Simple assay based on restriction fragment length polymorphism associated with IL28B in chronic hepatitis C patients

Abstract

Objective. Several studies recently revealed that single nucleotide polymorphisms (SNPs) in the interleukin28B (IL28B) region are associated with the response to pegylated interferon-alfa (PEG-IFN-alfa) and ribavirin (RBV) treatment among hepatitis C virus (HCV)-infected individuals of European, African and Asian ancestry. The purpose of the study was to establish methods for determining the SNP rs8099917 associated with IL28B, which might be useful for further research of the treatment of HCV. Material and methods. Blood samples obtained from 93 consecutive patients with chronic hepatitis C were examined. On the basis of the sequence data, a new simple genotyping assay based on a PCR–restriction fragment length polymorphism (RFLP) with two enzymes, BsrDI and Tsp45I, was developed. Results. The proportion of null virological responders in the combined TG/GG group was higher than that in the TT group (p = 0.015), suggesting that minor allele is one of the important factors playing crucial roles in IFN-resistance. Genotyping of rs8099917 by our new method showed results identical to PCR and sequence in 98.9% and 98.9% by BsrDI and Tsp45I, respectively. Using two enzymes, BsrDI and Tsp45I, it was possible to distinguish IL28B SNP rs8099917. Conclusion. This simple method using RFLP will provide the framework for further studies of HCV.

Key Words::

• Antiviral treatment
• HCV
• IL28B
• null virological response
• PCR–RFLP

Acknowledgements

We are grateful to Satomi Hasegawa for excellent technical assistance. This work was supported by grants for Scientific Research 21590829, 21590828, and 21390225 from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (TK, FI, and OY), a grant from the Viral Hepatitis Research Foundation of Japan (TK), and a grant from Chiba University Young Research-Oriented Faculty Member Development Program in Bioscience Areas (TK).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.