Sequence Analysis of the IL28A/IL28B Inverted Gene Duplication That Contains Polymorphisms Associated with Treatment Response in Hepatitis C Patients

Abstract

Several SNPs located in or around the IL28B gene are associated with response of patients infected with Hepatitis C virus to treatment with pegylated interferon-α +/− ribavirin or with spontaneous clearance of the virus. The results of such studies are so compelling that future treatment approaches are likely to involve clinical decisions being made on the basis of a patient's genotype. Since IL28B is a paralogue of IL28A with greater than 95% sequence identity, it is possible that without genotyping assay specificity, sequences in IL28A may contribute to genotype identification, and potentially confound treatment decisions. This study aimed to 1) examine DNA sequences in IL28B surrounding each of the reported associated SNPs and the corresponding regions in IL28A; and 2) develop a robust assay for rs12979860, the most ‘cosmopolitan’ SNP most strongly associated with treatment response across all global populations studied to date. Bioinformatic analysis of genomic regions surrounding IL28A and IL28B demonstrated that 3 SNPs were unique to IL28B, whereas the remaining 6 SNP regions shared >93% identity between IL28A and IL28B. Using a panel of DNA samples, PCR amplification followed by Sanger sequencing was used to examine IL28B SNPs and the corresponding regions in IL28A. For the overlapping SNPs, all 6 in IL28B were confirmed to be polymorphic whereas the corresponding positions in IL28A were monomorphic. Based upon IL28A and IL28B sequence data, a specific TaqMan® assay was developed for SNP rs12979860 that was 100% concordant to the sequence-derived genotypes. Analysis using a commercial assay identified one discordant result which led to a change in their genotype-calling algorithm. Where future treatment decisions are made upon the results of genotyping assays, it is very important that results are concordant with data from a sequence-based format. This is especially so in situations where designing specific PCR primers is a challenge.

A Single Nucleotide Polymorphism Associated with Hepatitis C Virus Infections Located in the Distal Region of the IL28B Promoter Influences NF-κB-Mediated Gene Transcription

“…Among the SNPs identified by these studies rs12979860 and rs8099917 are consistently associated with treatment response in several study groups post-GWAS [7]–[11]. In fact, rs12979860 is referred to as the “cosmopolitan” SNP due to its strong associations with treatment response against HCV infections in several ethnic groups [12]. This SNP lies about 3 kb upstream of IL28B coding region whereas rs8099917 lies about 7.5 kb upstream of IL28B coding region.…”

A Fast and Cost-Effective Method for Identifying a Polymorphism of Interleukin 28B Related to Hepatitis C

“…These assays as well as direct sequencing and other assays [10-12] allow for the screening of patient samples. However, for many laboratories, large routine analysis in a tertiary care hospital with reagent and equipment costs limits these implementations, especially in developing countries.…”