Figure 3.
Residual UBA1b translation defines a threshold for VEXAS syndrome pathogenesis. (A) Schematic overview of the translation reporter system used to determine UBA1b translation. The first cistron of these reporters is translated in a CAP-dependent mechanism and encodes for C-terminally HA-tagged UBA1 constructs varied in p.Met41 and mutated at p.Met1 and p.Met67 (to suppress UBA1a and UBA1c translation and thus allowing for sensitive quantification of UBA1b by anti-HA immunoblotting). The second cistron is translated using an IRES and encodes for a C-terminally HA-tagged MBP, which is used for normalization across different p.Met41 variants. (B) p.Met41Val supports less UBA1b translation than p.Met41Thr and p.Met41Leu. Anti-HA immunoblot analysis of HEK293T lysates expressing indicated UBA1b translation reporters. (C) Quantification of the experiment shown in panel B. Error bars denote SD, n = 12 (4 biological with 3 technical replicates each; technical replicates of each biological replicate are depicted in the same color and symbol), **P < .01; ****P < .0001, ordinary 1-way ANOVA. (D) The canonical VEXAS variants (p.Met41Val/Thr/Leu) support more UBA1b translation than any of the other possible single-nucleotide variants at the Met41 start codon. Anti-HA immunoblot analysis of HEK293T lysates expressing indicated UBA1b translation reporters. (E) Quantification of the experiment shown in panel D. Error bars denote SD, n = 4 (2 biological with 2 technical replicates each; technical replicates of each biological replicate are depicted in the same color and symbol), *P < .05; ****P < .0001, ordinary 1-way ANOVA. (F) Two novel VEXAS variants (c.121 A>T; p.Met41Leu and c.119 G>C; p.Gly40Ala) are present in a patient in cis on the same allele. Overview of the genome sequencing reads of the p.Met41LeuTTG/p.Gly40Ala patient. (G) The p.Gly40Ala variant rescues residual UBA1b translation of the p.Met41LeuTTG to the level of the canonical VEXAS variant p.Met41Val. Anti-HA immunoblot analysis of HEK293T lysates expressing indicated UBA1b translation reporters. (H) Quantification of the experiment shown in panel G. Error bars denote SD, n = 10 (2 biological with 5 technical replicates each; technical replicates of each biological replicate are depicted in the same color and symbol), **P < .01; ****P < .0001, ordinary 1-way ANOVA. Shaded area represents minimum threshold below p.Met41Val expression. SD, standard deviation.

Residual UBA1b translation defines a threshold for VEXAS syndrome pathogenesis. (A) Schematic overview of the translation reporter system used to determine UBA1b translation. The first cistron of these reporters is translated in a CAP-dependent mechanism and encodes for C-terminally HA-tagged UBA1 constructs varied in p.Met41 and mutated at p.Met1 and p.Met67 (to suppress UBA1a and UBA1c translation and thus allowing for sensitive quantification of UBA1b by anti-HA immunoblotting). The second cistron is translated using an IRES and encodes for a C-terminally HA-tagged MBP, which is used for normalization across different p.Met41 variants. (B) p.Met41Val supports less UBA1b translation than p.Met41Thr and p.Met41Leu. Anti-HA immunoblot analysis of HEK293T lysates expressing indicated UBA1b translation reporters. (C) Quantification of the experiment shown in panel B. Error bars denote SD, n = 12 (4 biological with 3 technical replicates each; technical replicates of each biological replicate are depicted in the same color and symbol), **P < .01; ****P < .0001, ordinary 1-way ANOVA. (D) The canonical VEXAS variants (p.Met41Val/Thr/Leu) support more UBA1b translation than any of the other possible single-nucleotide variants at the Met41 start codon. Anti-HA immunoblot analysis of HEK293T lysates expressing indicated UBA1b translation reporters. (E) Quantification of the experiment shown in panel D. Error bars denote SD, n = 4 (2 biological with 2 technical replicates each; technical replicates of each biological replicate are depicted in the same color and symbol), *P < .05; ****P < .0001, ordinary 1-way ANOVA. (F) Two novel VEXAS variants (c.121 A>T; p.Met41Leu and c.119 G>C; p.Gly40Ala) are present in a patient in cis on the same allele. Overview of the genome sequencing reads of the p.Met41LeuTTG/p.Gly40Ala patient. (G) The p.Gly40Ala variant rescues residual UBA1b translation of the p.Met41LeuTTG to the level of the canonical VEXAS variant p.Met41Val. Anti-HA immunoblot analysis of HEK293T lysates expressing indicated UBA1b translation reporters. (H) Quantification of the experiment shown in panel G. Error bars denote SD, n = 10 (2 biological with 5 technical replicates each; technical replicates of each biological replicate are depicted in the same color and symbol), **P < .01; ****P < .0001, ordinary 1-way ANOVA. Shaded area represents minimum threshold below p.Met41Val expression. SD, standard deviation.

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