A novel XNA-based Luminex assay to detect UBA1 somatic mutations associated with VEXAS syndrome

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.