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

International Journal of Molecular Sciences

Elena L. Enache²  Anca Sin²  Ligia Bancu²  Christophe Ramière¹  Olivier Diaz¹  Patrice André¹  Liviu S. Enache² 

[1] Université de Lyon, Université Lyon 1, Lyon F-69008, France; E-Mails:;University of Medicine and Pharmacy Tirgu Mures, 38 Gh. Marinescu st., Tirgu Mures 540142, Romania; E-Mails:

关键词: chronic hepatitis C;    personalized diagnostics;    duplex assay;    high-resolution melting;    interleukin 28B;    patatin-like phospholipase domain containing 3 (PNPLA3);    single nucleotide polymorphism;   

DOI  :  10.3390/ijms160922223

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.