![Somatic mutations in UBA1 cause VEXAS. (A) Electropherograms showing that the c.167C>T, p.Ser56Phe variant (denoted by an asterisk) is present in peripheral blood and is specifically enriched in sorted myeloid, but not lymphoid, cells. (B) In contrast to the p.Met41 variants, the p.Ser56Phe patient variant does not result in loss of UBA1b or gain of UBA1c, as revealed by immunoblot analysis of HEK293T cells transfected with the indicated UBA1FLAGHA patient variants. WT, wild-type. (C) The p.Ser56Phe mutation reduces catalytic activity of nuclear UBA1a and cytoplasmic UBA1b in a temperature-dependent manner. Ubiquitin thioester formation assays were performed by preincubating denoted recombinantly purified UBA1 variants at 4°C or 37°C for 30 minutes, followed by incubation with ubiquitin and adenosine triphosphate (ATP) for 30 minutes on ice and immunoblot analysis. (D) Quantification of relative ubiquitin thioester formation of UBA1 proteins shown in (C). Ubiquitin thioester formation was calculated as a normalized fraction of modified protein [Ub∼UBA1/(Ub∼UBA1+UBA1)], and wild-type (WT) protein was set to 1 (n = 6 independent experiments using 2 independently purified protein preparations). ***P < .001, standard Student t test. (E) Electropherogram of the c.118-1G>A mutation in peripheral blood of P10 showing the mutant allele, present at the intron 2 acceptor, to be the predominant allele. (F) Reverse transcriptase polymerase chain reaction of patient-derived RNA revealed that the c.118-1G>C variant results in a reduction in correctly spliced UBA1 and the formation of multiple incorrectly spliced products (expected band size = 250 bp). Because the patient is deceased, no fresh tissue was available; therefore, RNA was extracted from paraffin-embedded fixed tissue. (G-H) Representative bone marrow morphology from patient P9 with p.Ser56Phe mutation, stained with haematoxylin and eosin. Hypercellular trephine morphology shows erythroid expansion, reduced granulopoiesis, and scattered atypical megakaryocytes (blue arrows). ns, not significant.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/137/26/10.1182_blood.2020010286/7/bloodbld2020010286f1.png?Expires=1719130797&Signature=N0~k~fPeCGcmxpq0z9H6e46GPm5taxL6Uo-yh4zlFQRwHTuZQuIPX7veQZpF3lEqO8U1NpC4OdzcAMWTMXwb~NzBrQqSDbJWveCGS120Ay7Le~2QUgEI8Tjh3gNnx4IBrxDyZGFdgb5R3dCxsFRPH-c17Dp7DUmU4to39fZszQ6uhclZ7ktyTzgMlR67j6vZyqioL4puQL5kEhr-4Rwq9l45VjFxBmJpQ~cNBGGQ8WAgnuHFLsGbNMjzK51Q6eFu7uLFwrnJbvmuQrkjtji~lc9HvvVmwfNGfDKydYMBp4kJPqkQJ7NnfsRWFjEFZw8wea~tQN8iAqM-2vkjVQwQHQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Somatic mutations in UBA1 cause VEXAS. (A) Electropherograms showing that the c.167C>T, p.Ser56Phe variant (denoted by an asterisk) is present in peripheral blood and is specifically enriched in sorted myeloid, but not lymphoid, cells. (B) In contrast to the p.Met41 variants, the p.Ser56Phe patient variant does not result in loss of UBA1b or gain of UBA1c, as revealed by immunoblot analysis of HEK293T cells transfected with the indicated UBA1FLAGHA patient variants. WT, wild-type. (C) The p.Ser56Phe mutation reduces catalytic activity of nuclear UBA1a and cytoplasmic UBA1b in a temperature-dependent manner. Ubiquitin thioester formation assays were performed by preincubating denoted recombinantly purified UBA1 variants at 4°C or 37°C for 30 minutes, followed by incubation with ubiquitin and adenosine triphosphate (ATP) for 30 minutes on ice and immunoblot analysis. (D) Quantification of relative ubiquitin thioester formation of UBA1 proteins shown in (C). Ubiquitin thioester formation was calculated as a normalized fraction of modified protein [Ub∼UBA1/(Ub∼UBA1+UBA1)], and wild-type (WT) protein was set to 1 (n = 6 independent experiments using 2 independently purified protein preparations). ***P < .001, standard Student t test. (E) Electropherogram of the c.118-1G>A mutation in peripheral blood of P10 showing the mutant allele, present at the intron 2 acceptor, to be the predominant allele. (F) Reverse transcriptase polymerase chain reaction of patient-derived RNA revealed that the c.118-1G>C variant results in a reduction in correctly spliced UBA1 and the formation of multiple incorrectly spliced products (expected band size = 250 bp). Because the patient is deceased, no fresh tissue was available; therefore, RNA was extracted from paraffin-embedded fixed tissue. (G-H) Representative bone marrow morphology from patient P9 with p.Ser56Phe mutation, stained with haematoxylin and eosin. Hypercellular trephine morphology shows erythroid expansion, reduced granulopoiesis, and scattered atypical megakaryocytes (blue arrows). ns, not significant.