Unraveling the genetic predisposition of ribavirin-induced anaemia
Authors: Tarik Asselah, Eric Pasmant, Saïd Lyoumi
Journal: Journal of Hepatology (2010)

COMMENTARY ON: ITPA gene variants protect against anaemia in patients treated for chronic hepatitis C. Fellay et al. [10]. Copyright (2010) Nature Publishing Group. Abstract reprinted with permission from Nature. www.ncbi.nlm.nih.gov/pubmed/20173735 Abstract: Chronic infection with the hepatitis C virus (HCV) affects 170 million people worldwide and is an important cause of liver-related morbidity and mortality. The standard of care therapy combines pegylated interferon (peg-IFN) alpha and ribavirin (RBV), and is associated with a range of treatment-limiting adverse effects. One of the most important of these is RBV-induced haemolytic anaemia, which affects most patients and is severe enough to require dose modification in up to 15% of the patients. Here we show that genetic variants leading to inosine triphosphatase deficiency, a condition not thought to be clinically important, protect against haemolytic anaemia in hepatitis C-infected patients receiving RBV. Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. In addition to the viral and environmental behavioural factors, host genetic diversity is believed to contribute to the spectrum of the disease [[1]Asselah T. Bieche I. Sabbagh A. et al.Gene expression and hepatitis C virus infection.Gut. 2009; 58: 846-858Crossref PubMed Scopus (91) Google Scholar]. The standard of care (SOC) consists of the combination of pegylated interferon (Peg-IFN) plus ribavirin (RBV). However, this care only induces a sustained virological response (SVR) in half of genotype 1 patients. Several independent genome wide association studies (GWAS)s reported single nucleotide polymorphisms (SNP)s in the IL28B (IFN-λ3) region, associated with SVR [2Ge D. Fellay J. Thompson A.J. et al.Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance.Nature. 2009; 461: 399-401Crossref PubMed Scopus (3114) Google Scholar, 3Asselah T, Essioux L, Marcellin P, et al. A chromosome 19 SNP (RS12979860) predicts outcome (EVR/SVR) in HCV patients treated with interferon, independent of pegylation or ribavirin. J Hepatol 2010; EASL: A1180.Google Scholar]. The mechanisms by which RBV enhances the rate of SVR when added to IFNs remain unknown. Chances to achieve an SVR significantly decrease when the patients receive less than 80% of the total dose of peg-IFN and/or less than 80% of the total RBV and/or during less than 80% of the total treatment duration [[4]McHutchison J.G. Manns M. Patel K. et al.Adherence to combination therapy enhances sustained response in genotype-1-infected patients with chronic hepatitis C.Gastroenterology. 2002; 123: 1061-1069Abstract Full Text Full Text PDF PubMed Scopus (959) Google Scholar]. The current SOC causes a mean decline in haemoglobin (Hb) concentrations of approximately 3 g/dl. The RBV-induced anaemia most often occurs as a dose-dependent haemolytic anaemia, typically developing within the first 4 weeks of therapy [[5]Shiffman M.L. Ghany M.G. Morgan T.R. et al.Impact of reducing peginterferon alfa-2a and ribavirin dose during retreatment in patients with chronic hepatitis C.Gastroenterology. 2007; 132: 103-112Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar]. IFN can also contribute to the development of anaemia by suppressing the bone marrow production of erythrocytes. Dose reductions or the use of haematopoietic growth factors may help control RBV-induced anaemia, but the discontinuation of RBV is sometimes necessary. The conventional SOC for managing anaemia during the therapy has consisted in reducing the RBV dose if the haemoglobin level decreases to less than 10 g/dl, and to completely stop it if the haemoglobin level drops below 8.5 g/dl. A decrease in the RBV dose, especially in the first several months of treatment, can diminish the response rates considerably. The recombinant erythropoietin hormone, epoetin alfa (Epogen, Procrit), has emerged as an option for improving HCV treatment-related anaemia while supporting the optimal treatment doses of RBV and IFN. No study, however, has established that the use of erythropoietin increases the overall SVR rates associated with HCV therapy [[5]Shiffman M.L. Ghany M.G. Morgan T.R. et al.Impact of reducing peginterferon alfa-2a and ribavirin dose during retreatment in patients with chronic hepatitis C.Gastroenterology. 2007; 132: 103-112Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar]. Interestingly, viramidine (currently known as taribavirin), a prodrug of RBV taken up predominantly by hepatocytes and extensively converted to RBV by adenosine deaminase has been developed [[6]Marcellin P. Gish R.G. Gitlin N. et al.Safety and efficacy of viramidine versus ribavirin in ViSER2: randomized, double-blind study in therapy-naive hepatitis C patients.J Hepatol. 2010; 52: 32-38Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar]. It is known that viramidine does not accumulate in red blood cells, so this agent is likely to be associated with lower anaemia rates. Compared to ribavirine, viramidine did not meet the primary efficacy non-inferiority end-point but met the safety end-point [[6]Marcellin P. Gish R.G. Gitlin N. et al.Safety and efficacy of viramidine versus ribavirin in ViSER2: randomized, double-blind study in therapy-naive hepatitis C patients.J Hepatol. 2010; 52: 32-38Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar]. Determination of a viramidine dosage that would yield superior efficacy over RBV is needed. RBV is essential for improving SVR through preventing relapses and a breakthrough. Bronowicki et al. investigated the consequence of RBV cessation in genotype 1-infected patients who cleared HCV RNA at week 24 [[7]Bronowicki J.P. Ouzan D. Asselah T. et al.Effect of ribavirin in genotype 1 patients with hepatitis C responding to pegylated interferon alfa-2a plus ribavirin.Gastroenterology. 2006; 131: 1040-1048Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar]. They randomised patients to either combination therapy after 24 weeks or Peg-IFN alpha-2a alone. Patients who stopped RBV had significantly higher viral breakthroughs and SVR was reduced. However, stopping RBV at 24 weeks in patients who had experienced a rapid virologic response did not affect SVR. Similar data were also generated by the IDEAL study, which compared SVR amonst three groups: (i) Peg-IFN alpha-2b 1.5 g/kg/week + RBV 800–1400 mg/day; (ii) Peg-IFN alpha-2b 1.0 g/kg/week + RBV 800–1400 mg/day; and (iii) peginterferon-alpha-2a 180 g/kg/week + RBV 1000–1200 mg/day [[8]McHutchison J.G. Lawitz E.J. Shiffman M.L. et al.Peginterferon alfa-2b or alfa-2a with ribavirin for treatment of hepatitis C infection.N Engl J Med. 2009; 361: 580-593Crossref PubMed Scopus (1122) Google Scholar]. Increased exposure to RBV was associated with an increased SVR. Although anaemia requiring dose reduction and/or erythropoietin occurred in 28% of all patients, this did not reduce the SVR. In fact, patients with anaemia needing RBV dose reduction had a higher rate of SVR even when the RBV was reduced by as much as 50%. Anaemia is linked to RBV concentration, even if this relationship is highly variable. To optimise RBV utilisation, it has been proposed that the global exposure after the first dose of ribavirin is an early and robust pharmacokinetic predictor of SVR [[9]Loustaud-Ratti V. Alain S. Rousseau A. et al.Ribavirin exposure after the first dose is predictive of sustained virological response in chronic hepatitis C.Hepatology. 2008; 47: 1453-1461Crossref PubMed Scopus (81) Google Scholar]. Fellay et al. using DNA from consenting participants of the IDEAL study, performed a GWAS of determinants of treatment-related anaemia in individuals with chronic genotype 1 hepatitis C [[10]Fellay J. Thompson A.J. Ge D. et al.ITPA gene variants protect against anaemia in patients treated for chronic hepatitis C.Nature. 2010; 464: 405-408Crossref PubMed Scopus (415) Google Scholar]. Shortly, GWAS use dense maps of genetic markers that cover the human genome to look for allele frequency differences between cases and controls. A significant frequency difference suggests that the corresponding region of the genome contains functional DNA variants that influence the trait of interest. The strength of the genome-wide screening is its ability to reveal not only genes that would be expected to play a significant role, but also genes that would not, potentially adding a new insight into physiopathology. A total of 1286 individuals who were classified into three ethnic groups (988 European–Americans, 198 African–Americans and 100 Hispanics) were available for analyses. Among these 1286 patients, 542 developed clinically significant anaemia at week 4 (defined as either a decline in Hb of >3 g/dl and/or Hb levels <10 g/dl). The primary analysis focused on the quantitative change in Hb levels from the baseline to the fourth week of treatment, historically the point at which many patients begin erythropoietin treatment to stimulate red blood cell production. They tested each of 565,759 SNPs passing quality control measures in a linear regression model incorporating significant clinical covariates including the baseline Hb levels. Several SNPs on chromosome 20 (20p13 region) were found to be strongly associated with treatment-induced reduction in Hb at week 4, with the European–American sample showing overwhelming genome-wide significance (p = 1.1 × 10−45) for an association between quantitative Hb reduction at week 4 and the SNP rs6051702. These SNPs are located in a 250 kb region that contained five genes including the inosine triphosphatase (ITPA) gene. The protein encoded by this gene hydrolyzes inosine triphosphate (ITP) and deoxyinosine triphosphate (dITP), a central intermediate in purine metabolism. The encoded protein, which is a member of the HAM1 NTPase protein family, is found in the cytoplasm and acts as a homodimer. Defects in the encoded protein can result in inosine triphosphate pyrophosphorylase deficiency [[11]Sumi S. Marinaki A.M. Arenas M. et al.Genetic basis of inosine triphosphate pyrophosphohydrolase deficiency.Hum Genet. 2002; 111: 360-367Crossref PubMed Scopus (224) Google Scholar]. The putative role of ITPase is to recycle purines trapped in the form of ITP and to protect cell (including erythrocytes) from the accumulation of nucleotides such ITP, dITP, or xanthosine triphosphate (XTP) that may be incorporated into RNA and DNA. ITPA haplotype structure has shown that the two SNPs rs1127354 (proline-to-threonine substitution) and rs7270101 (splicing altering SNP) were the most relevant functional variants in determining ITPA low enzymatic activity and ITP accumulation in red blood cells [[11]Sumi S. Marinaki A.M. Arenas M. et al.Genetic basis of inosine triphosphate pyrophosphohydrolase deficiency.Hum Genet. 2002; 111: 360-367Crossref PubMed Scopus (224) Google Scholar]. Analysis of HapMap data from European parents found both of these low-activity variants to be preferentially associated with the protective rs6051702 C allele. This observation suggested the possibility that these two known functional ITPA variants were responsible for the observed association and conferred protection against anaemia. To test this possibility, Fellay et al. sequenced the entire coding region of the ITPA gene in a subset of 168 samples and found no other obvious reduced function mutations. Moreover, genotyping of the known functional SNPs rs1127354 and rs7270101 in the entire cohort confirmed that the two variants co-segregated with the protector rs6051702 minor allele C and entirely explained the association signal initially identified in the 20p13 region. These data confirmed that the two known functional variants conferring reduced ITPA activity were responsible for the protection against anaemia identified in the original GWAS. Moreover, the resulting difference in ITPA function explained nearly 30% of the variability in quantitative Hb reduction in the European–American sample. Fellay et al. also demonstrated that their observation had clinical relevance as ITPA deficiency was found to protect against clinically significant anaemia defined as either a decline in Hb of >3 g/dl or Hb levels <10 g/dl at week 4, which is the threshold at which RBV dose reduction is recommended (Fig. 1). However, no significant association has been observed between the ITPA deficiency variants and early or late anti-HCV treatment outcomes. A proposed model to explain these observations would be that the accumulation of ITP caused by ITPA deficiency competes with the triphosphate active form of ribavirin (RBV-TP) in unknown cellular processes, and thereby protects cells from the lytic effects of RBV-TP that also accumulates in red blood cells. However, the mechanism of RBV-induced haemolytic anaemia and the herein described protective effect of ITPA deficiency remains to be clearly understood. Because ITPA deficiency seems to be a benign condition, therapies that target this enzyme could help protect against RBV-induced anaemia and testing patients for the deficiency could help identify those at risk. This study by Fellay et al. constitutes a clear example of a synthetic association in which the effects of rarer functional variants are observed as an association for a more common variant present on a whole genome genotyping chip. Relatively common genetic variants have been identified by GWAS that explain very significant proportions of the population variation in drug responses, including the ITPA variants described here and the IL28B variants reported recently [2Ge D. Fellay J. Thompson A.J. et al.Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance.Nature. 2009; 461: 399-401Crossref PubMed Scopus (3114) Google Scholar, 3Asselah T, Essioux L, Marcellin P, et al. A chromosome 19 SNP (RS12979860) predicts outcome (EVR/SVR) in HCV patients treated with interferon, independent of pegylation or ribavirin. J Hepatol 2010; EASL: A1180.Google Scholar]. It is also worth noting that four GWAS have recently reported associations with a range of clinically relevant haematological traits [[12]Ganesh S.K. Zakai N.A. van Rooij F.J. et al.Multiple loci influence erythrocyte phenotypes in the CHARGE consortium.Nat Genet. 2009; 41: 1191-1198Crossref PubMed Scopus (284) Google Scholar]. These studies notably identified several SNPs in the hexokinase 1 gene (HK1 on chromosome 10) significantly associated with two erythrocyte traits (Hb concentration and haematocrit). Interestingly, HK1 SNPs were also identified by Fellay et al. in the context of RBV-induced anaemia, although the association did not reach GWAS significance [[10]Fellay J. Thompson A.J. Ge D. et al.ITPA gene variants protect against anaemia in patients treated for chronic hepatitis C.Nature. 2010; 464: 405-408Crossref PubMed Scopus (415) Google Scholar]. The study by Fellay et al. represents important additional progress in identifying candidate genes associated with diverse and clinically relevant blood-cell traits. The identification of ITPA deficiency as a major protective factor against RBV-induced haemolytic anaemia not only provides a valuable pharmacogenetic diagnostic and maybe pharmacological target, but also provides new insights into red blood cell biology. Prediction of ribavirin-induced anaemia is an important issue. Protease inhibitors are designed for genotype 1 and therefore will be first registered to be associated with PEG-IFN and ribavirin for genotype 1 infected patients [[13]Asselah T. Benhamou Y. Marcellin P. Protease and polymerase inhibitors for the treatment of hepatitis C.Liver Int. 2009; 29: 57-67Crossref PubMed Scopus (74) Google Scholar]. Ribavirin is a drug which is essential to produce higher SVR rates both with Peg-IFN and HCV protease inhibitors currently in Phase III clinical trials. For other genotypes (2, 3 and 4), the SOC (PEG-IFN plus ribavirin) will be used for several years. Thus, ribavirin will stay an important drug to achieve higher SVR rates in HCV infected persons, and the prediction of anaemia will remain an important issue. The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.