reported that polymorphism in the gene
encoding mitogen-activated protein kinase-activated pro-
tein kinase 3 (MAPKAPK3) is significantly associated with
IFN therapy in patients infected with HCV genotype 1b
(Tsukada et al., 2009). Recently, a positive association of a
polymorphism in the interleukin IL28B gene with the
outcome of PEG-IFN plus ribavirin therapy has been
reported (Ge et al., 2009), and this association has been
replicated by other investigators (Rauch et al., 2010;
Suppiah et al., 2009; Tanaka et al., 2009; Thomas et al.,
In this report, we have described the results of a large-scale
case–control association analysis for responsiveness to IFN
therapy in chronic hepatitis C (CHC) using gene-based
single-nucleotide polymorphisms (SNPs). We identified
common variants in the N-acetylgalactosaminyltransferase
8 (GALNT8) gene that were significantly associated with
treatment outcome. Moreover, we have provided func-
tional evidence suggesting that GALNT8 may modulate
signal transduction of IFN-a-mediated antiviral activity.
In the first screening using 65 sustained responders (SRs)
and 118 non-responders (NRs), the success rate of
genotyping was 90.3% (72739 SNPs). After quality-control
filtering, 58809 autosomal SNPs remained. The distri-
bution of association P values (Manhattan plot) of the first
screening is shown in Fig. S1 (available in JGV Online),
and the quantile–quantile plots in Fig. S2. To identify and
correct for possible population stratification, the genomic
control method was used (Lewis, 2002). The calculated
genomic control l was 1.04, indicating that the effect of
population stratification was minimal (Devlin & Roeder,
1999). The top 100 SNPs are listed in Table S1. The
quantile–quantile plot indicated a number of SNPs
showing a stronger association than would be expected
by chance but that were not statistically significant. We
selected 689 SNPs having P,0.01 under the allelic model
for the second screening, consisting of 417 SRs and 488
NRs. After the two-step screening, we found that SNP
rs2286580 [combined P53.961026; odds ratio (OR) 1.52;
95% confidence interval (CI) 1.29–1.82] located in intron 6
of GALNT8 and rs2267552 in intron 9 of NADH
dehydrogenase (ubiquinone) 1a subcomplex, subunit 9
(NDUFA9) (combined P57.861026; OR 1.49; 95% CI
1.32–1.78) exceeded the significance level of P,1.3661025
using the joint analysis (Skol et al., 2006) (Table 1). They
were both on chromosome 12 and were in strong
linkage disequilibrium (LD) with each other (r250.81)
(Fig. 1). This association was validated in an independent
replication cohort consisting of 94 SRs and 234 NRs
(P53.361023) (Table 1). The remaining SNPs carried
to the second screening eventually showed a lack of
According to the genotype data of Japanese individuals
from the Phase II HapMap database, rs2286580 and
rs2267552 are located within the same LD block (defined
by Gabriel et al., 2002) on chromosome 12p13, spanning
about 104 kb (Fig. 1, lower panel). Fourteen tag SNPs were
selected from 127 SNPs within the LD block surrounding
rs2286580 using HapMap JPT data (minor allele frequency
.0.05, r2.0.8; de Bakker et al., 2005) and were genotyped
in all subjects enrolled in the study. An additional 25 SNPs,
which were in the same bin as rs2286580 (r2.0.8), were
also genotyped and analysed because rs2286580 had the
lowest P value among these tag SNPs. Finally, the most
significant association (P52.261028) was observed at
rs10849138 in intron 6 of the GALNT8 gene (Fig. 1, upper
panel, and Table 2). We also examined associations with
respect to haplotype. None of the haplotypes showed
stronger associations than the single-marker association of
rs10849138 (data not shown).
HCV genotype-stratified analysis
Because each sample set except for the first set of the
screening stage contained various HCV genotypes, subjects
were stratified according to HCV genotype to evaluate
whether GALNT8 variants were associated with treatment
outcome independently of HCV genotypes. We found that
rs10849138 (C/G) was significantly associated within
subjects chronically infected with HCV genotypes 1b and
2a (OR51.51, P52.361024, and OR51.71 P55.061024,
respectively), whereas subjects chronically infected with
HCV genotype 2b showed the same tendency but this was
not significant (OR51.60, P50.064). No heterogeneity was
observed in the ORs among HCV genotypes for the SNP by
the Breslow–Day test. The estimated combined OR by the
Mantel–Haenszel method was 1.58 (95% CI 1.33–1.86)
(Table 3). We further genotyped rs3792323 of MAPKAPK3,
which we have reported previously in subjects treated with
IFN monotherapy (Tsukada et al., 2009) (Table S2). The
impacts of GALNT8 and MAPKAPK3 were comparable in
genotype 1b, whereas in non-1b genotypes, GALNT8
seemed to have a stronger effect on treatment than
MAPKAPK3. In contrast, IL28B showed the most signifi-
cant association in genotype 1b.
R. Nakano and others
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GALNT8 variants affect HCV response to IFN-a