Abstract
The genetic diagnosis of tuberous sclerosis complex is difficult because of its broad spectrum of mutations. In addition to point mutations in coding regions, intragenic or chromosomal-level large deletions, deep intronic splicing mutations, and mosaic mutations represent a significant proportion of the mutations. In this study, multimodular, long-range PCR-based next-generation sequencing assays were optimized and validated using >100 samples with known TSC1 and TSC2 variants. Multiplex, long-range PCR covering the entire genomic region of both genes detected all 138 known variants; however, it also yielded false-positive results. Intragenic large deletions were detected with accurate breakpoint sequences. Chromosomal-level deletions were estimated by discordant allele segregation in the family and confirmed by DNA microarray. Deep intronic mutations were verified using a combination of long-range DNA PCR and full-length mRNA sequencing. DNA samples were mixed to simulate mosaic mutations, and most variants were detected but could not be distinguished from equivalently detected false-positive results. Repeated false-positive results were classified, and the strategy of selecting the common variants detected in the duplicate analysis and eliminating known false-positive results improved the sensitivity (85.2%) and positive predictive value (96.6%) of a 10% mosaic simulation. Long-range PCRbased next-generation sequencing is a highly versatile genetic test; however, confirmation tests remain necessary for clinical use because false-positive results cannot be completely eliminated from single experiments.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.