Mutations in the iron-sulfur cluster biogenesis protein HSCB cause congenital sideroblastic anemia
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
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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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 2

HSCB depletion decreases Fe-S protein expression and impairs respiration.

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HSCB depletion decreases Fe-S protein expression and impairs respiration...
(A–D) Western blots of Fe-S cluster biogenesis, Fe-S, and lipoylated proteins (A) and respiratory proteins (B) in K562 erythroleukemia cells 10 days after control and HSCB-specific shRNA infection. Three independent experiments are shown. Western blots in A and B are quantified in C and D, respectively. Scrambled control (shSC) and HSCB‑specific shRNAs 1 (sh1) and 2 (sh2) are indicated by black, red, and blue, respectively. NI, noninfected. For all panels, mean expression ± SD is normalized to NI cells. ANOVA, *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. shSC. (E–G) Seahorse Extracellular Flux (XF) Analyzer Cell Energy Phenotype analyses of shRNA-treated K562 cells. Data presented are representative of at least 3 independent experiments. NI and shSC-treated K562 cells are indicated by black points with long-dashed and short-dashed lines, respectively. sh1- and sh2-treated K562 cells are indicated by red and blue, respectively. (E) Cell Mito Stress Test energy maps of basal (left points) and stressed (right points) oxygen consumption versus extracellular acidification, which is a measure of glycolysis. (F) Seahorse extracellular flux analysis. Oligomycin (O), carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), and rotenone/antimycin A (R/A) allow assessment of basal ATP production, maximal respiration, and non-mitochondrial respiration, respectively. (G) Basal respiration and residual respiratory capacity. Activity normalized to NI cells. ANOVA, ****P < 0.001 vs. shSC. ACO2, aconitase 2; ATP5A, ATP synthase F1 subunit α; COXII, mitochondrially encoded cytochrome c oxidase II; CS, citrate synthase; FECH, ferrochelatase; HSPA9, heat shock protein family A (Hsp70) member 9; KGDH, α-ketoglutarate dehydrogenase; MTCYB, mitochondrially encoded cytochrome b; NDUFB8, NADH:ubiquinone oxidoreductase subunit B8; PDH, pyruvate dehydrogenase; RC, respiratory complex; SHDA and SDHB, succinate dehydrogenase A and B subunits; UQCRC2, ubiquinol–cytochrome c reductase core protein 2. E2 subunits of PDH and KGDH were detected with an anti–lipoic acid antibody.