Genotyping of IL28B polymorphism rs12979860 by high resolution melting of unlabeled probe

Abstract

Introduction: The single nucleotide polymorphism (SNP) rs12979860 maps 3 kb upstream of the human interleukin 28B (IL28B) gene on chromosome 19, which encodes the type III interferon IFN-gamma3. Genotyping of the SNP, which comprises a C or T dimorphism, is clinically useful in individuals infected with HCV genotype 1 virus (HCV-1). Treatment of chronic HCV infection generally consists of a combination of PEG-Interferon-alpha-2a or 2b and ribavirin. Genome-wide association studies have demonstrated the C/C variant is associated with an improved response and spontaneous clearance to the drug regimen, and its identification may allow a more individualized therapy and an improved sustained virological response. An unlabeled probe-based high resolution melting (HRM) real-time PCR has been developed on the LightScanner 32(registered trademark) (LS 32) (Idaho Technology Inc., UT, USA), to serve as a rapid and inexpensive method to identify the rs12979860 polymorphism in patients infected with HCV-1. Methods: An unlabeled probe with a C3 blocker (Suprenom, Singapore) was designed to perfectly match the C allele of rs12979860. Real-time PCR amplification was performed with a ratio of 1:5:5 of forward primer, reverse primer and unlabeled probe in the LightScanner(registered trademark) Master Mix with incorporated LC Green(registered trademark) PLUS (Idaho Tech) on the LS 32, followed by a final denaturing/ re-annealing step to generate HRM profiles. The region of the unlabeled probe melt was manually selected and the fluorescence, normalized. The LS 32(registered trademark) software (v1.0.0.33) was used for genotype analysis, clustering and auto-grouping of the unknown samples. Forty archived nucleic acid extracts retrieved from previously processed HCV-1a and -1b infected patient plasma, were analyzed. The HRM results were validated using a co-developed sequencing-based method. Results: HRM generated unique melting profiles for all three (C/C, C/T, T/T) variants. Melt peaks for the unlabeled probe with a mismatched base (for T allele) and a perfectly matched base (for C allele) were clearly discernable at 64(degrees)C and 72(degrees)C, respectively. Normalized melt curves reproducibly showed distinct patterns for homozygous (C/C and T/T) and heterozygous (C/T) genotypes. Of the 40 clinical samples tested, 27 (67.5%) had C/C; 11 (27.5%), C/T; and 2 (5%), T/T genotypes. Conclusions: The above rapid and low-cost in-house real-time PCR assay was able to genotype the clinically important IL28B polymorphism in HCV-1 infected patients, and provides a good alternative to Sanger sequencing. The allelic frequencies may account for a difference in treatment response in different ethnic groups; this aspect will be further investigated in our local multi-ethnic population.