Predictive Potential of IL-18 - 607 and Osteopontin- 442 Polymorphism in Interferon-Based Therapy

of HCV Infection in the Pakistani Population

Muhammad Imran, Sobia Manzoor,* and Fahed Parvaiz

Abstract

The adaptive immune system plays an important role in response to interferon plus ribavirin treatment ofhepatitis C virus (HCV) infection. Cytokines play a significant role in the adaptive immune system. Theproduction of cytokines may be regulated by single nucleotide polymorphisms (SNPs). This study was designedto examine the correlation of some important SNPs of cytokines with interferon plus ribavirin treatment of HCVinfection in the Pakistani population. We followed 140 chronic HCV-infected patients in our study. All of thesepatients had completed their planned course of interferon plus ribavirin treatment. We also considered 120healthy subjects as controls. The detection of interleukin-18 (IL-18) SNPs was performed by tetra-primersamplification-refectory mutation system polymerase chain reaction, while for genotyping of osteopontin (OPN),transforming growth factor beta (TGFb), and N-acetylgalactosaminyltransferase 8 (GALNT8) SNPs, allele-specific polymerase chain reaction was performed. The distribution of the IL-18 - 607AA genotype variedsignificantly between healthy control and patient groups. Its distribution was significantly high in healthysubjects than HCV patients ( p = 0.031), signifying its potential involvement in the natural clearance of HCVinfection. The occurrence of the - 607AA genotype of IL-18 was also significantly higher in the sustainedvirological group (SVR) than in the nonresponder (NR) group ( p = 0.046), highlighting its protective in-volvement in the treatment outcome of chronic HCV infection. The frequency of the OPN - 442TT genotypewas higher in the SVR group than in the NR group ( p = 0.034), indicating a significant possible role of thisgenotype in therapy for HCV infection. No important association was found between TGFb and GALNT8genotypes and the natural clearance and treatment response of HCV infection. IL-18 - 607AA and OPN - 442TTgenotypes can be used as positive predictive markers of interferon plus ribavirin treatment of HCV infection in thePakistani population.

Introduction

Hepatitis C Virus (HCV) is the main cause of liverdisease, infecting about 200 million people across the

world (22). About 10 million people are infected in Pakistan(1). Statistical analysis shows that 10–20% of patients natu-rally clear the virus, while the remaining 80–90% of patientsprogress to chronic stage. One out of every three chronicHCV patients develops cirrhosis and hepatocellular carci-noma (HCC). This suggests that the response to infectionvaries from person to person. Recent studies have adequatelydemonstrated that HCV pathogenesis is a multifactorial phe-nomenon, involving both viral and host factors. Publisheddata have established that the outcome of HCV infection is

mainly dependent on the competence of innate and adaptivearms of the immune system (11,28,47). The current standardtreatment against the most difficult HCV genotype, genotype1 infection, is a triple therapy based on pegylated interferon,ribavirin, and a protease inhibitor, either boceprevir or tela-previr (23). Recently, the Food and Drug Administrationapproved two new oral antiviral agents, simeprevir and so-fosbuvir, into the drug regimen of HCV patients, along withpegylated interferon and ribavirin. Simeprevir is a proteaseinhibitor that is administered for 24 weeks. Its reported sus-tained virological (SVR) rate for HCV genotype 1 is 80%.Sofosbuvir is a polymerase inhibitor that is given for 12weeks. Its reported SVR rate for HCV genotypes 1, 4, 5, and 6is 90% (42).

Atta-ur-Rahman School of Applied Bio-Sciences, Department of Healthcare Biotechnology, National University of Sciences andTechnology (NUST), Islamabad, Pakistan.

*Corresponding Author and Principal Investigator.

VIRAL IMMUNOLOGYVolume 27, Number 8, 2014ª Mary Ann Liebert, Inc.Pp. 404–411DOI: 10.1089/vim.2014.0044

404

Interferon-based therapy is mostly effective against HCVgenotype 3, which is the most highly distributed genotypein the Pakistani population (2). Interferon-based therapydecreases serum HCV-RNA levels in a biphasic pattern.The rapid decline of HCV-RNA levels within 1–2 daysmight be attributed to the direct interaction of interferon onHCV production. On the other hand, the decline of HCV-RNA levels in the second phase might be attributed to cell-mediated immune response (35). The decline of HCV-RNAlevels in SVR patients is more prominent than non-SVRpatients, indicating that the efficacy of interferon-basedtherapy is not only dependent on viral factors but also onhost factors (54).

Previously it has been shown that vulnerability to HCVinfection and response to interferon plus ribavirin therapy isregulated at different function levels, such as the productionof cytokines by various cells, single nucleotide polymor-phisms (SNPs) of different genes, and antigen presentation(11,28,47). Cytokines are secreted by most of cells, forexample immune cells, endothelial cells, fibroblasts, and arange of stromal cells. Cytokines are important regulators ofimmune responses. There are inter-individual variations incytokines production, which may be attributed to SNPs inthe DNA of these cytokines. These variations influence thebalance between pro-inflammatory and anti-inflammatorycytokines, and might affect the severity as well as thetreatment outcome of HCV infection (6).

Interleukin-18 (IL-18) is an inflammatory cytokine that islargely secreted by monocytes, macrophages, and immaturedendritic cells. IL-18 synergizes the effect of IL-12 andinduces a Th-1-mediated immune response, while in theabsence of IL-12, it induces a Th2-mediated immune re-sponse. It also activates the secretion of two other importantcytokines: tumor necrosis factor alpha (TNF-a) and inter-feron gamma (IFN-c). TNF-a regulates the secretion ofIL-18 by stimulating the secretion of IL-18 binding protein(IL-18BP) (53). Interferon therapy of chronic HCV patientsleads to a 3–24-fold increased production of IL-18BP toregulate inflammation and fibrosis development (31). Gen-erally, the production of IL-18 is considerably higher inchronic HCV patients, suggesting its critical function indisease progression (53). The enhanced production of IL-18is associated with hepatic injury (48) and poor treatmentresponse to interferon therapy (3). It has been establishedthat IL-18 production remains low in SVR patients duringtreatment (52). Enhanced secretion of IL-18 and IL-18BPhas been reported in Chickungunya virus patients as com-pared with controls. Thus, virus infection leads to increasedproduction of IL-18 and IL-18BP (9). Earlier studies havereported that IL-18 production is mainly dependent on IL-18genetic variants -607C/A (rs1946518) and -137G/C (rs187238).These SNPs are linked with interferon plus ribavirin therapyof HCV (30), HBV (24,51), and human immunodeficiencyvirus (HIV) infection (41). We selected these two SNPs tofind their correlation with natural clearance and treatmentoutcome of HCV infection in our population.

TGF-b is an anti-inflammatory cytokine that suppressesthe development and cytotoxicity of natural killer (NK)cells. It blocks the secretion of IFN-c and IL-12. SNPs ofTGF-b are responsible for its variable production (18), livercirrhosis (37), and natural virus clearance (25). We con-sidered the correlation of TGF-b functional variants at co-

don 10 T/C (rs1982073) and codon 25 G/C (rs1800471) withHCV infection and treatment outcome.

OPN is an extracellular matrix (ECM) that is mainlyproduced by bone and kidney. It has a wide range of bio-logical activities, which involve cell adhesion, proliferation,and migration (16,44). Recently, the involvement of OPNpromoter variant in HCV infection has been investigated. Itwas found that elevated levels of OPN mRNA and proteinexpression were associated with hepatocellular carcinoma(20). Similar results for HBV infection were also found (45).It was concluded that increased plasma level of OPN is aprognostic marker of hepatocellular carcinoma, in bothHCV and HBV infection (49,55).

OPN plays a crucial role in liver fibrosis during the pro-cess of tissue remodeling (7). ECM is mainly deposited insubendothelial space by hepatic stellate cells (HSC) (8,40).It has been demonstrated that OPN is significantly involvedin the activation of HSC, as incubation of OPN with HSCleads to cell proliferation and migration. Additionally, ithas been shown that OPN directly induces the productionof collagen and type II TGFb receptor from HSCs, bothat transcriptional and translational levels. Thus, OPN has akey function in hepatic fibrosis and injury (29). We studiedthe influence of OPN promoter polymorphism - 442C/T(rs11730582) on natural clearance and interferon-basedtherapy of HCV infection.

Probable polypeptide GALNT8 is considered to catalyzethe early reaction in the biosynthesis of O-linked oligosac-charide. It transfers an N-acetyl-D-galactosamine residue tothe protein receptor. A single nucleotide variant in intron 6of GALNT8 (rs10849138) is shown to be involved in thetreatment response of combinational therapy for HCV in-fection in the Japanese population (34). We studied the as-sociation of this particular polymorphism of GALNT8 withHCV infection in our population.

The main objective of this study was to find the distri-bution of important SNPs of cytokines regarding chronicHCV infection, their association with natural clearance, andtreatment response in the Pakistani population.

Materials and Methods

Patients and healthy controls

We considered 140 patients in our study. Patient bloodsampling was performed in Saidu Group of Teaching Hos-pital (Khyber Pakhtun khwa) and the Gambat Institute ofMedical Sciences (Sindh). Seventy-five patients were fromSwat and its vicinity, and 65 patients were from Gambattand its vicinity. The inclusion criteria were the detection ofHCV-RNA by reverse transcription polymerase chain re-action (RT-PCR). HCV-RNA quantification was performedwith the MyiQ2 two-color real time PCR detection system(BioRad) by using the Gene Proof Hepatitis C Virus PCRKit designed for the detection of HCV-RNA. Patients withother infectious diseases, for example HBV, HIV, tubercu-losis (TB), malaria, or autoimmune liver disease, were notincluded in this study. Chronic HCV subjects possessing ge-notypes 1, 4, and co-infection were administered treatment ofinterferon alpha 2a and ribavirin for 1 year, while all otherswere mostly administered treatment for 6 months. Patientswho failed to complete a planned course of treatment for anyreason were not included in the study. We also enrolled 120

SNPS AS PREDICTORS OF IFN THERAPY OF HCV INFECTION 405

unrelated healthy adult subjects in this study as controls. Allthese subjects were from Islamabad, having no previoushistory of infectious HBV or HCV infection. The study wasagreed by the Ethic Committee of the institute, and writtenpermission was taken from all enrolled subjects for the de-tection of genetic variations of different cytokine genes.

PCR

DNA was extracted from all the collected blood samplesby the use of a Genomic DNA mini kit (Cat no. 1820-02;Invitrogen) in accordance with the manufacturer’s protocol.DNA was quantified by Nanodrop (Eppendorf Biophot-ometer) and stored at - 20�C before use. Amplification-refectory mutation system PCR (ARMS-PCR) for IL-18and allele-specific PCR (AS-PCR) for TGFb, OPN, andGALNT8 were performed by using the list of primersshown in Table 1. Both the ARMS-PCR (19) and AS-PCR(10) techniques are well established for the determinationof SNPs, and both produce dependable results.

PCR amplification for IL-18 was carried out in 27 lL ofPCR mixture, while for all other genes, it was carried out in25 lL of PCR mixture comprising 100–500 ng DNA. Ad-ditionally, the PCR mixture contained 11 lL NF H2O,200 mM dNTPs (Fermentas), 2.5 mM MgCl2 (Fermentas),2 · PCR buffer (Fermentas), and 2 units of Taq DNA poly-merase (Fermentas). Cycling conditions were as follows: longdenaturation at 95�C for 5 min, followed by 35 cycles of shortdenaturation at 95�C for 30 sec. Primers annealing of IL-18- 137 (rs1946518), OPN - 442 (rs11730582), and GALNT8

(rs10849138) genetic variants was carried out at 62�C for30 sec, while primers annealing of IL-18 - 137 (rs187238),TGFb 10 (rs1982073), and TGFb 25 (rs1800471) geneticvariants was carried out at 66�C for 30 sec. Cycling extensionwas performed at 72�C for 30 sec. The final extension wascarried out at 72�C for 10 min. PCR amplification was run on2% agarose gel to observe under UV light of the gel docsystem (Wealtec) and compared with 50 bp DNA marker(Fermentas) as shown in Figure 1, 2 and SupplementaryFigures S1–S12; Supplementary Data are available online atwww.liebertpub.com/vim.

Table 1. Gene, Primer Sequence, and Product Size for the Study of Various Gene Polymorphisms

Gene Primer sequence Product size (bp)

IL-18 - 607 polymorphismCommon forward primer 5¢-TAA CCT CAT TCA GGA CTT CC-3¢Sequence specific primer 1 5¢-GTT GCA GAA AGT GTA AAA ATT ATT AC-3¢ 196Sequence specific primer 2 5¢-GTT GCA GAA AGT GTA AAA ATT ATT AA-3¢ 196Common reverse primer 5¢-CTT TGC TAT CAT TCC AGG AA-3¢ 301

IL-18 - 137 polymorphismCommon forward primer 5¢-AGG AGG GCA AAA TGC ACT GG-3¢Sequence specific primer 1 5¢-CCC CAA CTT TTA CGG AAG AAA AG-3¢ 261Sequence specific primer 2 5¢-CCC CAA CTT TTA CGG AAG AAA AC-3¢ 261Common reverse primer 5¢-CCA ATA GGA CTG ATT ATT CCG CA-3¢ 446

OPN - 442 polymorphismReverse primer 1 5¢-TTG TTC AAG CCT GCA AGG AGT TCA GAA-3¢ 442Reverse primer 2 5¢-TTG TTC AAG CCT GCA AGG AGT TCA GAG-3¢Forward primer 5¢-GTC CTT AAG ATA CGC AGA GCA TTT GC-3¢TGFb polymorphism at codon 10Forward primer 1 5¢-TCC GGG CTG CGG CTG CTG CC-3¢ 297Forward primer 2 5¢-TCC GGG CTG CGG CTG CTG CT-3¢Common reverse primer 5¢-GTC GGC CTC AGG CTC GGG C-3¢TGFb polymorphism at codon 25Forward primer 1 5¢-TAC TGG TGC TGA CGC CTG GCC G-3¢ 254Forward primer 2 5¢-TAC TGG TGC TGA CGC CTG GCC C-3¢Common reverse primer 5¢-GTC GGC CTC AGG CTC GGG C-3¢GALNT8 polymorphism in intron 6 at rs10849138Forward primer 1 5¢-CTG GGT CAG ACC TGA AGG-3¢ 340Forward primer 2 5¢-CTG GGT CAG ACC TGA AGC-3¢Common reverse primer 5¢-GCA CTG GCC TCT AAC TTT GGA-3¢

FIG. 1. Demographic representation of IL-18 - 607ARMS-PCR amplified products. Lane M represents 50 bpladder. Lanes 1 and 2 show a heterozygous subject, whilelanes 3 and 4 represent a homozygous subject with IL-18- 607 genotypes.

406 IMRAN ET AL.

Statistical analysis

Statistical analysis of the data was performed using SPSSv13 software (SPSS, Inc.). The Hardy–Weinberg principlewas applied to all healthy subjects and patients to find theexpected prevalence of different alleles and genotypes ofstudied cytokine genetic variants in our population. Pearsonchi square analysis was performed to reveal any importantvariations in the prevalence of IL-18, OPN, TGFb, andGALNT8 genotypes in treatment responders and nonre-sponders. A two-tailed p-value of £ 0.05 was regarded asstatistically significant.

Results

IL-18 gene polymorphism

The IL-18 - 607 gene polymorphism was tested in 260individuals. The demographic and clinical profile of all in-dividuals is shown in Table 2.

Regarding IL-18 - 607 gene polymorphism, there were atotal of 87, 113, and 60 individuals for CC, CA, and AAgenotypes respectively. All subjects were categorized intothree groups: healthy controls, SVRs, and NRs. The distri-butions of IL-18 - 607 genotypes among these groups areshown in Table 3.

The distributions of all IL-18 - 607 genotypes were inaccordance with the Hardy–Weinberg principle in all threegroups. The IL-18 607AA genotype was more prevalent inhealthy individuals (29%) than chronic HCV patients (17.8%),signifying its possible protective function in the spontaneousclearance of HCV infection ( p = 0.031), as shown in Figure 3.

Pearson chi square analysis of SVR and NR groupsshowed that there was no considerable variation between

either group for the distribution of IL-18 - 607CC and CAgenotypes. However, the distribution of IL-18AA genotypesamong the SVR group was considerably higher than the NRgroup ( p = 0.046), as shown in Figure 4.

Regarding IL-18 - 137 genotypes, the total distributionsof GG, CG, and CC genotypes were 100, 131, and 29 re-spectively. The prevalence of these genotypes was in agree-ment with the Hardy–Weinberg principle. No considerablevariations in the prevalence of IL-18 - 137GG, CG, or CCgenotypes were noticed in any of the three groups (Table 3).

OPN - 442 polymorphism

The collective prevalence of the OPN - 442 polymor-phism in 260 subjects was 53, 118, and 89 for CC, CT, andTT genotypes respectively (Table 3). The distributions of allOPN - 442 genotypes were in accordance with the Hardy–Weinberg equilibrium. We found no considerable variationsin occurrence of all OPN - 442 genotypes among healthycontrols and patients. When the patients were further cate-gorized into SVR and NR groups, no significant variationswere found in the prevalence of OPN - 442CC and CTgenotypes, as shown in Figure 5. However, the distributionof the OPN - 442TT genotype was considerably higher inthe SVR group than the NR group, signifying its positivecorrelation with treatment response to combinational ther-apy ( p = 0.034). Moreover, we found that all IL-18 - 607genotypes were inherited and independent of OPN - 442genotypes. There was no coinheritance of any genotype ofIL-18 - 607 with any OPN - 442 genotype.

TGFb gene polymorphism

The distributions of TGFb codon 10 polymorphisms in260 individuals were 80, 140, and 40 for TT, CT, and TTgenotypes respectively, while the distributions of TGFbcodon 25 polymorphism were 124, 101, and 35 for geno-types GG, GC, and CC respectively (Table 3). Genotypedistributions of both polymorphisms in all three groups(control, SVR, and NR) were in agreement with the Hardy–Weinberg equilibrium and showed no significant differences.Pearson chi square analysis of the SVR and NR groupsshowed no significant correlation of any genotype of TGFbcodon 10 or TGFb codon 25 polymorphisms with the treat-ment response to HCV infection (Table 3).

GALNT8 gene polymorphism

The distributions of GALNT8 genotypes in 260 indi-viduals showed that their collective prevalence was 63,

FIG. 2. AS-PCR amplification of OPN. Lane M specifies50 bp ladder. Lanes 1 and 2 specify a heterozygous subject,lanes 3 and 4 specify a homozygous subject, while lanes5 and 6 specify a heterozygous subject with OPN - 442polymorphism.

Table 2. Demographic and Clinical Features 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 · 105)5.64 · 105

(0.4 · 105 - 98.7 · 105)— 0.241

*Age is shown as mean – SD.**Viral load is shown as median and ranges.SVRs, sustained virological groups; NRs, nonresponders.

SNPS AS PREDICTORS OF IFN THERAPY OF HCV INFECTION 407

133, and 64 for genotypes CC, CG, and GG respectively.The distribution of this polymorphism in all groups fol-lowed the Hardy–Weinberg equilibrium, with no importantvariations in the prevalence of GALNT8 polymorphism inany of the three groups (control, SVR, and NR). Pearsonchi square analysis of the SVR and NR groups showed nosignificant correlation of any GALNT8 genotype with re-

sponse to interferon plus ribavirin treatment of HCV in-fection (Table 3).

Discussion

Our recent efforts were focused on those host factors thatmay significantly predict treatment response of chronic

Table 3. Distribution of Various Gene Polymorphisms and Their Associations

with Treatment Response to Hepatitis C Virus Infection

Genotype Total (260) Control (120) SVR (115) NR (25) p-Value*

IL-18 - 607 polymorphismCC 87 (33.4%) 32 44 11 0.594CA 113 (43.4%) 53 47 13 0.308AA 60 (23%) 35 24 1 0.046

IL-18 - 137 polymorphismGG 100 (38.4%) 43 48 9 0.597GC 131 (50.3%) 61 57 13 0.825CC 29 (11.1%) 16 10 3 0.606

OPN - 442 polymorphismCC 53 (20.3%) 27 20 6 0.441CT 118 (45.3%) 52 51 15 0.155TT 89 (34.2%) 41 44 4 0.034TGFb polymorphism at codon 10TT 80 (30.6%) 34 38 8 0.920CT 140 (53.8%) 66 62 12 0.591CC 40 (15.3%) 20 15 5 0.368

TGFb polymorphism at codon 25GG 124 (47.6%) 55 59 10 0.306GC 101 (38.8%) 48 44 9 0.833CC 35 (13.4%) 17 12 6 0.066

GALNT8 polymorphism in intron 6 at, rs10849138CC 63 (24.2%) 31 25 7 0.499GC 133 (51.1%) 64 57 12 0.887GG 64 (24.6%) 25 33 6 0.635

*p-Values were calculated by Pearson chi square test for SVR and NR groups. The boldface typed p-values represent significant values.Thus, IL-18 –607AA and OPN –442TT genotypes were positively associated with interferon-based therapy of HCV infection.

FIG. 3. Percentage distributions and p-values of IL-18- 607 genotypes in healthy control and patients. p-Valuesfor IL-18 - 607 genotypes CC, CA, and AA were 0.032,0.832, and 0.031, respectively.

FIG. 4. Graphic representation of IL-18 - 607 genotypesdistributions in SVRs and NRs. p-Values for IL-18 - 607genotypes CC, CA, and AA were 0.594, 0.308, and 0.046,respectively. SVRs, sustained virological groups; NRs,nonresponders.

408 IMRAN ET AL.

HCV-infected subjects in the Pakistani population. Weconsidered some of the most important SNPs of IL-18, OPN,TGFb, and GALNT8 regarding natural clearance andtreatment response of HCV-infected subjects from Sindand Khyber Pakhtun khwa. We found no considerable dis-crepancies in the prevalence of host SNPs. The rate oftreatment response in chronic HCV-infected subjects from thetwo studied provinces was also comparable. Therefore, we didnot categorize patients on the basis of province. Our resultsshowed that, overall, there was a high SVR rate for interferon-based treatment of HCV infection in our population (82%).Published data from this region have also shown high SVRrates for interferon-based therapy of HCV infection (4).

SNPs of host genes contribute significantly to interferon-based therapy of HCV infection. Genome-wide associationstudies (GWAS) suggest a significant correlation of IL-28BSNPs (rs12979860, rs12980275, and rs8099917) in sponta-neous clearance and interferon plus ribavirin treatment ofHCV infection (14,39). The function of IL-28B SNPs inHCV genotype 2 and 3 is still controversial. However, thefunction of IL-28B SNPs in HCV genotype 1 is well es-tablished. Therefore, genetic testing of IL-28B is approvedonly for HCV genotype 1 by the European Association forthe Study of the Liver (EASL) and the American Associa-tion for the Study of Liver Diseases (AASLD) (5).

IL-18 with six exons and five introns is positioned onchromosome 11q22.2-q22.3 (27). Promoter polymorphismof IL-18 - 607 and - 137 may potentially have an effect onIFN-c (50). These promoter polymorphisms have been ob-served as susceptible loci for various diseases such as atopiceczema (36), type 1 diabetes (26,21), and rheumatoid arthritis(17,43). Our current findings suggest that IL-18 promotervariant (-607) is also associated with treatment outcome HCVinfection in the Pakistani population. Chronically infectedHCV subjects possessing AA genotype for IL-18 - 607 have ahigher SVR rate than other subjects possessing CC or CAgenotypes for interferon plus ribavirin treatment ( p = 0.046). Arecent report from Egypt has demonstrated that the occurrenceof IL-18 - 607AA genotype was considerably higher in

healthy controls than patients, pointing to the significant con-tribution of this genotype in the spontaneous clearance of HCVinfection (13). A recent study from India has also demon-strated that IL-18 - 607AA genotype has a protective role inHCV infection, as it is linked with a reduction in diseaseseverity (30). In our study, we not only found a significantvariation in the prevalence of the IL-18 - 607AA genotype inhealthy controls and patients ( p = 0.031), but we also foundthat the distribution of this genotype was very much higher inthe SVR group than the NR group ( p = 0.046). Hence, it is canbe concluded that individuals possessing the IL-18 - 607AAgenotype have a significantly higher SVR rate than individualspossessing the IL-18 - 607CA or CC genotypes. RegardingIL-18 - 137 gene polymorphism, we did not find any associ-ation with natural clearance and treatment outcome of HCVinfection (Table 3 and Supplementary Figures).

OPN polymorphism at nt - 442 is found to be closelyassociated with hepatitis activity in HCV subjects (32). Ourfindings suggest that HCV-infected subjects with the OPN- 442TT genotype demonstrate a high SVR rate to interferonand ribavirin treatment. The incidence of the OPN - 442TTgenotype was significantly higher in the SVR group than theNR group ( p = 0.034), signifying its positive correlationwith combinational therapy for HCV infection. We did notfind a statistically significant correlation of any other OPN- 442 genotypes in the natural clearance or treatment re-sponse of HCV infection (Table 3). A recent study has es-tablished that promoter polymorphism of OPN may be a usefulmarker for the treatment outcome of HCV infection (33).

Our data suggest that the occurrence of the IL-18 - 607AAgenotype in SVR subjects of HCV genotype 3a was two timesgreater than NR subjects of HCV genotype 3a (7.82% vs. 4%).On the other hand, the distribution of the OPN - 442TT ge-notype was even higher (six times) in SVR subjects of HCVgenotype 3a than NR subjects of HCV genotype 3a (25.2% and4%). Our results reveal that the OPN treatment favorable ge-notype - 442 TT was more frequent in HCV genotype 3a SVRsubjects than the IL-18 treatment favorable - 607AA genotype.

In the case of chronic HCV patients, it is demonstrated thatviral core protein upregulates TGFb transcription (46), which inturn leads to exacerbating liver fibrosis progression (25). TGFbpolymorphisms at codon 10 and codon 25 have been linked withfaster progression of liver fibrosis (15,38). Functional poly-morphism at codon 10 of TGFb is likely to interrupt the exportcapability of newly synthesized proteins (18). It has been es-tablished that genetic variant at codon 25 of TGFb is linked withthe development of graft fibrosis after liver transplantation (12).In the current study, we searched for the correlation of TGFbpolymorphism at codon 10 and codon 25 with HCV patho-genesis and treatment response. Our findings demonstratedthat there were no significant variations in the distributions ofTGFb functional variants at codon 10 or 25 in any of the threegroups (control, SVR, and NR as shown in SupplementaryFigures). Thus, there was no significant correlation of anyTGFb genotype with spontaneous clearance and treatmentresponse of HCV infection in our population (Table 3).

A recent report by Nakano et al. demonstrated an asso-ciation of GALNT8 polymorphism rs2286580 in intron 6 ofpolypeptide with interferon monotherapy of HCV infectionin the Japanese population (34). However, the results werenot replicated in patients of the same population receivingpegylated interferon and ribavirin treatment for HCV

FIG. 5. Graphic representation of OPN - 442 genotypesdistributions in SVRs and NRs. p-Values for OPN - 442genotypes CC, CT, and TT were 0.441, 0.155, and 0.034,respectively.

SNPS AS PREDICTORS OF IFN THERAPY OF HCV INFECTION 409

infection. The correlation peaked for the GALNT8 variantrs10849138 for combinational therapy of HCV infection.We studied the correlation of rs10849138 for combinationaltherapy of HCV infection. Our results showed no significantcorrelation of any genotype of rs10849138 with spontaneousclearance and response to interferon plus ribavirin treatmentof HCV infection in our population (Table 3 and Supple-mentary Figures S1–S12).

The main limitation of our study was the small samplesize, particularly when the data were evaluated on the basisof HCV genotypes or provinces. However, the sample sizefor HCV genotype 3a was sufficient to highlight its asso-ciation with IL-18 - 607AA and OPN - 442TT genotypes.

Conclusion

In conclusion, interferon-based therapy of chronic HCVinfection is associated with serious side effects and costeffects besides its long treatment duration. This study mayhelp health policy makers to suggest alternative treatmentoptions for those chronic HCV patients where the chance offailure of interferon and ribavirin treatment of HCV infec-tion is greater. Our study shows that IL-18 AA genotype at- 607 and OPN TT genotype at - 442 may be used aspositive predictive markers for combinational therapy ofHCV infection in the Pakistani population.

Acknowledgments

Financial support for this study was provided by theNational University of Sciences and Technology (NUST),H-12, Islamabad, Pakistan, and Higher Education Com-mission of Pakistan.

Author Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Dr. Sobia Manzoor

Atta-ur-Rahman School of Applied Bio-SciencesDepartment of Healthcare Biotechnology

National University of Sciences and Technology (NUST)Islamabad 44000

Pakistan

E-mail: dr.sobiamanzoor@asab.nust.edu.pk

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