Figure 2.
Bitopertin improves the erythroid cell maturation of marrow from patients with DBA. (A-B) Representative flow cytometry analysis of marrow mononuclear cells from a patient with DBA (D1) and a control subject (N1), cultured without or with 10 nM bitopertin after 10 days of culture. Lin– (lineage negative: CD3−, CD11b−, CD19−) cells from each culture were plotted as CD36 × GlyA (A) or CD71 × GlyA (B), showing the percentage of lin− cells in each quadrant. The largest improvement in the DBA marrow cultures is seen in the earliest precursors (CD36 or CD71 single positive), which are circled. (C-D) A total of 200 000 marrow mononuclear cells from control subjects or patients with DBA with mutations in RPS19, RPS26, or RPL11 were cultured for 7 to 13 days without or with bitopertin. The total number of erythroid cells in each culture was calculated from the number of cells in the highlighted quadrants using either CD36 × GlyA or CD71 × GlyA (see panels A-B). The day shown for each DBA sample is the day that each DBA sample showed peak improvement with bitopertin treatment, which was different for each patient’s marrow culture. Data are presented as the percentage of untreated which was calculated by dividing the numbers of erythroid cells recovered from the bitopertin treated cultures by the number of cells recovered from the untreated cultures individually for each control and patient culture (C) or as the mean ± standard error of the mean of all 4 patient and control cultures (D) at the day that each patient culture showed peak improvement. P values are shown for the comparison of the treated vs untreated (100%). (E) Relative heme content of bone marrow cultures from control subjects or patients with DBA after 7 days of culture without or with bitopertin. Samples were collected and analyzed for total heme content as before.4,11 Data are presented as the mean ± standard error of the mean (of 3 independent experiments) of the treated samples relative to the heme content of the untreated samples. (F) Relative amounts of globin protein in marrow cultures from control subjects or patients with DBA after 7 days of culture without or with bitopertin (left). Total protein was isolated from samples and analyzed via western blot (right) as before.4 Fluorescent images of the blots were captured on a Bio-Rad ChemiDoc MP and processed in Image Lab. Data are presented as a percentage of globin present in the untreated control sample after correcting for loading using actin (n = 2 independent experiments). ∗P < .05; ∗∗P < .01; ns, P > .05. ns, not significant.

Bitopertin improves the erythroid cell maturation of marrow from patients with DBA. (A-B) Representative flow cytometry analysis of marrow mononuclear cells from a patient with DBA (D1) and a control subject (N1), cultured without or with 10 nM bitopertin after 10 days of culture. Lin– (lineage negative: CD3−, CD11b−, CD19−) cells from each culture were plotted as CD36 × GlyA (A) or CD71 × GlyA (B), showing the percentage of lin− cells in each quadrant. The largest improvement in the DBA marrow cultures is seen in the earliest precursors (CD36 or CD71 single positive), which are circled. (C-D) A total of 200 000 marrow mononuclear cells from control subjects or patients with DBA with mutations in RPS19, RPS26, or RPL11 were cultured for 7 to 13 days without or with bitopertin. The total number of erythroid cells in each culture was calculated from the number of cells in the highlighted quadrants using either CD36 × GlyA or CD71 × GlyA (see panels A-B). The day shown for each DBA sample is the day that each DBA sample showed peak improvement with bitopertin treatment, which was different for each patient’s marrow culture. Data are presented as the percentage of untreated which was calculated by dividing the numbers of erythroid cells recovered from the bitopertin treated cultures by the number of cells recovered from the untreated cultures individually for each control and patient culture (C) or as the mean ± standard error of the mean of all 4 patient and control cultures (D) at the day that each patient culture showed peak improvement. P values are shown for the comparison of the treated vs untreated (100%). (E) Relative heme content of bone marrow cultures from control subjects or patients with DBA after 7 days of culture without or with bitopertin. Samples were collected and analyzed for total heme content as before.4,11 Data are presented as the mean ± standard error of the mean (of 3 independent experiments) of the treated samples relative to the heme content of the untreated samples. (F) Relative amounts of globin protein in marrow cultures from control subjects or patients with DBA after 7 days of culture without or with bitopertin (left). Total protein was isolated from samples and analyzed via western blot (right) as before.4 Fluorescent images of the blots were captured on a Bio-Rad ChemiDoc MP and processed in Image Lab. Data are presented as a percentage of globin present in the untreated control sample after correcting for loading using actin (n = 2 independent experiments). ∗P < .05; ∗∗P < .01; ns, P > .05. ns, not significant.

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