Siobán B. Keel

A heme export protein is required for red blood cell differentiation and iron homeostasis

Hemoproteins are critical for the function and integrity of aerobic cells. However, free heme is toxic. Therefore, cells must balance heme synthesis with its use. We previously demonstrated that the feline leukemia virus, subgroup C, receptor (FLVCR) exports cytoplasmic heme. Here, we show that FLVCR-null mice lack definitive erythropoiesis, have craniofacial and limb deformities resembling those of patients with Diamond-Blackfan anemia, and die in midgestation. Mice with FLVCR that is deleted neonatally develop a severe macrocytic anemia with proerythroblast maturation arrest, which suggests that erythroid precursors export excess heme to ensure survival. We further demonstrate that FLVCR mediates heme export from macrophages that ingest senescent red cells and regulates hepatic iron. Thus, the trafficking of heme, and not just elemental iron, facilitates erythropoiesis and systemic iron balance.

Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy

We report germline missense mutations in ETV6 segregating with the dominant transmission of thrombocytopenia and hematologic malignancy in three unrelated kindreds, defining a new hereditary syndrome featuring thrombocytopenia with susceptibility to diverse hematologic neoplasms. Two variants, p.Arg369Gln and p.Arg399Cys, reside in the highly conserved ETS DNA-binding domain. The third variant, p.Pro214Leu, lies within the internal linker domain, which regulates DNA binding. These three amino acid sites correspond to hotspots for recurrent somatic mutation in malignancies. Functional studies show that the mutations abrogate DNA binding, alter subcellular localization, decrease transcriptional repression in a dominant-negative fashion and impair hematopoiesis. These familial genetic studies identify a central role for ETV6 in hematopoiesis and malignant transformation. The identification of germline predisposition to cytopenias and cancer informs the diagnosis and medical management of at-risk individuals.

Genomic analysis of bone marrow failure and myelodysplastic syndromes reveals phenotypic and diagnostic complexity

Accurate and timely diagnosis of inherited bone marrow failure and inherited myelodysplastic syndromes is essential to guide clinical management. Distinguishing inherited from acquired bone marrow failure/myelodysplastic syndrome poses a significant clinical challenge. At present, diagnostic genetic testing for inherited bone marrow failure/myelodysplastic syndrome is performed gene-by-gene, guided by clinical and laboratory evaluation. We hypothesized that standard clinically-directed genetic testing misses patients with cryptic or atypical presentations of inherited bone marrow failure/myelodysplastic syndrome. In order to screen simultaneously for mutations of all classes in bone marrow failure/myelodysplastic syndrome genes, we developed and validated a panel of 85 genes for targeted capture and multiplexed massively parallel sequencing. In patients with clinical diagnoses of Fanconi anemia, genomic analysis resolved subtype assignment, including those of patients with inconclusive complementation test results. Eight out of 71 patients with idiopathic bone marrow failure or myelodysplastic syndrome were found to harbor damaging germline mutations in GATA2, RUNX1, DKC1, or LIG4. All 8 of these patients lacked classical clinical stigmata or laboratory findings of these syndromes and only 4 had a family history suggestive of inherited disease. These results reflect the extensive genetic heterogeneity and phenotypic complexity of bone marrow failure/myelodysplastic syndrome phenotypes. This study supports the integration of broad unbiased genetic screening into the diagnostic workup of children and young adults with bone marrow failure and myelodysplastic syndromes.

Novel germ line DDX41 mutations define families with a lower age of MDS/AML onset and lymphoid malignancies

Recently our group and others have identified DDX41 mutations both as germ line and acquired somatic mutations in families with multiple cases of late onset myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML), suggesting that DDX41 acts as a tumor suppressor. To determine whether novel DDX41 mutations could be identified in families with additional types of hematologic malignancies, our group screened two cohorts of families with a diverse range of hematologic malignancy subtypes. Among 289 families, we identified nine (3%) with DDX41 mutations. As previously observed, MDS and AML were the most common malignancies, often of the erythroblastic subtype, and 1 family displayed early-onset follicular lymphoma. Five novel mutations were identified, including missense mutations within important functional domains and start-loss and splicing mutations predicted to result in truncated proteins. We also show that most asymptomatic mutation carriers have normal blood counts until malignancy develops. This study expands both the mutation and phenotypic spectra observed in families with germ line DDX41 mutations. With an increasing number of both inherited and acquired mutations in this gene being identified, further study of how DDX41 disruption leads to hematologic malignancies is critical.

The Microcytic Red Cell and the Anemia of Inflammation

A recent description of an alternative messenger RNA transcript of the iron-export protein ferroportin opens the door to understanding how duodenal cells maintain dietary-iron absorption when the body is iron deficient and how mean corpuscular volume is regulated in the contexts of iron deficiency and inflammatory disorders.

Genetic features of myelodysplastic syndrome and aplastic anemia in pediatric and young adult patients

The clinical and histopathological distinctions between inherited versus acquired bone marrow failure and myelodysplastic syndromes are challenging. The identification of inherited bone marrow failure/myelodysplastic syndromes is critical to inform appropriate clinical management. To investigate whether a subset of pediatric and young adults undergoing transplant for aplastic anemia or myelodysplastic syndrome have germline mutations in bone marrow failure/myelodysplastic syndrome genes, we performed a targeted genetic screen of samples obtained between 1990–2012 from children and young adults with aplastic anemia or myelodysplastic syndrome transplanted at the Fred Hutchinson Cancer Research Center. Mutations in inherited bone marrow failure/myelodysplastic syndrome genes were found in 5.1% (5/98) of aplastic anemia patients and 13.6% (15/110) of myelodysplastic syndrome patients. While the majority of mutations were constitutional, a RUNX1 mutation present in the peripheral blood at a 51% variant allele fraction was confirmed to be somatically acquired in one myelodysplastic syndrome patient. This highlights the importance of distinguishing germline versus somatic mutations by sequencing DNA from a second tissue or from parents. Pathological mutations were present in DKC1, MPL, and TP53 among the aplastic anemia cohort, and in FANCA, GATA2, MPL, RTEL1, RUNX1, SBDS, TERT, TINF2, and TP53 among the myelodysplastic syndrome cohort. Family history or physical examination failed to reliably predict the presence of germline mutations. This study shows that while any single specific bone marrow failure/myelodysplastic syndrome genetic disorder is rare, screening for these disorders in aggregate identifies a significant subset of patients with inherited bone marrow failure/myelodysplastic syndrome.

Novel mechanisms of PIEZO1 dysfunction in hereditary xerocytosis

Mutations in PIEZO1 are the primary cause of hereditary xerocytosis, a clinically heterogeneous, dominantly inherited disorder of erythrocyte dehydration. We used next-generation sequencing-based techniques to identify PIEZO1 mutations in individuals from 9 kindreds referred with suspected hereditary xerocytosis (HX) and/or undiagnosed congenital hemolytic anemia. Mutations were primarily found in the highly conserved, COOH-terminal pore-region domain. Several mutations were novel and demonstrated ethnic specificity. We characterized these mutations using genomic-, bioinformatic-, cell biology-, and physiology-based functional assays. For these studies, we created a novel, cell-based in vivo system for study of wild-type and variant PIEZO1 membrane protein expression, trafficking, and electrophysiology in a rigorous manner. Previous reports have indicated HX-associated PIEZO1 variants exhibit a partial gain-of-function phenotype with generation of mechanically activated currents that inactivate more slowly than wild type, indicating that increased cation permeability may lead to dehydration of PIEZO1-mutant HX erythrocytes. In addition to delayed channel inactivation, we found additional alterations in mutant PIEZO1 channel kinetics, differences in response to osmotic stress, and altered membrane protein trafficking, predicting variant alleles that worsen or ameliorate erythrocyte hydration. These results extend the genetic heterogeneity observed in HX and indicate that various pathophysiologic mechanisms contribute to the HX phenotype.

Coordinate expression of heme and globin is essential for effective erythropoiesis.

Erythropoiesis requires rapid and extensive hemoglobin production. Heme activates globin transcription and translation; therefore, heme synthesis must precede globin synthesis. As free heme is a potent inducer of oxidative damage, its levels within cellular compartments require stringent regulation. Mice lacking the heme exporter FLVCR1 have a severe macrocytic anemia; however, the mechanisms that underlie erythropoiesis dysfunction in these animals are unclear. Here, we determined that erythropoiesis failure occurs in these animals at the CFU-E/proerythroblast stage, a point at which the transferrin receptor (CD71) is upregulated, iron is imported, and heme is synthesized--before ample globin is produced. From the CFU-E/proerythroblast (CD71(+) Ter119(-) cells) stage onward, erythroid progenitors exhibited excess heme content, increased cytoplasmic ROS, and increased apoptosis. Reducing heme synthesis in FLVCR1-defient animals via genetic and biochemical approaches improved the anemia, implying that heme excess causes, and is not just associated with, the erythroid marrow failure. Expression of the cell surface FLVCR1 isoform, but not the mitochondrial FLVCR1 isoform, restored normal rbc production, demonstrating that cellular heme export is essential. Together, these studies provide insight into how heme is regulated to allow effective erythropoiesis, show that erythropoiesis fails when heme is excessive, and emphasize the importance of evaluating Ter119(-) erythroid cells when studying erythroid marrow failure in murine models.

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

When heme synthesis exceeds globin synthesis, red blood cell differentiation fails, resulting in severe anemia. Putting the heme in anemia Diamond Blackfan anemia (DBA) and myelodysplastic syndrome (MDS) are both associated with impaired ribosome assembly; however, how the ribosomal defect connects to anemia remains unknown. Yang et al. report that ribosomal deficiencies in individuals with either DBA or Del(5q) MDS lead to insufficient globin protein synthesis but normal heme synthesis, which results in excess heme and reactive oxygen species in early erythroid precursors and proerythroblast cell death. Treating affected cells with an inhibitor of heme synthesis improves erythroid cell output in cell culture. Thus, blocking heme synthesis may serve as a therapeutic strategy in individuals with DBA or Del(5q) MDS. Diamond Blackfan anemia (DBA) and myelodysplastic syndrome (MDS) with isolated del(5q) are severe macrocytic anemias; although both are associated with impaired ribosome assembly, why the anemia occurs is not known. We cultured marrow cells from DBA (n = 3) and del(5q) MDS (n = 6) patients and determined how heme (a toxic chemical) and globin (a protein) are coordinated. We show that globin translation initiates slowly, whereas heme synthesis proceeds normally. This results in insufficient globin protein, excess heme and excess reactive oxygen species in early erythroid precursors, and CFU-E (colony-forming unit–erythroid)/proerythroblast cell death. The cells that can more rapidly and effectively export heme or can slow heme synthesis preferentially survive and appropriately mature. Consistent with these observations, treatment with 10 μM succinylacetone, a specific inhibitor of heme synthesis, improved the erythroid cell output of DBA and del(5q) MDS marrow cultures by 68 to 95% (P = 0.03 to 0.05), whereas the erythroid cell output of concurrent control marrow cultures decreased by 4 to 13%. Our studies demonstrate that erythropoiesis fails when heme exceeds globin. Our data further suggest that therapies that decrease heme synthesis (or facilitate heme export) could improve the red blood cell production of persons with DBA, del(5q) MDS, and perhaps other macrocytic anemias.

Evidence that the expression of transferrin receptor 1 on erythroid marrow cells mediates hepcidin suppression in the liver.

Hepcidin is the key regulator of iron absorption and recycling, and its expression is suppressed by red blood cell production. When erythropoiesis is expanded, hepcidin expression decreases. To gain insight into the stage of erythroid differentiation at which the regulation might originate, we measured serum hepcidin levels in archived pure red cell aplasia samples from patients whose block in erythroid differentiation was well defined by hematopoietic colony assays and marrow morphologic review. Hepcidin values are high or high normal in pure red cell aplasia patients in whom erythropoiesis is inhibited prior to the proerythroblast stage, but are suppressed in patients with excess proerythroblasts and few later erythroid cells. These data suggest that the suppressive effect of erythropoietic activity on hepcidin expression can arise from proerythroblasts, the stage at which transferrin receptor 1 expression peaks, prompting the hypothesis that transferrin receptor 1 expression on erythroid precursors is a proximal mediator of the erythroid regulator of hepcidin expression. Our characterization of erythropoiesis, iron status, and hepcidin expression in mice with global or hematopoietic cell-specific haploinsufficiency of transferrin receptor 1 provides initial supporting data for this model. The regulation appears independent of erythroferrone and growth differentiation factor 15, supporting the concept that several mechanisms signal iron need in response to an expanded erythron.