Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach

We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases

Autophagy facilitates organelle clearance during differentiation of human erythroblasts:Evidence for a role for ATG4 paralogs during autophagosome maturation

Wholesale depletion of membrane organelles and extrusion of the nucleus are hallmarks of mammalian erythropoiesis. Using quantitative EM and fluorescence imaging we have investigated how autophagy contributes to organelle removal in an ex vivo model of human erythroid differentiation. We found that autophagy is induced at the polychromatic erythroid stage, and that autophagosomes remain abundant until enucleation. This stimulation of autophagy was concomitant with the transcriptional upregulation of many autophagy genes: of note, expression of all ATG8 mammalian paralog family members was stimulated, and increased expression of a subset of ATG4 family members (ATG4A and ATG4D) was also observed. Stable expression of dominant-negative ATG4 cysteine mutants (ATG4B(C74A); ATG4D(C144A)) did not markedly delay or accelerate differentiation of human erythroid cells; however, quantitative EM demonstrated that autophagosomes are assembled less efficiently in ATG4B(C74A)-expressing progenitor cells, and that cells expressing either mutant accumulate enlarged amphisomes that cannot be degraded. The appearance of these hybrid autophagosome/endosome structures correlated with the contraction of the lysosomal compartment, suggesting that the actions of ATG4 family members (particularly ATG4B) are required for the control of autophagosome fusion with late, degradative compartments in differentiating human erythroblasts

Induction of adult levels of β-globin in human erythroid cells that intrinsically express embryonic or fetal globin by transduction with KLF1 and BCL11A-XL

A major barrier to the clinical use of erythrocytes generated in vitro from pluripotent stem cells or cord blood progenitors is failure of these erythrocytes to express adult hemoglobin. The key regulators of globin switching KLF1 and BCL11A are absent or at a lower level than in adult cells in K562 and erythroid cells differentiated in vitro from induced pluripotent stem cells and cord blood progenitors. Transfection or transduction of K562 and cord blood erythroid cells with either KLF1 or BCL11A-XL had little effect on β-globin expression. In contrast, transduction with both transcription factors stimulated β-globin expression. Similarly, increasing the level of BCL11A-XL in the induced pluripotent stem cell-derived erythroid cell line HiDEP-1, which has levels of endogenous KLF1 similar to adult cells but lacks BCL11A, resulted in levels of β-globin equivalent to that of adult erythroid cells. Interestingly, this increase in β-globin was coincident with a decrease in ε− and ζ−, but not γ-globin, implicating BCL11A in repression of embryonic globin expression. The data show that KLF1 and BCL11A-XL together are required, but sufficient to induce adult levels of β-globin in induced pluripotent stem cell and cord blood-derived erythroid cells that intrinsically express embryonic or fetal globin

Combined immunodeficiency associated with increased apoptosis of lymphocytes and radiosensitivity of fibroblasts

Severe immunodeficiency characterized by lymphopenia was found in two siblings, one of whom was examined in detail. The calcium flux, pattern of tyrosine phosphorylation of proteins, and interleukin 2 (IL-2) production and proliferation in response to mitogens suggested that the peripheral blood T cells activated normally. The peripheral blood T cells were shown to have an activated phenotype with increased expression of CD45RO+ and CD95/Fas, Increased spontaneous apoptosis occurred in unstimulated lymphocyte cultures. The elevated apoptosis was not due to alterations in expression or to mutations in Bcl-2, Bd-X-L, or Flip, nor could the spontaneous apoptosis be prevented by blocking Fas, suggesting that it was independent of Fas signaling. This is the first inherited combined immunodeficiency associated with impaired Lymphocyte survival. Fibroblasts derived from the patient showed appreciable radiosensitivity in clonal assays, but apoptosis was not elevated. Our results show that the fibroblasts represent a new radiosensitive phenotype not associated with cell cycle checkpoint defects, V(D)J recombination defects, or elevated chromosome breakage. We suggest that the affected gene plays a role in an undetermined damage response mechanism that results in elevated spontaneous apoptosis in lymphoid cells and radiosensitivity in fibroblasts