Fig. 2

In vitro studies support variant pathogenicity. a Western blot performed on patient-derived fibroblasts from patients 4 and 5 of family 3 carrying the L78Rfs*35/R942Q VARS variant showed almost 50% reduction in VARS protein. Values are mean of three separate experiments. Error bars represent SD. b RT-qPCR on the fibroblasts showed almost complete absence of the frameshift allele at mRNA level, treatment with cycloheximide caused a partial increase in expression of the frameshift allele, which was not seen with DMSO-treated control. Values are mean of three separate experiments performed in triplicate. Error bars represent SD. c Immunocytochemistry highlighted the nucleus (Hoechst), VARS and KDEL, a marker for endoplasmatic reticulum. VARS co-localizes with KDEL. There was no difference in localization between the control line and the patient fibroblasts. d VARS and TARS aminoacylation activity measured in extracts from the patient fibroblasts. The data were normalized to ATTC fibroblasts. VARS aminoacylation activity was measured in technical triplicate at three separate passages, and TARS activity was measured once in technical triplicate. Data are represented as mean-specific activity and error bars represent SEM. * indicates significant difference from control. e A haploid yeast strain deleted for endogenous VAS1 was transformed with a LEU2-bearing pRS315 vector containing wild-type VAS1, the indicated mutant form of VAS1, or no insert (empty). Cultures for each strain (labeled along the top) were either undiluted (UD) or diluted 1:10 or 1:100 and then spotted on solid medium containing 5-FOA to determine whether the VAS1 alleles complement loss of endogenous VAS1 at 30 °C. Only G822S shows absent growth indicating a functional null allele. f VARS and TARS aminoacylation activity measured in extracts from patient-derived lymphoblasts of patients 1 and 2 (L434V/G822S) and their parents and patient 9 (R404W). Data were normalized to the paternal cells. VARS aminoacylation activity was measured in technical triplicate at three separate passages, and TARS activity was measured once in technical triplicate. Data are represented as mean-specific activity and error bars represent SEM. * indicates significant difference from L434V paternal lymphoid cells. In a, b, d, and f one-way ANOVA with Tukey’s multiple comparisons test was used. Significant values are noted **p < 0.01, ***p < 0.001, and ****p < 0.0001