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. 2011 Jul;13(4):446-51.
doi: 10.1016/j.jmoldx.2011.03.008. Epub 2011 May 14.

Simultaneous genotyping of rs12979860 and rs8099917 variants near the IL28B locus associated with HCV clearance and treatment response

Roberta Melis  1 , Christiane Fauron, Gwendolyn McMillin, Elaine Lyon, Brian Shirts, Lindsey M Hubley, Patricia R Slev
Affiliations

Simultaneous genotyping of rs12979860 and rs8099917 variants near the IL28B locus associated with HCV clearance and treatment response

Roberta Melis et al. J Mol Diagn. 2011 Jul.

Abstract

Recent genome-wide association studies have identified two host single-nucleotide polymorphisms (SNPs) near the IL28B gene (rs12979860 C/T and rs8099917 T/G) that are associated with sustained virological response in patients infected with the hepatitis C virus. Herein, we describe a rapid multiplexed dual-color fluorescence resonance energy transfer (FRET) probe assay that accurately genotypes for both SNPs simultaneously. A single-nucleotide extension assay was also developed for verification of genotypes. Agreement (100%) was observed in genotype calls between the FRET and single-nucleotide extension methods for both SNPs, yielding 100% analytical sensitivity and specificity. By using the FRET assay, 443 samples of varying ethnic backgrounds were genotyped and six different compound genotypes (rs12979860/rs8099917) were detected in whites, Asians, Middle Easterners, Hispanics, and African Americans, at the following frequencies: CC/TT (39.2%, 78.9%, 40.0%, 33.9%, and 16.8%), CT/TT (20.8%, 0%, 40%, 9.3%, and 37.0%), TT/TT (2.4%, 0%, 0%, 3.4%, and 35.3%), CT/TG (24.0%, 19.7%, 20%, 39.8%, and 3.4%), TT/TG (8.0%, 1.4%, 0%, 3.4%, and 5.9%), and TT/GG (5.6%, 0%, 0%, 10.2%, and 1.7%), respectively. The multiplexed FRET assay can be used to effectively genotype for both SNPs in a single tube, with high analytical sensitivity and specificity.

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Figures

Figure 1
Figure 1
Multiplexed FRET rs12979860 and rs8099917 genotyping results. Melting analysis curves of multiplexed, asymmetric, real-time PCR and dual-color FRET probes of three individuals. A:rs12979860 homozygous for TT (blue) with Tm at 57.6°C, homozygous for CC (red) with Tm at 66.8°C, and heterozygous for TC (green) analyzed with the F2 channel. B:rs8099917 homozygous for TT (blue) with Tm at 61.9°C, homozygous for GG (red) with Tm at 69.3°C, and heterozygous for TG (green) analyzed with the F3 channel. The gray line indicates negative (no template) control.
Figure 2
Figure 2
Multiplexed SNE genotyping results of rs12979860 and rs8099917. Electropherograms of the SNE reaction products are shown for three different individuals: rs12979860 heterozygous for CT; rs8099917 heterozygous for GT (A); rs12979860 homozygous for CC; rs8099917 homozygous for TT (B); and rs12979860 homozygous TT; rs8099917 homozygous for GG (C).
Figure 3
Figure 3
Histogram of frequencies of rs12979860/rs8099917 compound genotypes observed across ethnic populations. Genotype frequencies were obtained for whites (CAU; n = 125), Asians (AS; n = 71), Middle Easterners (ME; n = 10), Hispanics (HIS; n = 118), and African Americans (AA; n = 119). Six different rs12979860/rs8099917 compound genotypes are shown on the x axis. The rs12979860 C and the rs8099917 T favorable alleles associated with efficacy in virus clearance and treatment outcome.
Figure 4
Figure 4
Frequency comparisons of rs12979760 CC and rs8099917 TT favorable genotypes, associated with efficacy in HCV virus clearance and treatment outcome; and rs12979760 CT + TT and rs8099917 TG + GG risk genotypes across different ethnic groups. CAU indicates white; AS, Asian; ME, Middle Eastern; HIS, Hispanic; and AA, African American.
Figure 5
Figure 5
The distribution of rs12979860/rs8099917 haplotypes in whites (CAU), Asians (AS), Middle Easterners (ME), Hispanics (HIS), and African Americans (AA). Haplotype frequencies were obtained from the analysis of 886 chromosomes. Frequencies of C-T, T-T, and T-G haplotypes are shown on the x axis. The C-G haplotype was not observed. F indicates that a favorable haplotype is associated with efficacy in virus clearance and treatment outcome. The rs12979860 C and rs8099917 T favorable alleles are represented in bold and gray shaded. I indicates indeterminate haplotype with the presence of one risk allele (rs12979860 T) and the presence of the rs8099917 T favorable allele. R indicates that the risk haplotype is associated with the presence of two risk alleles (rs12979860 T and rs8099917 G).
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