Elsevier

Journal of Hepatology

Volume 54, Issue 4, April 2011, Pages 629-639
Journal of Hepatology

Research Article
A single nucleotide polymorphism in activated cdc42 associated tyrosine kinase 1 influences the interferon therapy in hepatitis C patients

https://doi.org/10.1016/j.jhep.2010.07.021Get rights and content

Background & Aims

Cdc42 is a Rho family GTPase protein and was recently implicated in mediating hepatitis C virus (HCV) infectivity. This study examines the association between Cdc42-related gene and interferon (IFN) therapy in HCV patients.

Methods

We analyzed the associations between the outcome of IFN therapy and 17 tagging single nucleotide polymorphisms (SNPs) within two genes involved in Cdc42 signaling (CDC42 and ACK1). A total of 295 out of the 409 study subjects were sustained responders (SR) and 114 were non-responders (NR). Replication was performed using an independent set of 794 IFN-treated patients.

Results

SNP rs2278034 [A/G] in intron 11 of activated Cdc42 associated tyrosine kinase (ACK) 1 was associated with the outcome of IFN therapy (p = 6.4 × 10−4). Replication analysis confirmed the association (p = 2.2 × 10−3) for patients treated with IFN monotherapy, but the association was not significant for pegylated-IFN-plus ribavirin therapy. Analysis using published HapMap expression data revealed that ACK1 expression correlates with IFN-stimulated gene (ISG) expression independently of ethnicity, but the relationship between rs2278034 and ACK1 expression was observed only within Asian populations. Over-expression of ACK1, but not the kinase-inactive mutant, increased ISG transcription in Huh7 cells. ACK1 expression enhanced the IFN-stimulated response element (ISRE) and interferon-γ-activated site (GAS) promoter activity through tyrosine phosphorylation of signal transducers and activators of transcription (STAT) 1. Furthermore, ACK1 over-expression in HCV-N replicon cells inhibited HCV replication.

Conclusions

SNP rs2278034 in ACK1 is associated with IFN therapy outcome in patients with HCV. ACK1 may play a role in innate and IFN-induced antiviral action against HCV.

Introduction

Hepatitis C virus (HCV) infection is a major cause of liver disease. Nearly 200 million individuals worldwide are infected by HCV [1], [2], the majority of whom develop persistent infection that may lead to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. It is well known that the efficacy of interferon (IFN)-based therapies depends on the HCV genotype and serum HCV-RNA level [3]. The current antiviral therapeutic regimen is pegylated IFNα used in combination with the nucleotide analog ribavirin. However, although ribavirin improves the effect of IFN, the eradication rate of HCV is only about 50% in patients infected with genotype 1b and approximately 80% in patients infected with other genotypes [4], [5], [6].

Several host factors that influence the efficacy of IFN therapies have been identified recently, including age, sex, liver fibrosis, and obesity [7], [8]. Recent studies, including our own, also demonstrated that the response to IFN therapy is associated with several single nucleotide polymorphisms (SNPs) in genes including: myxovirus resistance protein A (MxA), mannose-binding lectin (MBL), low-molecular-mass polypeptide 7 (LMP7), osteopontin (OPN), IFNα receptor 1 (IFNAR1), mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3), and interleukin 28B (IL28B) [9], [10], [11], [12], [13], [14], [15].

During the initial steps of infection, HCV virus is taken up by hepatocytes in the liver in a multistep process involving a number of factors [16]. A better understanding of the mechanisms of HCV entry into the host cells could have important implications for developing strategies to prevent infection and therapies to treat chronic HCV disease. Initial host cell attachment seems to involve glycosaminoglycans, low-density lipoprotein receptor, scavenger receptor class B type I, CD81, and claudin-1 [17], [18], [19], [20], [21]. It was demonstrated recently that CD81 increases the levels of three Rho-family GTPase proteins; Rac1, Rho, and Cdc42 [22]. Rho GTPases are molecular switches that cycle between an inactive GDP-bound state and an active GTP-bound state. Inhibition of Rac1 and Cdc42 with specific small interfering RNAs (siRNAs) or inhibitors significantly reduced the infectivity of HCV, with Cdc42 knockdown producing the most dynamic reduction in infectivity. These findings implicated Cdc42 as a key molecule directing HCV entry and suggesting that polymorphisms in the CDC42 gene that modulate protein expression or function might affect the response to HCV therapies.

In addition, non-receptor protein tyrosine kinase (PTK) proteins such as Janus kinase, which are essential for IFN activity, transmit signals into the host cells. ACK1 is one such PTK that also binds selectively to Cdc42 in humans [23], [24], [25]. ACK1 can phosphorylate several proteins and mediate EGF signaling to Rho-family GTPases including Cdc42 [26]. However, whether ACK1 gene expression affects the outcome of IFN therapy for HCV infection remains unknown.

The present study examined whether CDC42 and ACK1 SNPs are associated with the different response levels to IFN therapy using a tagging-SNP approach. The results provide genetic and functional evidence for the role of the ACK1 gene activity in the responsiveness to IFN therapy.

Section snippets

Patient and DNA samples

We examined 1203 patients with chronic HCV infection who were referred to the Hiroshima University Hospital, hospitals belonging to the Hiroshima Liver Study Group (http://home.hiroshima-u.ac.jp/naika1/hepatology/english/study.html) or Toranomon Hospital for IFN monotherapy. Each patient was treated with 6 million units of IFNα administered intramuscularly every day for 8 weeks, followed by the same dose twice a week for 16 weeks, for a total dose of 528 million units. In the association study, we

Association between tagging-SNPs in ACK1 and outcome of IFN therapy

We analyzed the 17 tagging SNPs for CDC42 and ACK1 in the first set of 114 SR (SR-1) and 295 NR (NR-1) subjects, all of which were successfully genotyped (Table 2). All SNPs passed the Hardy–Weinberg equilibrium test. Only one SNP at intron 11 of ACK1 (rs2278034) was significantly associated with the therapeutic efficiency of IFNα. The allele dose-dependent association of rs2278034 with responsiveness to IFN therapy is shown in Table 3. Under a recessive model for the G allele of rs2278034, the

Discussion

Cdc42 has been implicated in mediating the entry of HCV into hepatocytes, but how Cdc42 affects the response of HCV patients to IFN therapy is unknown. This study therefore examined the genetic association between Cdc42-related genes and efficacy of IFN therapy and identified the ACK1 gene at chromosome 3q29 as a new candidate susceptibility gene.

Association analysis revealed that HCV-infected Japanese carriers of the homozygous ACK1 rs2278034 GG genotype were more likely to achieve sustained

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Financial support

This study was partly supported by RIKEN and by Dainippon Sumitomo Pharma Co., Ltd.

Acknowledgments

This work was supported by the Japanese Millennium project. We thank all members of the Center for Genomic Medicine of RIKEN and Dainippon-Sumitomo Pharma Co., Ltd. for supporting this study. We are also grateful to members of the Hiroshima Liver Research Group and Toranomon Hospital, Japan, for their support of this study.

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