Correlation of OAS1 Gene Polymorphismat Exon 7 Splice Accepter Site with Interferon-Based

Therapy of HCV Infection in Pakistan

Muhammad Imran, Sobia Manzoor,* Nasir Mahmood Khattak, Muqddas Tariq, Madiha Khalid,Farakh Javed, and Shameem Bhatti

Abstract

The most useful treatment for HCV infection worldwide is peg-interferon plus ribavirin, although the responsevaries from person to person. Hence, host genetics are significantly involved in the treatment response to HCVinfection. The 2¢-5¢ oligoadenylate synthetase (OAS) is one of the most important components of the immunesystem having significant antiviral functions. The aim of this study was to investigate the role of single nucleotidepolymorphism (SNP) at the exon 7 splice acceptor site (SAS) of OAS1 to interferon-based therapy of HCVinfection. OAS1 genotyping was performed in 140 HCV patients by restriction fragment length polymorphismpolymerase chain reaction method (RFLP-PCR). These patients were enrolled for the study in 2010–2013. OAS1SNP was also established in 120 healthy controls. Correlation of HCV genotypes, OAS1 SNP, and other factorswith response to interferon therapy were statistically analyzed by SPSS 13 software. There were no significantdifferences in the distribution of OAS1 genotypes between healthy and patients subjects. The distribution of AGand AA genotypes of OAS1 genotypes between sustained virological responders (SVRs) and the non-responders(NRs) group were also comparable. However, Pearson chi square analysis indicated that the patients possessing aGG genotype of the OAS1 gene at exon 7 SAS demonstrated significantly positive association with treatmentresponse to HCV infection ( p = 0.039). This study determined that SNP at exon 7 SAS of OAS1 was significantlyassociated with response to interferon-based therapy of HCV infection in our population.

Introduction

Hepatitis C virus (HCV) was discovered in 1989 (6).It is placed in the family Flaviviridae of viruses. This

plus-stranded RNA virus has infected about 200 millionpeople worldwide (16,44). Acute HCV infection occurswithin the initial 6 months of viral attack. After 6 months, ifthe immune system fails to clear the virus, the mild infectionleads to chronic infection. This virus is a leading cause ofchronic hepatitis in 50% to 80% of individuals (1). HCV is asilent killer as 70%–80% cases of HCV infection are withoutsymptoms. The viral infection is diagnosed after many yearsof infection, when the stage of decompensated liver occurs.Approximately 20% of chronic HCV cases progress to livercirrhosis, while approximately the same percentage ofchronic HCV patients develop hepatocellular carcinoma(7,15).

The current standard treatment for chronic HCV genotype3 (HCVG3) infections is pegylated alpha interferon (IFN)

with weight-based ribavirin, which is given for 24–48 weeks(37). Increasing data support evidence that serological andclinical factors may predict a long-term response to inter-feron-based therapy of HCV infection. These features in-volve both viral and host factors (10,18,23,28,40,41,45).Among the viral factors, genotypes and HCV RNA levelsare significantly associated with the treatment response,while among the host factors, disease duration, patient age,stage of liver fibrosis, and other genetic factors can ad-versely influence the treatment response (8,11,40). The hostgenetic factors have an imperative role in the building of aunique immune response by controlling differences amongindividuals in the stimulation of cytokines or other intra-cellular antiviral proteins (3,13). One of the most importantreasons for these differences among individuals in cytokinesproduction is attributed to single nucleotide polymorphisms(SNPs) in the human genome. SNPs also have a significantrole in the development of chronic HCV infection and re-sponse to combinational therapy of HCV infection (35,39).

Atta-ur-Rahman School of Applied Bio-Sciences, National University of Sciences and Technology, Islamabad 44000, Pakistan.*corresponding author

VIRAL IMMUNOLOGYVolume 27, Number 3, 2014ª Mary Ann Liebert, Inc.Pp. 105–111DOI:10.1089/vim.2013.0107

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Recently, genome wide association studies (GWAS) ofmore than 1600 patients revealed a strong association ofIL28B polymorphism at rs12979860 with pegylated inter-feron and ribavirin treatment. It was shown that patientswith a CC genotype at this particular position have a 2.5times or even better response to interferon-based therapy.This association was also confirmed in other ethnic groupsworldwide. Additionally, the C allele was also more prev-alent in healthy subjects as compared to chronically infectedsubjects in multi-ethnic populations, pointing its potentialrole in natural clearance of HCV infection (38).

IFN therapy stimulates several intracellular antiviralproteins. The OAS1 is one of the most important intracel-lular antiviral proteins that are stimulated by IFN therapy(21,23). The OAS1 is an IFN-stimulated enzyme that con-verts ATP into 2¢–5¢ linked oligomers of adenosine in thepresence of double-stranded (ds) RNA (31). Latent ribonu-clease L (RNaseL) is activated by 2¢–5¢ linked oligoade-nylate to degrade viral RNA and inhibit protein synthesis.Thus, 2¢–5¢ OAS protein has discriminating activity againstseveral viruses.

The 2¢–5¢ OAS gene family is comprised of OAS1, OAS2,and OAS3 as important members. The OAS1 gene is placedon human chromosome 12q24.2 encoding four isoformsp42, p44, p46, and p48 (17). Many recent studies havedemonstrated a significant role of 2¢-5¢ OAS-RNaseL systemfor clearing viral infections (22,24,30). In HCV infection,the core protein has been shown to activate the 40/46-kDaOAS1 gene promoter specifically in various human hepato-cyte-derived cell lines. The promoter induction of OAS1gene was further enhanced in the cells treated with inter-feron alpha (27). Thus, any SNP with an effect on OAS1activity may also have a role in determination of resistance/susceptibility to HCV infection and also interferon-basedtherapy of HCV infection.

Although the OAS1 gene has a very important contribu-tion in antiviral interactions, there are many variations in itssequence. Approximately 36 SNPs have been noted in thisgene. The strongest SNP that is associated with OAS1 ac-tivity is found at exon 7 SAS. This SNP (rs 10774671) isalso suggested to influence susceptibility to type 1 diabetes(5). The normal splicing at exon 7 of OAS1 gene is achievedby the presence of AG sequence at this position. The Gallele at this position retains the splice site and confershigher enzymatic activity (p46), whereas the presence of Aallele at this position (AA at acceptor site) ablates the splicesite and confers lower enzymatic activity (p48 and p52) tothe OAS1 gene (4,20). Thus, the genetic variation at exon 7SAS of OAS1 makes it an excellent candidate gene that cansignificantly predict the response to IFN-based therapy inHCV or HBV patients (20,36).

During the last decade, there is a continuous struggle fordiscovering new treatment options of HCV infection, sinceinterferon therapy is long, expensive, and associated withsevere side effects. Different approaches for searching newtreatment options of HCV infection involve small organiccompounds, siRNA, shRNA, peptide methods, and targetingvarious host factors that have important role in HCV lifecycle. Recently, two NS3 protease inhibitors, Telaprevir andBoceprevir, have been added to peg-interferon plus ribavirintreatment of HCV genotype 1 (19). Many direct actingagents such as TMC-435, BI 201335, ABT-333, and Debio

025 are in stage III of clinical development (16). How thesesmall genetic variations would affect therapy will requirefurther research.

Materials and Methods

Subjects

The blood samples of chronic HCV patients were col-lected from the Gambat Institute of Medical Sciences(Province; Sindh) and Saidu Group of Teaching Hospital(Province; Khyber Pakhtun Khwa). Initially, 186 chronicHCV patients were enrolled in the study. Patients receivedIFN + ribavirin treatment for 6 months, except HCV geno-type 1 and 4 patients who received the combinational therapyfor 6 more months. Subjects who discontinued treatmentdue to any reason were excluded from the study. Finally,140 chronic HCV infected patients remained. One hundredand twenty healthy controls (negative for HCV Ab and RNAas well as hepatitis B surface antigen) were also involved inthe study. The institutional Ethics Committee gave permissionfor the study. The consent of both the patients and healthysubjects were taken for the genetic testing of OAS1 geneprior to enrollment in the study. The patients were moni-tored for 6 months after the end of therapy and were groupedinto SVR and IFN non-responder (NR-IFN) on the basis oftreatment response. All patients who tested negative forHCV RNA (using both nested RT-PCR and real-time PCR)after 6 months of treatment completion were grouped intoSVR, while those patients who did not show SVR weregrouped in NR.

Determination of OAS1 gene polymorphism

The blood samples of all healthy controls and HCV-infected subjects going through IFN-based therapy werecollected in EDTA tubes. DNA extraction was performed byusing a Genomic DNA mini kit (Invitrogen; Cat no. 1820-02) according to manufacturer’s instruction, followed by itsquantification with Nanodrop (Eppendorf Biophotometer,Germany). RFLP-PCR was used for genotyping of OAS1gene. The technique is widely used for mutation detectionworldwide (2,43). We used reported primers to amplify203 bp PCR fragments of OAS1 gene. Primer sequencesused were as follows: rs10774671; forward, 5¢-TGCAATGCAGGAAGACTCC-3¢ and reverse, 5¢-TGCAGGTCCAGTCCTCTTCT-3¢ (9). This product also covers ourdesired SNP. PCR amplification was carried out in 25 lLmixture containing 100–500 ng DNA, 2.5 mM MgCl2(Fermentas), 10 pM of each primer, 2x PCR buffer (Fer-mentas), 200 mM dNTP’s (Fermentas), and 2 units of TaqDNA polymerase (Fermentas). Cycling condition involvedlong denaturation at 95�C for 5 min, followed by 35 cycles,each at 94�C for 30 sec; annealing at 62�C for 30 sec; andextension at 72�C for 1 min. Cycling condition was followedby a final extension step at 72�C for 10 min.

The restriction enzyme, AluI (Fermentas), was used forthe digestion of amplified product. 8 lL of the PCR productwas digested by 5 units of AluI enzyme in a total volumeof 20 lL. The incubation period was for 4 h at 37�C. 10 lLof the digested product was run on a 3% agarose gelstained with ethidium bromide. The electrophoresedproduct was observed under UV light of Wealtec gell doc

106 IMRAN ET AL.

system. HCV genotyping was performed according toOhno (29). The protocol is already optimized protocol inour laboratory (32).

Statistical analysis

Hardy-Weinberg equilibrium was applied to determinethe distribution of OAS1 genotypes in both patients andhealthy controls. Statistical analysis between the SVR andNR groups were determined by SPSS 13. Pearson Chisquare test was applied on treatment responders and non-responders to determine association between treatment re-sponse and OAS1 genotypes. A two-tailed p value £ 0.05was considered to be statistically significant.

Results

Patients and healthy subjects

All the patients and controls were descendents ofPakistani origin. Groups of SVR and NR chronic HCV pa-tient groups were compared to reveal any of the clinical,pathological, biochemical, or virological parameters asso-ciated with specific patterns of treatment response. Datawere analyzed as mean – SD. The univariate analysis ofclinical and virological factors was compared between SVRand NR; no significant differences were revealed betweenthe two groups (Table 1).

Analysis of OAS1 polymorphism by RFLP-PCR

The presence of the AA genotype at the exon 7 SAS wasspecified by complete digestion of the 203 bp PCR productinto two fragments: 150 bp and 53 bp by AluI enzyme(AGCT). The GG genotype at the exon 7 SAS was specifiedby absence of AluI site where A to G polymorphism createdan indigestible sequence (GGCT) with a retained intact of203 bp PCR fragment. The heterozygous (AG) condition atthe exon 7 SAS of OAS 1 gene was represented by threefragments of 203 bp, 150 bp and 53 bp on agarose gelelectrophoresis (Fig. 1).

Frequency of OAS1 genotypes in healthyand patient groups

One hundred and twenty healthy subjects from both sexeswere also enrolled in this study to determine whether the

tested SNP represents an important genetic component ofthe Pakistani population. The frequency of OAS1 genepolymorphism at exon 7 SAS was tested in both healthy andpatient groups to unravel any association of this particularSNP with chronic HCV infection. The distribution fre-quency of OAS1 genotypes among the healthy subjectswere; 62%, 33%, and 5% for genotypes GG, GA, and AA,respectively, while the frequency distribution of OAS1 ge-notypes among the patient subjects were; 69%, 25%, and6% for OAS1 genotypes GG, GA, and AA. The resultsshowed that G allele (70%) tends to be more frequent than Aallele (30%) in both control and infected individuals. Therewere no significant differences in OAS1 genotypes distri-bution between healthy and patient groups. This indicatesthat there is no association of any OAS1 genotype with thenatural clearance of HCV infection (Fig. 2).

Frequency of OAS1 genotypes in SVR and NR groups

The patient group was further divided into SVR and NRgroups in order to find any correlation of this particular SNPwith interferon-based therapy of HCV infection in ourpopulation. The distribution frequency of OAS1 genotypesbetween the two groups were compared and analyzed byPearson Chi square analysis. It was found that the frequencyof GG genotype was significantly higher in SVR group thanNR group (73% vs. 53%; p = 0.039). The frequency of othertwo genotypes of OAS1 gene (i.e., AG and AA) at thisparticular position were comparable in both SVR and NRgroups, representing a nonsignificant association of thesegenotypes with interferon-based therapy of HCV infection.The high prevalence of GG in SVR group suggests thatindividuals possessing this genotype at exon 7 SAS of OAS1gene are better responders to interferon-based therapy ofHCV infection (Fig. 3).

HCV genotypes vs. OAS1 genotypes

The most common HCV genotype was 3. Previous studieshave also shown that HCV genotype 3 is the most prevalentgenotype in Pakistan (32). For genotype 3 (both 3a and3b) most of the patients possessing OAS1 GG genotypeachieved SVR (i.e., 39 out of 41 patients). Patients withHCV genotype 3 and OAS1 AG genotype also succeeded inachieving SVR (39 out of 46) (Table 2).

Table 1. Demographic Profile of Patients and Healthy Controls

Variables SVRs NRs Healthy Controls P** values

Numbers 115 25 120 –Age* 36.28 – 12.11 37.32 – 8.24 25.11 – 8.21 0.608Gender 72 male; 43 female 11 male; 14 female 60 male; 60 female 0.086Baseline viral

load***1.11 · 105

(0.045 · 105 – 54.7 · 1055.64 · 105

(0.4 · 105 – 98.7 · 105)– 0.241

rs10774671genotype

GG 84 13 74 0.039AG 26 9 40 0.161AA 5 3 6 0.135

*Age is taken as STDEV – Mean; Baseline viral load*** is taken as median and ranges; **p values for age are calculated by independentt-test, for gender by Spearman correlation, for genotype by Pearson chi square test.

OAS1 GENE POLYMORPHISM 107

Discussion

HCV infection is a major global health problem. An es-timated 10 million people (6%) in Pakistan are infected withHCV (42). Only 20%–30% of patients are expected to befree of the virus within 6 months of infection. 70%–80% ofacute HCV infections become chronic. During the last 2decades, extensive research work has been done to unravelthe association of pathogen and host’s genetics on patho-genesis of common diseases. For this purpose, SNPs in thehuman genome are specifically targeted to find their asso-ciations with susceptibility, resistance, or treatment responseto various diseases (12,26). Both host and viral factors play

an important role in outcome of HCV infection and itsresponse to interferon-based therapy. HCV possesses ex-tensive sequence diversity in its genome because of itserror-prone RNA-dependent RNA polymerase resulting indifferent subtypes and genotypes (34). OAS1 gene is animportant component of the signaling pathway of interferon.Its homolog in mice has demonstrated susceptibility againstflaviviruses (25). Our results suggest that there is a signifi-cant association of OAS1 gene polymorphism at exon 7 SASwith interferon-based therapy of HCV infections, confirm-ing the previous reports of El-Awady et al. (9). Patientswith GG genotype at this position achieved more SVR( p = 0.039) on interferon-based therapy as compared to thepatients possessing either GA or AA genotypes. Our resultssuggest that G allele conferred better functional capability toOAS1 enzyme towards the viral clearance. These resultsestablished the earlier reports that G allele at exon 7 SAS ofOAS1 gene is associated with higher enzyme activity (P46).On the other hand, A allele at this site (AA genotype) re-sulted in enzyme with higher weight but lower activity(P48). Furthermore, it was shown that patients possessingAA genotype are poorer responders to interferon-basedtherapy in an Egyptian population. The response rate tointerferon-based therapy of HCV infection for OAS1 genepolymorphism at exon 7 SAS was gradually increased fromAA to AG and then to GG genotype (2). A recent reportfrom China has shown that three SNPs of OAS1 gene(rs2660 G/A, rs10774671 G/A, and rs3741981 G/A) are inlinkage disequilibrium. The A allele at these three loci wassignificantly more common in chronic HCV patients ascompared to healthy controls ( p = 0.001). Moreover, it wasfound that A allele at these loci also increased the risk ofchronic HCV infection (46). The minor allele frequency(MAF) (i.e., G allele) in our population was 0.496. Datafrom Ensembl genome browser show that MAF in Africa,America, and Han Chinese in Bejing, China is 0.587, 0.301,and 0.309, respectively.

There is substantial number of studies showing that hu-man genetic differences have an important role in diseaseprogression and treatment response. Recently, it has been

FIG. 1. Electrophoresis pattern of three different indi-viduals by RFLP-PCR analysis. Lane M, 50 bp marker; Lane1, homozygous A genotype; Lane 2, homozygous G geno-type; Lane 3, heterozygous A/G genotype of OAS1 gene.

FIG. 2. Comparison of OAS1 polymorphism distribution between healthycontrols and chronic hepatitis C patients.

108 IMRAN ET AL.

discovered that SNPs of the interferon lambda group arestrongly associated with interferon plus ribavirin therapy ofHCV infection (12). Further investigation of IL28Brs12979860 has demonstrated that this variant is actuallylocated within IFNL4. There are two alternative forms ofIFNL4: IFNL4 deltaG that produces protein while theother alternative form is without deletion and produces noprotein. IFNL4 deltaG was found to be more prevalent inAfrican American and European American populations. Thepresence of IFNL4 deltaG protein was responsible forpoorer response and viral clearance in this population. Itwas concluded that in African Americans and EuropeanAmericans, IFNL4 deltaG is better predictor of pegylatedinterferon plus ribvirin treatment of HCV infection thanIL28B genotype. However, currently new direct actingagents against HCV infection are being developed andthe role of IFNL4 deltaG in the era of DAA is still to bedetermined (14).

The present study confirmed that mutation at exon 7 SASof OAS1 gene is significantly associated with interferon-based therapy of chronic HCV infection. However, the mainlimitation of our study was a small sample size. Althoughthe sample size of HCV genotype 3 was larger, the samplesize of other genotype was not large enough to apply sta-

tistics on each genotype separately. Moreover, we did notstudy other SNPs of OAS1 gene that are in linkage dis-equilibrium with this particular SNP. The haplotype study ofOAS1 gene may further highlight the importance of OAS1gene in interferon-based therapy of HCV infection. How-ever, our pilot study highlighted the importance of this SNPfor interferon-based therapy of HCV infection in our pop-ulation. Currently, there are discoveries of new therapeuticoptions for HCV infection; the importance of these SNPs inassociation with these new therapeutic options will also bestudied.

Conclusion

The current SOC for HCV infection is interferon plusribavirin but it is expensive and associated with serious sideeffects. It is very necessary to have some predicativemarkers for the treatment response. We evaluated the dis-tribution of OAS1 genotypes and their response to interferonplus ribavirin treatment in chronically infected HCV pa-tients of the Pakistani population. There were no significantdifferences in distribution of OAS1 polymorphism betweenpatients and healthy subjects. However, the frequency ofOAS1 GG genotypes was higher in the SVR group than NRgroup, suggesting the protective role of this genotype inresponse to interferon-based therapy of HCV infection. Inconclusion, OAS1 polymorphism at exon 7 SAS is an in-dependent determinant that can predict outcome of inter-feron plus ribavirin therapy in case of HCV infection in ourpopulation.

Acknowledgments

SM and MI conceived the study, and did the analysis. MIand NMK performed all experiments. MI searched the lit-erature and drafted the manuscript. NMK, MT, MK, and FJhelped MI in sample collection and data analysis. SM crit-ically reviewed the manuscript. SM supervised and designedthe study, gave final approval and helped MI in performingthe research. All authors have read and approved the finalversion of the manuscript.

FIG. 3. Comparison of OAS1 polymorphism distribution between SVRs andNRs. The p values were calculated by Pearson Chi square test.

Table 2. Prevalence of OAS1 Genotypes

in Chronic HCV Patients

HCVgenotypes OAS1 genotypes

Total number GG SVR + NR AG SVR + NR AA SVR + NR3a (79) 27 + 1 37 + 5 8 + 11a (14) 3 + 0 4 + 4 1 + 23b (21) 12 + 1 2 + 1 4 + 11b (5) 1 + 1 2 + 0 0 + 1

Untyped (9) 2 + 0 3 + 3 1 + 0Co-infection

(10)1 + 1 4 + 2 1 + 1

4 (2) 1 + 0 1 + 0 0 + 0

OAS1 GENE POLYMORPHISM 109

Funding: Financial support in part by National Universityof Sciences and Technology (NUST), H-12, Islamabad,Pakistan, and Higher Education Commission of Pakistan ishighly acknowledged.

Author Disclosure Statement

The authors declare that they have no competing interests.

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Address correspondence to:Dr. Sobia Manzoor

Atta-ur-Rahman School of Applied Bio-SciencesNational University of Sciences and Technology

Islamabad 44000Pakistan

E-mail: lcianunique@yahoo.com;dr.sobiamanzoor@asab.nust.edu.pk

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