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A rapid, accessible real-time PCR approach to identify UBA1 somatic mutations in VEXAS syndrome
Luisa Agnello1,2, Caterina Maria Gambino1,2, Lidia La Barbera3
1Department of Biomedicine, Neurosciences and Advanced Diagnostics, Institute of Clinical Biochemistry, Clinical Molecular Medicine, and Clinical Laboratory Medicine, University of Palermo, Palermo, Italy.
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.
