Diagnostic guidelines for high-resolution melting curve (HRM) analysis: An interlaboratory validation of BRCA1 mutation scanning using the 96-well LightScanner™

Abstract

Genetic analysis of BRCA1 by sequencing is often preceded by a scanning method like denaturing gradient gel electrophoresis (DGGE), protein truncation test (PTT) or DHPLC. High-resolution melting curve (HRM) analysis is a promising and economical method for high-throughput mutation scanning. The EuroGentest network (www.eurogentest.org) aims to assist with the introduction of novel technologies in the diagnostic setting. Therefore, we have performed a thorough and highstandard interlaboratory evaluation and validation of HRM, in collaboration with Idaho Technology, the manufacturer of the LightScanner™ (LS). Through this detailed study of 170 variants, we have generated guidelines for easy setup and implementation of HRM as a scanning technique for new genes, which are adaptable to the quality system of an individual diagnostic laboratory. This validation study includes the description of a BRCA1-specific mutation screening test using the 96-well LS. This assay comprises 40 amplicons and was evaluated using a statistically significant elaborate panel of variants and control DNA samples. All heterozygous variants were detected. Moreover, genotype analysis for nine common polymorphisms created a fast screening and detection method for these frequently occurring nonpathogenic variants. A blind study using a total of 28 patient-derived DNA samples resulted also in 100% detection and showed an average specificity of 98%, indicating a low incidence of false positives (FPs). © 2009 Wiley-Liss, Inc.