Establishing high resolution melting analysis: method validation and evaluation for c-RET proto-oncogene mutation screening

Martin Benej; Bela Bendlova; Eliska Vaclavikova; Martina Poturnajova

doi:10.1515/cclm.2011.730

Article

Establishing high resolution melting analysis: method validation and evaluation for c-RET proto-oncogene mutation screening

Martin Benej , Bela Bendlova , Eliska Vaclavikova and Martina Poturnajova

Published/Copyright: October 6, 2011

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From the journal Clinical Chemistry and Laboratory Medicine Volume 50 Issue 1

Abstract

Background: Reliable and effective primary screening of mutation carriers is the key condition for common diagnostic use. The objective of this study is to validate the method high resolution melting (HRM) analysis for routine primary mutation screening and accomplish its optimization, evaluation and validation. Due to their heterozygous nature, germline point mutations of c-RET proto-oncogene, associated to multiple endocrine neoplasia type 2 (MEN2), are suitable for HRM analysis. Early identification of mutation carriers has a major impact on patients’ survival due to early onset of medullary thyroid carcinoma (MTC) and resistance to conventional therapy.

Methods: The authors performed a series of validation assays according to International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines for validation of analytical procedures, along with appropriate design and optimization experiments. After validated evaluation of HRM, the method was utilized for primary screening of 28 pathogenic c-RET mutations distributed among nine exons of c-RET gene.

Results: Validation experiments confirm the repeatability, robustness, accuracy and reproducibility of HRM. All c-RET gene pathogenic variants were detected with no occurrence of false-positive/false-negative results.

Conclusions: The data provide basic information about design, establishment and validation of HRM for primary screening of genetic variants in order to distinguish heterozygous point mutation carriers among the wild-type sequence carriers. HRM analysis is a powerful and reliable tool for rapid and cost-effective primary screening, e.g., of c-RET gene germline and/or sporadic mutations and can be used as a first line potential diagnostic tool.

Keywords: c-RET proto-oncogene; high resolution melting analysis; medullary thyroid carcinoma; method evaluation; molecular diagnostics

Corresponding author: Martina Poturnajova, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava, Slovakia Phone: +421 2 59327 407, Fax: +421 2 59327 250

Received: 2011-5-25

Accepted: 2011-9-5

Published Online: 2011-10-06

Published in Print: 2012-01-01

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https://doi.org/10.1515/cclm.2011.730

Keywords for this article

c-RET proto-oncogene; high resolution melting analysis; medullary thyroid carcinoma; method evaluation; molecular diagnostics