Dataset Information

High-Resolution Melting Assay for Genotyping Variants of the CYP2C19 Enzyme and Predicting Voriconazole Effectiveness.

ABSTRACT: Voriconazole is a triazole antifungal agent recommended as primary treatment for invasive aspergillosis, as well as some other mold infections. However, it presents some pharmacokinetic singularities that lead to a great variability intra- and interindividually, nonlinear pharmacokinetics, and a narrow therapeutic range. Most experts have recommended tracing the levels of voriconazole in patients when receiving treatment. This azole is metabolized through the hepatic enzyme complex cytochrome P450 (CYPP450), with the isoenzyme CYP2C19 being principally involved. Allelic variations (polymorphisms) of the gene that encodes this enzyme are known to contribute to variability in voriconazole exposure. Three different allelic variants, CYP2C19*17, CYP2C19*2, and CYP2C19*3, could explain most of the phenotypes related to the voriconazole metabolism and some of its pharmacokinetic singularities. We designed a rapid molecular method based on high-resolution melting to characterize these polymorphisms in a total of 142 samples, avoiding sequencing. Three PCRs were designed with similar cycling conditions to run simultaneously. The results showed that our method represents a fast, accurate, and inexpensive means to study these variants related to voriconazole metabolism. In clinical practice, this could offer a useful tool to individually optimize therapy and reduce expenses in patients with fungal infections.

SUBMITTER: Bernal-Martinez L

PROVIDER: S-EPMC6535561 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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High-Resolution Melting Assay for Genotyping Variants of the CYP2C19 Enzyme and Predicting Voriconazole Effectiveness.

Bernal-Martínez L L Alcazar Fuoli L L Miguel-Revilla B B Carvalho A A Cuétara Garcia M S MS Garcia-Rodriguez J J Cunha C C Gómez-García de la Pedrosa E E Gomez-Lopez A A

Antimicrobial agents and chemotherapy 20190524 6

Voriconazole is a triazole antifungal agent recommended as primary treatment for invasive aspergillosis, as well as some other mold infections. However, it presents some pharmacokinetic singularities that lead to a great variability intra- and interindividually, nonlinear pharmacokinetics, and a narrow therapeutic range. Most experts have recommended tracing the levels of voriconazole in patients when receiving treatment. This azole is metabolized through the hepatic enzyme complex cytochrome P4 ...[more]

PMID: 30910893

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Project description:BackgroundTogether single nucleotide substitutions and small insertion/deletion variants are the most common form of sequence variation in the human gene pool.High-resolution SNP profile and/or haplotype analyses enable the identification of modest-risk susceptibility genes to common diseases, genes that may modulate responses to pharmaceutical agents, and SNPs that can affect either their expression or function. In addition, sensitive techniques for germline or somatic mutation detection are important tools for characterizing sequence variations in genes responsible for tumor predisposition. Cost-effective methods are highly desirable. Many of the recently developed high-throughput technologies are geared toward industrial scale genetic studies and arguably do not provide useful solutions for small laboratory investigator-initiated projects. Recently, the use of new fluorescent dyes allowed the high-resolution analysis of DNA melting curves (HRM).ResultsHere, we compared the capacity of HRM, applicable to both genotyping and mutation scanning, to detect genetic variations in the tumor suppressor gene TP53 with that of mutation screening by full resequencing. We also assessed the performance of a variety of available HRM-based genotyping assays by genotyping 30 TP53 SNPs. We describe a series of solutions to handle the difficulties that may arise in large-scale application of HRM to mutation screening and genotyping at the TP53 locus. In particular, we developed specific HRM assays that render possible genotyping of 2 or more, sometimes closely spaced, polymorphisms within the same amplicon. We also show that simultaneous genotyping of 2 SNPs from 2 different amplicons using a multiplex PCR reaction is feasible; the data can be analyzed in a single HRM run, potentially improving the efficiency of HRM genotyping workflows.ConclusionThe HRM technique showed high sensitivity and specificity (1.0, and 0.8, respectively, for amplicons of <400 bp) for mutation screening and provided useful genotyping assays as assessed by comparing the results with those obtained with Sanger sequencing. Thus, HRM is particularly suitable for either performing mutation scanning of a large number of samples, even in the situation where the amplicon(s) of interest harbor a common variant that may disturb the analysis, or in a context where gathering common SNP genotypes is of interest.

Dataset Information

High-Resolution Melting Assay for Genotyping Variants of the CYP2C19 Enzyme and Predicting Voriconazole Effectiveness.

Publications

High-Resolution Melting Assay for Genotyping Variants of the CYP2C19 Enzyme and Predicting Voriconazole Effectiveness.

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