Research ArticleSNP rs8099917 in Gene IL28B Might Be Associated with Risk ofChronic Infection by HCV but Not with Response to Treatment
Simone Regina Souza da Silva Conde12 Julius Caesar Mendes Soares Monteiro2
Bruna Tereza Silva dos Santos2 Nathaacutelia Karla Fonseca Filgueiras2
Pedro Alves de Almeida Lins2 Felipe Bonfim Freitas1 Ednelza da Silva Graccedila1
Sacircmia Demachki3 Marialva Tereza Ferreira de Arauacutejo3
Ricardo Ishak1 and Antonio Carlos Rosaacuterio Vallinoto1
1 Laboratory of Virology Institute of Biological Sciences Federal University of Para Guama 66075-110 Belem PA Brazil2 School of Medicine Institute of Health Sciences Federal University of Para Praca Camilo Salgado No 01 Belem PA Brazil3 Service of Anatomic Pathology School of Medicine Joao de Barros Barreto University Hospital Federal University of ParaBelem PA Brazil
Correspondence should be addressed to Antonio Carlos Rosario Vallinoto vallinotoufpabr
Received 9 October 2013 Revised 23 December 2013 Accepted 3 January 2014 Published 11 February 2014
Academic Editor Jorge Fabian Quarleri
Copyright copy 2014 Simone Regina Souza da Silva Conde et al This is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited
Aim The aim of this study was to characterize the genetic profile of patients with chronic hepatitis C virus (HCV) infection relativeto polymorphisms rs12979860 and rs8099917 in gene IL28B and the association of those polymorphisms with the response totreatment with pegylated interferon and ribavirin performed at a reference center in Brazilian Amazonia Methods A total of 75individuals with chronic hepatitis C and 98 healthy individuals from both genders over 18 years old were assessed DNA sampleswere collected from leukocytes and subjected to real-time polymerase chain reaction to genotype polymorphisms rs12979860and rs8099917 Results Analysis of the allelic and genotypic frequencies of the investigated polymorphisms showed that bothgroups were in Hardy-Weinberg equilibrium polymorphism rs12979860 exhibited no significant difference between the groupsFor polymorphism rs8099917 allele T was significantly less frequent (119875 = 00195) among the patients (633) than the controls(755) and the patients were 17 times as likely to exhibit allele G No difference in response to treatment was associated with SNPpatterns Conclusion The results suggest a possible association of SNP rs8099917 with higher odds of chronic HCV infection butdo not indicate a putative influence of the investigated SNPs on the sustained virologic response
1 Introduction
Hepatitis C is considered to be an insidious disease and itsnatural history has not yet been fully elucidated Accordingto some studies 55 to 85 of the individuals affected by theacute form of disease remain infected for over six monthsand become chronic carriers [1] In these patients the delayeddiagnosis and the fact that the infectionmight have remainedasymptomatic over a long period of time result in advancedstages of liver cirrhosis and corresponding complicationssuch as bleeding esophageal varices ascites spontaneous
bacterial peritonitis and hepatocellular carcinoma (HCC)[2 3]
In individuals who present with spontaneous viral clear-ance the immune response is mediated by proinflammatorycomponents of type 1 immunity [4] the type III interferonclass in particular which includes IL-29 (IFN-1205821) IL-28A(IFN-1205822) and IL-28B (IFN-1205823) These cytokines exhibit sig-nificant antiviral antiproliferative and antitumor activity andare expressed by mononuclear cells monocyte derivativesand dendritic cells when viral infection occurs [5] Single-nucleotide polymorphisms (SNPs) that change the molecular
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 748606 6 pageshttpdxdoiorg1011552014748606
2 BioMed Research International
expression andprotein structure have been found in the genesthat encode the abovementioned cytokines SNPs have alsobeen associated with the risk of infection and the response totreatment [4 6 7]
The treatment of chronic hepatitis C aims at inhibitingviral replication and eliminating the virus from the organismThe currently recommended regimen is based on the useof pegylated interferon (PEG-IFN) combined with ribavirin(RBV) for 24 to 72 weeks as a function of the virus genotypethe virologic response in the course of treatment and thepatientrsquos tolerance [8] The aim of the regimen is to achievea sustained virologic response (SVR) which is defined as theabsence of detectable viral RNA 24 weeks after the end oftreatment [9]
Countless factors related to the host virus and viralkinetics have been associated with the outcome of antivi-ral treatment [10] Polymorphisms in gene IL28B such asrs12979860 and rs8099917 are strongly associated with theSVR [11ndash13]
Based on the data above and the lack of studies conductedin the Amazonian area the aim of this study was to charac-terize the genetic profile of the SNPs in gene IL28B and theirassociate with SVR to treatment
2 Materials and Methods
21 Case Series Theparticipants were recruited from January2004 to June 2012 at the liver disease outpatient clinic of theHoly House of Mercy Foundation of Para (Fundacao SantaCasa deMisericordia do ParamdashFSCMPA) which is a regionalreference in hepatology The study protocol was approved bythe institutional ethics committee (0482011 and 1082011)
A total of 75 consecutive patients with chronic hepatitisC from both genders (41 males and 34 females) older than18 years old (average of 5256 plusmn 1005 years) and positive forviral RNA (PCR-RNA-HCV) were nonrandomly included Atotal of 41 individuals in that samplewere subjected to specifictreatment with PEG-IFN combined with RBV according tothe standard protocol recommended by the Brazilian HealthMinistry [8] Those cases corresponded to treatment-naiveindividuals or to retreatment after the failure of conventionalIFN+RBV combination therapy and had received at least onedose of the antiviral regimen (intention-to-treat analysis)Two PEG-IFN regimens were randomly indicated PEG-IFN-1205722b (15 120583gkgweek) or PEG-IFN-1205722a (180 120583gkgweek)concomitant to RBV (15mgkgday)
The viral kinetics of the participants were assessedaccording to the following criteria (i) null response (NR)defined by the reduction of the viral RNA level less than twolog by week 12 of treatment (ii) end-of-treatment response(ETR) defined by the absence of viral RNA at week 48of treatment and (iii) sustained virologic response (SVR)defined by undetectable viral RNA 24 weeks after the endof treatment Individuals coinfected with hepatitis B virus(HBV) andor human immunodeficiency virus (HIV) wereexcluded
The control group comprised 98 healthy individuals fromboth genders (39 males and 59 females) who were older than
18 years old (average of 3778 plusmn 1509 years) and seronegativefor HCV HBV and HIV
In the present study we were careful to include only indi-viduals from the same ethnic background residents in urbanor rural area of the state of Para Caucasians Amerindiansand Afro-Brazilian communities were excluded in order toavoid and reduce biases
22 Sample Collection After the participants expressed theiragreement to participate in the study and signed an informedconsent form the epidemiological clinical laboratory andhistopathological data in their clinical records were enteredinto a standard form Samples of peripheral blood (5mL)were collected for the extraction of total DNA from leuko-cytes using a modification of the phenol-chloroformmethoddescribed by Sambrook and Russel [14] followed by cell lysisprotein precipitation and DNA precipitation and hydrationSamples of viral RNA were obtained based on the extractionof total RNA from the plasma using the Total RNA Purifica-tion Kit (NORGEN Biotek Corporation Canada)
23 Genotyping Polymorphisms rs12979860 (C gt T) and rs-8099917 (T gt G) in gene IL28B were genotyped by real-timePCR (qPCR) using a Step One PLUS Sequence Detector(Applied Biosystems Foster City CA USA)
Each polymorphism assay contained one pair of primersand one pair of probes and each allele of the polymor-phisms was labeled with VIC or FAMThe rs12979860 targetsequence was sent to the Applied Biosystems company whichdesigned the following primer and probe sequences 51015840-GCCTGT CGT GTA CTG AAC CA-31015840 (forward) 51015840-GCG CGGAGTTGCAATTCAAC-31015840 (reverse) 51015840VIC-TGGTTCGCGCTT C-31015840 (allele C probe) and 51015840FAM-CTGGTT CACGCCTTC-31015840 (allele T probe) For polymorphism rs8099917 anassay predesigned by Applied Biosystems and identified asC 11710096 10 was used
Each reaction included [1X] TaqMan Universal PCRMaster Mix [2X] [1X] TaqMan Assay [20X] and 20 ng ofDNA in a total volume of 10 120583L The following parameterswere used in amplification and allele detection one cycle at60∘C for 30 seconds followed by one cycle at 95∘C for 10minutes and 50 cycles at 92∘C for 30 seconds and 60∘C for90 seconds
24 Viral Load Genotyping and Histopathological Examina-tion The viral load and genotyping tests were performedat Central Laboratory using the qualitative and quantita-tive PCR-HCV-RNA method (Roche-COBAS AMPLICORHepatitis C Virus (HCV) Test version 20 v) For the pur-pose of analysis the viral load values were dichotomizedas low (lt600000 IUmL) or high (gt600000 IUmL) Thehistopathological assessment followed the French Metavirclassification [15]
25 Statistical Analysis Descriptive and inferential analyseswere performed as a function of the normality of the dataParametric ANOVA and Studentrsquos t-test were used for quan-titative data and the nonparametric chi-square test (with
BioMed Research International 3
Table 1 Genotypic and allelic frequencies of SNP rs12979860 and rs8099917 in gene IL28B in individuals with chronic hepatitis C virusinfection and the control group Belem 2012
the Yates correction) Fisherrsquos exact test and G-test (withWilliamsrsquo correction) were used for qualitative nominal orordinal dataThegenotypic and allelic frequencies foundwereassessed using the Hardy-Weinberg equilibrium
The degree of association between genotypes and viralinfection was measured using the odds ratio (OR) andcorresponding 95 confidence intervals (CI) The analyseswere performed using the software Epi Info 353 [16] andBioEstat 53 [17] with alpha le 5 established as the level toreject the null hypothesis
3 Results
Clinical and laboratory assessment of the stage of hepatitisshowed that 76 (5775) of the participants did not exhibitliver cirrhosis while 24 (1875) did
The viral genotype was established in 70 participantswith genotype 1 in 771 (5470) of the cases and genotype3 in 229 (1670) Among the participants subjected totreatment 805 (3341) exhibited genotype 1 and 195(841) exhibited genotype 3
The viral load was measured in 61 participants and wasless than 60000 IUmL in 639 (3961) of the cases
Liver biopsy was performed in 59 participants histopath-ological analysis showed that 61 (3659) of the biopsiesexhibited activity periportal or periseptal scores of zero toone and 593 exhibited fibrosis scores of two to four
Analysis of the SNP rs12979860 allelic and genotypicfrequencies showed that both groups (patients and controls)were in Hardy-Weinberg equilibrium (Table 1) A tendency(119875 = 00813) to a higher frequency of allele Twas found in thegroup of patients compared to the controls but no significant
difference in the genotypic frequency (119875 = 01865) was foundbetween the two groups
Analysis of the polymorphism rs8099917 allelic frequency(Table 1) showed that both groups (patients and controls)were in Hardy-Weinberg equilibrium Allele T was lessfrequent in the group of patients (633) compared to thecontrols (755) and the probability of patients exhibitingallele G was 17-fold higher than in the controls Thesedifferences were statistically significant (119875 = 00195) Inaddition a tendency (119875 = 00600) to a higher proportionof genotype TT (119875 = 00413) was found in the group ofcontrols (582) compared to the patients (413) whereasthe frequency of genotype GG was twice as high among thepatients (147) compared to the controls (71)
Among all patients treated with PEG-IFN + RBV 61(2541) were undergoing their first treatment for hepatitis Cand 39 (1641) were being retreated due to the failure oftreatment with conventional IFN
Intention-to-treat analysis resulted in 5121 of ERT(2141) and 39 of SVR (1641) Treatment discontinuancedue to NR or non-ERT occurred in 414 of the cases(1741) and intolerance to side effects occurred in 97 (441)(Figure 1) Neither SNP rs12979860 nor rs8099917 exhibiteda statistically significant correlation with SVR (Table 2)
4 Discussion
In this study no significant difference was found in thegenotype and allele proportions of SNP rs12979860 possiblydue to the small size of the sample of individuals withhepatitis C Nevertheless genotype CT predominated amongthe patients (493) which agrees with the results reportedby Ge et al [11] for North-American patients with hepatitis
4 BioMed Research International
Table 2 Association of SNPs rs12979860 and rs8099917 with sustained virologic response in individuals with chronic hepatitis C treated withPEG-IFN and RBV
T 24 (429) 32 (571) 56lowastOR (CC versus CT versus TT) a119866-test bchi-square testSVR sustained virologic response ETR end-of-treatment response NR null response
ETRRVFNon-ETR
Discontinuation dueto intolerance
Non-ETR
Null response
Week 12
Onset of treatment
cEVR658
pEVR170
Week 48560
Week 48170
Week 24after treatment
4358
Non-SVR24
SVR24
Non-SVR97
SVR341
Non-SVR0
SVR24
ETR
Non-ETR
Week 24after treatment
512
100 (n = 41)
100 (n = 41)
97 (n = 4) (n = 27) (n = 7)
170 (n = 7)
121 (n = 5)
(n = 23) (n = 7)
73 (n = 3)463 (n = 18) 48 (n = 2) 121 (n = 5)
24 (n = 1) (n = 17) (n = 2)
(n = 1) (n = 0) (n = 14) (n = 4) (n = 1) (n = 1)
Figure 1 Follow-up of the treatment of individuals with chronichepatitis C treated with PEG-IFN and RBV at FSCMPA fromJanuary 2004 to January 2012 cEVR complete early virologicresponse pEVR partial early virologic response ERT end-of-treatment response SVR sustained virologic response
C whereby genotype CT was found in 497 of the indi-viduals of European ancestry 476 of African-Americansand 467 of Hispanics In addition Bochud et al [7] andLunge et al [18] found a higher prevalence of genotype CT inCaucasian Europeans (49) and Brazilians (606) infectedbyHIV-1 respectively In contrast the prevalence of genotypeCC is high in theBrazilian (514) Taiwanese (898) South-Korean (877) and Japanese (768) populations [3 619 20] Those differences might be attributed to the ethnicbackground of each particular population or to the selectivepressure exerted by environmental factors
The allelic distribution of polymorphism rs12979860 inthe group of patients was similar to the distribution describedby Venegas et al [21] relative to individuals treated for HCVin Chile Additionally Ge et al [11] found a high frequencyof allele T in Afro-Americans (605) differing from otherhuman populations [19 20 22]
Analysis of the SNP rs8099917 genotypes revealed atendency (119875 = 006) to a higher frequency of genotype TTamong the controls and GT among the patients The higherfrequency of the heterozygous genotypeGT and homozygousgenotype GG among the patients agrees with the resultsreported by Venegas et al [21] However it disagrees with thefindings of other studies conducted with individuals infectedby HCV according to which the frequency of genotype TTwas higher than any other genotype in Caucasian Europeans[22 23] Japanese [3] and Taiwanese [6]
For polymorphism rs8099917 the distribution of allele Gexhibited a significant difference between the groups beinghigher among the patients than the controls and the presence
BioMed Research International 5
of that allele was associated with 18 greater odds of becomingchronically infected byHCVThe results of this study point toa possible association between the presence of that SNP andgreater odds of susceptibility to chronic infection by HCVHowever to confirm this hypothesis it would be necessaryto analyze a group of individuals who were exposed toHCV but subsequently cleared the virus Only one study byVenegas et al [21] has reported a high frequency of thatallele while in other studies it varied from 52 [6] to 255[24] Nevertheless the variation of the allelic frequency ofthis SNP among different human populations suggests thatthe presence of an unfavorable genotype (GG) alone does notaccount for the susceptibility to infection and other geneticfactors not investigated in this present study might also beinvolved in that phenomenon
The rate of SVR exhibited by the treated patients was 39which is lower than the rate found in the previous studiesby Hadziyannis et al [25] using PEG-IFN alfa-2b whichwas 63 However it is similar to the rates found in studiesconducted with patients with only genotype 1 of HCV (398ndash46) which agrees with the predominance of that genotypeamong the group of treated patients in the present study[10 26 27]
5 Conclusions
We did not find a significant association of SNPs rs12979860and rs8099917 with SVR thus disagreeing with studies thatfound an association between genotype CC (rs12979860) andSVR in individuals with genotype 1 [10 11 27] 2 and 3 [13]as well as between genotype TT (rs8099917) and SVR inindividuals with genotype 1 [12]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors thank the patients and send them their bestwishes This study was partially supported by a grant fromthe Brazilian National Council for Scientific and Technolog-ical Development (CNPq) and the Dean of Research andGraduate Studies Office (Pro-Reitoria de Pesquisa e Pos-Graduacao-PROPESPUFPA)Research Support and Devel-opment Foundation (Fundacao de Apoio e Desenvolvimentoda PesquisamdashFADESP)
References
[1] M G Ghany D B Strader D L Thomas and L B SeeffldquoDiagnosis management and treatment of hepatitis C anupdaterdquo Hepatology vol 49 no 4 pp 1335ndash1374 2009
[2] A Ascione T Tartaglione andG G Costanzo ldquoNatural historyof chronic hepatitis C virus infectionrdquo Digestive and LiverDisease vol 39 supplement 1 pp S4ndashS7 2007
[3] M Kobayashi F Suzuki N Akuta et al ldquoAssociation oftwo polymorphisms of the IL28B gene with viral factors and
treatment response in 1518 patients infected with hepatitis Cvirusrdquo Journal of Gastroenterology vol 47 no 5 pp 596ndash6052012
[4] A Balagopal D L Thomas and C L Thio ldquoIL28B and thecontrol of hepatitis C virus infectionrdquoGastroenterology vol 139no 6 pp 1865ndash1876 2010
[5] S Li P Hu Q-Q Zhang et al ldquoSingle nucleotide polymor-phisms of the IL28B and sustained virologic response of patientswith chronic hepatitis C to PEG-interferonribavirin therapya meta-analysisrdquo Hepatitis Monthly vol 11 no 3 pp 163ndash1722011
[6] J-Y Chen C-Y Lin C-M Wang et al ldquoIL28B genetic varia-tions are associated with high Sustained Virological Response(SVR) of interferon-120572 plus ribavirin therapy in Taiwanesechronic HCV infectionrdquo Genes and Immunity vol 12 no 4 pp300ndash309 2011
[7] P-Y Bochud S Bibert Z Kutalik et al ldquoIL28B alleles associatedwith poor Hepatitis C Virus (HCV) clearance protect againstinflammation and fibrosis in patients infected with non-1 HCVgenotypesrdquo Hepatology vol 55 no 2 pp 384ndash394 2012
[8] Ministerio da Saude Brasil Boletim Epidemiologico HepatiteVirais Ministerio da Saude Brasılia 2011
[9] A Kau J Vermehren and C Sarrazin ldquoTreatment predictors ofa sustained virologic response in hepatitis B and Crdquo Journal ofHepatology vol 49 no 4 pp 634ndash651 2008
[10] A J Thompson A J Muir M S Sulkowski et al ldquoInterleukin-28B polymorphism improves viral kinetics and is the strongestpretreatment predictor of sustained virologic response in geno-type 1 hepatitis C virusrdquoGastroenterology vol 139 no 1 pp 120ndash129 2010
[11] D Ge J Fellay A J Thompson et al ldquoGenetic variation inIL28B predicts hepatitis C treatment-induced viral clearancerdquoNature vol 461 no 7262 pp 399ndash401 2009
[12] Y Tanaka N Nishida M Sugiyama et al ldquoGenome-wideassociation of IL28B with response to pegylated interferon-120572and ribavirin therapy for chronic hepatitis Crdquo Nature Geneticsvol 41 no 10 pp 1105ndash1109 2009
[13] A Mangia A J Thompson R Santoro et al ldquoAn IL28Bpolymorphism determines treatment response of hepatitis Cvirus genotype 2 or 3 patients who do not achieve a rapidvirologic responserdquoGastroenterology vol 139 no 3 pp 821ndash8272010
[14] J Sambrook and D W Russell ldquoPurification of nucleic acidsby extraction with phenolchloroformrdquo Cold Spring HarborProtocols 2006
[15] P Bedossa and T Poynard ldquoAn algorithm for the grading ofactivity in chronic hepatitis Crdquo Hepatology vol 24 no 2 pp289ndash293 1996
[16] ldquoCenters for Diseases Control and Prevention Epi Infordquo version3 5 3 httpwwwncdcgovepiinfohtmlvendorshtm
[17] M Ayres M Ayres Jr D L Ayres and A S Santos Bioestat5 0mdashAplicacoes Estatısticas Nas Areas Das Ciencias Biologicas eMedicas Sociedade Civil Mamiraua Belem Brazil 2007
[18] V R Lunge D B Da Rocha J U Beria D C Tietzmann AT Stein and D Simon ldquoIL28B polymorphism associated withspontaneous clearance of hepatitis C infection in a SouthernBrazilian HIV type 1 populationrdquo AIDS Research and HumanRetroviruses vol 28 no 2 pp 215ndash219 2012
[19] K LyooWHur J E Choi et al ldquoPolymorphism near the IL28Bgene in Korean hepatitis C virus-infected patients treated withpeg-interferon plus ribavirinrdquo Journal of Clinical Virology vol52 no 4 pp 363ndash366 2011
6 BioMed Research International
[20] L N Cavalcante K Abe-Sandes A L D Angelo et alldquoIL28B polymorphisms aremarkers of therapy response and areinfluenced by genetic ancestry in chronic hepatitis C patientsfrom an admixed populationrdquo Liver International vol 32 no 3pp 476ndash486 2012
[21] M Venegas R A Villanueva K Gonzalez and J BrahmldquoIL28B polymorphisms associated with therapy response inChilean chronic hepatitis C patientsrdquo World Journal of Gas-troenterology vol 17 no 31 pp 3636ndash3639 2011
[22] A F Stattermayer R Stauber H Hofer et al ldquoImpact ofIL28B genotype on the early and sustained virologic responsein treatment-naıve patients with chronic hepatitis Crdquo ClinicalGastroenterology andHepatology vol 9 no 4 pp 344ndash350 2011
[23] A Rauch Z Kutalik P Descombes et al ldquoGenetic variationin IL28B is associated with chronic hepatitis C and treatmentfailure a genome-wide association studyrdquoGastroenterology vol138 no 4 pp 1338ndash1344 2010
[24] F Marabita A Aghemo S de Nicola et al ldquoGenetic variationin the interleukin-28B gene is not associated with fibrosisprogression in patients with chronic hepatitis C and known dateof infectionrdquo Hepatology vol 54 no 4 pp 1127ndash1134 2011
[25] S JHadziyannisH Sette Jr T RMorgan et al ldquoPeginterferon-1205722a and ribavirin combination therapy in chronic hepatitis Ca randomized study of treatment duration and ribavirin doserdquoAnnals of Internal Medicine vol 140 no 5 pp 346ndash355 2004
[26] J G McHutchison E J Lawitz M L Shiffman et al ldquoPeginter-feron alfa-2b or alfa-2a with ribavirin for treatment of hepatitisC infectionrdquoThe New England Journal of Medicine vol 361 no6 pp 580ndash593 2009
[27] C D Howell A Gorden K A Ryan et al ldquoSingle nucleotidepolymorphism upstream of interleukin 28B associated withphase 1 and phase 2 of early viral kinetics in patients infectedwith HCV genotype 1rdquo Journal of Hepatology vol 56 no 3 pp557ndash563 2012
expression andprotein structure have been found in the genesthat encode the abovementioned cytokines SNPs have alsobeen associated with the risk of infection and the response totreatment [4 6 7]
The treatment of chronic hepatitis C aims at inhibitingviral replication and eliminating the virus from the organismThe currently recommended regimen is based on the useof pegylated interferon (PEG-IFN) combined with ribavirin(RBV) for 24 to 72 weeks as a function of the virus genotypethe virologic response in the course of treatment and thepatientrsquos tolerance [8] The aim of the regimen is to achievea sustained virologic response (SVR) which is defined as theabsence of detectable viral RNA 24 weeks after the end oftreatment [9]
Countless factors related to the host virus and viralkinetics have been associated with the outcome of antivi-ral treatment [10] Polymorphisms in gene IL28B such asrs12979860 and rs8099917 are strongly associated with theSVR [11ndash13]
Based on the data above and the lack of studies conductedin the Amazonian area the aim of this study was to charac-terize the genetic profile of the SNPs in gene IL28B and theirassociate with SVR to treatment
2 Materials and Methods
21 Case Series Theparticipants were recruited from January2004 to June 2012 at the liver disease outpatient clinic of theHoly House of Mercy Foundation of Para (Fundacao SantaCasa deMisericordia do ParamdashFSCMPA) which is a regionalreference in hepatology The study protocol was approved bythe institutional ethics committee (0482011 and 1082011)
A total of 75 consecutive patients with chronic hepatitisC from both genders (41 males and 34 females) older than18 years old (average of 5256 plusmn 1005 years) and positive forviral RNA (PCR-RNA-HCV) were nonrandomly included Atotal of 41 individuals in that samplewere subjected to specifictreatment with PEG-IFN combined with RBV according tothe standard protocol recommended by the Brazilian HealthMinistry [8] Those cases corresponded to treatment-naiveindividuals or to retreatment after the failure of conventionalIFN+RBV combination therapy and had received at least onedose of the antiviral regimen (intention-to-treat analysis)Two PEG-IFN regimens were randomly indicated PEG-IFN-1205722b (15 120583gkgweek) or PEG-IFN-1205722a (180 120583gkgweek)concomitant to RBV (15mgkgday)
The viral kinetics of the participants were assessedaccording to the following criteria (i) null response (NR)defined by the reduction of the viral RNA level less than twolog by week 12 of treatment (ii) end-of-treatment response(ETR) defined by the absence of viral RNA at week 48of treatment and (iii) sustained virologic response (SVR)defined by undetectable viral RNA 24 weeks after the endof treatment Individuals coinfected with hepatitis B virus(HBV) andor human immunodeficiency virus (HIV) wereexcluded
The control group comprised 98 healthy individuals fromboth genders (39 males and 59 females) who were older than
18 years old (average of 3778 plusmn 1509 years) and seronegativefor HCV HBV and HIV
In the present study we were careful to include only indi-viduals from the same ethnic background residents in urbanor rural area of the state of Para Caucasians Amerindiansand Afro-Brazilian communities were excluded in order toavoid and reduce biases
22 Sample Collection After the participants expressed theiragreement to participate in the study and signed an informedconsent form the epidemiological clinical laboratory andhistopathological data in their clinical records were enteredinto a standard form Samples of peripheral blood (5mL)were collected for the extraction of total DNA from leuko-cytes using a modification of the phenol-chloroformmethoddescribed by Sambrook and Russel [14] followed by cell lysisprotein precipitation and DNA precipitation and hydrationSamples of viral RNA were obtained based on the extractionof total RNA from the plasma using the Total RNA Purifica-tion Kit (NORGEN Biotek Corporation Canada)
23 Genotyping Polymorphisms rs12979860 (C gt T) and rs-8099917 (T gt G) in gene IL28B were genotyped by real-timePCR (qPCR) using a Step One PLUS Sequence Detector(Applied Biosystems Foster City CA USA)
Each polymorphism assay contained one pair of primersand one pair of probes and each allele of the polymor-phisms was labeled with VIC or FAMThe rs12979860 targetsequence was sent to the Applied Biosystems company whichdesigned the following primer and probe sequences 51015840-GCCTGT CGT GTA CTG AAC CA-31015840 (forward) 51015840-GCG CGGAGTTGCAATTCAAC-31015840 (reverse) 51015840VIC-TGGTTCGCGCTT C-31015840 (allele C probe) and 51015840FAM-CTGGTT CACGCCTTC-31015840 (allele T probe) For polymorphism rs8099917 anassay predesigned by Applied Biosystems and identified asC 11710096 10 was used
Each reaction included [1X] TaqMan Universal PCRMaster Mix [2X] [1X] TaqMan Assay [20X] and 20 ng ofDNA in a total volume of 10 120583L The following parameterswere used in amplification and allele detection one cycle at60∘C for 30 seconds followed by one cycle at 95∘C for 10minutes and 50 cycles at 92∘C for 30 seconds and 60∘C for90 seconds
24 Viral Load Genotyping and Histopathological Examina-tion The viral load and genotyping tests were performedat Central Laboratory using the qualitative and quantita-tive PCR-HCV-RNA method (Roche-COBAS AMPLICORHepatitis C Virus (HCV) Test version 20 v) For the pur-pose of analysis the viral load values were dichotomizedas low (lt600000 IUmL) or high (gt600000 IUmL) Thehistopathological assessment followed the French Metavirclassification [15]
25 Statistical Analysis Descriptive and inferential analyseswere performed as a function of the normality of the dataParametric ANOVA and Studentrsquos t-test were used for quan-titative data and the nonparametric chi-square test (with
BioMed Research International 3
Table 1 Genotypic and allelic frequencies of SNP rs12979860 and rs8099917 in gene IL28B in individuals with chronic hepatitis C virusinfection and the control group Belem 2012
the Yates correction) Fisherrsquos exact test and G-test (withWilliamsrsquo correction) were used for qualitative nominal orordinal dataThegenotypic and allelic frequencies foundwereassessed using the Hardy-Weinberg equilibrium
The degree of association between genotypes and viralinfection was measured using the odds ratio (OR) andcorresponding 95 confidence intervals (CI) The analyseswere performed using the software Epi Info 353 [16] andBioEstat 53 [17] with alpha le 5 established as the level toreject the null hypothesis
3 Results
Clinical and laboratory assessment of the stage of hepatitisshowed that 76 (5775) of the participants did not exhibitliver cirrhosis while 24 (1875) did
The viral genotype was established in 70 participantswith genotype 1 in 771 (5470) of the cases and genotype3 in 229 (1670) Among the participants subjected totreatment 805 (3341) exhibited genotype 1 and 195(841) exhibited genotype 3
The viral load was measured in 61 participants and wasless than 60000 IUmL in 639 (3961) of the cases
Liver biopsy was performed in 59 participants histopath-ological analysis showed that 61 (3659) of the biopsiesexhibited activity periportal or periseptal scores of zero toone and 593 exhibited fibrosis scores of two to four
Analysis of the SNP rs12979860 allelic and genotypicfrequencies showed that both groups (patients and controls)were in Hardy-Weinberg equilibrium (Table 1) A tendency(119875 = 00813) to a higher frequency of allele Twas found in thegroup of patients compared to the controls but no significant
difference in the genotypic frequency (119875 = 01865) was foundbetween the two groups
Analysis of the polymorphism rs8099917 allelic frequency(Table 1) showed that both groups (patients and controls)were in Hardy-Weinberg equilibrium Allele T was lessfrequent in the group of patients (633) compared to thecontrols (755) and the probability of patients exhibitingallele G was 17-fold higher than in the controls Thesedifferences were statistically significant (119875 = 00195) Inaddition a tendency (119875 = 00600) to a higher proportionof genotype TT (119875 = 00413) was found in the group ofcontrols (582) compared to the patients (413) whereasthe frequency of genotype GG was twice as high among thepatients (147) compared to the controls (71)
Among all patients treated with PEG-IFN + RBV 61(2541) were undergoing their first treatment for hepatitis Cand 39 (1641) were being retreated due to the failure oftreatment with conventional IFN
Intention-to-treat analysis resulted in 5121 of ERT(2141) and 39 of SVR (1641) Treatment discontinuancedue to NR or non-ERT occurred in 414 of the cases(1741) and intolerance to side effects occurred in 97 (441)(Figure 1) Neither SNP rs12979860 nor rs8099917 exhibiteda statistically significant correlation with SVR (Table 2)
4 Discussion
In this study no significant difference was found in thegenotype and allele proportions of SNP rs12979860 possiblydue to the small size of the sample of individuals withhepatitis C Nevertheless genotype CT predominated amongthe patients (493) which agrees with the results reportedby Ge et al [11] for North-American patients with hepatitis
4 BioMed Research International
Table 2 Association of SNPs rs12979860 and rs8099917 with sustained virologic response in individuals with chronic hepatitis C treated withPEG-IFN and RBV
T 24 (429) 32 (571) 56lowastOR (CC versus CT versus TT) a119866-test bchi-square testSVR sustained virologic response ETR end-of-treatment response NR null response
ETRRVFNon-ETR
Discontinuation dueto intolerance
Non-ETR
Null response
Week 12
Onset of treatment
cEVR658
pEVR170
Week 48560
Week 48170
Week 24after treatment
4358
Non-SVR24
SVR24
Non-SVR97
SVR341
Non-SVR0
SVR24
ETR
Non-ETR
Week 24after treatment
512
100 (n = 41)
100 (n = 41)
97 (n = 4) (n = 27) (n = 7)
170 (n = 7)
121 (n = 5)
(n = 23) (n = 7)
73 (n = 3)463 (n = 18) 48 (n = 2) 121 (n = 5)
24 (n = 1) (n = 17) (n = 2)
(n = 1) (n = 0) (n = 14) (n = 4) (n = 1) (n = 1)
Figure 1 Follow-up of the treatment of individuals with chronichepatitis C treated with PEG-IFN and RBV at FSCMPA fromJanuary 2004 to January 2012 cEVR complete early virologicresponse pEVR partial early virologic response ERT end-of-treatment response SVR sustained virologic response
C whereby genotype CT was found in 497 of the indi-viduals of European ancestry 476 of African-Americansand 467 of Hispanics In addition Bochud et al [7] andLunge et al [18] found a higher prevalence of genotype CT inCaucasian Europeans (49) and Brazilians (606) infectedbyHIV-1 respectively In contrast the prevalence of genotypeCC is high in theBrazilian (514) Taiwanese (898) South-Korean (877) and Japanese (768) populations [3 619 20] Those differences might be attributed to the ethnicbackground of each particular population or to the selectivepressure exerted by environmental factors
The allelic distribution of polymorphism rs12979860 inthe group of patients was similar to the distribution describedby Venegas et al [21] relative to individuals treated for HCVin Chile Additionally Ge et al [11] found a high frequencyof allele T in Afro-Americans (605) differing from otherhuman populations [19 20 22]
Analysis of the SNP rs8099917 genotypes revealed atendency (119875 = 006) to a higher frequency of genotype TTamong the controls and GT among the patients The higherfrequency of the heterozygous genotypeGT and homozygousgenotype GG among the patients agrees with the resultsreported by Venegas et al [21] However it disagrees with thefindings of other studies conducted with individuals infectedby HCV according to which the frequency of genotype TTwas higher than any other genotype in Caucasian Europeans[22 23] Japanese [3] and Taiwanese [6]
For polymorphism rs8099917 the distribution of allele Gexhibited a significant difference between the groups beinghigher among the patients than the controls and the presence
BioMed Research International 5
of that allele was associated with 18 greater odds of becomingchronically infected byHCVThe results of this study point toa possible association between the presence of that SNP andgreater odds of susceptibility to chronic infection by HCVHowever to confirm this hypothesis it would be necessaryto analyze a group of individuals who were exposed toHCV but subsequently cleared the virus Only one study byVenegas et al [21] has reported a high frequency of thatallele while in other studies it varied from 52 [6] to 255[24] Nevertheless the variation of the allelic frequency ofthis SNP among different human populations suggests thatthe presence of an unfavorable genotype (GG) alone does notaccount for the susceptibility to infection and other geneticfactors not investigated in this present study might also beinvolved in that phenomenon
The rate of SVR exhibited by the treated patients was 39which is lower than the rate found in the previous studiesby Hadziyannis et al [25] using PEG-IFN alfa-2b whichwas 63 However it is similar to the rates found in studiesconducted with patients with only genotype 1 of HCV (398ndash46) which agrees with the predominance of that genotypeamong the group of treated patients in the present study[10 26 27]
5 Conclusions
We did not find a significant association of SNPs rs12979860and rs8099917 with SVR thus disagreeing with studies thatfound an association between genotype CC (rs12979860) andSVR in individuals with genotype 1 [10 11 27] 2 and 3 [13]as well as between genotype TT (rs8099917) and SVR inindividuals with genotype 1 [12]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors thank the patients and send them their bestwishes This study was partially supported by a grant fromthe Brazilian National Council for Scientific and Technolog-ical Development (CNPq) and the Dean of Research andGraduate Studies Office (Pro-Reitoria de Pesquisa e Pos-Graduacao-PROPESPUFPA)Research Support and Devel-opment Foundation (Fundacao de Apoio e Desenvolvimentoda PesquisamdashFADESP)
References
[1] M G Ghany D B Strader D L Thomas and L B SeeffldquoDiagnosis management and treatment of hepatitis C anupdaterdquo Hepatology vol 49 no 4 pp 1335ndash1374 2009
[2] A Ascione T Tartaglione andG G Costanzo ldquoNatural historyof chronic hepatitis C virus infectionrdquo Digestive and LiverDisease vol 39 supplement 1 pp S4ndashS7 2007
[3] M Kobayashi F Suzuki N Akuta et al ldquoAssociation oftwo polymorphisms of the IL28B gene with viral factors and
treatment response in 1518 patients infected with hepatitis Cvirusrdquo Journal of Gastroenterology vol 47 no 5 pp 596ndash6052012
[4] A Balagopal D L Thomas and C L Thio ldquoIL28B and thecontrol of hepatitis C virus infectionrdquoGastroenterology vol 139no 6 pp 1865ndash1876 2010
[5] S Li P Hu Q-Q Zhang et al ldquoSingle nucleotide polymor-phisms of the IL28B and sustained virologic response of patientswith chronic hepatitis C to PEG-interferonribavirin therapya meta-analysisrdquo Hepatitis Monthly vol 11 no 3 pp 163ndash1722011
[6] J-Y Chen C-Y Lin C-M Wang et al ldquoIL28B genetic varia-tions are associated with high Sustained Virological Response(SVR) of interferon-120572 plus ribavirin therapy in Taiwanesechronic HCV infectionrdquo Genes and Immunity vol 12 no 4 pp300ndash309 2011
[7] P-Y Bochud S Bibert Z Kutalik et al ldquoIL28B alleles associatedwith poor Hepatitis C Virus (HCV) clearance protect againstinflammation and fibrosis in patients infected with non-1 HCVgenotypesrdquo Hepatology vol 55 no 2 pp 384ndash394 2012
[8] Ministerio da Saude Brasil Boletim Epidemiologico HepatiteVirais Ministerio da Saude Brasılia 2011
[9] A Kau J Vermehren and C Sarrazin ldquoTreatment predictors ofa sustained virologic response in hepatitis B and Crdquo Journal ofHepatology vol 49 no 4 pp 634ndash651 2008
[10] A J Thompson A J Muir M S Sulkowski et al ldquoInterleukin-28B polymorphism improves viral kinetics and is the strongestpretreatment predictor of sustained virologic response in geno-type 1 hepatitis C virusrdquoGastroenterology vol 139 no 1 pp 120ndash129 2010
[11] D Ge J Fellay A J Thompson et al ldquoGenetic variation inIL28B predicts hepatitis C treatment-induced viral clearancerdquoNature vol 461 no 7262 pp 399ndash401 2009
[12] Y Tanaka N Nishida M Sugiyama et al ldquoGenome-wideassociation of IL28B with response to pegylated interferon-120572and ribavirin therapy for chronic hepatitis Crdquo Nature Geneticsvol 41 no 10 pp 1105ndash1109 2009
[13] A Mangia A J Thompson R Santoro et al ldquoAn IL28Bpolymorphism determines treatment response of hepatitis Cvirus genotype 2 or 3 patients who do not achieve a rapidvirologic responserdquoGastroenterology vol 139 no 3 pp 821ndash8272010
[14] J Sambrook and D W Russell ldquoPurification of nucleic acidsby extraction with phenolchloroformrdquo Cold Spring HarborProtocols 2006
[15] P Bedossa and T Poynard ldquoAn algorithm for the grading ofactivity in chronic hepatitis Crdquo Hepatology vol 24 no 2 pp289ndash293 1996
[16] ldquoCenters for Diseases Control and Prevention Epi Infordquo version3 5 3 httpwwwncdcgovepiinfohtmlvendorshtm
[17] M Ayres M Ayres Jr D L Ayres and A S Santos Bioestat5 0mdashAplicacoes Estatısticas Nas Areas Das Ciencias Biologicas eMedicas Sociedade Civil Mamiraua Belem Brazil 2007
[18] V R Lunge D B Da Rocha J U Beria D C Tietzmann AT Stein and D Simon ldquoIL28B polymorphism associated withspontaneous clearance of hepatitis C infection in a SouthernBrazilian HIV type 1 populationrdquo AIDS Research and HumanRetroviruses vol 28 no 2 pp 215ndash219 2012
[19] K LyooWHur J E Choi et al ldquoPolymorphism near the IL28Bgene in Korean hepatitis C virus-infected patients treated withpeg-interferon plus ribavirinrdquo Journal of Clinical Virology vol52 no 4 pp 363ndash366 2011
6 BioMed Research International
[20] L N Cavalcante K Abe-Sandes A L D Angelo et alldquoIL28B polymorphisms aremarkers of therapy response and areinfluenced by genetic ancestry in chronic hepatitis C patientsfrom an admixed populationrdquo Liver International vol 32 no 3pp 476ndash486 2012
[21] M Venegas R A Villanueva K Gonzalez and J BrahmldquoIL28B polymorphisms associated with therapy response inChilean chronic hepatitis C patientsrdquo World Journal of Gas-troenterology vol 17 no 31 pp 3636ndash3639 2011
[22] A F Stattermayer R Stauber H Hofer et al ldquoImpact ofIL28B genotype on the early and sustained virologic responsein treatment-naıve patients with chronic hepatitis Crdquo ClinicalGastroenterology andHepatology vol 9 no 4 pp 344ndash350 2011
[23] A Rauch Z Kutalik P Descombes et al ldquoGenetic variationin IL28B is associated with chronic hepatitis C and treatmentfailure a genome-wide association studyrdquoGastroenterology vol138 no 4 pp 1338ndash1344 2010
[24] F Marabita A Aghemo S de Nicola et al ldquoGenetic variationin the interleukin-28B gene is not associated with fibrosisprogression in patients with chronic hepatitis C and known dateof infectionrdquo Hepatology vol 54 no 4 pp 1127ndash1134 2011
[25] S JHadziyannisH Sette Jr T RMorgan et al ldquoPeginterferon-1205722a and ribavirin combination therapy in chronic hepatitis Ca randomized study of treatment duration and ribavirin doserdquoAnnals of Internal Medicine vol 140 no 5 pp 346ndash355 2004
[26] J G McHutchison E J Lawitz M L Shiffman et al ldquoPeginter-feron alfa-2b or alfa-2a with ribavirin for treatment of hepatitisC infectionrdquoThe New England Journal of Medicine vol 361 no6 pp 580ndash593 2009
[27] C D Howell A Gorden K A Ryan et al ldquoSingle nucleotidepolymorphism upstream of interleukin 28B associated withphase 1 and phase 2 of early viral kinetics in patients infectedwith HCV genotype 1rdquo Journal of Hepatology vol 56 no 3 pp557ndash563 2012
Table 1 Genotypic and allelic frequencies of SNP rs12979860 and rs8099917 in gene IL28B in individuals with chronic hepatitis C virusinfection and the control group Belem 2012
the Yates correction) Fisherrsquos exact test and G-test (withWilliamsrsquo correction) were used for qualitative nominal orordinal dataThegenotypic and allelic frequencies foundwereassessed using the Hardy-Weinberg equilibrium
The degree of association between genotypes and viralinfection was measured using the odds ratio (OR) andcorresponding 95 confidence intervals (CI) The analyseswere performed using the software Epi Info 353 [16] andBioEstat 53 [17] with alpha le 5 established as the level toreject the null hypothesis
3 Results
Clinical and laboratory assessment of the stage of hepatitisshowed that 76 (5775) of the participants did not exhibitliver cirrhosis while 24 (1875) did
The viral genotype was established in 70 participantswith genotype 1 in 771 (5470) of the cases and genotype3 in 229 (1670) Among the participants subjected totreatment 805 (3341) exhibited genotype 1 and 195(841) exhibited genotype 3
The viral load was measured in 61 participants and wasless than 60000 IUmL in 639 (3961) of the cases
Liver biopsy was performed in 59 participants histopath-ological analysis showed that 61 (3659) of the biopsiesexhibited activity periportal or periseptal scores of zero toone and 593 exhibited fibrosis scores of two to four
Analysis of the SNP rs12979860 allelic and genotypicfrequencies showed that both groups (patients and controls)were in Hardy-Weinberg equilibrium (Table 1) A tendency(119875 = 00813) to a higher frequency of allele Twas found in thegroup of patients compared to the controls but no significant
difference in the genotypic frequency (119875 = 01865) was foundbetween the two groups
Analysis of the polymorphism rs8099917 allelic frequency(Table 1) showed that both groups (patients and controls)were in Hardy-Weinberg equilibrium Allele T was lessfrequent in the group of patients (633) compared to thecontrols (755) and the probability of patients exhibitingallele G was 17-fold higher than in the controls Thesedifferences were statistically significant (119875 = 00195) Inaddition a tendency (119875 = 00600) to a higher proportionof genotype TT (119875 = 00413) was found in the group ofcontrols (582) compared to the patients (413) whereasthe frequency of genotype GG was twice as high among thepatients (147) compared to the controls (71)
Among all patients treated with PEG-IFN + RBV 61(2541) were undergoing their first treatment for hepatitis Cand 39 (1641) were being retreated due to the failure oftreatment with conventional IFN
Intention-to-treat analysis resulted in 5121 of ERT(2141) and 39 of SVR (1641) Treatment discontinuancedue to NR or non-ERT occurred in 414 of the cases(1741) and intolerance to side effects occurred in 97 (441)(Figure 1) Neither SNP rs12979860 nor rs8099917 exhibiteda statistically significant correlation with SVR (Table 2)
4 Discussion
In this study no significant difference was found in thegenotype and allele proportions of SNP rs12979860 possiblydue to the small size of the sample of individuals withhepatitis C Nevertheless genotype CT predominated amongthe patients (493) which agrees with the results reportedby Ge et al [11] for North-American patients with hepatitis
4 BioMed Research International
Table 2 Association of SNPs rs12979860 and rs8099917 with sustained virologic response in individuals with chronic hepatitis C treated withPEG-IFN and RBV
T 24 (429) 32 (571) 56lowastOR (CC versus CT versus TT) a119866-test bchi-square testSVR sustained virologic response ETR end-of-treatment response NR null response
ETRRVFNon-ETR
Discontinuation dueto intolerance
Non-ETR
Null response
Week 12
Onset of treatment
cEVR658
pEVR170
Week 48560
Week 48170
Week 24after treatment
4358
Non-SVR24
SVR24
Non-SVR97
SVR341
Non-SVR0
SVR24
ETR
Non-ETR
Week 24after treatment
512
100 (n = 41)
100 (n = 41)
97 (n = 4) (n = 27) (n = 7)
170 (n = 7)
121 (n = 5)
(n = 23) (n = 7)
73 (n = 3)463 (n = 18) 48 (n = 2) 121 (n = 5)
24 (n = 1) (n = 17) (n = 2)
(n = 1) (n = 0) (n = 14) (n = 4) (n = 1) (n = 1)
Figure 1 Follow-up of the treatment of individuals with chronichepatitis C treated with PEG-IFN and RBV at FSCMPA fromJanuary 2004 to January 2012 cEVR complete early virologicresponse pEVR partial early virologic response ERT end-of-treatment response SVR sustained virologic response
C whereby genotype CT was found in 497 of the indi-viduals of European ancestry 476 of African-Americansand 467 of Hispanics In addition Bochud et al [7] andLunge et al [18] found a higher prevalence of genotype CT inCaucasian Europeans (49) and Brazilians (606) infectedbyHIV-1 respectively In contrast the prevalence of genotypeCC is high in theBrazilian (514) Taiwanese (898) South-Korean (877) and Japanese (768) populations [3 619 20] Those differences might be attributed to the ethnicbackground of each particular population or to the selectivepressure exerted by environmental factors
The allelic distribution of polymorphism rs12979860 inthe group of patients was similar to the distribution describedby Venegas et al [21] relative to individuals treated for HCVin Chile Additionally Ge et al [11] found a high frequencyof allele T in Afro-Americans (605) differing from otherhuman populations [19 20 22]
Analysis of the SNP rs8099917 genotypes revealed atendency (119875 = 006) to a higher frequency of genotype TTamong the controls and GT among the patients The higherfrequency of the heterozygous genotypeGT and homozygousgenotype GG among the patients agrees with the resultsreported by Venegas et al [21] However it disagrees with thefindings of other studies conducted with individuals infectedby HCV according to which the frequency of genotype TTwas higher than any other genotype in Caucasian Europeans[22 23] Japanese [3] and Taiwanese [6]
For polymorphism rs8099917 the distribution of allele Gexhibited a significant difference between the groups beinghigher among the patients than the controls and the presence
BioMed Research International 5
of that allele was associated with 18 greater odds of becomingchronically infected byHCVThe results of this study point toa possible association between the presence of that SNP andgreater odds of susceptibility to chronic infection by HCVHowever to confirm this hypothesis it would be necessaryto analyze a group of individuals who were exposed toHCV but subsequently cleared the virus Only one study byVenegas et al [21] has reported a high frequency of thatallele while in other studies it varied from 52 [6] to 255[24] Nevertheless the variation of the allelic frequency ofthis SNP among different human populations suggests thatthe presence of an unfavorable genotype (GG) alone does notaccount for the susceptibility to infection and other geneticfactors not investigated in this present study might also beinvolved in that phenomenon
The rate of SVR exhibited by the treated patients was 39which is lower than the rate found in the previous studiesby Hadziyannis et al [25] using PEG-IFN alfa-2b whichwas 63 However it is similar to the rates found in studiesconducted with patients with only genotype 1 of HCV (398ndash46) which agrees with the predominance of that genotypeamong the group of treated patients in the present study[10 26 27]
5 Conclusions
We did not find a significant association of SNPs rs12979860and rs8099917 with SVR thus disagreeing with studies thatfound an association between genotype CC (rs12979860) andSVR in individuals with genotype 1 [10 11 27] 2 and 3 [13]as well as between genotype TT (rs8099917) and SVR inindividuals with genotype 1 [12]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors thank the patients and send them their bestwishes This study was partially supported by a grant fromthe Brazilian National Council for Scientific and Technolog-ical Development (CNPq) and the Dean of Research andGraduate Studies Office (Pro-Reitoria de Pesquisa e Pos-Graduacao-PROPESPUFPA)Research Support and Devel-opment Foundation (Fundacao de Apoio e Desenvolvimentoda PesquisamdashFADESP)
References
[1] M G Ghany D B Strader D L Thomas and L B SeeffldquoDiagnosis management and treatment of hepatitis C anupdaterdquo Hepatology vol 49 no 4 pp 1335ndash1374 2009
[2] A Ascione T Tartaglione andG G Costanzo ldquoNatural historyof chronic hepatitis C virus infectionrdquo Digestive and LiverDisease vol 39 supplement 1 pp S4ndashS7 2007
[3] M Kobayashi F Suzuki N Akuta et al ldquoAssociation oftwo polymorphisms of the IL28B gene with viral factors and
treatment response in 1518 patients infected with hepatitis Cvirusrdquo Journal of Gastroenterology vol 47 no 5 pp 596ndash6052012
[4] A Balagopal D L Thomas and C L Thio ldquoIL28B and thecontrol of hepatitis C virus infectionrdquoGastroenterology vol 139no 6 pp 1865ndash1876 2010
[5] S Li P Hu Q-Q Zhang et al ldquoSingle nucleotide polymor-phisms of the IL28B and sustained virologic response of patientswith chronic hepatitis C to PEG-interferonribavirin therapya meta-analysisrdquo Hepatitis Monthly vol 11 no 3 pp 163ndash1722011
[6] J-Y Chen C-Y Lin C-M Wang et al ldquoIL28B genetic varia-tions are associated with high Sustained Virological Response(SVR) of interferon-120572 plus ribavirin therapy in Taiwanesechronic HCV infectionrdquo Genes and Immunity vol 12 no 4 pp300ndash309 2011
[7] P-Y Bochud S Bibert Z Kutalik et al ldquoIL28B alleles associatedwith poor Hepatitis C Virus (HCV) clearance protect againstinflammation and fibrosis in patients infected with non-1 HCVgenotypesrdquo Hepatology vol 55 no 2 pp 384ndash394 2012
[8] Ministerio da Saude Brasil Boletim Epidemiologico HepatiteVirais Ministerio da Saude Brasılia 2011
[9] A Kau J Vermehren and C Sarrazin ldquoTreatment predictors ofa sustained virologic response in hepatitis B and Crdquo Journal ofHepatology vol 49 no 4 pp 634ndash651 2008
[10] A J Thompson A J Muir M S Sulkowski et al ldquoInterleukin-28B polymorphism improves viral kinetics and is the strongestpretreatment predictor of sustained virologic response in geno-type 1 hepatitis C virusrdquoGastroenterology vol 139 no 1 pp 120ndash129 2010
[11] D Ge J Fellay A J Thompson et al ldquoGenetic variation inIL28B predicts hepatitis C treatment-induced viral clearancerdquoNature vol 461 no 7262 pp 399ndash401 2009
[12] Y Tanaka N Nishida M Sugiyama et al ldquoGenome-wideassociation of IL28B with response to pegylated interferon-120572and ribavirin therapy for chronic hepatitis Crdquo Nature Geneticsvol 41 no 10 pp 1105ndash1109 2009
[13] A Mangia A J Thompson R Santoro et al ldquoAn IL28Bpolymorphism determines treatment response of hepatitis Cvirus genotype 2 or 3 patients who do not achieve a rapidvirologic responserdquoGastroenterology vol 139 no 3 pp 821ndash8272010
[14] J Sambrook and D W Russell ldquoPurification of nucleic acidsby extraction with phenolchloroformrdquo Cold Spring HarborProtocols 2006
[15] P Bedossa and T Poynard ldquoAn algorithm for the grading ofactivity in chronic hepatitis Crdquo Hepatology vol 24 no 2 pp289ndash293 1996
[16] ldquoCenters for Diseases Control and Prevention Epi Infordquo version3 5 3 httpwwwncdcgovepiinfohtmlvendorshtm
[17] M Ayres M Ayres Jr D L Ayres and A S Santos Bioestat5 0mdashAplicacoes Estatısticas Nas Areas Das Ciencias Biologicas eMedicas Sociedade Civil Mamiraua Belem Brazil 2007
[18] V R Lunge D B Da Rocha J U Beria D C Tietzmann AT Stein and D Simon ldquoIL28B polymorphism associated withspontaneous clearance of hepatitis C infection in a SouthernBrazilian HIV type 1 populationrdquo AIDS Research and HumanRetroviruses vol 28 no 2 pp 215ndash219 2012
[19] K LyooWHur J E Choi et al ldquoPolymorphism near the IL28Bgene in Korean hepatitis C virus-infected patients treated withpeg-interferon plus ribavirinrdquo Journal of Clinical Virology vol52 no 4 pp 363ndash366 2011
6 BioMed Research International
[20] L N Cavalcante K Abe-Sandes A L D Angelo et alldquoIL28B polymorphisms aremarkers of therapy response and areinfluenced by genetic ancestry in chronic hepatitis C patientsfrom an admixed populationrdquo Liver International vol 32 no 3pp 476ndash486 2012
[21] M Venegas R A Villanueva K Gonzalez and J BrahmldquoIL28B polymorphisms associated with therapy response inChilean chronic hepatitis C patientsrdquo World Journal of Gas-troenterology vol 17 no 31 pp 3636ndash3639 2011
[22] A F Stattermayer R Stauber H Hofer et al ldquoImpact ofIL28B genotype on the early and sustained virologic responsein treatment-naıve patients with chronic hepatitis Crdquo ClinicalGastroenterology andHepatology vol 9 no 4 pp 344ndash350 2011
[23] A Rauch Z Kutalik P Descombes et al ldquoGenetic variationin IL28B is associated with chronic hepatitis C and treatmentfailure a genome-wide association studyrdquoGastroenterology vol138 no 4 pp 1338ndash1344 2010
[24] F Marabita A Aghemo S de Nicola et al ldquoGenetic variationin the interleukin-28B gene is not associated with fibrosisprogression in patients with chronic hepatitis C and known dateof infectionrdquo Hepatology vol 54 no 4 pp 1127ndash1134 2011
[25] S JHadziyannisH Sette Jr T RMorgan et al ldquoPeginterferon-1205722a and ribavirin combination therapy in chronic hepatitis Ca randomized study of treatment duration and ribavirin doserdquoAnnals of Internal Medicine vol 140 no 5 pp 346ndash355 2004
[26] J G McHutchison E J Lawitz M L Shiffman et al ldquoPeginter-feron alfa-2b or alfa-2a with ribavirin for treatment of hepatitisC infectionrdquoThe New England Journal of Medicine vol 361 no6 pp 580ndash593 2009
[27] C D Howell A Gorden K A Ryan et al ldquoSingle nucleotidepolymorphism upstream of interleukin 28B associated withphase 1 and phase 2 of early viral kinetics in patients infectedwith HCV genotype 1rdquo Journal of Hepatology vol 56 no 3 pp557ndash563 2012
Table 2 Association of SNPs rs12979860 and rs8099917 with sustained virologic response in individuals with chronic hepatitis C treated withPEG-IFN and RBV
T 24 (429) 32 (571) 56lowastOR (CC versus CT versus TT) a119866-test bchi-square testSVR sustained virologic response ETR end-of-treatment response NR null response
ETRRVFNon-ETR
Discontinuation dueto intolerance
Non-ETR
Null response
Week 12
Onset of treatment
cEVR658
pEVR170
Week 48560
Week 48170
Week 24after treatment
4358
Non-SVR24
SVR24
Non-SVR97
SVR341
Non-SVR0
SVR24
ETR
Non-ETR
Week 24after treatment
512
100 (n = 41)
100 (n = 41)
97 (n = 4) (n = 27) (n = 7)
170 (n = 7)
121 (n = 5)
(n = 23) (n = 7)
73 (n = 3)463 (n = 18) 48 (n = 2) 121 (n = 5)
24 (n = 1) (n = 17) (n = 2)
(n = 1) (n = 0) (n = 14) (n = 4) (n = 1) (n = 1)
Figure 1 Follow-up of the treatment of individuals with chronichepatitis C treated with PEG-IFN and RBV at FSCMPA fromJanuary 2004 to January 2012 cEVR complete early virologicresponse pEVR partial early virologic response ERT end-of-treatment response SVR sustained virologic response
C whereby genotype CT was found in 497 of the indi-viduals of European ancestry 476 of African-Americansand 467 of Hispanics In addition Bochud et al [7] andLunge et al [18] found a higher prevalence of genotype CT inCaucasian Europeans (49) and Brazilians (606) infectedbyHIV-1 respectively In contrast the prevalence of genotypeCC is high in theBrazilian (514) Taiwanese (898) South-Korean (877) and Japanese (768) populations [3 619 20] Those differences might be attributed to the ethnicbackground of each particular population or to the selectivepressure exerted by environmental factors
The allelic distribution of polymorphism rs12979860 inthe group of patients was similar to the distribution describedby Venegas et al [21] relative to individuals treated for HCVin Chile Additionally Ge et al [11] found a high frequencyof allele T in Afro-Americans (605) differing from otherhuman populations [19 20 22]
Analysis of the SNP rs8099917 genotypes revealed atendency (119875 = 006) to a higher frequency of genotype TTamong the controls and GT among the patients The higherfrequency of the heterozygous genotypeGT and homozygousgenotype GG among the patients agrees with the resultsreported by Venegas et al [21] However it disagrees with thefindings of other studies conducted with individuals infectedby HCV according to which the frequency of genotype TTwas higher than any other genotype in Caucasian Europeans[22 23] Japanese [3] and Taiwanese [6]
For polymorphism rs8099917 the distribution of allele Gexhibited a significant difference between the groups beinghigher among the patients than the controls and the presence
BioMed Research International 5
of that allele was associated with 18 greater odds of becomingchronically infected byHCVThe results of this study point toa possible association between the presence of that SNP andgreater odds of susceptibility to chronic infection by HCVHowever to confirm this hypothesis it would be necessaryto analyze a group of individuals who were exposed toHCV but subsequently cleared the virus Only one study byVenegas et al [21] has reported a high frequency of thatallele while in other studies it varied from 52 [6] to 255[24] Nevertheless the variation of the allelic frequency ofthis SNP among different human populations suggests thatthe presence of an unfavorable genotype (GG) alone does notaccount for the susceptibility to infection and other geneticfactors not investigated in this present study might also beinvolved in that phenomenon
The rate of SVR exhibited by the treated patients was 39which is lower than the rate found in the previous studiesby Hadziyannis et al [25] using PEG-IFN alfa-2b whichwas 63 However it is similar to the rates found in studiesconducted with patients with only genotype 1 of HCV (398ndash46) which agrees with the predominance of that genotypeamong the group of treated patients in the present study[10 26 27]
5 Conclusions
We did not find a significant association of SNPs rs12979860and rs8099917 with SVR thus disagreeing with studies thatfound an association between genotype CC (rs12979860) andSVR in individuals with genotype 1 [10 11 27] 2 and 3 [13]as well as between genotype TT (rs8099917) and SVR inindividuals with genotype 1 [12]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors thank the patients and send them their bestwishes This study was partially supported by a grant fromthe Brazilian National Council for Scientific and Technolog-ical Development (CNPq) and the Dean of Research andGraduate Studies Office (Pro-Reitoria de Pesquisa e Pos-Graduacao-PROPESPUFPA)Research Support and Devel-opment Foundation (Fundacao de Apoio e Desenvolvimentoda PesquisamdashFADESP)
References
[1] M G Ghany D B Strader D L Thomas and L B SeeffldquoDiagnosis management and treatment of hepatitis C anupdaterdquo Hepatology vol 49 no 4 pp 1335ndash1374 2009
[2] A Ascione T Tartaglione andG G Costanzo ldquoNatural historyof chronic hepatitis C virus infectionrdquo Digestive and LiverDisease vol 39 supplement 1 pp S4ndashS7 2007
[3] M Kobayashi F Suzuki N Akuta et al ldquoAssociation oftwo polymorphisms of the IL28B gene with viral factors and
treatment response in 1518 patients infected with hepatitis Cvirusrdquo Journal of Gastroenterology vol 47 no 5 pp 596ndash6052012
[4] A Balagopal D L Thomas and C L Thio ldquoIL28B and thecontrol of hepatitis C virus infectionrdquoGastroenterology vol 139no 6 pp 1865ndash1876 2010
[5] S Li P Hu Q-Q Zhang et al ldquoSingle nucleotide polymor-phisms of the IL28B and sustained virologic response of patientswith chronic hepatitis C to PEG-interferonribavirin therapya meta-analysisrdquo Hepatitis Monthly vol 11 no 3 pp 163ndash1722011
[6] J-Y Chen C-Y Lin C-M Wang et al ldquoIL28B genetic varia-tions are associated with high Sustained Virological Response(SVR) of interferon-120572 plus ribavirin therapy in Taiwanesechronic HCV infectionrdquo Genes and Immunity vol 12 no 4 pp300ndash309 2011
[7] P-Y Bochud S Bibert Z Kutalik et al ldquoIL28B alleles associatedwith poor Hepatitis C Virus (HCV) clearance protect againstinflammation and fibrosis in patients infected with non-1 HCVgenotypesrdquo Hepatology vol 55 no 2 pp 384ndash394 2012
[8] Ministerio da Saude Brasil Boletim Epidemiologico HepatiteVirais Ministerio da Saude Brasılia 2011
[9] A Kau J Vermehren and C Sarrazin ldquoTreatment predictors ofa sustained virologic response in hepatitis B and Crdquo Journal ofHepatology vol 49 no 4 pp 634ndash651 2008
[10] A J Thompson A J Muir M S Sulkowski et al ldquoInterleukin-28B polymorphism improves viral kinetics and is the strongestpretreatment predictor of sustained virologic response in geno-type 1 hepatitis C virusrdquoGastroenterology vol 139 no 1 pp 120ndash129 2010
[11] D Ge J Fellay A J Thompson et al ldquoGenetic variation inIL28B predicts hepatitis C treatment-induced viral clearancerdquoNature vol 461 no 7262 pp 399ndash401 2009
[12] Y Tanaka N Nishida M Sugiyama et al ldquoGenome-wideassociation of IL28B with response to pegylated interferon-120572and ribavirin therapy for chronic hepatitis Crdquo Nature Geneticsvol 41 no 10 pp 1105ndash1109 2009
[13] A Mangia A J Thompson R Santoro et al ldquoAn IL28Bpolymorphism determines treatment response of hepatitis Cvirus genotype 2 or 3 patients who do not achieve a rapidvirologic responserdquoGastroenterology vol 139 no 3 pp 821ndash8272010
[14] J Sambrook and D W Russell ldquoPurification of nucleic acidsby extraction with phenolchloroformrdquo Cold Spring HarborProtocols 2006
[15] P Bedossa and T Poynard ldquoAn algorithm for the grading ofactivity in chronic hepatitis Crdquo Hepatology vol 24 no 2 pp289ndash293 1996
[16] ldquoCenters for Diseases Control and Prevention Epi Infordquo version3 5 3 httpwwwncdcgovepiinfohtmlvendorshtm
[17] M Ayres M Ayres Jr D L Ayres and A S Santos Bioestat5 0mdashAplicacoes Estatısticas Nas Areas Das Ciencias Biologicas eMedicas Sociedade Civil Mamiraua Belem Brazil 2007
[18] V R Lunge D B Da Rocha J U Beria D C Tietzmann AT Stein and D Simon ldquoIL28B polymorphism associated withspontaneous clearance of hepatitis C infection in a SouthernBrazilian HIV type 1 populationrdquo AIDS Research and HumanRetroviruses vol 28 no 2 pp 215ndash219 2012
[19] K LyooWHur J E Choi et al ldquoPolymorphism near the IL28Bgene in Korean hepatitis C virus-infected patients treated withpeg-interferon plus ribavirinrdquo Journal of Clinical Virology vol52 no 4 pp 363ndash366 2011
6 BioMed Research International
[20] L N Cavalcante K Abe-Sandes A L D Angelo et alldquoIL28B polymorphisms aremarkers of therapy response and areinfluenced by genetic ancestry in chronic hepatitis C patientsfrom an admixed populationrdquo Liver International vol 32 no 3pp 476ndash486 2012
[21] M Venegas R A Villanueva K Gonzalez and J BrahmldquoIL28B polymorphisms associated with therapy response inChilean chronic hepatitis C patientsrdquo World Journal of Gas-troenterology vol 17 no 31 pp 3636ndash3639 2011
[22] A F Stattermayer R Stauber H Hofer et al ldquoImpact ofIL28B genotype on the early and sustained virologic responsein treatment-naıve patients with chronic hepatitis Crdquo ClinicalGastroenterology andHepatology vol 9 no 4 pp 344ndash350 2011
[23] A Rauch Z Kutalik P Descombes et al ldquoGenetic variationin IL28B is associated with chronic hepatitis C and treatmentfailure a genome-wide association studyrdquoGastroenterology vol138 no 4 pp 1338ndash1344 2010
[24] F Marabita A Aghemo S de Nicola et al ldquoGenetic variationin the interleukin-28B gene is not associated with fibrosisprogression in patients with chronic hepatitis C and known dateof infectionrdquo Hepatology vol 54 no 4 pp 1127ndash1134 2011
[25] S JHadziyannisH Sette Jr T RMorgan et al ldquoPeginterferon-1205722a and ribavirin combination therapy in chronic hepatitis Ca randomized study of treatment duration and ribavirin doserdquoAnnals of Internal Medicine vol 140 no 5 pp 346ndash355 2004
[26] J G McHutchison E J Lawitz M L Shiffman et al ldquoPeginter-feron alfa-2b or alfa-2a with ribavirin for treatment of hepatitisC infectionrdquoThe New England Journal of Medicine vol 361 no6 pp 580ndash593 2009
[27] C D Howell A Gorden K A Ryan et al ldquoSingle nucleotidepolymorphism upstream of interleukin 28B associated withphase 1 and phase 2 of early viral kinetics in patients infectedwith HCV genotype 1rdquo Journal of Hepatology vol 56 no 3 pp557ndash563 2012
of that allele was associated with 18 greater odds of becomingchronically infected byHCVThe results of this study point toa possible association between the presence of that SNP andgreater odds of susceptibility to chronic infection by HCVHowever to confirm this hypothesis it would be necessaryto analyze a group of individuals who were exposed toHCV but subsequently cleared the virus Only one study byVenegas et al [21] has reported a high frequency of thatallele while in other studies it varied from 52 [6] to 255[24] Nevertheless the variation of the allelic frequency ofthis SNP among different human populations suggests thatthe presence of an unfavorable genotype (GG) alone does notaccount for the susceptibility to infection and other geneticfactors not investigated in this present study might also beinvolved in that phenomenon
The rate of SVR exhibited by the treated patients was 39which is lower than the rate found in the previous studiesby Hadziyannis et al [25] using PEG-IFN alfa-2b whichwas 63 However it is similar to the rates found in studiesconducted with patients with only genotype 1 of HCV (398ndash46) which agrees with the predominance of that genotypeamong the group of treated patients in the present study[10 26 27]
5 Conclusions
We did not find a significant association of SNPs rs12979860and rs8099917 with SVR thus disagreeing with studies thatfound an association between genotype CC (rs12979860) andSVR in individuals with genotype 1 [10 11 27] 2 and 3 [13]as well as between genotype TT (rs8099917) and SVR inindividuals with genotype 1 [12]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors thank the patients and send them their bestwishes This study was partially supported by a grant fromthe Brazilian National Council for Scientific and Technolog-ical Development (CNPq) and the Dean of Research andGraduate Studies Office (Pro-Reitoria de Pesquisa e Pos-Graduacao-PROPESPUFPA)Research Support and Devel-opment Foundation (Fundacao de Apoio e Desenvolvimentoda PesquisamdashFADESP)
References
[1] M G Ghany D B Strader D L Thomas and L B SeeffldquoDiagnosis management and treatment of hepatitis C anupdaterdquo Hepatology vol 49 no 4 pp 1335ndash1374 2009
[2] A Ascione T Tartaglione andG G Costanzo ldquoNatural historyof chronic hepatitis C virus infectionrdquo Digestive and LiverDisease vol 39 supplement 1 pp S4ndashS7 2007
[3] M Kobayashi F Suzuki N Akuta et al ldquoAssociation oftwo polymorphisms of the IL28B gene with viral factors and
treatment response in 1518 patients infected with hepatitis Cvirusrdquo Journal of Gastroenterology vol 47 no 5 pp 596ndash6052012
[4] A Balagopal D L Thomas and C L Thio ldquoIL28B and thecontrol of hepatitis C virus infectionrdquoGastroenterology vol 139no 6 pp 1865ndash1876 2010
[5] S Li P Hu Q-Q Zhang et al ldquoSingle nucleotide polymor-phisms of the IL28B and sustained virologic response of patientswith chronic hepatitis C to PEG-interferonribavirin therapya meta-analysisrdquo Hepatitis Monthly vol 11 no 3 pp 163ndash1722011
[6] J-Y Chen C-Y Lin C-M Wang et al ldquoIL28B genetic varia-tions are associated with high Sustained Virological Response(SVR) of interferon-120572 plus ribavirin therapy in Taiwanesechronic HCV infectionrdquo Genes and Immunity vol 12 no 4 pp300ndash309 2011
[7] P-Y Bochud S Bibert Z Kutalik et al ldquoIL28B alleles associatedwith poor Hepatitis C Virus (HCV) clearance protect againstinflammation and fibrosis in patients infected with non-1 HCVgenotypesrdquo Hepatology vol 55 no 2 pp 384ndash394 2012
[8] Ministerio da Saude Brasil Boletim Epidemiologico HepatiteVirais Ministerio da Saude Brasılia 2011
[9] A Kau J Vermehren and C Sarrazin ldquoTreatment predictors ofa sustained virologic response in hepatitis B and Crdquo Journal ofHepatology vol 49 no 4 pp 634ndash651 2008
[10] A J Thompson A J Muir M S Sulkowski et al ldquoInterleukin-28B polymorphism improves viral kinetics and is the strongestpretreatment predictor of sustained virologic response in geno-type 1 hepatitis C virusrdquoGastroenterology vol 139 no 1 pp 120ndash129 2010
[11] D Ge J Fellay A J Thompson et al ldquoGenetic variation inIL28B predicts hepatitis C treatment-induced viral clearancerdquoNature vol 461 no 7262 pp 399ndash401 2009
[12] Y Tanaka N Nishida M Sugiyama et al ldquoGenome-wideassociation of IL28B with response to pegylated interferon-120572and ribavirin therapy for chronic hepatitis Crdquo Nature Geneticsvol 41 no 10 pp 1105ndash1109 2009
[13] A Mangia A J Thompson R Santoro et al ldquoAn IL28Bpolymorphism determines treatment response of hepatitis Cvirus genotype 2 or 3 patients who do not achieve a rapidvirologic responserdquoGastroenterology vol 139 no 3 pp 821ndash8272010
[14] J Sambrook and D W Russell ldquoPurification of nucleic acidsby extraction with phenolchloroformrdquo Cold Spring HarborProtocols 2006
[15] P Bedossa and T Poynard ldquoAn algorithm for the grading ofactivity in chronic hepatitis Crdquo Hepatology vol 24 no 2 pp289ndash293 1996
[16] ldquoCenters for Diseases Control and Prevention Epi Infordquo version3 5 3 httpwwwncdcgovepiinfohtmlvendorshtm
[17] M Ayres M Ayres Jr D L Ayres and A S Santos Bioestat5 0mdashAplicacoes Estatısticas Nas Areas Das Ciencias Biologicas eMedicas Sociedade Civil Mamiraua Belem Brazil 2007
[18] V R Lunge D B Da Rocha J U Beria D C Tietzmann AT Stein and D Simon ldquoIL28B polymorphism associated withspontaneous clearance of hepatitis C infection in a SouthernBrazilian HIV type 1 populationrdquo AIDS Research and HumanRetroviruses vol 28 no 2 pp 215ndash219 2012
[19] K LyooWHur J E Choi et al ldquoPolymorphism near the IL28Bgene in Korean hepatitis C virus-infected patients treated withpeg-interferon plus ribavirinrdquo Journal of Clinical Virology vol52 no 4 pp 363ndash366 2011
6 BioMed Research International
[20] L N Cavalcante K Abe-Sandes A L D Angelo et alldquoIL28B polymorphisms aremarkers of therapy response and areinfluenced by genetic ancestry in chronic hepatitis C patientsfrom an admixed populationrdquo Liver International vol 32 no 3pp 476ndash486 2012
[21] M Venegas R A Villanueva K Gonzalez and J BrahmldquoIL28B polymorphisms associated with therapy response inChilean chronic hepatitis C patientsrdquo World Journal of Gas-troenterology vol 17 no 31 pp 3636ndash3639 2011
[22] A F Stattermayer R Stauber H Hofer et al ldquoImpact ofIL28B genotype on the early and sustained virologic responsein treatment-naıve patients with chronic hepatitis Crdquo ClinicalGastroenterology andHepatology vol 9 no 4 pp 344ndash350 2011
[23] A Rauch Z Kutalik P Descombes et al ldquoGenetic variationin IL28B is associated with chronic hepatitis C and treatmentfailure a genome-wide association studyrdquoGastroenterology vol138 no 4 pp 1338ndash1344 2010
[24] F Marabita A Aghemo S de Nicola et al ldquoGenetic variationin the interleukin-28B gene is not associated with fibrosisprogression in patients with chronic hepatitis C and known dateof infectionrdquo Hepatology vol 54 no 4 pp 1127ndash1134 2011
[25] S JHadziyannisH Sette Jr T RMorgan et al ldquoPeginterferon-1205722a and ribavirin combination therapy in chronic hepatitis Ca randomized study of treatment duration and ribavirin doserdquoAnnals of Internal Medicine vol 140 no 5 pp 346ndash355 2004
[26] J G McHutchison E J Lawitz M L Shiffman et al ldquoPeginter-feron alfa-2b or alfa-2a with ribavirin for treatment of hepatitisC infectionrdquoThe New England Journal of Medicine vol 361 no6 pp 580ndash593 2009
[27] C D Howell A Gorden K A Ryan et al ldquoSingle nucleotidepolymorphism upstream of interleukin 28B associated withphase 1 and phase 2 of early viral kinetics in patients infectedwith HCV genotype 1rdquo Journal of Hepatology vol 56 no 3 pp557ndash563 2012
[20] L N Cavalcante K Abe-Sandes A L D Angelo et alldquoIL28B polymorphisms aremarkers of therapy response and areinfluenced by genetic ancestry in chronic hepatitis C patientsfrom an admixed populationrdquo Liver International vol 32 no 3pp 476ndash486 2012
[21] M Venegas R A Villanueva K Gonzalez and J BrahmldquoIL28B polymorphisms associated with therapy response inChilean chronic hepatitis C patientsrdquo World Journal of Gas-troenterology vol 17 no 31 pp 3636ndash3639 2011
[22] A F Stattermayer R Stauber H Hofer et al ldquoImpact ofIL28B genotype on the early and sustained virologic responsein treatment-naıve patients with chronic hepatitis Crdquo ClinicalGastroenterology andHepatology vol 9 no 4 pp 344ndash350 2011
[23] A Rauch Z Kutalik P Descombes et al ldquoGenetic variationin IL28B is associated with chronic hepatitis C and treatmentfailure a genome-wide association studyrdquoGastroenterology vol138 no 4 pp 1338ndash1344 2010
[24] F Marabita A Aghemo S de Nicola et al ldquoGenetic variationin the interleukin-28B gene is not associated with fibrosisprogression in patients with chronic hepatitis C and known dateof infectionrdquo Hepatology vol 54 no 4 pp 1127ndash1134 2011
[25] S JHadziyannisH Sette Jr T RMorgan et al ldquoPeginterferon-1205722a and ribavirin combination therapy in chronic hepatitis Ca randomized study of treatment duration and ribavirin doserdquoAnnals of Internal Medicine vol 140 no 5 pp 346ndash355 2004
[26] J G McHutchison E J Lawitz M L Shiffman et al ldquoPeginter-feron alfa-2b or alfa-2a with ribavirin for treatment of hepatitisC infectionrdquoThe New England Journal of Medicine vol 361 no6 pp 580ndash593 2009
[27] C D Howell A Gorden K A Ryan et al ldquoSingle nucleotidepolymorphism upstream of interleukin 28B associated withphase 1 and phase 2 of early viral kinetics in patients infectedwith HCV genotype 1rdquo Journal of Hepatology vol 56 no 3 pp557ndash563 2012
![Page 1: Research Article SNP rs8099917 in Gene IL28B Might Be ...C was used. Each reaction included [1X ] TaqMan Universal PCR Master Mix [2X ], [1X ] TaqMan Assay [20 X ],andngof DNA in a](https://reader033.documents.pub/reader033/viewer/2022060907/60a2275aea9180250f33defb/html5/thumbnails/1.jpg)
![Page 2: Research Article SNP rs8099917 in Gene IL28B Might Be ...C was used. Each reaction included [1X ] TaqMan Universal PCR Master Mix [2X ], [1X ] TaqMan Assay [20 X ],andngof DNA in a](https://reader033.documents.pub/reader033/viewer/2022060907/60a2275aea9180250f33defb/html5/thumbnails/2.jpg)
![Page 3: Research Article SNP rs8099917 in Gene IL28B Might Be ...C was used. Each reaction included [1X ] TaqMan Universal PCR Master Mix [2X ], [1X ] TaqMan Assay [20 X ],andngof DNA in a](https://reader033.documents.pub/reader033/viewer/2022060907/60a2275aea9180250f33defb/html5/thumbnails/3.jpg)
![Page 4: Research Article SNP rs8099917 in Gene IL28B Might Be ...C was used. Each reaction included [1X ] TaqMan Universal PCR Master Mix [2X ], [1X ] TaqMan Assay [20 X ],andngof DNA in a](https://reader033.documents.pub/reader033/viewer/2022060907/60a2275aea9180250f33defb/html5/thumbnails/4.jpg)
![Page 5: Research Article SNP rs8099917 in Gene IL28B Might Be ...C was used. Each reaction included [1X ] TaqMan Universal PCR Master Mix [2X ], [1X ] TaqMan Assay [20 X ],andngof DNA in a](https://reader033.documents.pub/reader033/viewer/2022060907/60a2275aea9180250f33defb/html5/thumbnails/5.jpg)
![Page 6: Research Article SNP rs8099917 in Gene IL28B Might Be ...C was used. Each reaction included [1X ] TaqMan Universal PCR Master Mix [2X ], [1X ] TaqMan Assay [20 X ],andngof DNA in a](https://reader033.documents.pub/reader033/viewer/2022060907/60a2275aea9180250f33defb/html5/thumbnails/6.jpg)
![Page 7: Research Article SNP rs8099917 in Gene IL28B Might Be ...C was used. Each reaction included [1X ] TaqMan Universal PCR Master Mix [2X ], [1X ] TaqMan Assay [20 X ],andngof DNA in a](https://reader033.documents.pub/reader033/viewer/2022060907/60a2275aea9180250f33defb/html5/thumbnails/7.jpg)
