chronic hepatitis c ( chc ) is a major health care burden and a leading cause of end - stage liver disease and hepatocellular carcinoma ( hcc ) worldwide . although non - uniform distributions of chc in certain areas complicate the establishment of global and regional epidemiology , the global prevalence of chc has been estimated as 2.35% , affecting approximately 160 million people ( 1 ) . in taiwan , seroprevalences of hepatitis c virus ( hcv ) antibody positivity were estimated as 4.4% to 8.6% ( 2 , 3 ) . upwards of 53% of chc patients are infected with hcv genotype 1 ( g1 ) ( 4 ) . either in most resource - limited areas worldwide or in most of the asian countries , pegylated interferon ( pegifn ) plus ribavirin ( rbv ) combination therapy remains the first - line standard of care ( soc ) for chc . the national health insurance program of taiwan reimburses the cost of soc for chc . however , severe rbv - induced hemolytic anemia severely complicates chc patients during soc by causing suboptimal tolerance , poor compliance and early withdrawal from therapy ( 5 ) . the risks of anemia have been shown to be higher in asian than non - asian patients ( 6 ) . extensive accumulation of rbv in erythrocytes causes membrane oxidative damage , premature hemolysis and subsequent anemia ( 7 ) . the haptoglobin phenotype , pretreatment platelet level ( 8) , impaired renal function ( 9 ) , high dose / body weight ratio , old age and female sex ( 10 ) have been previously demonstrated to be associated with anemia . recent genome - wide association studies have advanced to a strong association between rbv - induced anemia and single - nucleotide polymorphisms ( snps ) in the inosine triphosphate pyrophosphatase ( itpa ) gene on chromosome 20 in chc patients on soc ( 11 ) . identifying patients at high risk of anemia is crucial for improving patient compliance and soc outcomes for chc patients ( 12 ) . novel treatment strategies or algorithms based on a combination of relevant pharmacogenetics and host factors may facilitate patient counseling prior to anemia events ( 13 ) . however , limited studies included predictive modeling of severe anemia ( hemoglobin , hb < 10 g / dl ) based on an index incorporating the strong predictor , itpa snp status ( 14 , 15 ) . therefore , we estimated the effect of itpa snp status on severe anemia and treatment responses to construct a clinically practical predictive index for severe anemia during chc combination therapy . the study protocol conforms to the ethical guidelines of the 1975 declaration of helsinki and was approved by the institutional review board of china medical university hospital . informed consent was obtained from each patient included in the study . from september 2005 to september 2013 , consecutive east asian patients with chc g1 were screened and enrolled in a prospective cohort to analyze antiviral treatment responses . we defined chc g1 as positive result for serum anti - hcv antibody ( abbott laboratories , abbott park , il , usa ) for more than six months with detectable serum hcv g1 rna ( cobas amplicor hcv monitor 2.0 ; roche diagnostics , branchburg , nj , usa ) . patients with a history of any of the following conditions were excluded from the cohort ; age < 20 years or > 75 years , treatment discontinued in less than four months after starting soc with no severe anemia event , hepatitis b virus coinfection , human immunodeficiency virus coinfection , hcc , alcoholic liver disease , primary biliary cirrhosis , primary sclerosing cholangitis , wilson s disease , autoimmune hepatitis , hemochromatosis , previous ifn - based therapy , baseline hb < 13 g / dl in men , hb < 12 g / dl in women , nucleoside- or nucleotide - analog therapies other than rbv , decompensated cirrhosis and end - stage renal disease . eligible patients were randomly assigned to the development and validation cohorts at a ratio of 4:1 . patients received pegifn -2a ( pegasys , hoffmann - la roche , basel , switzerland ) at a dosage of 180 g / week or pegifn -2b ( peg - intron , schering - plough , kenilworth , nj , usa ) at a dosage of 1.5 g / kg / week subcutaneously . oral rbv was prescribed for all patients at a daily dose of 1000 mg ( body weight < 75 kg ) or 1200 mg ( body weight  75 kg ) for 24 weeks or 48 weeks based on the baseline hcv rna ( < versus  6 log10 copies / ml ) and virological response at week 4 ( 16 , 17 ) . baseline and mean doses of rbv ( mg / kg / day ) on treatment were calculated for each patient . erythropoietin ( epo ) was considered if hb fell below 10 g / dl during the treatment . blood biochemistry ( beckman coulter , ca , usa ) and complete blood count analyses ( sysmex hst - series , kanogawa , japan ) were performed in the central laboratory at the medical center . hcv rna load was quantified at baseline and monitored at weeks 4 , 12 , 24 and 48 . sustained virological response ( svr ) was defined as undetectable hcv rna at or before the end of treatment and at 24 weeks after the end of treatment . monthly distributions of patients with severe anemia ( hemoglobin < 10 g / dl ) on treatment were compared between c / c versus  a / a or c / a  genotypes . participant genomic dna was extracted from peripheral blood mononuclear cells using a qiagen dna blood mini kit ( qiagen , valencia , ca , usa ) . genotyping for one functional missense itpa variant in exon 2 at rs1127354 on chromosome 20 ( 18 , 19 ) was performed using 20 l of faststart universal probe master mix ( roche diagnostics , branchburg , nj , usa ) containing 500 nmol rs1127354 forward primer ( 5-tcttggaacaggtcgttcagattcta-3 ) , 500 nmol rs1127354 reverse primer ( 5-aggaagacagagaaatccaaccatc-3 ) , 250 nmol c allele probe ( fam - agtttccatgcactttggtgg - bbq ) and 250 nmol a allele probe ( yak - agtttacatgcactttggtggc - bbq ) . the reaction mixture was denatured at 95c for 10 minutes before thermal cycling at 95c for 15 seconds and 60c for 1 minute for 40 cycles . genotypes were analyzed using steponetm software version 2.0 ( life technologies , carlsbad , ca , usa ) . therefore , patients were not genotyped for itpa snp rs7270101 ( 18 ) . the cohort was also genotyped for interleukin 28b ( il28b ) polymorphisms at rs8099917 and rs12979860 , as previously described ( 20 ) . both kolmogorov - smirnov and shapiro - wilk tests showed that ( p < 0.05 ) none of the continuous variables in this study had a normal distribution . therefore , continuous variables were expressed as median ( interquartile range , iqr ) and estimated using mann - whitney u test . categorical variables were estimated using the chi - square test or fisher s exact test . univariate logistic regression identified the variables ( p < 0.25 ) associated with severe anemia for subsequent multiple regressions . by expressing the likelihood of severe anemia as a probability ranging from 0 to 1 , an index for identifying severe anemia was constructed by incorporating significant independent associated factors and the beta coefficients acquired through the final multiple logistic regression for severe anemia ( 14 ) . the diagnostic performance of the predictive index was evaluated by the area under the receiver operating - characteristic curves ( aucs ) . the odds ratios ( ors ) of significant associations were determined based on a 95% confidence interval ( ci ) . the study protocol conforms to the ethical guidelines of the 1975 declaration of helsinki and was approved by the institutional review board of china medical university hospital . from september 2005 to september 2013 , consecutive east asian patients with chc g1 were screened and enrolled in a prospective cohort to analyze antiviral treatment responses . we defined chc g1 as positive result for serum anti - hcv antibody ( abbott laboratories , abbott park , il , usa ) for more than six months with detectable serum hcv g1 rna ( cobas amplicor hcv monitor 2.0 ; roche diagnostics , branchburg , nj , usa ) . patients with a history of any of the following conditions were excluded from the cohort ; age < 20 years or > 75 years , treatment discontinued in less than four months after starting soc with no severe anemia event , hepatitis b virus coinfection , human immunodeficiency virus coinfection , hcc , alcoholic liver disease , primary biliary cirrhosis , primary sclerosing cholangitis , wilson s disease , autoimmune hepatitis , hemochromatosis , previous ifn - based therapy , baseline hb < 13 g / dl in men , hb < 12 g / dl in women , nucleoside- or nucleotide - analog therapies other than rbv , decompensated cirrhosis and end - stage renal disease . eligible patients were randomly assigned to the development and validation cohorts at a ratio of 4:1 . patients received pegifn -2a ( pegasys , hoffmann - la roche , basel , switzerland ) at a dosage of 180 g / week or pegifn -2b ( peg - intron , schering - plough , kenilworth , nj , usa ) at a dosage of 1.5 g / kg / week subcutaneously . oral rbv was prescribed for all patients at a daily dose of 1000 mg ( body weight < 75 kg ) or 1200 mg ( body weight  75 kg ) for 24 weeks or 48 weeks based on the baseline hcv rna ( < versus  6 log10 copies / ml ) and virological response at week 4 ( 16 , 17 ) . baseline and mean doses of rbv ( mg / kg / day ) on treatment were calculated for each patient . erythropoietin ( epo ) was considered if hb fell below 10 g / dl during the treatment . blood biochemistry ( beckman coulter , ca , usa ) and complete blood count analyses ( sysmex hst - series , kanogawa , japan ) were performed in the central laboratory at the medical center . hcv rna load was quantified at baseline and monitored at weeks 4 , 12 , 24 and 48 . sustained virological response ( svr ) was defined as undetectable hcv rna at or before the end of treatment and at 24 weeks after the end of treatment . monthly distributions of patients with severe anemia ( hemoglobin < 10 g / dl ) on treatment were compared between c / c versus  a / a or c / a  genotypes . participant genomic dna was extracted from peripheral blood mononuclear cells using a qiagen dna blood mini kit ( qiagen , valencia , ca , usa ) . genotyping for one functional missense itpa variant in exon 2 at rs1127354 on chromosome 20 ( 18 , 19 ) was performed using 20 l of faststart universal probe master mix ( roche diagnostics , branchburg , nj , usa ) containing 500 nmol rs1127354 forward primer ( 5-tcttggaacaggtcgttcagattcta-3 ) , 500 nmol rs1127354 reverse primer ( 5-aggaagacagagaaatccaaccatc-3 ) , 250 nmol c allele probe ( fam - agtttccatgcactttggtgg - bbq ) and 250 nmol a allele probe ( yak - agtttacatgcactttggtggc - bbq ) . the reaction mixture was denatured at 95c for 10 minutes before thermal cycling at 95c for 15 seconds and 60c for 1 minute for 40 cycles . genotypes were analyzed using steponetm software version 2.0 ( life technologies , carlsbad , ca , usa ) . the cohort was also genotyped for interleukin 28b ( il28b ) polymorphisms at rs8099917 and rs12979860 , as previously described ( 20 ) . both kolmogorov - smirnov and shapiro - wilk tests showed that ( p < 0.05 ) none of the continuous variables in this study had a normal distribution . therefore , continuous variables were expressed as median ( interquartile range , iqr ) and estimated using mann - whitney u test . categorical variables were estimated using the chi - square test or fisher s exact test . univariate logistic regression identified the variables ( p < 0.25 ) associated with severe anemia for subsequent multiple regressions . by expressing the likelihood of severe anemia as a probability ranging from 0 to 1 , an index for identifying severe anemia was constructed by incorporating significant independent associated factors and the beta coefficients acquired through the final multiple logistic regression for severe anemia ( 14 ) . the diagnostic performance of the predictive index was evaluated by the area under the receiver operating - characteristic curves ( aucs ) . the odds ratios ( ors ) of significant associations were determined based on a 95% confidence interval ( ci ) . excluded patients included those with baseline hepatitis b virus coinfection ( n = 9 ) , hcc ( n = 5 ) , a history of ifn - based therapy ( n = 11 ) , men with baseline hb < 13 g / dl or women < 12 g / dl ( n = 8) and end - stage renal disease ( n = 2 ) . therefore , the chc g1 cohort consisted of 418 eligible participants who intended to receive combination therapy . nine patients were also excluded , because the treatment was discontinued within four months from the treatment baseline for reasons other than severe anemia . the cohort was randomized into a development cohort ( n = 324 ) and a validation cohort ( n = 81 ) . among them , 367 ( 90.6% ) of 405 patients had liver histology data . a total of 148 ( 36.5% ) patients received combination therapy for 24 weeks and 257 ( 63.5% ) patients for 48 weeks . patients per protocol ( n = 357 ) entered the analysis for svr ( 41 patients discontinued before the intended eot and seven patients were lost to follow - up for svr visit ) . the median patient age was 53.0 ( 13.0 ) years and 49.6% of patients were men . the c / c genotype was present in 66.4% of patients and 33.6% had the a / a or the c / a allelic variants (  a / a or c / a  as a combo variable ) at rs1127354 . the development cohort ( n = 324 ) and validation cohort ( n = 81 ) did not differ significantly in a univariate analysis . monthly distributions of severe anemia events are demonstrated in figure 1 . among patients who received 24-week treatment and developed severe anemia , a significantly higher percentage of patients with c / c genotype did so during the first month on treatment than those with  a / a or c / a  genotype ( 37.1% versus 0.0% , p = 0.0135 ) . likewise , among patients who received 48-week treatment and developed severe anemia , a significantly higher percentage of patients with c / c genotype did so during the first month on treatment than those with  a / a or c / a  genotype ( 22.9% versus 2.9% , p = 0.0079 ) . in total , 166 patients ( 166/324 , 51.2% ) in the development cohort developed severe anemia on treatment . multiple logistic regressions identified age (  50 y : or = 9.7 , 95% ci = 5.0 - 18.6 ) , itpa rs1127354 ( c / c : or = 3.3 , 95% ci = 1.8 - 5.8 ) and baseline hemoglobin ( < 14.0 g / dl : or 6.4 , 95% ci = 3.3 - 12.1 ; 14.0 - 14.9 : or = 2.4 , 95% ci = 1.2 - 4.6 ) as predictors of severe anemia on treatment ( tables 1 and 2 ) . there was no significant dose effect between itpa snp status ( c / a versus a / a , p = 0.5387 ) and severe anemia . therefore , the predictive index derived from the development cohort incorporating age ( y ) , itpa snp status and baseline hb ( g / dl ) was expressed as 1/(1 + exp ( - x ) ) , where x = - 3.2835 + 2.2709  a + 1.1822  i + 1.8848  h1 + 0.8684  h2 ( a = 1 if age  50 ; a = 0 if age < 50 ; i = 1 if itpa snp = c / c ; i = 0 if itpa snp =  a / a or c / a  ; h1 = 1 if hb < 14.0 ; h1 = 0 if hb  14.0 ; h2 = 1 if hb = 14.0 - 14.9 ; h2 = 0 if hb < 14.0 or  15.0 ) . to dichotomize the anemia status ( with versus without severe anemia ) in the development , the cutoff value of 0.5 was optimal when maximizing the values of the predictive index yielded a diagnostic accuracy ( auc ) of 0.830 ( 95% ci = 0.783 - 0.871 ) in the development ( n = 324 ) and 0.902 ( 0.826 - 0.925 ) in the validation ( n = 81 ) cohorts to dichotomize the anemia status , respectively ( figure 2 ) . in total , 135 ( 135/357 per protocol , 37.8% ) patients achieved rvr . seventy - three of them ( 73/135 , 54.1% ) had a median baseline hcv rna of 5.29 ( 0.76 ) log10 copies / ml and received 24-week treatment . the remaining 62 patients ( 62/135 , 45.9% ) received 48-week treatment . liver pathology was acquired in 69 of 73 ( 94.5% ) patients with rvr and 24-week treatment . among them , metavir fibrosis stages 0 - 2 were noted in 54 ( 54/69 , 78.3% ) , and 3 - 4 in 15 ( 15/69 , 21.7% ) . in total , 255 ( 255/357 per protocol , 71.4% ) patients achieved svr . the rvr ( or = 8.2 ; 95% ci = 3.519.1 ; p < .0001 ) , t / t il28b genotype at rs8099917 ( or = 3.0 ; 95% ci = 1.2 - 7.4 ; p = 0.0156 ) , the metavir fibrosis stages 0 - 2 ( or = 1.9 ; 95% ci = 1.0 - 3.5 ; p = 0.0450 ) , baseline hcv rna ( or = 0.4 ; 95% ci = 0.2 - 0.7 ; p = 0.0008 ) and 48-week treatment duration ( or = 1.1 ; 95% ci = 1.0 - 1.1 ; p = 0.0003 ) were independently associated with svr . anemia events and epo treatment were not independently associated with svr . in patients with either itpa c / c or a / a or c / a  genotypes , the mean rbv dose exposures were not significantly different between patients with svr versus non - svr . the il28b snp ( rs12979860 ) was not included in the final multiple regression because of collinearity with the other il28b snp ( rs8099917 ) ( table 3 ) . in addition , through univariate analyses , the itpa snp status was significantly associated with rbv dose reduction in the development cohort ( n = 324 ) ( mann - whitney u test , p = 0.0035 ) . patients ( n = 103/324 , 31.8% ) with  a / a or c / a  genotype received 100% ( 8% ) intended rbv doses . in contrast , patients ( n = 221/324 , 68.2% ) with c / c genotype received 97% ( 1.4% ) intended rbv doses . however , itpa snp status was not significantly associated with epo use ( chi - square test , p = 0.1617 ) . forty - five of 103 patients ( 45/103 , 43.7% ) with  a / a or c / a  genotype received epo treatment . in contrast , 115 of 221 patients ( 115/221 , 52.0% ) with c / c genotype received epo treatment . a ) among patients who received 24-week treatment and developed severe anemia , a significantly higher percentage of patients with c / c genotype did so during the first month than those with  a / a or c / a  genotype ( p = 0.0135 ) . b ) ; also , among patients who received 48-week treatment ( p = 0.0079 ) . data are presented for hemoglobin < 10 g / dl and development cohort ( n = 324 ) . excluded patients included those with baseline hepatitis b virus coinfection ( n = 9 ) , hcc ( n = 5 ) , a history of ifn - based therapy ( n = 11 ) , men with baseline hb < 13 g / dl or women < 12 g / dl ( n = 8) and end - stage renal disease ( n = 2 ) . therefore , the chc g1 cohort consisted of 418 eligible participants who intended to receive combination therapy . nine patients were also excluded , because the treatment was discontinued within four months from the treatment baseline for reasons other than severe anemia . the cohort was randomized into a development cohort ( n = 324 ) and a validation cohort ( n = 81 ) . among them , 367 ( 90.6% ) of 405 patients had liver histology data . a total of 148 ( 36.5% ) patients received combination therapy for 24 weeks and 257 ( 63.5% ) patients for 48 weeks . patients per protocol ( n = 357 ) entered the analysis for svr ( 41 patients discontinued before the intended eot and seven patients were lost to follow - up for svr visit ) . the median patient age was 53.0 ( 13.0 ) years and 49.6% of patients were men . the c / c genotype was present in 66.4% of patients and 33.6% had the a / a or the c / a allelic variants (  a / a or c / a  as a combo variable ) at rs1127354 . the development cohort ( n = 324 ) and validation cohort ( n = 81 ) did not differ significantly in a univariate analysis . monthly distributions of severe anemia events are demonstrated in figure 1 . among patients who received 24-week treatment and developed severe anemia , a significantly higher percentage of patients with c / c genotype did so during the first month on treatment than those with  a / a or c / a  genotype ( 37.1% versus 0.0% , p = 0.0135 ) . likewise , among patients who received 48-week treatment and developed severe anemia , a significantly higher percentage of patients with c / c genotype did so during the first month on treatment than those with a / a or c / a  genotype ( 22.9% versus 2.9% , p = 0.0079 ) . in total , 166 patients ( 166/324 , 51.2% ) in the development cohort developed severe anemia on treatment . multiple logistic regressions identified age (  50 y : or = 9.7 , 95% ci = 5.0 - 18.6 ) , itpa rs1127354 ( c / c : or = 3.3 , 95% ci = 1.8 - 5.8 ) and baseline hemoglobin ( < 14.0 g / dl : or 6.4 , 95% ci = 3.3 - 12.1 ; 14.0 - 14.9 : or = 2.4 , 95% ci = 1.2 - 4.6 ) as predictors of severe anemia on treatment ( tables 1 and 2 ) . there was no significant dose effect between itpa snp status ( c / a versus a / a , p = 0.5387 ) and severe anemia . therefore , the predictive index derived from the development cohort incorporating age ( y ) , itpa snp status and baseline hb ( g / dl ) was expressed as 1/(1 + exp ( - x ) ) , where x = - 3.2835 + 2.2709  a + 1.1822  i + 1.8848  h1 + 0.8684  h2 ( a = 1 if age  50 ; a = 0 if age < 50 ; i = 1 if itpa snp = c / c ; i = 0 if itpa snp =  a / a or c / a  ; h1 = 1 if hb < 14.0 ; h1 = 0 if hb  14.0 ; h2 = 1 if hb = 14.0 - 14.9 ; h2 = 0 if hb < 14.0 or  15.0 ) . to dichotomize the anemia status ( with versus without severe anemia ) in the development , the cutoff value of 0.5 was optimal when maximizing the values of the predictive index yielded a diagnostic accuracy ( auc ) of 0.830 ( 95% ci = 0.783 - 0.871 ) in the development ( n = 324 ) and 0.902 ( 0.826 - 0.925 ) in the validation ( n = 81 ) cohorts to dichotomize the anemia status , respectively ( figure 2 ) . seventy - three of them ( 73/135 , 54.1% ) had a median baseline hcv rna of 5.29 ( 0.76 ) log10 copies / ml and received 24-week treatment . liver pathology was acquired in 69 of 73 ( 94.5% ) patients with rvr and 24-week treatment . among them , metavir fibrosis stages 0 - 2 were noted in 54 ( 54/69 , 78.3% ) , and 3 - 4 in 15 ( 15/69 , 21.7% ) . in total , 255 ( 255/357 per protocol , 71.4% ) patients achieved svr . the rvr ( or = 8.2 ; 95% ci = 3.519.1 ; p < .0001 ) , t / t il28b genotype at rs8099917 ( or = 3.0 ; 95% ci = 1.2 - 7.4 ; p = 0.0156 ) , the metavir fibrosis stages 0 - 2 ( or = 1.9 ; 95% ci = 1.0 - 3.5 ; p = 0.0450 ) , baseline hcv rna ( or = 0.4 ; 95% ci = 0.2 - 0.7 ; p = 0.0008 ) and 48-week treatment duration ( or = 1.1 ; 95% ci = 1.0 - 1.1 ; p = 0.0003 ) were independently associated with svr . anemia events and epo treatment were not independently associated with svr . in patients with either itpa c / c or a / a or c / a  genotypes , the mean rbv dose exposures were not significantly different between patients with svr versus non - svr . the il28b snp ( rs12979860 ) was not included in the final multiple regression because of collinearity with the other il28b snp ( rs8099917 ) ( table 3 ) . in addition , through univariate analyses , the itpa snp status was significantly associated with rbv dose reduction in the development cohort ( n = 324 ) ( mann - whitney u test , p = 0.0035 ) . patients ( n = 103/324 , 31.8% ) with  a / a or c / a  genotype received 100% ( 8% ) intended rbv doses . in contrast , patients ( n = 221/324 , 68.2% ) with c / c genotype received 97% ( 1.4% ) intended rbv doses . however , itpa snp status was not significantly associated with epo use ( chi - square test , p = 0.1617 ) . forty - five of 103 patients ( 45/103 , 43.7% ) with  a / a or c / a  genotype received epo treatment . in contrast , 115 of 221 patients ( 115/221 , 52.0% ) with c / c genotype received epo treatment . a ) among patients who received 24-week treatment and developed severe anemia , a significantly higher percentage of patients with c / c genotype did so during the first month than those with b ) ; also , among patients who received 48-week treatment ( p = 0.0079 ) . data are presented for hemoglobin < 10 g / dl and development cohort ( n = 324 ) . this incidence was higher than that reported ( 39.3% ) in a previous study of 466 east asian patients diagnosed with chc ( 5 ) . the higher anemia frequency in our study can be attributed to the higher rbv dosage in patients infected with hcv g1 . consistent with hb kinetics reported in previous studies ( 5 , 12 , 19 , 22 - 25 ) , the anemia events in our study accumulated during the first few months of treatment and remained relatively stable thereafter ( figure 1 ) . the accumulated itp was substituted for gtp during the biosynthesis of atp and protected against rbv - induced hemolytic anemia . . methods of gene expression analysis such as mrna transcripts or western blotting were not used in our study . however , the results of snp genotyping of itpa that we performed correlated closely with phenotypes or predicted itpa activities of these allelic variants ( 26 - 28 ) . although rbv pharmacokinetics in serum or in erythrocytes or predicted itpa activities were not applicable in our current study ( 29 ) , this index may serve as a clinically practical one based on dichotomized parameters available in clinical settings . future studies can develop algorithms or scoring systems based on age , sex , body weight , renal function , hb and itpa snp status for modifying rbv dosage to achieve an optimal steady - state concentration range or for administering epo prior to the development of anemia , rather than reducing the rbv dose following an anemia event ( 30 , 31 ) . recent studies indicated that itpa allelic variants  a / a or c / a  at rs1127354 are less likely associated with rbv dose reduction ( 19 , 22 , 23 , 32 ) . future studies can also use a time - to - event analysis to identify significant predictors for rbv dose reduction . the effects of older age and female sex on severe anemia ( < 11 g / dl ) during the first four weeks of treatment were also reported in a study of 61 hcv g1-infected japanese patients , in which 49 patients exhibited rbv - sensitive itpa c / c genotype at rs1127354 and 12 patients exhibited rbv - resistant  a / a or c / a  variants ( 19 ) . therefore , older female patients require stringent monitoring of anemia during combination therapy . however , sex was not included in the predictive index through multiple logistic regressions in the current study because of collinearity with baseline hb . therefore , it was not applicable to adopt creatinine level in multiple regression modeling for predictive index of severe anemia . in the study by tsubota et al . , estimated glomerular filtration rate was found to be one of the significant associated factors to construct a predictive model for rbv - induced anemia ( 14 ) . our future studies would recruit participants in larger sample sizes receiving more uniform treatment durations to allow time - dependent analysis of anemia events for constructing a predictive index through cox regression analysis . based on our results , we suggest that subjects with a predictive index greater than 0.5 might receive epo treatment early after the start of pegifn and rbv combination therapy to facilitate quality of life , prevent rbv dose reduction and avoid compromised treatment responses . however , anticipated benefits associated with early implementation of epo treatment need to be confirmed by further studies . both rvr and t / t il28b genotype at rs8099917 remained significant predictors for svr in our chc g1 cohort ( 33 ) . also consistent with recent studies ( 18 , 19 , 22 - 24 , 34 ) , itpa snp status was a non - significant predictor of svr in our study . however , the reported itpa - svr correlations varied ( 35 ) . in a recent report by rembeck et al . ( 27 ) , itpa allelic variants encoding reduced itpa activity was demonstrated to correlate with svr and non - relapse , unrelated to rbv adherence or protection against anemia , although the molecular mechanisms require further elucidation . although previous reports yielded conflicting results regarding the significance of anemia - svr correlation ( 5 , 12 , 36 , 37 ) , most recent studies on itpa snps have not estimated anemia - svr correlation ( 18 , 19 , 22 - 25 , 32 , 38 ) . in our study , svr was not associated with severe anemia or hb decline (  4 g / dl ) on treatment ( table 3 ) . recent anemia - svr correlations have also been reported based on the divergent results of non - genetic ( 5 , 23 , 36 ) and molecular studies ( 11 , 14 ) . molecular studies indicated that anemia might not only be a surrogate for rbv exposure , because anemia - svr association was attenuated when adjusting for rbv levels ( 39 ) , but also represent the first molecular evidence connecting anemia - svr association to novel genes and pathways through statistical analyses on gene expressions and mrna signatures ( 11 ) . in conclusion , itpa snp status is a significant predictor of rbv - induced hemolytic anemia in patients diagnosed with chc g1 and receiving pegifn and rbv combination therapy . the itpa snp - based index is an accurate and practical predictive solution for severe anemia in clinical practice . information on itpa snp status of patients may assist in maximizing the tolerability of soc for patients with chc .