Interlaboratory diagnostic validation of conformation-sensitive capillary electrophoresis for mutation scanning.

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.

Design and validation of a conformation sensitive capillary electrophoresis-based mutation scanning system and automated data analysis of the more than 15 kbp-spanning coding sequence of the SACS gene.

In this study, we developed and analytically validated a fully automated, robust confirmation sensitive capillary electrophoresis (CSCE) method to perform mutation scanning of the large SACS gene. This method facilitates a rapid and cost-effective molecular diagnosis of autosomal recessive spastic ataxia of Charlevoix-Saguenay. Critical issues addressed during the development of the CSCE system included the position of a DNA variant relative to the primers and the CG-content of the amplicons. The validation was performed in two phases; a retrospective analysis of 32 samples containing 41 different known DNA variants and a prospective analysis of 20 samples of patients clinically suspected of having autosomal recessive spastic ataxia of Charlevoix-Saguenay. These 20 samples appeared to contain 73 DNA variants. In total, in 32 out of the 45 amplicons, a DNA variant was present, which allowed verification of the detection capacity during the validation process. After optimization of the original design, the overall analytical sensitivity of CSCE for the SACS gene was 100%, and the analytical specificity of CSCE was 99.8%. In conclusion, CSCE is a robust technique with a high analytical sensitivity and specificity, and it can readily be used for mutation scanning of the large SACS gene. Furthermore this technique is less time-consuming and less expensive, as compared with standard automated sequencing.