Interlaboratory Diagnostic Validation of Conformation-Sensitive Capillary Electrophoresis for Mutation Scanning

Abstract

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.

Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes

“…In our unit, a multistep workflow including conformation-sensitive capillary electrophoresis 9 as a prescreening method for analysis of BRCA mutations was used (Supplementary Figure 1). A total of 28 DNA samples previously characterized by this workflow were used as a Training Set to setup our NGS workflow, and 14 new DNAs were used as a Validation Set (see Experimental design in the Results section).…”

“…6 Although direct Sanger sequencing is considered the gold standard for the analysis of BRCA1 and BRCA2 mutations, their large size (5592 bp and 10257 bp, respectively), and lack of mutation hot spots (see Breast Cancer Information Core database: http://www.research.nhgri.nih.gov/bic/) mean useful prescreening strategies. [7][8][9] Moreover, large genomic rearrangements (LGRs) of these genes require the use of other complementary techniques. 10,11 The development of cost-effective BRCA mutation detection workflows will not only benefit the genetic counseling process for patients with HBOCS but will also enhance the process of selecting patients for personalized treatments, as could be the case of PARP inhibitors, for example.…”

EMMA, a cost- and time-effective diagnostic method for simultaneous detection of point mutations and large-scale genomic rearrangements: application to BRCA1 and BRCA2 in 1,525 patients

“…This does not constitute an issue for genes with a very low polymorphic content such as RB1 but is a major hurdle for BRCA genes, which exhibit a large number of polymorphisms throughout their sequence. In this case, the advantages of prescreening methods are lost if each variant peak has to be sequenced (Mattocks, et al 2010). The situation becomes much simpler if the method allows correct recognition of polymorphisms according to their profile.…”

“…However, its broad diffusion was hampered by design constraints and lack of sensitivity (Rozycka, et al, 2000). The recently described Heteroduplex Analysis by Capillary Array Electrophoresis (Perez-Cabornero, et al, 2009), also called Conformation-Sensitive Capillary Electrophoresis (CSCE) (Mattocks, et al 2010) represents a real improvement, but it also presents certain disadvantages. It uses a home-made polymer recipe that does not allow batch-to-batch reproducibility.…”

BRCA1 and BRCA2 mutations in males with familial breast and ovarian cancer syndrome. Results of a Spanish multicenter study

“…Briefly, all coding exons and exonintron boundaries of BRCA1/BRCA2 were amplified by PCR followed in some laboratories by direct sequencing. The remaining laboratories performed a BRCA1/2 mutation pre-screening based on abnormal pattern detection using either conformation sensitive gel electrophoresis (CSGE) [14], heteroduplex analysis by capillary array electrophoresis (HA-CAE) [15], conformation sensitive capillary electrophoresis (CSCE) [16], high performance liquid chromatography (HPLC) or high resolution melting (HRM) [17,18] methods followed by sequencing of the abnormal patterns. Large genomic rearrangements were studied by multiplex ligation dependent probe amplification (MLPA; MRC Holland, Amsterdam, The Netherlands) [19].…”