Duplex High-Resolution Melting Assay for the Simultaneous Genotyping of IL28B rs12979860 and PNPLA3 rs738409 Polymorphisms in Chronic Hepatitis C Patients

Abstract

Chronic hepatitis C (CHC) is a major burden for public health worldwide. Although newer direct-acting antivirals show good efficacy, their cost precludes their wide adoption in resource-limited regions. Thus, strategies are being developed to help identify patients with high susceptibility to response to classic PEG-interferon + ribavirin therapy. IL28B polymorphism rs12979860 C/T is an important predictor for an efficient response to interferon-based therapy. A genetic variant in adiponutrin (PNPLA3) gene, rs738409 C/G, is associated with steatosis, severity, and progression of liver fibrosis in CHC patients, and predicts treatment outcome in difficult-to-cure HCV-infected patients with advanced fibrosis. We developed a rapid and inexpensive assay based on duplex high-resolution melting (HRM) for the simultaneous genotyping of these two polymorphisms. The assay validation was performed on synthetic DNA templates and 132 clinical samples from CHC patients. When compared with allele-specific PCR and sequencing, our assay showed 100% (95% CI: 0.9724–1) accuracy, with 100% sensitivity and specificity. Our assay was robust against concentration and quality of DNA samples, melting curve normalization intervals, HRM analysis algorithm, and sequence variations near the targeted SNPs (single nucleotide polymorphism). This duplex assay should provide useful information for patient-oriented management and clinical decision-making in CHC.

PNPLA3 Gene Polymorphisms in HCV/HIV-Coinfected Individuals

“…Enache and colleagues also developed an HRM method which differs from ours in terms of the primer set and concentrations, reaction mix, and cycling conditions, but which also yielded 100% concordance in classification when compared to traditional sequencing methods. 7 Regardless of protocol, this methodology allows for rapid and relatively inexpensive characterization of PNPLA3 gene polymorphisms without need for expensive sequence analysis.…”

“…Realtime qPCR was then performed on a BioRad CFX96 thermal cycler using the extracted genomic DNA, utilizing a primer pair modified from Enache, et al, which flanks the SNP region of interest, at the corresponding human genomic region 43928824–43928869 on chromosome 22. 7 The forward primer (5’-GCCTTGGTATGTTCCTGCTTC-3’) was utilized with an adaptation of the reverse primer to include a degenerate base near the 3’ end, to accommodate a variant (rs738408 A/G) at this location, (5’-GGATAAGGCCACTGTAGAARG-3’). Following qPCR amplification, a high resolution melt curve was generated by heating the qPCR products from 65–80° in 0.2° increments (10 seconds/step).…”

Triplex High-Resolution Melting Assay for the Simultaneous Assessment of IFNL3 rs12979860, ABCB11 rs2287622, and RNF7 rs16851720 Genotypes in Chronic Hepatitis C Patients

“…y Primers for rs12979860 were developed in our previous study. 25 F, forward; HRM, high-resolution melting assays; o., overlapping primers; OE-PCR, overlap-extension PCR; R, reverse; SNP, single-nucleotide polymorphism. jmd.amjpathol.org -The Journal of Molecular Diagnostics combinations thereof, were tested along with melting standards, in 3 consecutive days.…”

Hepatitis C and the Impact of Host Genetics

“…This study extends their previous work which described a 2-plex SNP detection HRM method (IL28B and PNPLAs SNPs). 30 Taken together, the authors have designed HRM primer sets to determine four HCV relevant SNPs, three of which can be performed in a single tube. Whether they have included the most clinically relevant SNPs in their 3-plex panel remains to be determined.…”