A rapid, accessible real-time PCR approach to identify UBA1 somatic mutations in VEXAS syndrome

Abstract

Background: VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a severe adult-onset autoinflammatory disease caused by somatic mutations in the X-linked UBA1 gene, most commonly affecting codon 41. Early molecular confirmation is essential, but sequencing-based methods may be limited by turnaround time, cost, and sensitivity for low-level somatic variants. We aimed to validate a rapid, accessible allele-specific real-time PCR assay for detection of the most frequent UBA1 hotspot mutations associated with VEXAS syndrome. Methods: In this prospective monocentric study conducted at the University Hospital "P. Giaccone" (Palermo, Italy), 17 adults were enrolled: six patients with high clinical suspicion of VEXAS syndrome and eleven healthy controls. UBA1 variants c.121A>G, c.121A>C, and c.122T>C were screened using a SYBR Green-based allele-specific real-time PCR kit with mutation-specific reaction mixes and an internal housekeeping control, followed by melting curve analysis for variant discrimination. All PCR-positive samples were confirmed by Sanger sequencing. Results: Allele-specific real-time PCR identified UBA1 mutations in 5/6 (83.3%) suspected VEXAS cases. Sanger sequencing confirmed all real-time PCR-positive results, demonstrating 100% concordance between methods. Conclusion: This allele-specific real-time PCR assay enables rapid and reliable detection of the most common UBA1 codon 41 mutations associated with VEXAS syndrome using standard real-time PCR platforms. The approach provides a practical, cost-effective screening strategy to support timely diagnosis in patients with high clinical suspicion.