Three UBA1 clones for a unique VEXAS syndrome

Dear Editor, The VEXAS syndrome (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) is a recently described autoinflammatory disease characterized by mutations in the E1 ubiquitin-activating enzyme encoded by the UBA1 gene [1]. Clinico-biological findings include haematological abnormalities, treatment-refractory inflammatory syndrome, skin lesions, thromboembolic events, pulmonary infiltrate and chondritis [1–3]. To date, hundreds of patients have been described in the literature, with most subjects harbouring a single mutation in UBA1 [4–6]. In their first cohort, Beck et al. identified recurrent UBA1 mutations all affecting methionine 41 of exon 3 of UBA1: p. M41T (c.122T>C), p. M41V (c.121A>G) and p. M41L (c.121A>C) [1]. Since this initial description, other mutations have been reported, such as splice region mutations at exon 3 (c.118-2A>C, c.118-1G>C and C.118-9_118-2del) as well as a mutation affecting serine 56 in exon 3 (c.167C>T) [7]. The mutation of the UBA1 gene is considered to be the initiating event of VEXAS syndrome. However, the mechanisms leading to its emergence are unknown and are therefore, by default, mainly considered as a Darwinian process with random occurrence and clonal expansion in haematopoietic cells as a product of natural selection.

Here we describe a rare case of VEXAS syndrome with independent occurrences of UBA1 mutations in at least three distinct clones, highlighting that the molecular pathophysiology of the disease may be more complex than expected.

A 67-year-old man with polychondritis and no blood count abnormality was referred to our department. He received high-dose prednisone for 6 months, followed by MTX 20 mg per week because of a corticoid-refractory inflammatory syndrome. Blood counts showed macrocytic anaemia (haemoglobin 8.7 g/dl; mean corpuscular volume 107 fl) associated with thrombocytopenia (platelet count 73 × 10⁹/l). The white blood cell count was 6.3 × 10⁹/l including 4.5 × 10⁹/l neutrophils, 0.1 × 10⁹/l basophils, 1.3 × 10⁹/l lymphocytes and 0.4 × 10⁹/l monocytes. Bone marrow (BM) was of high cellularity without excess blasts but signs of dysmyelopoiesis (Fig. 1A–D). Vacuoles were observed in granulocytic and erythroid lineage progenitors. Cytological pattern was compatible with a myelodysplastic syndrome with multilineage dysplasia. Conventional cytogenetics showed no abnormality. A 90-myeloid gene panel was screened by high throughput sequencing on BM sample and revealed three distinct mutations in UBA1 (NM_003334.4) without mutations or copy-number abnormality in other genes, leading to the diagnosis of VEXAS syndrome. A major clone, with the highest variant allele frequency (VAF), corresponded to the most described mutation at codon 41 (c.121A>G, p. M41V; VAF 73%). Two other mutations with lower VAFs involved the exon 3 splice-site (c.118-1G>C; VAF 20%) and the codon 41 (c.122T>C, p. M41T; VAF 2%). The analysis of read alignments demonstrated these mutations were carried by distinct reads (Fig. 1E). Given the location of the UBA1 gene on the X chromosome, it was thus possible to determine that each mutation represented a distinct clone in this male patient and that each VAF could be directly related to the fraction of BM haematopoietic cells carrying the mutation. Overall, the three distinct UBA1-mutated clones were considered together to represent ∼95% of all BM haematopoietic cells. To exclude sequencing artifacts, the presence of each mutation was checked by droplet digital PCR on the same sample as well as two other follow-up samples using three distinct assays.

Figure 1.

(A–D) Morphological evaluation of bone marrow with multilineage dysplasia. (A) Multinucleated erythroblast and hypogranular granulocytic lineage. (B) Micromegakaryocyte. (C and D) Myeloid progenitors with multiple vacuoles. (E) Integrative Genomics Viewer (IGV) interface of sequencing. Analysis of UBA1 sequencing reads shows that three mutations are supported by different reads (red, blue, purple)

Open in new tabDownload slide

The patient received ruxolitinib 20 mg twice per day plus low-dose prednisone. At 2 months, the patient became highly transfusion-dependent. Recombinant erythropoietin administration was inefficient. A treatment with tocilizumab was initiated 6 months later (8 mg/kg every 4 weeks) without discontinuing ruxolitinib. After three cycles, the biological inflammatory syndrome disappeared, but a significant need for transfusions persists. Cutaneous and articular lesions have considerably improved, yet there remains pronounced asthenia due to severe anaemia.

This report questions the pathophysiology of VEXAS syndrome. Indeed, the concurrent emergence of several UBA1-mutated clones raises the possibility of an underlying process impacting the selection of these clones. This could imply a specific condition of the haematopoietic stem cell or its microenvironment, certain selection pressure factors (such as the immune system, environmental pollutants or exposure to specific drugs), or synergistic effects of the different clones in the pathology, promoting their simultaneous emergence. Interestingly, another patient harbouring both p.M41T and c.118-2A>C mutations was recently reported [6]. Since most previous literature reports did not use high throughput sequencing for UBA1 screening, these cases suggest that this condition may be underestimated. Furthermore, the screening of a large panel including genes involved in age-related clonal haematopoiesis, which have been reported to pre-exist or co-occur with UBA1 mutations in some patients [6, 8], was negative in the present case.

In conclusion, we described a rare case of VEXAS syndrome with three distinct clones presenting different mutations of UBA1, questioning the pathophysiology of the disease. Moreover, this case highlights the value of UBA1 screening by high-throughput sequencing to investigate clonal architecture and dynamics of VEXAS syndrome under therapy.

Data availability

Data are available upon reasonable request by any qualified researchers who engage in rigorous, independent scientific research, and will be provided following review and approval of a research proposal and Statistical Analysis Plan (SAP) and execution of a Data Sharing Agreement (DSA). All data relevant to the study are included in the article.

Contribution statement

N.D., C.P. and A.M.-R. performed genetics assays. B.P. and O.N. performed cytological analysis. L.T. and N.C. provided clinical data. P.R. performed cytogenetical assays. B.P., N.D., O.N. and L.T. wrote the manuscript, which was approved by all authors.

Funding

No specific funding was received from any bodies in the public, commercial or not-for-profit sectors to carry out the work described in this article.

Disclosure statement: The authors have declared no conflicts of interest.

Ethics: Informed consent has been received for the publication of this case report.

References