Human Mutation METHODS Diagnostic Guidelines for High-Resolution Melting Curve (HRM) Analysis: An Interlaboratory Validation of BRCA1 Mutation Scanning Using the 96-Well LightScanner TM For the Focus Section on HRMA Technology

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 TM (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)

EuroGentest: A Collaborative Network Aimed at Improving the Quality of Genetic Testing in Europe and Beyond

The EuroGentest network aims to improve and harmonize the quality of genetic testing services in Europe, from test development through to information for patients. The network encompasses Biochemical, Clinical, Cyto- and Molecular Genetics, Genetic counselling and patients groups. the Unit 1 of the network has disseminted information on laboratory accreditation, created a database on the current QA programmes and status in European genetic testing services, and reference materials producers and providers.JRC.D.2-Reference material