Research ArticleGenetics

Delayed globin synthesis leads to excess heme and the macrocytic anemia of Diamond Blackfan anemia and del(5q) myelodysplastic syndrome

See allHide authors and affiliations

Science Translational Medicine  11 May 2016:
Vol. 8, Issue 338, pp. 338ra67
DOI: 10.1126/scitranslmed.aaf3006
  • Fig. 1. Heme and globin during normal erythropoiesis, hypothesis, and DBA patient 1’s marrow aspirate.

    (A) Heme and globin synthesis during normal erythropoiesis. When erythroid progenitor cells mature, cell surface proteins are sequentially expressed, beginning with the erythropoietin receptor (green). The presence or absence of these markers can thus be used to determine the stage of differentiation (see text for details). CFU-E and early proerythroblasts up-regulate TfR expression (CD71; orange), allowing the uptake of transferrin-bound iron. CD36 (yellow) is also up-regulated at this stage (12, 13), whereas glycophorin A (GlyA; pink) is expressed later in red blood cell maturation. Iron induces heme synthesis (red arrow) because the 5′ untranslated region (5′UTR) of ALAS2, the initial and rate-limiting step of the heme synthetic pathway, contains an iron-responsive element (IRE). Heme then induces globin transcription and translation by binding the inhibitors BACH1 (15, 16) and eIF2α kinase (17), respectively, so as soon as heme is present, globin synthesis (blue arrow) begins. FLVCR serves as a safety valve to export excess heme and protect erythroid cells from heme toxicities during the period of time (gray bar) that heme synthesis is robust, but globin levels are low (20, 21). Hypothesis: In DBA and del(5q) MDS, globin synthesis initiates slowly because of insufficient ribosome availability or function, whereas heme synthesis proceeds normally. The capacity of FLVCR to export heme from early erythroid precursors is exceeded. This results in toxic quantities of intracellular free heme, CFU-E/proerythroblast cell death, a low reticulocyte count, and a severe macrocytic anemia. IRPs, iron regulatory proteins. (B) Flow cytometric analysis of a marrow aspirate from DBA patient 1. Progenitors were identified by intermediate CD45 expression and low side scatter. There is a relative expansion of CD34+/CD38 low progenitors (61% of bright CD34+; red) that lack immunophenotypic evidence of lineage commitment (top arrow) in comparison to normal marrow (24% of bright CD34+; red). In addition, erythroid precursors (high CD71; blue) are almost completely absent and the few present are arrested at the proerythroblast stage [still express CD117 (c-kit)] (bottom arrow) in comparison to normal marrow where most CD71-positive cells lack CD117 expression. These data suggest that DBA cells die at or just before the proerythroblast stage when they first express CD71 (the TfR), import iron, and begin heme synthesis. APC, allophycocyanin; PE, phycoerythrin.

  • Fig. 2. Representative studies of marrow cells at culture day 10.

    Marrow culture studies confirm that DBA erythropoiesis fails by the early proerythroblast stage. (A) As shown in this study of DBA patient 2 and a concurrent normal control, there is a relative increase (25.3% versus 12.1%) in the percentage of DBA versus normal cells in stage I and a relative decrease in later stages (III and IV). (B) Similarly, the absolute numbers of DBA cells fall significantly between stages I and II. Although the number of DBA cells in stage II is low, these numbers progressively increase in stages III and IV, suggesting that those few DBA cells, which reach stage II, can expand in number and mature to stages III and IV. Flow percentages were multiplied by the total number of cells in culture and then expressed as that number of cells derived from 2 × 105 bone marrow mononuclear cells (BMMNCs) placed in culture on day 0. Similar patterns were seen in cultures of marrow cells from the two other DBA patients and the six del(5q) MDS patients. In DBA, globin synthesis initiates slowly and heme accumulates. (C) Heme synthesis begins normally as indicated by the levels of ALAS2, the first and rate-limiting step in heme synthesis. (D) However, there is substantially more heme in stage I DBA cells than in stage I control cells (4.01 ± 0.11 versus 2.78 ± 0.02 ng per 104 cells) in this study of DBA patient 1. (E) Although the amount of heme in her stage I cells is high, the amount of globin protein is sufficiently low that it is not detectable by Western blot. (F and G) Her α-globin and β-globin mRNAs are also low; however, they are 35 to 65% of control values and are thus not as low as globin protein. α-Globin and β-globin mRNA levels were comparably decreased in stage I to IV cells from the two other DBA patients and three del(5q) MDS patients at culture day 10, but quantities of marrow cells were not sufficient for Western blot analyses.

  • Fig. 3. Cumulative data from DBA and del(5q) MDS day 10 marrow cultures.

    Erythroid differentiation fails from heme toxicity. (A) In cumulative studies, DBA patients’ (n = 3) erythroid cells expand poorly (n = 3; P = 0.009, t test), and cell death occurs at or before stage II as shown in Fig. 2B. (B) Stage I cells contain increased heme (n = 3; P = 0.036, t test). (C) The DBA cells in stages III and IV express significantly lower ALAS2 than stage III and IV control cells. In contrast, stage I DBA cells express equal amounts of ALAS2 as control cells (2.53 ± 0.72 versus 3.61 × 104 ± 0.93 × 104 copies; n = 3; P = 0.29, t test). (D) Similarly, the DBA cells in stages III and IV express significantly higher FLVCR than stage III and IV control cells. In contrast, stage I DBA cells express equal amounts of FLVCR mRNA as control cells (3.67 ± 0.10 versus 2.73 × 103 ± 0.57 × 103 copies; n = 3; P = 0.29, t test). These data suggest that stage I DBA cells able to down-regulate their heme content by decreasing heme synthesis (lower ALAS2) or increasing heme export (higher FLVCR) survive, whereas stage I cells with excessive heme die. The studies of marrow cells from five del(5q) MDS patients resemble the studies in the three DBA patients. There is poor cell expansion at culture day 10 (A), significantly lower ALAS2 mRNA in stage III and IV cells (C), and extremely high FLVCR mRNA in stage III and IV cells (D). There was also increased heme in the stage I cells of the two del(5q) MDS patients from whom sufficient marrow was available for this assay (B).

  • Fig. 4. Longitudinal studies of erythroid maturation.

    Sequential observations of marrow cultures confirm the hypothesis diagrammed in fig. S3. (A) The flow cytometric patterns of day 13 and 17 DBA patient 1’s cells are equivalent to controls, confirming that the late erythroid maturation of DBA cells proceeds normally. (B and C) ALAS2 protein and globin protein levels at culture days 3, 10, 13, and 17. Western blot analyses are shown in (B), and their quantifications are shown in (C). At culture day 3 (green arrow), the amount of ALAS2 in DBA patient 1’s cells is comparable to (actually higher than) control. However, globin production initiates slowly (second green arrow) and is much lower than control at day 10, leading to a longer than normal time interval when heme is present but globin is absent or low. We interpret the decrease in ALAS2 protein at culture days 13 and 17 to reflect the preferential survival of DBA cells with less excessive heme, given that increased cell death occurs by day 7 (see text). ALAS2, globin, and FLVCR mRNA data are shown in fig. S4A and are consistent with these findings. (D) At culture day 7, increased numbers of DBA cells and del(5q) MDS cells have cytoplasmic ROS (62.8 and 74.1%, respectively, versus 44.7% of control) and the MFI, representing the quantities of ROS per cell, is also higher (1.42× and 3.54×, respectively) than control. When the DBA patient study was repeated, the results were comparable. (E) Increased numbers of DBA cells and del(5q) MDS cells stain positively for annexin V (19.4 and 19.8%, respectively, versus 12.1% of control cells). When the DBA patient study was repeated, the results were comparable. (F) The erythroid differentiation of DBA and del(5q) MDS cells improves when heme synthesis is slowed. Marrow cells from DBA and del(5q) MDS patients and concurrent controls were grown in the presence or absence of a low concentration of succinylacetone (SA; 10 μM) (29), a potent and specific competitive inhibitor of the second step of the heme synthetic pathway. Succinylacetone significantly improved erythroid cell expansion in the DBA culture at days 7 and 10, whereas the erythroid cell expansion of control cells mildly decreased. Specifically, the DBA cell expansion at culture day 7 (expressed as number of erythroid cells derived from 2 × 105 BMMNCs placed in culture on day 0) increased 74.6% from 1.34 × 105 to 2.34 × 105 (n = 2; P = 0.04, t test). This increased 95.4% from 0.99 × 105 to 1.93 × 105 in a second independent study (n = 2; P = 0.03, t test). Erythroid cell expansion in the del(5q) MDS culture increased 67.7% from 0.6 × 105 to 1.0 × 105 (n = 2; P = 0.03, t test). (G) Treatment with hemopexin (HPX) to facilitate heme export through FLVCR. Exposure to 1.5 μM hemopexin improved the erythroid cell expansion of the DBA culture by 63.3% at day 14 (n = 2; P = 0.05, t test), whereas the erythroid cell expansion in the control culture decreased by 26.1% (n = 2; P = 0.10, t test). When the study was repeated, the results were comparable. (H to J) Sequential observations of marrow cultures from del(5q) MDS patient 6 also support our hypothesis. The late erythroid maturation of del(5q) MDS cells proceeds equivalently to the concurrent normal control (H). Western blot analyses of ALAS2 protein and globin protein levels (normalized to actin) at culture days 10, 13, and 17 are shown in (I) and their quantifications are in (J). ALAS2, globin, and FLVCR mRNA levels are shown in fig. S4B; there were insufficient numbers of cells for studies at culture day 3. The data resemble the DBA patient study (Fig. 4, A to C, and fig. S4A). Heme synthesis initiates normally, but globin synthesis is delayed.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/338/338ra67/DC1

    Patient information

    Fig. S1. The in vitro erythroid differentiation of normal human BMMNCs mimics in vivo differentiation.

    Fig. S2. Heme availability is highest in early erythroid cells.

    Fig. S3. Preferential selection during erythropoiesis.

    Fig. S4. Sequential assessment of mRNA in marrow cultures from DBA patient 1 and del(5q) MDS patient 6.

    Fig. S5. Hemopexin facilitates heme export through FLVCR to improve the erythroid differentiation of DBA patient 1’s marrow cells.

    Fig. S6. Studies of MDS patients with RARS and RCMD.

    Fig. S7. P53 activation, heme excess, and ferroptosis.

    Fig. S8. Isotype controls and gating strategy for flow cytometry.

    Fig. S9. Unmodified complete Western blots.

    Table S1. Clinical data from the MDS patients.

    Table S2. Probe and primer set for quantitative real-time PCR.

    Table S3. Source data and statistical calculations for Fig. 3.

    Table S4. Source data and statistical calculations for Fig. 4.

    Reference (48)

  • Supplementary Material for:

    Delayed globin synthesis leads to excess heme and the macrocytic anemia of Diamond Blackfan anemia and del(5q) myelodysplastic syndrome

    Zhantao Yang, Siobán B. Keel, Akiko Shimamura, Li Liu, Aaron T. Gerds, Henry Y. Li, Brent L. Wood, Bart L. Scott, Janis L. Abkowitz*

    *Corresponding author. Email: janabk{at}uw.edu

    Published 11 May 2016, Sci. Transl. Med. 8, 338ra67 (2016)
    DOI: 10.1126/scitranslmed.aaf3006

    This PDF file includes:

    • Patient information
    • Fig. S1. The in vitro erythroid differentiation of normal human BMMNCs mimics in vivo differentiation.
    • Fig. S2. Heme availability is highest in early erythroid cells.
    • Fig. S3. Preferential selection during erythropoiesis.
    • Fig. S4. Sequential assessment of mRNA in marrow cultures from DBA patient 1 and del(5q) MDS patient 6.
    • Fig. S5. Hemopexin facilitates heme export through FLVCR to improve the erythroid differentiation of DBA patient 1’s marrow cells.
    • Fig. S6. Studies of MDS patients with RARS and RCMD.
    • Fig. S7. P53 activation, heme excess, and ferroptosis.
    • Fig. S8. Isotype controls and gating strategy for flow cytometry.
    • Fig. S9. Unmodified complete Western blots.
    • Table S1. Clinical data from the MDS patients.
    • Table S2. Probe and primer set for quantitative real-time PCR.
    • Table S3. Source data and statistical calculations for Fig. 3.
    • Table S4. Source data and statistical calculations for Fig. 4.
    • Reference (48)

    [Download PDF]

Navigate This Article