Rapid and sensitive detection of BRCA1/2 mutations in a diagnostic setting: comparison of two high-resolution melting platforms.
BACKGROUND
High-resolution melting is an emerging technique for detection of nucleic acid sequence variations. Developments in instrumentation and saturating intercalating dyes have made accurate high-resolution melting analysis possible and created opportunities to use this technology in diagnostic settings. We evaluated 2 high-resolution melting instruments for screening BRCA1 and BRCA2 mutations.


METHODS
To cover the complete coding region and splice sites, we designed 112 PCR amplicons (136-435 bp), amplifiable with a single PCR program. LCGreen Plus was used as the intercalating dye. High-resolution melting analysis was performed on the 96-well Lightscanner (Idaho Technology Inc.) and the 96-well LightCycler 480 (Roche) instruments. We evaluated sensitivity by analyzing 212 positive controls scattered over almost all amplicons and specificity by blind screening of 22 patients for BRCA1 and BRCA2. In total, we scanned 3521 fragments.


RESULTS
All 212 known heterozygous sequence variants were detected on the Lightscanner by analysis on normal sensitivity setting. On the LightCycler 480, the standard instrument sensitivity setting of 0.3 had to be increased to 0.7 to detect all variants, decreasing the specificity to 95.9% (vs 98.7% for the Lightscanner).


CONCLUSIONS
Previously, we screened BRCA1/2 by direct sequencing of the large exon 11 and denaturing gel gradient electrophoresis (DGGE) for all other coding exons. Since the introduction of high-resolution melting, our turnaround time has been one third of that with direct sequencing and DGGE, as post-PCR handling is no longer required and the software allows fast analyses. High-resolution melting is a rapid, cost-efficient, sensitive method simple enough to be readily implemented in a diagnostic laboratory.
De Leeneer
Kim
K
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.
Coene
Ilse
I
Poppe
Bruce
B
De Paepe
Anne
A
Claes
Kathleen
K
D051017:Apoptosis Regulatory Proteins; D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics; D004198:Disease Susceptibility; D005260:Female; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D044366:Transition Temperature
D051017:Apoptosis Regulatory Proteins; C118024:BLID protein, human; D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human