Somatic Mutations in UBA1 and Severe Adult-Onset Autoinflammatory Disease

PARTICIPANTS

Three affected men, each with a somatic UBA1 variant, were identified by analysis of exome data from genetic databases at the National Institutes of Health (NIH) Clinical Center. Fifteen additional men in observational cohorts at the NIH Clinical Center were identified on the basis of clinical similarities to the previous three affected men: their somatic UBA1 variants were confirmed by Sanger sequencing. Seven other participants were identified in study populations at University College London and Leeds Teaching Hospitals NHS Trust in the United Kingdom. All participants were enrolled in research studies that had been approved by the respective institutional review boards and provided written informed consent. Clinical diagnoses were defined according to standard criteria.^10-16

GENETIC AND FUNCTIONAL ANALYSIS

We sequenced the exomes of the first 3 participants and 3 additional participants and sequenced UBA1 in the other 19 participants and their unaffected family members. We prioritized genes involved in the post-translational process of ubiquitylation because of its regulation of innate immune responses.¹⁷ We analyzed fractionated cell types in blood and fibroblast samples obtained from the participants using digital droplet polymerase-chain-reaction assay, immunoblotting, immunohistochemical testing, electron microscopy, flow cytometry, and cytokine profiling. To study the effect of presumed etiologic variants on UBA1 function, we edited the genome of zebrafish using the CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 (CRISPR-associated protein 9) system. The Supplementary Appendix, available with the full text of this article at NEJM.org, includes a description of the methods used for these procedures.

STATISTICAL ANALYSIS

Continuous variables are presented as means with standard deviations or medians with interquartile ranges and were compared with the use of parametric or nonparametric tests as appropriate. Categorical variables are expressed as absolute numbers and percentages and were compared with the use of the chi-square test. Statistical methods for the transcriptomic analyses are described in the Supplementary Appendix. Post hoc Bonferroni correction was performed for experiments with multiple comparisons, and the results are presented as adjusted P values.

GENOTYPE-FIRST SCREENING TO IDENTIFY THE VEXAS SYNDROME

We screened the exomes and genomes of 1477 persons referred because of undiagnosed recurrent fevers, systemic inflammation, or both and 1083 persons affected by atypical, unclassified disorders who were identified through the Undiagnosed Diseases Program.¹⁸ The combined population of participants was roughly balanced with respect to sex and spanned a wide range of ages (Fig. 1A). We identified 3 men, all with novel (absent from public databases including the Genome Aggregation Database), predicted deleterious, and apparently heterozygous variants at the same codon, methionine-41 (p.Met41) of the X-linked gene UBA1, which is highly intolerant to haploinsufficiency (as indicated by a “probability of being loss-of-function intolerant” [pLI] score of 1.0, on a range of 0.0 to 1.0, with a higher score indicating a greater degree of intolerance for loss-of-function variants in healthy persons). The seemingly heterozygous variants seen in these 3 affected men raised questions of mosaicism or aneuploidy, because males usually have only one copy of X-chromosomal genes. Mindful of the potential role of somatic mutations in adult-onset disease, we evaluated all the variants, including those with a lower-than-expected allele frequency.

We confirmed all UBA1 variants of interest using Sanger sequencing (Fig. 1A and Fig. S1 in the Supplementary Appendix) and determined that the variants were absent from fibroblasts in the tested participants (Fig. S2). None of the 25 affected men had affected family members, and all 8 available relatives tested negative for the mutation. The participants did not have aneuploidy on karyotype analysis, nor were X-chromosomal copy-number variations present, as determined by high-density single nucleotide polymorphism array analysis (Fig. S2C). We therefore predicted that these apparently heterozygous variants were somatic (also known as mosaic or postzygotic) mutations, with genetically heterogenous cells carrying either hemizygous wild-type or mutated UBA1.²⁰

To corroborate these findings, we interrogated exome data primarily from whole-blood samples obtained from 141,600 persons (77,162 unaffected adults and 64,438 persons who had been referred for diagnostic testing, mostly for neurodevelopmental diseases) who had undergone sequencing at a diagnostic company (GeneDx). None of the unaffected persons had a UBA1 p.Met41 allele fraction exceeding 5%. As for the affected persons, only 5%, the majority of whom were children, had immune phenotypes. However, we identified 3 men with UBA1 p.Met41 variants exceeding 71% variant allele frequency (Table S1), all with late-onset inflammatory disease. Thus, both screens identified UBA1 p.Met41 somatic mutations exclusively in men with adult-onset inflammatory disorders.

GENETIC FEATURES OF THE VEXAS SYNDROME

To characterize mosaicism, we isolated and sequenced different hematopoietic cell populations. The participants with UBA1 mosaic mutations had predominantly wild-type lymphocytes (T and B cells) and predominantly mutant myeloid cells (neutrophils and monocytes) in peripheral blood (Fig. 1B and 1C). Despite UBA1 mutations being lineage-restricted in the blood, early marrow progenitor cells showed mosaicism. Hematopoietic stem cells and multipotent progenitors, granulocyte–monocyte progenitors, megakaryocyte–erythroid progenitors, and lymphoid progenitors isolated from bone marrow had abundant mutant cells, but mutations were absent in mature lymphocytes. The participants had decreased peripheral lymphocyte counts, which suggests that the mutant cells either did not proliferate or were eliminated, an outcome that resulted in an increased proportion of the wild-type genotype (Fig. S3).

CLINICAL FEATURES OF THE VEXAS SYNDROME

The first 3 participants were men in whom severe inflammatory syndromes had developed in the fifth to seventh decade of life. Common clinical features included alveolitis, ear and nose chondritis, skin lesions, and thromboembolic disease (Fig. 2). Each participant had progressive hematologic abnormalities, including macrocytic anemia, thrombocytopenia, and myeloid dyspoiesis, but none had an overt hematologic malignant condition. The only common genetic lesion among these 3 participants was a nonsynonymous missense mutation at UBA1 p.Met41 (Fig. S4). We identified 22 additional participants who had overlapping clinical phenotypes; 7 of these participants were from an existing cohort of patients with relapsing polychondritis (ClinicalTrials.gov number, NCT02257866) (Table 1; see also the Supplementary Appendix). Among 18 participants who had undergone genetic screening on the basis of their clinical characteristics (male with adult-onset inflammatory syndrome and cytopenia, as well as chondritis, vasculitis, neutrophilic dermatosis, or a combination of these), 14 (78%) were found to have UBA1 mutations.

The 25 participants with confirmed mutations in UBA1 underwent extensive clinical assessment (Tables S2 through S8). All were male with a median age at disease onset of 64 years. Each of these participants had one of three somatic variants in codon 41 in UBA1 (predicting amino acid substitutions p.Met41Val [NM_003334.3:c.121A→G; ClinVar accession number, SCV001438043], p.Met41Thr [NM_003334.3:c.122T→C; ClinVar accession number, SCV001438045], or p.Met41Leu [NM_003334.3:c.121A→C; ClinVar accession number, SCV001438044]). The majority of these participants had recurrent fevers, pulmonary involvement, dermatologic manifestations (including neutrophilic dermatoses and cutaneous vasculitis), macrocytic anemia, hematopoietic dyspoiesis, and bone marrow vacuolization restricted to myeloid and erythroid precursor cells (Table 1, Fig. 2, Table S3, and Figs. S5 and S6). Electron microscopy revealed myeloid cells that were undergoing cell death and had vacuoles consisting of lipid droplets and disordered cellular organelles, including degenerating mitochondria.

Subgroups of participants met or partially met established diagnostic or classification criteria for a number of clinical conditions (Table 1 and Table S2). Transformation into a myelodysplastic syndrome with excess blasts or acute myeloid leukemia did not occur in any participant. All 25 participants had highly elevated levels of acutephase reactants and had no response to multiple disease-modifying antirheumatologic drugs (Tables S4 and S5). Glucocorticoids, often administered in high doses, were the only treatment that consistently ameliorated severe inflammatory symptoms. Of the 25 participants, 10 (40%) died from disease-related causes (respiratory failure or progressive anemia) or complications related to treatment.

IMMUNOLOGIC FEATURES OF THE VEXAS SYNDROME

Transcriptome analysis of the peripheral blood of participants revealed a shared gene expression signature consistent with the activation of multiple innate immune pathways (Fig. S7A and S7B and Table S9). Gene-expression profiling of isolated monocytes and neutrophils that were studied during a clinically quiescent period during which the participants had been receiving minimal treatment showed highly activated inflammatory signatures in multiple pathways including tumor necrosis factor, interleukin-6, and interferon-γ, a finding that is consistent with cell-intrinsic severe myeloid inflammation (Fig. S7C and Tables S10 through S12). Activation of pathways affecting the unfolded protein response and integrated stress response was identified only in myeloid cells (Fig. S8).²¹ Elevated levels of multiple cytokines in the serum were consistent with the findings from the transcriptome analysis (Fig. 3). Peripheral B cells and monocytes showed atypical differentiation, with loss of immature B cells and nonclassical and intermediate monocyte populations (Fig. S9). Functional studies of neutrophils obtained from the participants with the VEXAS syndrome, as compared with those from age- and sex-matched healthy controls, showed preserved phagocytic capacity in mutant cells but enhanced spontaneous neutrophil extracellular trap formation, findings that are consistent with dysregulated proinflammatory neutrophil activation (Fig. S10).

VEXAS SYNDROME AND LOSS OF CYTOPLASMIC UBA1 FUNCTION

UBA1 encodes the major E1-activating enzyme required for initiation of all cellular ubiquitin signaling.^22-25 UBA1 is expressed as two isoforms differing in translation start site — nuclear UBA1a (initiated at p.Met1) and cytoplasmic UBA1b (initiated at p.Met41) (Fig. 4A). Unexpectedly, monocytes obtained from the participants with the VEXAS syndrome, as compared with those from the controls, and ectopically expressed UBA1 Met41Val, as compared with the wild-type enzyme, had similar nuclear and cytoplasmic localization, despite loss of the initiating methionine for UBA1b (Fig. S11A through S11C).

We next analyzed the expression of UBA1 Met41 variants in human embryonic kidney (HEK293T) cells by immunoblotting, which revealed loss of UBA1b and an unanticipated faster-migrating band (Fig. 4B and Fig. S11B). We hypothesized that the protein generating the faster-migrating band might be an isoform generated by translation initiation from a downstream start codon (Fig. 4A). Indeed, introduction of a mutation targeting the Met67 codon resulted in the disappearance of the faster-migrating band, suggesting that the transcription of the messenger RNA of this novel isoform of UBA1, denoted here as UBA1c, is initiated from the Met67 codon. UBA1c localized to the cytoplasm (Fig. S11A), a finding consistent with our observations regarding monocytes from the participants with the VEXAS syndrome. Purified recombinant UBA1c was catalytically impaired in thioester assays, as compared with purified recombinant UBA1a and UBA1b, a finding that was indicated by the predominance of the faster-migrating uncharged form in the UBA1c lane in contrast to the excess of charged forms with other tested UBA1 proteins (Fig. 4C). We therefore concluded that Met41 variants result in the reduction of cytoplasmic UBA1 function by favoring the production of a catalytically deficient UBA1c over that of a catalytically proficient UBA1b.

We then attempted to confirm this cytoplasmic isoform switch by investigating the consequences of such a switch in the cells from the participants. Purified T cells from the participants, which were predominantly wild type, and from the unaffected controls had equivalent amounts of UBA1a and UBA1b proteins (Fig. 4D). In contrast, monocytes, which predominantly carried mutated UBA1 variants in the participants, had decreased levels of UBA1b and detectable levels of UBA1c, which confirmed the results of the transfection studies (Fig. 4D). Mutant monocytes were defective in ubiquitylation, as shown by a loss of polyubiquitin species and a concomitant increase in free ubiquitin (Fig. 4D). These cells also showed increased eIF2α phosphorylation and altered LC3 levels, which suggests that loss of ubiquitylation activates cellular stress responses that lead to up-regulation of the unfolded-protein response (as indicated by increased eIF2α phosphorylation) and dysregulation of autophagy (as indicated by altered LC3 levels) (Fig. 4D).

To study the contribution of nuclear and cytoplasmic UBA1 isoforms to inflammatory disease in vivo, we established CRISPR-Cas9–edited zebrafish models (Table S13). Zebrafish uba1 and human UBA1 are highly homologous (Fig. S12). Because uba1 is essential for viability,^26-28 we assessed inflammation during early development. Homozygous loss of all isoforms of Uba1 (Δuba1 [Δ indicates loss of protein]) or loss of Uba1b alone (Δuba1b) but not Uba1a (Δuba1a) in transgenic Tg(mpx:EGFP [mpx promoter controlling expression of enhanced green fluorescent protein]) zebrafish led to lower numbers of neutrophils than the numbers in sibling control heterozygous or wild-type fish (Fig. S13). All three zebrafish lines deficient in uba1 also showed growth abnormalities and early death between 7 and 21 days after fertilization, a finding that may be due in part to the germline nature of these mutations as compared with the somatic variants found in the participants with the VEXAS syndrome. Finally, Δuba1 or Δuba1b, but not Δuba1a, led to up-regulation of the expression of inflammatory genes in zebrafish, similar to that seen in the participants (Fig. 4E). Taken together, these results support the concept that systemic inflammation results from disruption of cytoplasmic UBA1 and its zebrafish homologue.