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Objectives: Patients with VEXAS syndrome carry mutations of UBA1 gene coding for the E1 enzyme. The three most frequent mutations are p.M41T(122T > C), p.M41V (c.121A > G), and p.M41L (c.121A > C) in codon 41 of exon 3. Currently, sanger sequencing was mainly used to detect these mutations, which has low sensitivity and low throughput. There is a need of high sensitivity, simple and high throughput method to characterize patients with VEXAS syndrome. Methods: Based on our proprietary XNA technology, we have developed a QClamp® Plex platform to detect eight mutations in a single reaction using the Luminex xMap technology. The assay sensitivity, specificity and precision were subsequently evaluated. Furthermore, the reference interval and clinical sensitivity/specificity were estimated using clinical healthy/positive DNA samples and the sanger sequencing method was used for comparison. Results: With spiking synthetic mutant DNA in wildtype GM24385 cell line DNA, this assay can detect UBA1 mutations with a detection limit of variant allele frequency (VAF) at 0.1-5%. Our assay shows 100% concordance with Sanger sequencing results when used for analyzing 15 positive and 19 negative clinical samples. Conclusions: The QClamps® Plex UBA1 Mutation Detection Assay is a quicker, simpler, and more sensitive assay that can accurately detect the UBA1 mutations even at early stages with low mutation frequency.
RESUMO
BACKGROUND: Colorectal cancer (CRC) is among the most frequently occurring cancers worldwide and its incidence is forecasted to increase. Testing for KRAS (Kirsten rat sarcoma viral oncogene homolog) mutations in colorectal tissue biopsy samples has become a crucial tool to guide therapeutic decisions for personalized treatment. OBJECTIVE: The objective of this study was to determine the diagnostic sensitivity and specificity of the IntelliPlex™ KRAS G12/13 Mutation Kit using clinical specimens compared to Sanger sequencing as the reference method. METHODS: A total of 248 formalin-fixed paraffin-embedded (FFPE) tissue samples, with CRC tumors comprising more than 10% of the whole tissue sample, were included in the study and analyzed for specific KRAS mutations in codons 12 and 13. For samples with discordant results between Sanger sequencing and the IntelliPlex™ KRAS G12/13 Mutation Kit, Pyrosequencing was utilized to resolve the KRAS mutational status. RESULTS: Sequencing determined 153 specimens as KRAS wild-type genotype while the IntelliPlex™ KRAS G12/13 Mutation Kit confirmed 139 of the wild-type cases, resulting in a clinical specificity of 90.8% (95% confidence interval (CI) 85.12-94.91). All 95 specimens with a reported mutation in codons 12 or 13 of KRAS by sequencing were also reported as non-wild-type by the IntelliPlex™ KRAS G12/13 Mutation Kit, resulting in a clinical sensitivity to detect KRAS mutations of 100% (95% CI 96.19-100). CONCLUSIONS: The IntelliPlex™ KRAS G12/13 Mutation Kit demonstrates suitable specificity and sensitivity for use in clinical laboratories to determine the mutational status of KRAS codons 12 and 13.