title: 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.
authors.affiliation: University of Medicine and Pharmacy Tirgu Mures, 38 Gh. Marinescu st., Tirgu Mures 540142, Romania. dincaluminita@yahoo.com.
authors.forename: Elena L
authors.initials: EL
authors.lastname: Enache
authors.affiliation: University of Medicine and Pharmacy Tirgu Mures, 38 Gh. Marinescu st., Tirgu Mures 540142, Romania. anka_sinn@yahoo.com.|Emergency County Clinical Hospital, 50 Gh. Marinescu st., Tirgu Mures 540136, Romania. anka_sinn@yahoo.com.
authors.forename: Anca
authors.initials: A
authors.lastname: Sin
authors.affiliation: University of Medicine and Pharmacy Tirgu Mures, 38 Gh. Marinescu st., Tirgu Mures 540142, Romania. ligiabancu@yahoo.com.|Emergency County Clinical Hospital, 50 Gh. Marinescu st., Tirgu Mures 540136, Romania. ligiabancu@yahoo.com.
authors.forename: Ligia
authors.initials: L
authors.lastname: Bancu
authors.affiliation: Université de Lyon, Université Lyon 1, Lyon F-69008, France. christophe.ramiere@inserm.fr.|Inserm U1111, 21 Avenue Tony Garnier, Lyon F-69007, France. christophe.ramiere@inserm.fr.|CIRI, Centre International de Recherche en Infectiologie, Université de Lyon, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France. christophe.ramiere@inserm.fr.|Ecole Normale Supérieure de Lyon, 15 parvis René Descartes, BP 7000 69342 Lyon Cedex 07, France. christophe.ramiere@inserm.fr.|CNRS, UMR5308, 21 avenue Tony Garnier, 69365 Lyon Cedex 07, France. christophe.ramiere@inserm.fr.|Hospices Civils de Lyon, Hôpital de la Croix Rousse, Laboratoire de Virologie, Lyon F-69004, France. christophe.ramiere@inserm.fr.
authors.forename: Christophe
authors.initials: C
authors.lastname: Ramière
authors.affiliation: Université de Lyon, Université Lyon 1, Lyon F-69008, France. olivier.diaz@inserm.fr.|Inserm U1111, 21 Avenue Tony Garnier, Lyon F-69007, France. olivier.diaz@inserm.fr.|CIRI, Centre International de Recherche en Infectiologie, Université de Lyon, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France. olivier.diaz@inserm.fr.|Ecole Normale Supérieure de Lyon, 15 parvis René Descartes, BP 7000 69342 Lyon Cedex 07, France. olivier.diaz@inserm.fr.|CNRS, UMR5308, 21 avenue Tony Garnier, 69365 Lyon Cedex 07, France. olivier.diaz@inserm.fr.
authors.forename: Olivier
authors.initials: O
authors.lastname: Diaz
authors.affiliation: Université de Lyon, Université Lyon 1, Lyon F-69008, France. patrice.andre@inserm.fr.|Inserm U1111, 21 Avenue Tony Garnier, Lyon F-69007, France. patrice.andre@inserm.fr.|CIRI, Centre International de Recherche en Infectiologie, Université de Lyon, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France. patrice.andre@inserm.fr.|Ecole Normale Supérieure de Lyon, 15 parvis René Descartes, BP 7000 69342 Lyon Cedex 07, France. patrice.andre@inserm.fr.|CNRS, UMR5308, 21 avenue Tony Garnier, 69365 Lyon Cedex 07, France. patrice.andre@inserm.fr.|Hospices Civils de Lyon, Hôpital de la Croix Rousse, Laboratoire de Virologie, Lyon F-69004, France. patrice.andre@inserm.fr.
authors.forename: Patrice
authors.initials: P
authors.lastname: André
authors.affiliation: University of Medicine and Pharmacy Tirgu Mures, 38 Gh. Marinescu st., Tirgu Mures 540142, Romania. enachesliviu@yahoo.com.|Emergency County Clinical Hospital, 50 Gh. Marinescu st., Tirgu Mures 540136, Romania. enachesliviu@yahoo.com.
authors.forename: Liviu S
authors.initials: LS
authors.lastname: Enache
mesh_terms: D000328:Adult; D016022:Case-Control Studies; D005260:Female; D060005:Genotyping Techniques / Q000379:methods*; D019698:Hepatitis C, Chronic / Q000235:genetics*; D006801:Humans; D007372:Interferons; D007378:Interleukins / Q000235:genetics*; D008049:Lipase / Q000235:genetics*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D008875:Middle Aged; D009691:Nucleic Acid Denaturation; D020641:Polymorphism, Single Nucleotide; D000095022:Interferon Lambda; D000217:Acyltransferases; D054512:Phospholipases A2, Calcium-Independent
chemical_list: D007372:Interferons; D007378:Interleukins; D008049:Lipase; D008565:Membrane Proteins; C000624353:interferon-lambda, human; D000095022:Interferon Lambda; C533682:PNPLA3 protein, human; D000217:Acyltransferases; D054512:Phospholipases A2, Calcium-Independent
keywords: chronic hepatitis C; duplex assay; high-resolution melting; interleukin 28B; patatin-like phospholipase domain containing 3 (PNPLA3); personalized diagnostics; single nucleotide polymorphism