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Fig. 2 Characterization of erythroid maturation defects in family 1 from cells cultured in vitro. (A) Cell growth curves during erythroid cell differentiation; 1-II-2, 1-II-4, 1-III-3, and 1-III-5 harbor the RPL17 c.217-3C>G variant; 1-II-5 is a healthy control (married-in spouse); D, day of culture. (B) Time course of CD34 and CD36 labelling. FACS analysis at D7 showed no consistent change in the percentage of BFU-e (CD34+/ CD36?) or CFU-e (CD34?/ CD36+) progenitor cells with RPL17 variants. The gradual loss of CD36 labelling from D12 to D15, indicative of terminal differentiation stages, occurred with similar kinetics in cells from both affected and unaffected individuals. Profound cell death of sample 1-III-3 prohibited study beyond D9. (C) Quantification of GPA+ cells (erythroid specific marker) by FACS from D7 to D15. (D) Erythropoietic differentiation was assessed by co-detection of ?4-integrin and Band3 in GPA+ cells. Increase in Band3 labelling, a marker of terminal erythroid cells, is paralleled by a decrease of ?4-integrin during erythroid differentiation (35). (E) Quantification of the early apoptotic marker, Annexin V from D7 to D15 shows no significant apoptosis in patient-derived cells compared with control cells.