Background/Purpose: Vacuoles, E1 ubiquitin-activating enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome is caused by somatic mutations of the ubiquitin-like modifier activating enzyme 1 (UBA1) gene and characterized by widespread inflammation, a high mortality rate, and escalating hematologic manifestations. Mutations in UBA1 appear within hematopoietic stem cells and become lineage restricted to myeloid populations. In most patients, 50-90% of circulating neutrophils carry the UBA1 mutation, yet there is limited understanding of neutrophils in VEXAS. The aim of this study was to evaluate the transcriptomes of neutrophils in peripheral blood and marrow from patients with VEXAS.

Methods: We conducted single-cell RNA sequencing (scRNA-seq) on neutrophils from 17 VEXAS patients with somatic pathogenic variants in UBA1 at p.Met41. Neutrophils were negatively isolated from paired bone marrow and peripheral blood samples. Cells were captured using 10X Genomics Chromium platform using 3’ 3.1 chemistry and sequenced on the Illumina Novaseq 6000/X plus. Reads were aligned and quantified using Cell Ranger v7.2.0; dimensional reduction, clustering, and gene expression analysis was conducted using R package Seurat v5.0.1. Neutrophil subsets in peripheral blood were categorized in reference to previously defined genetic markers identified in healthy and disease controls (Wigerblad, G., et al. 2022).

Results: The canonical mutation types among the recruited patients were p.Met41Thr (n=6), p.Met41Leu (n=7), and p.Met41Val (n=4). Mutation burden varied within the cohort (variant allele fraction (VAF) median 66%, range 41-89%). Clinical characteristics of the cohort reflected the spectrum of VEXAS-associated disease manifestations. Complete blood count (CBC) data revealed reduced lymphocyte, monocyte, and natural killer cell counts with preserved to elevated neutrophil counts. Immature neutrophils and myelocytes were commonly detected in peripheral blood. scRNA-seq of over 300,00 captured cells confirmed enrichment of granulocyte precursor cells in peripheral blood in patients with VEXAS.  These premature neutrophils were characterized by increased transcription of genes associated with neutrophil maturation processes, such as granule formation. Differences in neutrophil clustering patterns were observed in VEXAS compared to controls and within the different VEXAS-associated genotypes.  Shared and distinct neutrophil subsets were identified in VEXAS patients compared to controls. Upregulation of genes related to neutrophil growth and survival, inflammation, and regulation were observed in specific subsets of neutrophils from patients with VEXAS. In particular, increased expression of interferon response genes and inflammasome-related genes were observed in VEXAS neutrophils.

Conclusion: Neutrophils are a key effector cell in VEXAS syndrome, contributing to inflammation and poor clinical outcomes. Results from this study are informative about neutrophil heterogeneity in VEXAS syndrome and may lead to discovery of novel myeloid-directed therapeutic targets.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/neutrophil-transcriptomics-in-vexas-syndrome/