TY - JOUR
T1 - Rapid screening for targeted genetic variants via high-resolution melting curve analysis
AU - Chambliss, Allison B.
AU - Resnick, Molly
AU - Petrides, Athena K.
AU - Clarke, William A.
AU - Marzinke, Mark A.
N1 - Funding Information:
The authors gratefully acknowledge Dr. and Mrs. Cornelius Borman for their generous gift that supported the purchase of the instrumentation used in this study. This publication resulted in part from research supported by the Johns Hopkins University Center for AIDS Research, an NIH funded program (P30AI094189), which is supported by the following NIH Co-Funding and Participating Institutes and Centers: NIAID, NCI, NICHD, NHLBI, NIDA, NIMH, NIA, FIC, NIGMS, NIDDK, and OAR. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We also acknowledge the Johns Hopkins Genetic Resources Core Facility for their services. In particular, we acknowledge Roxann Ashworth of the Johns Hopkins Genetic Resources Core Facility and Katie Beierl of the Johns Hopkins Molecular Diagnostics Laboratory for their guidance.
Publisher Copyright:
© 2017 Walter de Gruyter GmbH, Berlin/Boston.
PY - 2017/3/1
Y1 - 2017/3/1
N2 - Background: Current methods for the detection of single nucleotide polymorphisms (SNPs) associated with aberrant drug-metabolizing enzyme function are hindered by long turnaround times and specialized techniques and instrumentation. In this study, we describe the development and validation of a high-resolution melting (HRM) curve assay for the rapid screening of variant genotypes for targeted genetic polymorphisms in the cytochrome P450 enzymes CYP2C9, CYP2C19, and CYP3A5. Methods: Sequence-specific primers were customdesigned to flank nine SNPs within the genetic regions of aforementioned drug metabolizing enzymes. PCR amplification was performed followed by amplicon denaturation by precise temperature ramping in order to distinguish genotypes by melting temperature (Tm). A standardized software algorithm was used to assign amplicons as 'reference' or 'variant' as compared to duplicate reference sequence DNA controls for each SNP. Results: Intra-assay (n = 5) precision of Tms for all SNPs was ≤ 0.19%, while inter-assay (n = 20) precision ranged from 0.04% to 0.21%. When compared to a reference method of Sanger sequencing, the HRM assay produced no false negative results, and overcall frequency ranged from 0% to 26%, depending on the SNP. Furthermore, HRM genotyping displayed accuracy over input DNA concentrations ranging from 10 to 200 ng/μL. Conclusions: The presented assay provides a rapid method for the screening for genetic variants in targeted CYP450 regions with a result of 'reference' or 'variant' available within 2 h from receipt of extracted DNA. The method can serve as a screening approach to rapidly identify individuals with variant sequences who should be further investigated by reflexed confirmatory testing for aberrant cytochrome P450 enzymatic activity. Rapid knowledge of variant status may aid in the avoidance of adverse clinical events by allowing for dosing of normal metabolizer patients immediately while identifying the need to wait for confirmatory testing in those patients who are likely to possess pharmacogenetically-relevant variants.
AB - Background: Current methods for the detection of single nucleotide polymorphisms (SNPs) associated with aberrant drug-metabolizing enzyme function are hindered by long turnaround times and specialized techniques and instrumentation. In this study, we describe the development and validation of a high-resolution melting (HRM) curve assay for the rapid screening of variant genotypes for targeted genetic polymorphisms in the cytochrome P450 enzymes CYP2C9, CYP2C19, and CYP3A5. Methods: Sequence-specific primers were customdesigned to flank nine SNPs within the genetic regions of aforementioned drug metabolizing enzymes. PCR amplification was performed followed by amplicon denaturation by precise temperature ramping in order to distinguish genotypes by melting temperature (Tm). A standardized software algorithm was used to assign amplicons as 'reference' or 'variant' as compared to duplicate reference sequence DNA controls for each SNP. Results: Intra-assay (n = 5) precision of Tms for all SNPs was ≤ 0.19%, while inter-assay (n = 20) precision ranged from 0.04% to 0.21%. When compared to a reference method of Sanger sequencing, the HRM assay produced no false negative results, and overcall frequency ranged from 0% to 26%, depending on the SNP. Furthermore, HRM genotyping displayed accuracy over input DNA concentrations ranging from 10 to 200 ng/μL. Conclusions: The presented assay provides a rapid method for the screening for genetic variants in targeted CYP450 regions with a result of 'reference' or 'variant' available within 2 h from receipt of extracted DNA. The method can serve as a screening approach to rapidly identify individuals with variant sequences who should be further investigated by reflexed confirmatory testing for aberrant cytochrome P450 enzymatic activity. Rapid knowledge of variant status may aid in the avoidance of adverse clinical events by allowing for dosing of normal metabolizer patients immediately while identifying the need to wait for confirmatory testing in those patients who are likely to possess pharmacogenetically-relevant variants.
KW - CYP450
KW - high resolution melting
KW - single nucleotide polymorphisms
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U2 - 10.1515/cclm-2016-0603
DO - 10.1515/cclm-2016-0603
M3 - Article
C2 - 27732553
AN - SCOPUS:85015995646
SN - 1434-6621
VL - 55
SP - 507
EP - 516
JO - Zeitschrift fur klinische Chemie und klinische Biochemie
JF - Zeitschrift fur klinische Chemie und klinische Biochemie
IS - 4
ER -