Figure 1.
PUS1 p.P175fs mutation leads to abnormal erythroid differentiation. (A) Representative image of BM iron stain of the patient with MLASA. Black arrows indicate ring sideroblasts. (B) Blood routine data of the patient from 2013 to 2019. Red dotted lines define the normal ranges. (C) Pedigree tree of the patient’s family (left panel) and chromatograms of Sanger sequencing results (right panels). The patient has a homozygous PUS1 mutation (c.523delC) is indicated by blackened symbols. Her parents carrying the same but heterozygous mutation are also indicated. The mutant proline at position 175 is marked with red, and the red arrow points to the location of the missing cytosine at position 523. (D) The schematic diagram of 3-stage erythroid differentiation from iPSCs. Green line: stage of tiling iPSC colony formation; orange line: stage of hemogenic induction; and blue line: stage of erythroid differentiation. (E) Analysis of 3-stage erythroid differentiation efficiency at different stages. Representative images of iPSC colonies (i). Flow cytometry analysis of hemogenic endothelium cells (HEs) (ii) and erythroblasts (iii). Cell pellets of CD71+CD235+ cells and CD71–CD235- cells produced by HEs in vitro for 7 days (iv). (F-H) Quantification of the sizes of iPSC colonies (F; normal, n = 5; MLASA, n = 5; MLASA-Res, n = 6), the percentages of HE cells (G; CD34+CD31+, n = 3) and erythroblasts (H; CD71+CD235a+, n = 3). (I) The schematic diagram of iPSCs normoxia strategy 1. Purple line: stage of hemogenic induction; and brown line: stage of erythroid differentiation. (J-K) Quantification of the flow cytometry analysis of HE cells (J; normal, n = 4; MLASA, n = 5; MLASA-Res, n = 6), and erythroblasts (K; normal, n = 2; MLASA, n = 3; MLASA-Res, n = 3) derived from iPSCs under normoxia induction strategy 1. Values in all panels denote mean ± standard deviation (SD; ∗P < .05; ∗∗P < .01); 1-way analysis of variance (ANOVA). ERY, erythropoiesis; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; PLT, platelet; RDW-CV, RBC volume distribution width-coefficient of variation; WBC, white blood cell.

PUS1 p.P175fs mutation leads to abnormal erythroid differentiation. (A) Representative image of BM iron stain of the patient with MLASA. Black arrows indicate ring sideroblasts. (B) Blood routine data of the patient from 2013 to 2019. Red dotted lines define the normal ranges. (C) Pedigree tree of the patient’s family (left panel) and chromatograms of Sanger sequencing results (right panels). The patient has a homozygous PUS1 mutation (c.523delC) is indicated by blackened symbols. Her parents carrying the same but heterozygous mutation are also indicated. The mutant proline at position 175 is marked with red, and the red arrow points to the location of the missing cytosine at position 523. (D) The schematic diagram of 3-stage erythroid differentiation from iPSCs. Green line: stage of tiling iPSC colony formation; orange line: stage of hemogenic induction; and blue line: stage of erythroid differentiation. (E) Analysis of 3-stage erythroid differentiation efficiency at different stages. Representative images of iPSC colonies (i). Flow cytometry analysis of hemogenic endothelium cells (HEs) (ii) and erythroblasts (iii). Cell pellets of CD71+CD235+ cells and CD71–CD235- cells produced by HEs in vitro for 7 days (iv). (F-H) Quantification of the sizes of iPSC colonies (F; normal, n = 5; MLASA, n = 5; MLASA-Res, n = 6), the percentages of HE cells (G; CD34+CD31+, n = 3) and erythroblasts (H; CD71+CD235a+, n = 3). (I) The schematic diagram of iPSCs normoxia strategy 1. Purple line: stage of hemogenic induction; and brown line: stage of erythroid differentiation. (J-K) Quantification of the flow cytometry analysis of HE cells (J; normal, n = 4; MLASA, n = 5; MLASA-Res, n = 6), and erythroblasts (K; normal, n = 2; MLASA, n = 3; MLASA-Res, n = 3) derived from iPSCs under normoxia induction strategy 1. Values in all panels denote mean ± standard deviation (SD; ∗P < .05; ∗∗P < .01); 1-way analysis of variance (ANOVA). ERY, erythropoiesis; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; PLT, platelet; RDW-CV, RBC volume distribution width-coefficient of variation; WBC, white blood cell.

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