2014 Volume 60 Issue 6 Pages 568-575
Objective: The single nucleotide polymorphisms (SNPs) close to the IL28B gene are strong predictors of response to interferon-based therapy for chronic hepatitis C patients. However, approximately 20% of patients have discordant responses to the therapy, suggesting that undiscovered variants of the SNPs may be present near to this disease-associated gene. We sought to develop a practical approach to explore rare variants involved in the treatment response, based on next-generation sequencing (NGS) technology.
Materials: Eight patients with the favorable genotype of the IL28B SNP (TT of rs8099917) who underwent 48 week of pegylated interferon-α and ribavirin therapy were enrolled in the study. They were categorized in two groups according to their virological response, 5 achieved an early virological response (EVR) while the others had a null virological response (NVR).
Methods: PCR primers were developed to amplify specifically IL28B and the relevant IL28A genes. The amplicons were sequenced by an NGS and capillary sequencing was used to validate the variations identified by NGS.
Measurements and Results: Target regions around IL28B and IL28A were specifically amplified by the in house primer sets. Real-time PCR was introduced to control the number of sequence reads on each sample before NGS analysis. Four candidate rare variants were identified through comparative NGS analyses of the EVR and NVR groups. In order to validate the results of the NGS, we subsequently used capillary sequencing but failed to confirm the existence of these rare variants.
Conclusions: We have established a technical serial sequencing platform with NGS, potentially enabling the discovery of rare variant SNPs in genes of interest, such as IL28B in HCV infection.