Belinda K. Singleton

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We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories.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 (ATG4BC74A; ATG4DC144A) did not markedly delay or accelerate differentiation of human erythroid cells; however, quantitative EM demonstrated that autophagosomes are assembled less efficiently in ATG4BC74A-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.

Mutations in the second zinc finger of human EKLF reduce promoter affinity but give rise to benign and disease phenotypes

Mutations in the human erythroid Krüppel-like factor (EKLF) can lead to either anemia or the benign InLu phenotype. To elucidate the relationship between these mutations and the differing phenotypes, we prepared recombinant forms of wild-type and 5 mutant EKLF proteins and quantitated their binding affinity to a range of EKLF-regulated genes. Missense mutants (R328H, R328L, and R331G) from persons with InLu phenotype did not bind DNA. Hence, as with the heterozygous loss of function nonsense (L127X, S270X, and K292X) and frameshift (P190Lfs and R319Efs) EKLF mutations, monoallelic loss of EKLF does not result in haploinsufficiency at all loci. In contrast, K332Q has a slightly reduced DNA binding affinity (∼ 2-fold) for all promoters examined but exhibits a phenotype only in a compound heterozygote with a nonfunctional allele. E325K also has a reduced, but significant, binding affinity, particularly for the β-globin gene but results in a disease phenotype even with the wild-type allele expressed, although not as a classic dominant-negative mutant. E325K protein may therefore actively interfere with EKLF-dependent processes by destabilizing transcription complexes, providing a rational explanation for the severity of the disease phenotype. Our study highlights the critical role of residues within the second EKLF zinc finger domain.

A novel GATA1 mutation (Stop414Arg) in a family with the rare X-linked blood group Lu(a-b-) phenotype and mild macrothrombocytic thrombocytopenia.

116, 140–143. Raanani, P., Trakhtenbrot, L., Rechavi, G., Rosenthal, E., Avigdor, A., Brok-Simoni, F., Leiba, M., Amariglio, N., Nagler, A. & Ben-Bassat, I. (2005) Philadelphia-chromosome-positive Tlymphoblastic leukemia: acute leukemia or chronic myelogenous leukemia blastic crisis. Acta Haematologica, 113, 181–189. Vardiman, J.W., Thiele, J., Arber, D.A., Brunning, R.D., Borowitz, M.J., Porwit, A., Harris, N.L., Le Beau, M.M., Hellstrom-Lindberg, E., Tefferi, A. & Bloomfield, C.D. (2009) The 2008 revision of the world health organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood, 114, 937–951.

Blood group phenotypes resulting from mutations in erythroid transcription factors.

Purpose of reviewThis review describes the genetics of unusual blood group phenotypes, particularly those with altered expression of Lutheran antigens, and how this area of study has informed our understanding of erythropoiesis in general and haemoglobin switching in particular. Recent findingsMutations in erythroid transcription factors GATA1 (GATA1 binding protein 1) and KLF1 (Kruppel-like factor 1) cause benign and disease phenotypes in humans [X-linked Lu(a−b−) phenotype, In(Lu) blood group phenotype, hereditary persistence of foetal haemoglobin, borderline HbA2, and congenital dyserythropoietic anaemia (CDA)]. These studies explain the occurrence of rare blood group phenotypes with simultaneous altered expression of antigens from several blood group systems and illuminate the role of KLF1 in gamma and delta globin gene regulation. SummaryThe study of rare blood group phenotypes is a potent tool for discovery of mutations in human genes. Elucidation of the molecular basis of the rare In(Lu) phenotype revealed the first mutations in human KLF1. Subsequently, numerous additional mutations have been described, one of which causes a rare form of CDA. Analysis of the X-linked Lu(a−b−) phenotype revealed a mutation in the C-terminal domain of human GATA1. The apparent sensitivity of the Lutheran glycoprotein to alterations in GATA1 and KLF1 activity suggest that it could be a useful biomarker of erythroid transcription factor mutation.

McLeod syndrome resulting from a novel XK mutation

Summary. McLeod Syndrome (MLS) is a rare X‐linked disorder characterized by haemopoietic abnormalities and late‐onset neurological and muscular defects. The McLeod blood group phenotype is typically associated with erythrocyte acanthocytosis, absence of the Kx antigen and reduced expression of Kell system antigens. MLS is caused by hemizygosity for mutations in the XK gene. We describe a patient with MLS who first showed symptoms in 1989 (aged 51 years). As the disease progressed, the patient developed a slight dementia, aggressive behaviour and choreatic movements. A cardiomyopathy was also diagnosed. An electroneuromyography showed neuropathic and myopathic changes. Liver enzymes were elevated and a blood smear showed acanthocytes. MLS was confirmed by serological analysis of the Kell antigens. Analysis of red blood cells by flow cytometry revealed the patient and his grandson to have reduced Kell antigen expression. The patients daughters had two populations of red cells, consistent with them being heterozygous for an XK0 allele. The molecular basis of MLS in this family is a novel mutation consisting of a 7453‐bp deletion that includes exon 2 of the XK gene. This confirms that the patients 7‐year‐old grandson, who is currently asymptomatic, also has the XK0 allele and is therefore likely to develop MLS.