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Rapid screening for targeted genetic variants via high-resolution melting curve analysis

  • Allison B. Chambliss , Molly Resnick , Athena K. Petrides , William A. Clarke und Mark A. Marzinke EMAIL logo
Veröffentlicht/Copyright: 12. Oktober 2016
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Clinical Chemistry and Laboratory Medicine (CCLM)
Aus der Zeitschrift Clinical Chemistry and Laboratory Medicine (CCLM) Band 55 Heft 4

Abstract

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 custom-designed 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.

Keywords: CYP450; high resolution melting; single nucleotide polymorphisms

Acknowledgments

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.

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: None declared.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Supplemental Material:

The online version of this article (DOI: 10.1515/cclm-2016-0603) offers supplementary material, available to authorized users.

Received: 2016-7-7
Accepted: 2016-9-2
Published Online: 2016-10-12
Published in Print: 2017-3-1

©2017 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2016-0603
Schlagwörter für diesen Artikel
CYP450; high resolution melting; single nucleotide polymorphisms

Artikel in diesem Heft

  1. Frontmatter
  2. Editorial
  3. The central role of external quality assurance in harmonisation and standardisation for laboratory medicine
  4. Review
  5. Fecal calprotectin in inflammatory bowel diseases: update and perspectives
  6. Mini Review
  7. Factor VIIa-antithrombin complex: a possible new biomarker for activated coagulation
  8. Opinion Papers
  9. Improving quality in the preanalytical phase through innovation, on behalf of the European Federation for Clinical Chemistry and Laboratory Medicine (EFLM) Working Group for Preanalytical Phase (WG-PRE)
  10. Metabolite profiling can change health-care delivery to obese patients with fatty liver disease: the search for biomarkers
  11. Genetics and Molecular Diagnostics
  12. Rapid screening for targeted genetic variants via high-resolution melting curve analysis
  13. Evaluation of the new cobas® HCV genotyping test based on real-time PCRs of three different HCV genome regions
  14. General Clinical Chemistry and Laboratory Medicine
  15. Harmonisation of serum dihydrotestosterone analysis: establishment of an external quality assurance program
  16. Spanish Preanalytical Quality Monitoring Program (SEQC), an overview of 12 years’ experience
  17. Peer groups splitting in Croatian EQA scheme: a trade-off between homogeneity and sample size number
  18. Prevalence of pseudonatremia in a clinical laboratory – role of the water content
  19. Retrospective validation of a β-trace protein interpretation algorithm for the diagnosis of cerebrospinal fluid leakage
  20. Economic evaluation of procalcitonin-guided antibiotic therapy in acute respiratory infections: a Chinese hospital system perspective
  21. Performance evaluation of ImmunoCAP® ISAC 112: a multi-site study
  22. Clinical autoantibody detection by microarray
  23. Cardiovascular Diseases
  24. Kinetics of troponin I in patients with myocardial injury after noncardiac surgery
  25. Infectious Diseases
  26. P35 and P22 Toxoplasma gondii antigens abbreviate regions to diagnose acquired toxoplasmosis during pregnancy: toward single-sample assays
  27. Letters to the Editor
  28. Deciphering a macro-troponin I complex; a case report
  29. Interference of laboratory disinfection with trichloro-isocyanuric acid on cardiac troponin I measurement using the Vitros immunoassay system
  30. Massive interference in free T4 and free T3 assays misleading clinical judgment
  31. In defense of aldosterone measurement by immunoassay: a broader perspective
  32. The prevalence of hemolysis – a survey using hemolysis index
  33. Detection of BRAFV600K mutant tumor-derived DNA in the pleural effusion from a patient with metastatic melanoma
  34. Evaluation of the accuracy of the Greiner Bio-One FC Mix Glucose tube
  35. Congress Abstracts
  36. 4th EFLM-BD European Conference on Preanalytical Phase Amsterdam (NL), 24–25 March 2017
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