To the Editor:

We read with interest the article by Sun et al.1 evaluating the influence of naturally occurring polymorphisms on the potency of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) replication complex inhibitor BMS-790052 (daclatasvir). To date, all substitutions resistant to Daclatasvir have been mapped to the first 100 amino acids of NS5A.2 The authors replaced the NS5A coding region of the HCV-1a and HCV-1b laboratory strains, H77c and Con1, with the corresponding regions of baseline (BL) specimens of 10 HCV-1a and HCV-1b-infected subjects. By this method the authors found that the BL-linked variant, Q30R-E62D, was responsible for the resistant phenotype of one HCV-1a subject, whereas the linked variant Q54H-Y93H displayed minimal resistance in one HCV-1b subject. Therefore, the authors suggest that BL polymorphisms in NS5A may significantly affect the emergence of resistance, providing additional challenges for the evaluation of variants associated with clinical failures.

To assess the naturally occurring rate of these resistant variants, we analyzed a cohort of HCV-1 null responders to pegylated-interferon (PegIFN) plus ribavirin (RBV) therapy. These patients are optimal candidates for a daclatasvir regimen as shown by phase II studies.3 By direct sequencing we analyzed the N-terminal region of the NS5A protein in 8 HCV-1a and 28 HCV-1b subjects. Viral RNA was isolated from the plasma and the NS5A gene was amplified by reverse transcriptase (RT) nested-polymerase chain reaction (PCR) using genotype-specific primers and Platinum Taq high-fidelity DNA polymerase (Invitrogen, Carlsbad, CA). Then, bidirectional DNA sequencing was performed using the BigDye Terminator v. 3.1 Cycle Sequencing Kit and ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). We found multiple substitutions in the first 129 amino acids of NS5A with no known resistance to daclatasvir (Table 1). No HCV-1a subject had the Q30R-E62D linked variant identified by Sun et al., while we found the Q54H-Y93H linked variant in one HCV-1b subject. Finally, all NS5A sequences from HCV-1b patients harbored changes at codon 28 and 30, which are of unknown significance.4 In conclusion, due to the low rates of naturally occurring resistant variants in the NS5A region found in HCV-1 null responders to PegIFN plus RBV, we do not support routine direct sequencing of HCV before starting daclatasvir.

Table 1. NS5A Polymorphisms in HCV 1a and HCV-1b-Infected Patients
HCV Genotype/ SubtypePolymorphisms in the NS5A Region Associated With Resistance to DaclatasvirPolymorphisms in the NS5A Region Without Known Resistance to Daclatasvir
GT-1aR6K; I12V; V37L; R48K; R78K; S85N
GT-1bL31M; Q54H; Y93HT14A; T17A/S; K26R; F37L; K44R; R48Q; T56A/I; T64A; R78K; H81R; A92T; R108K


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  • 1
    Sun JH, O'Boyle DR II, Zhang Y, Wang C, Nower P, Valera L, et al. Impact of a baseline polymorphism on the emergence of resistance to the hepatitis C virus nonstructural protein 5a replication complex inhibitor, BMS-790052. HEPATOLOGY 2012; 55: 1692-1699.
  • 2
    Fridell RA, Wang C, Sun J-H, O'Boyle DR II, Nower P, Valera L, et al. Genotypic and phenotypic analysis of variants resistant to HCV NS5A replication complex inhibitor BMS-790052: in vitro and in vivo correlations. HEPATOLOGY 2011; 54: 1924-1935.
  • 3
    Aghemo A, Colombo M. Interferon-free oral therapy for hepatitis C: “faraway, so close!”. Gastroenterology 2012; 142: 1253-1254.
  • 4
    Plaza Z, Soriano V, Vispo E, Del Mar Gonzalez M, Barreiro P, Seclén E, et al. Prevalence of natural polymorphisms at the HCV NS5A gene associated with resistance to daclatasvir, an NS5A inhibitor. Antivir Ther 2012 doi: 10.3851/IMP2091.

Enrico Galmozzi Ph.D.*, Alessio Aghemo M.D.*, Massimo Colombo M.D.*, * First Division of Gastroenterology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy.