In addition, we checked Hgb level and RBV dose before and after 4, 8, and 12 weeks of anti-viral treatment, and checked serum HCV RNA levels before and after 4 and 12 weeks of anti-viral treatment, and at 24 weeks after terminating anti-viral treatment to investigate rapid virologic response (RVR), early virologic response (EVR), cause and sustained virologic response (SVR). ITPA and IL28B genotyping Blood was collected into EDTA tubes, and genomic DNA was extracted from whole blood using the QIAamp? DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA qualities were assessed by calculating absorbance ratios (OD 260 nm/280 nm) using a NanoDrop model ND-1000 (Thermo Fisher Scientific Inc.

, Wilmington, DE, USA) and DNA quantities were estimated using the Quibit? dsDNA BR Assay kit using a Quibit? Fluorometer (Invitrogen, Carlsbad, CA, USA). ITPA variants rs1127354 C>A, rs7270101 A>C were determined in whole blood samples using the validated Pyrosequencing? assay. Primers were designed using PQS Assay Design software (Qiagen, Hilden, Germany) to target two SNP regions. The primers used were rs1127354: 5′-biotin-CGTGCTCACATGGAGAATCA-3′ (forward primer), 5′-TTTTCTGTGCCACCAAAGTG-3′ (reverse primer), and rs7270101: 5′-TTGGTGGCACAGAAAATTGAC-3′ (forward primer), 5′-biotin-GGGAAACAGACACACAGAAAGTCA-3′ (reverse primer). PCR reactions were carried out by adding 20 ng of template DNA, 5 ��L of 10�� PCR buffer, 5 ��L of 2.5 mM dNTPs, 1 ��L of each 10 ��M forward and reverse primer (one primer was biotinylated), and 0.

5 ��L of Blend Taq plus DNA polymerase (Toyobo, Osaka, Japan) in a 50 ��L reaction mix in a 96-well plate. Amplification was performed under the following conditions: rs1127354-initial denaturation at 94�� for 2 min followed by 45 cycles of 94�� for 30 sec, 60�� for 30 sec, 72�� for 30 sec; rs7270101-initial denaturation at 94�� for 2 min followed by 45 cycles of 94�� for 30 sec, 58�� for 30 sec, and 72�� for 30 sec. IL28B variant rs8099917 T>G was identified in whole blood using validated Pyrosequencing? assays. The primers used were rs8099917: 5′-biotin-TCCTCCTTTTGTTTTCCTTTCTG-3′ (forward primer), 5′-AAAAAGCCAGCTACCAAACTGT-3′ (reverse primer). Amplification was performed under the following conditions: rs8099917-initial denaturation at 94�� for 2 min followed by 45 cycles of 94�� for 30 sec, 60�� for 30 sec, and 72�� for 30 sec.

Pyrosequencing was performed took using an automated PSQ 96 MA instrument and the PyroMark Gold Q96 reagent kit for SNP genotyping and mutation analysis (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The sequencing primers used to detect short DNA sequences around SNPs of interest were: rs8099917-5′-TTCCAATTTGGGTGA-3′, rs1127354-5′-TTCAGATTCTAGGAGATAAGTT-3′, and Drug_discovery rs7270101-5′-GAAATCCAACCATCTTTTA-3′.