Test Id : UBA1Q

This test is intended for patients with clinical symptoms and other pertinent laboratory findings raising concern for VEXAS syndrome. These may include, but are not limited to, systemic or localized (eg, ear, orbital, skin) tissue inflammation presenting as rheumatologic disease, abnormal (usually low) whole blood counts, macrocytic anemia, characteristic microscopic changes in the bone marrow, as well as others.

1. Both refrigerated and ambient specimens must arrive within 7 days of collection.

2. Collect and package specimen as close to shipping time as possible.

The following information is required:

1. Pertinent clinical history

2. Date of collection

3. Specimen source (blood or bone marrow)

Submit only 1 of the following specimens:

Specimen Type: Whole blood

Container/Tube:

Preferred: Lavender top (EDTA)

Acceptable: Yellow top (ACD-B) or green top (heparin)

Specimen Volume: 4 mL

Collection Instructions:

1. Invert several times to mix blood.

2. Send whole blood specimen in original tube. Do not aliquot.

3. Label specimen as blood.

Specimen Stability: Refrigerated 7 days/Ambient 7 days

Specimen Type: Bone marrow aspirate

Container/Tube:

Preferred: Lavender top (EDTA)

Acceptable: Yellow top (ACD-B) or green top (heparin)

Specimen Volume: 2 mL

Collection Instructions:

1. Invert several times to mix bone marrow.

2. Send bone marrow specimen in original tube. Do not aliquot.

3. Label specimen as bone marrow.

Specimen Stability: Refrigerated 7 days/Ambient 7 days

Specimen Type: Extracted DNA from blood or bone marrow

Container/Tube: 1.5- to 2-mL tube with indication of volume and concentration of DNA

Specimen Volume: Entire specimen

Collection Instructions:

1. Label specimen as extracted DNA and source of specimen

2. Indicate volume and concentration of DNA on label. The required volume of DNA is at least 50 mcL at a concentration of 50 ng/mcL

Specimen Stability: Frozen (preferred)/Refrigerated

• Hematopathology Patient Information

1. Hematopathology Patient Information (T676)

2. If not ordering electronically, complete, print, and send a Hematopathology/Cytogenetics Test Request (T726) with the specimen.

Whole blood: 4 mL

Bone marrow: 2 mL

Extracted DNA: 50 mcL at 50 ng/mcL

Identification of pathogenic variant(s) in the UBA1 gene in patients presenting with symptoms concerning for or consistent with VEXAS syndrome

A highly sensitive quantitative assay for the detection of 7 UBA1 mutations (c.122T>C, p.Met41Thr; c.121A>G, p.Met41Val; c.121A>C, p.Met41Leu; c.118-1G>C, p.?; c.118-2A>C, p.?; c.118-9_118-2del, p.?; and c.167C>T, p.Ser56Phe).

VEXAS syndrome is caused by somatic mutations in the UBA1 gene, which is located on the X-chromosome and encodes ubiquitin-activating enzyme E1, an important component in the protein ubiquitylation process. This syndrome, identified in 2020, is characterized by adult-onset rheumatologic and hematologic manifestations. Inflammatory disease can present systemically, as well with more localized findings involving the orbit, skin or ears. Hematologic abnormalities are frequently present, including low blood counts, macrocytic anemia, and characteristic vacuolated myeloid and erythroid precursor cells in the bone marrow.(1) VEXAS syndrome occurs overwhelmingly in male patients, suggesting that biologic females who might acquire a UBA1 mutation may be relatively protected by the presence of the remaining normal wild type allele.(2) To date, nearly all documented cases of VEXAS syndrome have been caused by seven mutations that occur at three different sites within exon 3 of the UBA1 gene: the intron 2 acceptor splice site and p.Met41 codon region (c.118-122), and the p.Ser56 codon (c.167).(1-4) Importantly, a significant number of VEXAS patients may have predisposition to develop bone marrow failure, clonal hematopoiesis, myelodysplastic syndrome, or other hematologic neoplasms (eg, plasma cell neoplasms).(2-4) Patients with UBA1 mutation and features of bone marrow failure appear to be associated with poor prognosis and may not respond to standard immunosuppressive and hypomethylating agents typically utilized in the treatment of autoinflammatory disorders and myelodysplastic syndrome.(3,4) Testing for these mutations will serve to identify VEXAS syndrome patients who should be considered for alternative therapies or clinical trials.

An interpretive report will be provided.

The assay is reported as positive or negative. In positive cases, the mutation type and its variant allele fraction (VAF) are reported.

VAF%= (mutant copy number)/(mutant copy number + wild-type number)

The precision of this quantitative assay is very high but inter-assay variability may occur such that quantitative changes should not be considered significant if 2 single measurements differ by less than 0.5 log (3.16-fold).

Other potential UBA1 variants outside the 7 assay targets are not detected by this assay. The absence of UBA1 mutation does not exclude other causes of inflammatory disorders or clonal myeloid processes. Although most patients with VEXAS syndrome have high UBA1 mutation variant fractions, this assay may not identify very low mutation burden cases below the limit of detection.

1. Beck DB, Ferrada MA, Sikora KA, et al. Somatic mutations in UBA1 and severe adult-onset autoinflammatory disease. N Engl J Med. 2020;383(27):2628-2638

2. Grayson PC, Patel BA, Young NS. VEXAS syndrome. Blood. 2021;137(26):3591-3594

3. Huang H, Zhang W, Cai W, et al. VEXAS syndrome in myelodysplastic syndrome with autoimmune disorder. Exp Hematol Oncol. 2021;10(1):23

4. Gutierrez-Rodrigues F, Kusne Y, Fernandez J, et al. Spectrum of clonal hematopoiesis in VEXAS syndrome. Blood. 2023;142(3):244-259

5. Koster MJ, Warrington KJ. VEXAS within the spectrum of rheumatologic disease. Semin Hematol. 2021;58(4):218-225

6. Obiorah IE, Patel BA, Groarke EM, et al. Benign and malignant hematologic manifestations in patients with VEXAS syndrome due to somatic mutations in UBA1. Blood Adv. 2021;5(16):3203-3215

7. Oganesyan A, Hakobyan Y, Terrier B, Georgin-Lavialle S, Mekinian A. Looking beyond VEXAS: Coexistence of undifferentiated systemic autoinflammatory disease and myelodysplastic syndrome. Semin Hematol. 2021;58(4):247-253

8. Poulter JA, Savic S. Genetics of somatic auto-inflammatory disorders. Semin Hematol. 2021;58(4):212-217

9. Shaukat F, Hart M, Burns T, Bansal P. UBA1 and DNMT3A mutations in VEXAS syndrome. A case report and literature review. Mod Rheumatol Case Rep. 2022;6(1):134-139. doi:10.1093/mrcr/rxab021

10. Zakine E, Schell B, Battistella M, et al. UBA1 variations in neutrophilic dermatosis skin lesions of patients with VEXAS syndrome. JAMA Dermatol. 2021;157(11):1349-1354

This test is performed using a droplet digital polymerase chain reaction (ddPCR) system. DNA extracted from patient samples is PCR-amplified using oligonucleotide primers and mutant- and wildtype-specific fluorescently labeled probes directed to the genomic target regions. Results are analyzed using dedicated software and Poisson statistics to provide absolute quantification of mutant target and wild type copies. Calculated results are reported as mutant fractional abundance (variant allele fraction %).(Unpublished Mayo method)

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• Prospective clients should contact their account representative. For assistance, contact Customer Service.

This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

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