Faculty Bibliography

BACKGROUND:Adult-onset inflammatory syndromes often manifest with overlapping clinical features. Variants in ubiquitin-related genes, previously implicated in autoinflammatory disease, may define new disorders. METHODS:We analyzed peripheral-blood exome sequence data independent of clinical phenotype and inheritance pattern to identify deleterious mutations in ubiquitin-related genes. Sanger sequencing, immunoblotting, immunohistochemical testing, flow cytometry, and transcriptome and cytokine profiling were performed. CRISPR-Cas9-edited zebrafish were used as an in vivo model to assess gene function. RESULTS:lies on the X chromosome.) In such patients, an often fatal, treatment-refractory inflammatory syndrome develops in late adulthood, with fevers, cytopenias, characteristic vacuoles in myeloid and erythroid precursor cells, dysplastic bone marrow, neutrophilic cutaneous and pulmonary inflammation, chondritis, and vasculitis. Most of these 25 patients met clinical criteria for an inflammatory syndrome (relapsing polychondritis, Sweet's syndrome, polyarteritis nodosa, or giant-cell arteritis) or a hematologic condition (myelodysplastic syndrome or multiple myeloma) or both. Mutations were found in more than half the hematopoietic stem cells, including peripheral-blood myeloid cells but not lymphocytes or fibroblasts. Mutations affecting p.Met41 resulted in loss of the canonical cytoplasmic isoform of UBA1 and in expression of a novel, catalytically impaired isoform initiated at p.Met67. Mutant peripheral-blood cells showed decreased ubiquitylation and activated innate immune pathways. Knockout of the cytoplasmic UBA1 isoform homologue in zebrafish caused systemic inflammation. CONCLUSIONS:Using a genotype-driven approach, we identified a disorder that connects seemingly unrelated adult-onset inflammatory syndromes. We named this disorder the VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. (Funded by the NIH Intramural Research Programs and the EU Horizon 2020 Research and Innovation Program.).

Somatic mutations in UBA1 cause VEXAS (Vacuoles, E1 ubiquitin activating enzyme, X-linked, Autoinflammatory Somatic) syndrome, an adult-onset inflammatory disease with an overlap of hematologic manifestations. VEXAS syndrome is characterized by a high mortality rate and significant clinical heterogeneity. We sought to determine independent predictors of survival in VEXAS and to understand the mechanistic basis for these factors. We analyzed 83 patients with somatic pathogenic variants in UBA1 at p.Met41 (p.Met41Leu/Thr/Val), the start codon for translation of the cytoplasmic isoform of UBA1 (UBA1b). Patients with the p.Met41Val genotype were most likely to have an undifferentiated inflammatory syndrome. Multivariate analysis showed ear chondritis was associated with increased survival, while transfusion dependence and the p.Met41Val variant were independently associated with decreased survival. Using in vitro models and patient-derived cells, we demonstrate that p.Met41Val variant supports less UBA1b translation than either p.Met41Leu or p.Met41Thr, providing a molecular rationale for decreased survival. In addition, we show that these three canonical VEXAS variants produce more UBA1b than any of the six other possible single nucleotide variants within this codon. Finally, we report a patient, clinically diagnosed with VEXAS syndrome, with two novel mutations in UBA1 occurring in cis on the same allele. One mutation (c.121 A>T; p.Met41Leu) caused severely reduced translation of UBA1b in a reporter assay, but co-expression with the second mutation (c.119 G>C; p.Gly40Ala) rescued UBA1b levels to those of canonical mutations. We conclude that regulation of residual UBA1b translation is fundamental to the pathogenesis of VEXAS syndrome and contributes to disease prognosis.

Ubiquitylation is an essential post-translational modification that regulates intracellular signalling networks by triggering proteasomal substrate degradation, changing the activity of substrates or mediating changes in proteins that interact with substrates. Hundreds of enzymes participate in reversible ubiquitylation of proteins, some acting globally and others targeting specific proteins. Ubiquitylation is essential for innate immune responses, as it facilitates rapid regulation of inflammatory pathways, thereby ensuring sufficient but not excessive responses. A growing number of inborn errors of immunity are attributed to dysregulated ubiquitylation. These genetic disorders exhibit broad clinical manifestations, ranging from susceptibility to infection to autoinflammatory and/or autoimmune features, lymphoproliferation and propensity to malignancy. Many autoinflammatory disorders result from disruption of components of the ubiquitylation machinery and lead to overactivation of innate immune cells. An understanding of the disorders of ubiquitylation in autoinflammatory diseases could enable the development of novel management strategies.

OBJECTIVE:Somatic mutations in UBA1 have recently been causally linked to a severe adult-onset inflammatory condition referred to as VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. UBA1 is of fundamental importance to the modulation of ubiquitin homeostasis and to the majority of downstream ubiquitylation-dependent cellular processes. Direct sequencing analysis of exon 3 containing prevalent variants p.Met41Leu, p.Met41Val, and/or p.Met41Thr is usually used to confirm the disease associated mutations. METHODS:We studied clinical, biochemical and molecular genetic characteristics of a fifty-nine-year-old male with two-year history of arthritis, fever, night sweats, nonspecific skin rash, lymphadenopathy, and myelodysplastic syndrome with multilineage dysplasia. RESULTS:The mutational analysis revealed a hitherto undescribed sequence variant c.1430G>C in exon 14 (p.Gly477Ala) in UBA1 gene. In vitro enzymatic analyses showed that p.Gly477Ala led to both decreased E1 ubiquitin thioester formation and E2 enzyme charging. CONCLUSION/CONCLUSIONS:We herein report a case of a patient of European ancestry with clinical manifestations of VEXAS syndrome associated with a newly identified dysfunctional variant UBA1 enzyme. Due to insufficient response to various immunosuppressive treatments, allogeneic hematopoietic stem cell transplantation was performed, which resulted in significant improvement of clinical and laboratory manifestations of the disease.

IMPORTANCE:VEXAS (vacuoles, E1-ubiquitin-activating enzyme, X-linked, autoinflammatory, somatic) syndrome is a disease with rheumatologic and hematologic features caused by somatic variants in UBA1. Pathogenic variants are associated with a broad spectrum of clinical manifestations. Knowledge of prevalence, penetrance, and clinical characteristics of this disease have been limited by ascertainment biases based on known phenotypes. OBJECTIVE:To determine the prevalence of pathogenic variants in UBA1 and associated clinical manifestations in an unselected population using a genomic ascertainment approach. DESIGN, SETTING, AND PARTICIPANTS:This retrospective observational study evaluated UBA1 variants in exome data from 163 096 participants within the Geisinger MyCode Community Health Initiative. Clinical phenotypes were determined from Geisinger electronic health record data from January 1, 1996, to January 1, 2022. EXPOSURES:Exome sequencing was performed. MAIN OUTCOMES AND MEASURES:Outcome measures included prevalence of somatic UBA1 variation; presence of rheumatologic, hematologic, pulmonary, dermatologic, and other findings in individuals with somatic UBA1 variation on review of the electronic health record; review of laboratory data; bone marrow biopsy pathology analysis; and in vitro enzymatic assays. RESULTS:In 163 096 participants (mean age, 52.8 years; 94% White; 61% women), 11 individuals harbored likely somatic variants at known pathogenic UBA1 positions, with 11 of 11 (100%) having clinical manifestations consistent with VEXAS syndrome (9 male, 2 female). A total of 5 of 11 individuals (45%) did not meet criteria for rheumatologic and/or hematologic diagnoses previously associated with VEXAS syndrome; however, all individuals had anemia (hemoglobin: mean, 7.8 g/dL; median, 7.5 g/dL), which was mostly macrocytic (10/11 [91%]) with concomitant thrombocytopenia (10/11 [91%]). Among the 11 patients identified, there was a pathogenic variant in 1 male participant prior to onset of VEXAS-related signs or symptoms and 2 female participants had disease with heterozygous variants. A previously unreported UBA1 variant (c.1861A>T; p.Ser621Cys) was found in a symptomatic patient, with in vitro data supporting a catalytic defect and pathogenicity. Together, disease-causing UBA1 variants were found in 1 in 13 591 unrelated individuals (95% CI, 1:7775-1:23 758), 1 in 4269 men older than 50 years (95% CI, 1:2319-1:7859), and 1 in 26 238 women older than 50 years (95% CI, 1:7196-1:147 669). CONCLUSIONS AND RELEVANCE:This study provides an estimate of the prevalence and a description of the clinical manifestations of UBA1 variants associated with VEXAS syndrome within a single regional health system in the US. Additional studies are needed in unselected and genetically diverse populations to better define general population prevalence and phenotypic spectrum.

OBJECTIVES/OBJECTIVE:, is an autoinflammatory disease. METHODS:mutations on protein function and immune signalling. RESULTS:T237M mutation exhibited subclinical inflammation.Clinical features not conventionally attributed to inflammation were also common in the cohort and included short dental roots, enamel defects and decreased salivary flow. CONCLUSION/CONCLUSIONS:and some features of disease are amenable to immunomodulatory therapy.

VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome is a newly described disease due to somatic mutations in UBA1, encoding ubiquitin-activating enzyme 1. Patients with VEXAS syndrome have hematologic features, such as macrocytic anemia and myelodysplastic syndrome, and systemic inflammation, including cutaneous involvement. Our aim was to characterize the spectrum of skin manifestations in a large cohort of patients with VEXAS. 87 males and 1 female with genetically confirmed VEXAS syndrome were included in this retrospective study. Mean age at disease onset was 64 years (range 39-78). Most patients (n=71, 81%) had skin involvement. Initial disease presentation was confined to skin in 16 patients (18.2%) or to skin with systemic features in 19 (21%) patients. Skin lesions were painful/tender (n=15, 21.1%) or pruritic (n=13, 18.3%), characterized by erythema (n=20, 28.2%) or nodules (n=17, 23.9%), and occurred most frequently in an acral distribution (n=25, 35.2%). 110 skin biopsies were performed in 45 patients. Skin biopsies were interpreted as small or medium vessel vasculitis (n=29, 26.4%), Sweet syndrome (n=24, 21.8%), connective tissue disease (n=9, 8.2%), or erythema nodosum (n=7, 6.4%). Histologic review of 11 cases identified a neutrophilic predominance (n=6, 54.5%) in the epidermis (n=2, 18.2%), papillary dermis (n=6, 54.5%) and reticular dermis (n=5, 45.5%). Skin lesions improved with glucocorticoids in 85.5% of patients (n=47/55), but relapse after tapering was common (60%, n=33/55). Severe injection site reactions to anakinra were frequent (64.3%, n=9/14). Skin lesions are a common, early feature of VEXAS syndrome and share similarities in clinical and histologic presentation with other inflammatory skin disease. Appreciation of the spectrum of skin findings in VEXAS will facilitate early diagnosis.
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Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome was recently identified by genotype-driven approach. This syndrome is characterised by the presence of somatic mutations affecting methionine-41 (p.Met41) in UBA1 gene. All the identified patients were adult males who had recurrent fevers, cytopenias, dysplastic bone marrow with vacuoles in myeloid and erythroid precursors, neutrophilic skin and lung tissue inflammation and often had treatment refractory and fatal disease course. The various phenotypes reported with VEXAS syndrome include relapsing polychondritis, giant cell arteritis, polyarteritis nodosa, Sweet's syndrome, myelodysplastic syndrome and multiple myeloma.