Background: Indirect alternatives to sequencing as a method for mutation scanning are of interest to diagnostic laboratories because they have the potential for considerable savings in both time and costs. Ideally, such methods should be simple, rapid, and highly sensitive, and they should be validated formally to a very high standard. Currently, most reported methods lack one or more of these characteristics. We describe the optimization and validation of conformation-sensitive capillary electrophoresis (CSCE) for diagnostic mutation scanning.
Methods: We initially optimized the performance of CSCE with a systematic panel of plasmid-based controls. We then compared manual analysis by visual inspection with automated analysis by BioNumerics software (Applied Maths) in a blinded interlaboratory validation with 402 BRCA1 (breast cancer 1, early onset) and BRCA2 (breast cancer 1, early onset) variants previously characterized by Sanger sequencing.
Results: With automated analysis, we demonstrated a sensitivity of >99% (95% CI), which is indistinguishable from the sensitivity for conventional sequencing by capillary electrophoresis. The 95% CI for specificity was 90%-93%; thus, CSCE greatly reduces the number of fragments that need to be sequenced to fully characterize variants. By manual analysis, the 95% CIs for sensitivity and specificity were 98.3%-99.4% and 93.1%-95.5%, respectively.
Conclusions: CSCE is amenable to a high degree of automation, and analyses can be multiplexed to increase both capacity and throughput. We conclude that once it is optimized, CSCE combined with analysis with BioNumerics software is a highly sensitive and cost-effective mutation-scanning technique suitable for routine genetic diagnostic analysis of heterozygous nucleotide substitutions, small insertions, and deletions.