Spanish cohort of VEXAS syndrome: clinical manifestations, outcome of treatments and novel evidences about UBA1 mosaicism.

BACKGROUND: The vacuoles, E1-enzyme, X linked, autoinflammatory and somatic (VEXAS) syndrome is an adult-onset autoinflammatory disease (AID) due to postzygotic UBA1 variants. OBJECTIVES: To investigate the presence of VEXAS syndrome among patients with adult-onset undiagnosed AID. Additional studies evaluated the mosaicism distribution and the circulating cytokines. METHODS: Gene analyses were performed by both Sanger and amplicon-based deep sequencing. Patients' data were collected from their medical charts. Cytokines were quantified by Luminex. RESULTS: Genetic analyses of enrolled patients (n=42) identified 30 patients carrying UBA1 pathogenic variants, with frequencies compatible for postzygotic variants. All patients were male individuals who presented with a late-onset disease (mean 67.5 years; median 67.0 years) characterised by cutaneous lesions (90%), fever (66.7%), pulmonary manifestations (66.7%) and arthritis (53.3%). Macrocytic anaemia and increased erythrocyte sedimentation rate and ferritin were the most relevant analytical abnormalities. Glucocorticoids ameliorated the inflammatory manifestations, but most patients became glucocorticoid-dependent. Positive responses were obtained when targeting the haematopoietic component of the disease with either decitabine or allogeneic haematopoietic stem cell transplantation. Additional analyses detected the UBA1 variants in both haematopoietic and non-haematopoietic tissues. Finally, analysis of circulating cytokines did not identify inflammatory mediators of the disease. CONCLUSION: Thirty patients with adult-onset AID were definitively diagnosed with VEXAS syndrome through genetic analyses. Despite minor interindividual differences, their main characteristics were in concordance with previous reports. We detected for the first time the UBA1 mosaicism in non-haematopoietic tissue, which questions the previous concept of myeloid-restricted mosaicism and may have conceptual consequences for the disease mechanisms.

Flow cytometric-based isolation of nucleated erythroid cells during maturation: an approach to cell surface antigen studies.

Nucleated red blood cells (NRBCs) are involved in normal physiologic processes, as well as in several malignancies. They are usually counted manually under the microscope. However, blood sample manipulation may be a source of variability and manual counting is imprecise, time-consuming, and subjective. To improve identification of CD45-negative cells, we used a flow cytometry technique that avoids the addition of lysing reagents and stains viable cell nuclei. We applied this method for counting and isolating NRBC subpopulations in whole blood samples, using DNA/RNA viable staining to discriminate nonnucleated erythroid cells and debris. NRBC counts gave 197.95 cells per mm(3) in mobilized peripheral blood samples (1.00%, n = 20), 3897.59 cells per mm(3) in leukapheresis products (3.08%, n = 20), and 765.21 cells per mm(3) in cord blood samples (6.09%, n = 20). Normal bone marrow counts were 5449.42 cells per mm(3) (11.76%, n = 20). Scatter profiles showed three distinct populations, from early to late-stage erythroblasts, consisting of erythroblasts, orthochromatic erythroblasts, and ejected nuclei, as confirmed by Wright-Giemsa staining. In addition, flow cytometry immunophenotyping showed that glycophorin A was expressed dimly on NRBCs during maturation. These findings point to the feasibility of live NRBCs studies, which offer great potential for a wide range of disciplines.