Mutations in the iron-sulfur cluster biogenesis protein HSCB cause congenital sideroblastic anemia
Andrew Crispin, … , Mark D. Fleming, Sarah Ducamp
Andrew Crispin, … , Mark D. Fleming, Sarah Ducamp
Published July 7, 2020
Citation Information: J Clin Invest. 2020;130(10):5245-5256. https://doi.org/10.1172/JCI135479.
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Research Article Genetics Hematology

Mutations in the iron-sulfur cluster biogenesis protein HSCB cause congenital sideroblastic anemia

Abstract

The congenital sideroblastic anemias (CSAs) can be caused by primary defects in mitochondrial iron-sulfur (Fe-S) cluster biogenesis. HSCB (heat shock cognate B), which encodes a mitochondrial cochaperone, also known as HSC20 (heat shock cognate protein 20), is the partner of mitochondrial heat shock protein A9 (HSPA9). Together with glutaredoxin 5 (GLRX5), HSCB and HSPA9 facilitate the transfer of nascent 2-iron, 2-sulfur clusters to recipient mitochondrial proteins. Mutations in both HSPA9 and GLRX5 have previously been associated with CSA. Therefore, we hypothesized that mutations in HSCB could also cause CSA. We screened patients with genetically undefined CSA and identified a frameshift mutation and a rare promoter variant in HSCB in a female patient with non-syndromic CSA. We found that HSCB expression was decreased in patient-derived fibroblasts and K562 erythroleukemia cells engineered to have the patient-specific promoter variant. Furthermore, gene knockdown and deletion experiments performed in K562 cells, zebrafish, and mice demonstrate that loss of HSCB results in impaired Fe-S cluster biogenesis, a defect in RBC hemoglobinization, and the development of siderocytes and more broadly perturbs hematopoiesis in vivo. These results further affirm the involvement of Fe-S cluster biogenesis in erythropoiesis and hematopoiesis and define HSCB as a CSA gene.

Authors

Andrew Crispin, Chaoshe Guo, Caiyong Chen, Dean R. Campagna, Paul J. Schmidt, Daniel Lichtenstein, Chang Cao, Anoop K. Sendamarai, Gordon J. Hildick-Smith, Nicholas C. Huston, Jeanne Boudreaux, Sylvia S. Bottomley, Matthew M. Heeney, Barry H. Paw, Mark D. Fleming, Sarah Ducamp

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Figure 1

HSCB expression is decreased by CSA patient-specific mutations.

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HSCB expression is decreased by CSA patient-specific mutations. (A) Pedi...
(A) Pedigree of the family and HSCB variants: WT allele, c.–134A>C promoter variant (P), and c.259dup/p.Thr87Asnfs*27 frameshift allele (FS). (B) Perls Prussian blue iron staining of the proband’s bone marrow demonstrating characteristic ring sideroblasts. Original magnification, ×1000. (C and D) Sequencing traces of the P and FS variants, respectively. Asterisk indicates a common promoter polymorphism. (E) Representation of the HSCB promoter for the WT or P alleles. Predicted ETS binding site and the consequential reduction in transcription caused by the G/A substitution are indicated by the gray boxes and red arrow, respectively. (F) Representative Western blots of total protein extracts from skin fibroblasts from the proband (PT) and 2 unrelated female control fibroblast lines (C1 and C2). Actin (ACT) and heat shock cognate 70 (HSC70) were used as total protein controls, whereas IMMT (mitofilin) and citrate synthase (CS) controlled for mitochondrial content. (G) Quantification of PT and control (C) fibroblast HSCB expression. n = 3, unpaired t test, ***P < 0.001. (H) Western blot of CRISPR/Cas9–edited K562 clones homozygous for the WT (G/G) or the P allele (P-MUT A/A). (I) ChIP analysis of the HSCB promoter region in K562 cells using an ETS1-specific antibody demonstrating specific binding of ETS1 to the c.–134A region. Unpaired t test, ****P < 0.0001 vs. IgG alone. (J) Luciferase promoter activity in HeLa cells transfected with a promoterless luciferase expression construct or a similar plasmid containing the HSCB promoter with either the WT (c.–134G) or P (c.–134A) variants. Data are normalized to the activity of the promoterless construct cotransfected with 5 μg of the ETS1 plasmid. ANOVA, ****P < 0.0001 vs. no promoter.