Course of patient's disease and functional evaluation of the SLC19A1 mutation. (A) The graphs show the results of laboratory tests obtained after the patient’s first and second disease attacks and the course of treatment. The x-axis represents days from his first hospital admission (interrupted for a period between day 100 and 1150, when he was monitored in the outpatient department, and laboratory results were normal). The hemoglobin levels (g/L; red) are plotted on the left y-axis, and total bilirubin (μmol/L; blue) is plotted on the right y-axis. The serum folate levels (nmol/L; green) are plotted in the lower part of the graph, with homocysteine (μmol/L; orange) on the left y-axis and vitamin B₁₂ levels (ng/L; blue) on the right y-axis. Reference ranges are marked by bars in the corresponding colors on both axes. Treatment is indicated between both graphs, with erythrocyte transfusions marked by red arrows and the number of units given. (B) MTX uptake assay into wild-type and monoclonal gene-edited K562 model cell lines. The graph shows the amount of [3H]MTX per 1 million cells after 2-minute incubation with 0.5 μmol/L [3H]MTX. MTX uptake into the SLC19A1^KO and SLC19A1^mut cells was significantly decreased in comparison with the SLC19A1^wt. The data points were obtained from 3 independent experiments (each with 3 technical replicates) and tested by two-way analysis of variance with Bonferroni corrections; horizontal lines in the boxplots indicate medians and the 25th and 75th percentiles. (C) Sensitivity of wild-type and monoclonal gene-edited K562 model cell lines to MTX. Cell proliferation assays of the K562 cell lines with CRISPR/Cas9 introduced homozygous (SLC19A1^mut) mutation found in the patient compared with a wild-type control and to a complete SLC19A1 KO (2 representative clones carrying different frameshift mutations). The assay was performed in 3 separate experiments, each of which was performed in 6 replicates. The results are shown as the mean and standard deviation of all values, which were normalized to the appropriate controls in each experiment. ns, not significant. ***P < .001.