Inherited and Acquired Disorders of Hematopoiesis

A major focus of our laboratory has been in the study of inherited disorders of the erythrocyte, including disorders of erythrocyte shape (spherocytosis, elliptocytosis, pyropoikilocytosis, ovalocytosis, acanthocytosis) and metabolism (G6PD deficiency, disorders of glycolysis such as pyruvate kinase, hexokinase and glucose phosphate isomerase deficiency, and nucleotide metabolism). Other disorders under study include hemoglobin abnormalities (thalassemia syndromes and unstable hemoglobinopathies), congenital dyserythropoietic anemias, and marrow failure-associated disorders such as Diamond Blackfan anemia, Shwachman-Diamond syndrome, and Fanconi anemia. The genetic basis of congenital erythrocytosis/familial polycythemia is another area under study. A special emphasis of this work has focused on the manifestation of these disorders in the fetus and newborn and on genetic diagnosis of the patient with transfusion dependent anemia. These studies utilize genomic strategies including targeted and genome wide copy number analyses, and gene specific, targeted, whole exome and whole genome gene sequencing.

Determination of the precise genetic bases, structural characteristics, and functional consequences of disorders of the spectrin-based membrane skeleton in inherited erythrocyte disorders has been a major focus of the laboratory. Intensive work has centered on the disorders hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikilocytosis and Southeast Asian Ovalocytosis, a heterogeneous group of disorders associated with abnormalities of red blood cell shape, varying degrees of hemolytic anemia, and abnormal erythrocyte membranes. The laboratory has studied affected patients, performed mutation detection, and characterized the effects on gene expression and/or protein structure and function. These studies have generated important information about the genetic bases of these disorders and provided important insights into membrane protein interactions.

In hereditary spherocytosis, most mutations are heterogeneous and private, i.e. mutations are unique to a given family, whereas most cases of hereditary elliptocytosis are associated with specific spectrin mutations in persons of similar genetic backgrounds, suggesting a "founder effect" for these mutations. Mutation analyses have identified critical amino acids in alpha and beta spectrin involved in protein-protein interactions critical for spectrin structure and function and demonstrated that some of these mutations may influence previously undiscovered long-range membrane protein interactions. A very important clinical/translational observation is that abnormalities of erythrocyte spectrin can lead to recurrent fetal loss or severe neonatal anemia with hydrops fetalis. A parallel and complementary focus has been the study of the genes and candidate genes for these disorders. This includes gene cloning and mapping, characterization of gene structure and function. Finally, the neuroacanthocytoses, inherited disorders associated with abnormal erythrocyte shape, have been a focus of study in the laboratory.

Related work includes study of the genetic bases of volume regulation and ion transport in the erythrocyte in normal and disease states such as hereditary xerocytosis, stomatocytosis, hydrocytosis, and sickle cell disease. Our studies identified the primary hereditary xerocytosis gene as FAM38A encoding PIEZO1, the first report of mutations in a mammalian mechanosensory transduction channel. We have shown PIEZO1 mutations lead to hereditary xerocytosis via a number of different mechanisms including altering channel kinetics and influencing membrane trafficking of mutant PIEZO1 proteins. We and others later identified mutations in the Gardos channel, a critical contributor to dehydration in sickle cell disease, as a rare cause of a xerocytosis phenotype. We utilize physiologic, biochemical, cell biology, genetic, genomic and proteomic-based techniques to study erythrocyte hydration.

Erythrocyte dehydration is a major contributor to disease pathology in sickle cell disease. However, the control of hydration, governed by water and solute content, is poorly understood even in normal erythrocytes. Several studies indicate that the Gardos channel, a calcium-activated potassium channel, and the KCl cotransporters are candidate modifiers of erythrocyte hydration, as are likely PIEZO1 and its regulators. The genetic heterogeneity in these genes in normal and sickle cell patients indicates they may contribute to and influence the variable disease phenotype observed in patients with sickle cell disease. Studies of these candidate modifiers may allow pharmacogenomic approaches to identify and treat sickle cell patients with strong dehydration-associated phenotypes by manipulation of channel activity and erythrocyte hydration.

The mature erythrocyte must maintain adequate supplies of ATP, it must produce reducing substances to act as antioxidants, and it must control oxygen affinity of hemoglobin by producing 2,3-diphosphoglycerate (2,3-DPG). Because the mature erythrocyte does not have the ability to perform oxidative phosphorylation, its energy source is supplied by anaerobic glycolysis primarily via the Embden-Meyerhof pathway, by oxidative glycolysis through the HMP shunt, and through nucleotide salvage pathways. Inherited abnormalities of key enzymes and regulatory proteins involved in erythrocyte metabolism may lead to hemolytic anemia with erythrocyte susceptibility to oxidative stress. In some cases, symptomatology is present in other tissues such as brain and muscle. We have diagnosed numerous metabolic disorders of the erythrocyte, especially patients with pyruvate kinase deficiency, often in patients who have gone without a diagnosis for many years.

Some patients experience elevated red blood cell counts associated with increased sensitivity of erythroid progenitor cells to erythropoietin, upregulated production of erythropoietin, or both. Thus, abnormalities of the erythropoietin receptor, LNK, JAK2, VHL (Chuvash polycythemia), EHLN1, and EPAS1/HIF2A may lead to elevated red blood cell counts. Some patients experience altered hemoglobin levels due to alteration in hemoglobin oxygen affinity, alterations in bisphosphoglycerate mutase or NADH-cytochrome b5 reductase. In many patients with either inherited or acquired elevated red blood cell counts, the mechanism responsible is unknown. Molecular diagnosis and study of patients with erythrocytosis/polycythemia is a long-term focus of the laboratory.