Genome-wide association studies have demonstrated that SNPs found in the region surrounding IL28B
were associated with response of patients infected with HCV to treatment with pegylated interferon-α (with or without ribavirin) 
and with spontaneous clearance of HCV infection 
. Over the last two years, the number of publications examining IL28B
SNPs has increased tremendously and treatment options based upon a patient's IL28B
SNP genotype are being considered 
The IL28B gene has a close paralogue, IL28A, with which it shares 97.5% sequence identity. This raised the possibility that sequences in IL28A may contribute to the determination of IL28B SNP genotypes. Therefore, understanding genotype assay specificity would be vitally important to consider when using IL28B genotypes to make treatment decisions. Clinicians need to be confident that ‘homebrew’ assays are robust and fully validated to allow prospective genotyping to make treatment decisions on single subjects rather than assays for retrospective cohort-based analyses.
The IL28A and IL28B genes likely arose due to an ancestral gene duplication and inversion event, with IL28A being located on the positive DNA strand of chromosome 19 and IL28B 24 kb downstream on the negative strand. Although these genes are 97.5% identical across their 1.4 kb length, a region of >95% sequence identity stretches a total of 7.3 kb in both 5′ and 3′ directions and it includes 6 of the 9 reported treatment response associated SNPs. Our observation that in all cases, one of the two alleles of the IL28B SNPs also corresponded to identical nucleotides in IL28A led us to take a sequence-based approach in order to confirm SNP genotypes. Sequencing specific regions from a panel of DNA samples from 48 individuals representing CEU, JPT and YRI origins demonstrated that all 9 SNPs in the IL28B region were polymorphic, whereas the corresponding sequences in IL28A were all monomorphic. Despite this finding, there was still the potential for IL28A sequences to contribute to genotyping calls if assays were not sufficiently specific.
As a general rule when developing PCR-based genotyping assays, the greater the number of mismatches between the target primers and other homologous sequences, the greater the assay specificity. SNP rs12979860 currently appears to be the best single choice variant for diagnostic purposes across global populations or for use in clinical trials 
and as a result, it has now become the most studied SNP of the 9 treatment response associated SNPs that were initially identified. Our TaqMan® assay for rs12979860 used two PCR primers that contained 3 and 4 base mismatches per primer compared to the corresponding IL28A
sequences. This assay gave 100% concordant results to the sequence-derived genotypes. Whilst not implying that published rs12979860 assays have not been technically validated, examining a selection of different types of rs12979860 genotyping assays found varying numbers of base mismatches in the PCR amplification primers. For example, one TaqMan® assay had 0 and 3 mismatches in the amplification primer pairs 
, a PCR/Sybr green assay had 0 and 2 mismatches 
, and a MELT-MAMA PCR assay had 0 and 5 mismatches 
. In these three examples, the specificity for IL28B
is only driven by a single primer. Two commercially available rs12979860 assays from LabCorp Inc and Quest Diagnostics Inc. have not reported details of their respective assay components. Analysis of a 24 sample subset of the 48 Coriell DNAs revealed one discordant genotype using the LabCorp assay; interestingly in a sample that had its genotype missing in the HapMap data. Re-analysis of this discordant data-point led to a modification of the genotype calling algorithm now used at Monogram Biosciences Inc.
Given the importance of the studies being used worldwide to examine associations between SNP rs12979860 and response to treatment in various hepatitis populations, it is vitally important that robust and well validated assays are used. This is particularly important as future hepatitis patient treatment decisions will likely be dependent upon results obtained from such genotyping assays.