Daniela Maria Ribeiro

Hemoglobin: Structure, Synthesis and Oxygen Transport

Human hemoglobin (Hb) is the erythrocyte hemeprotein resulting from the combination of one pair of α-like (α or ζ) chains and another pair of β-like (β, δ, γ or e) chains. Each of these chains is associated with a heme prosthetic group, a tetrapyrrole ring (protoporphyrin IX) containing a central ferrous atom (Fe2+), which can reversibly bind to a molecule of O2, being, therefore, responsible for its transport from the lungs to the tissues. This introductory chapter summarizes these important aspects, including findings of protein structure, synthesis and function, as well as its gene organization and regulation. We also describe the developmental switches in globin chain production (from the embryonic period until hematological adult life), heme synthesis and globin gene expression/regulation, besides functional aspects of the hemoglobin molecule. The chapter also includes models that predict the mechanisms of Hb-O2 ligation, mediated by the presence of allosteric effectors, such as H+/CO2, Cl− and organic phosphates, such as 2,3-biphosphoglycerate (2,3-BPG, from erythrocyte metabolism).

The CCR5Δ32 Polymorphism in Brazilian Patients with Sickle Cell Disease

Background. Previous studies on the role of inflammation in the pathophysiology of sickle cell disease (SCD) suggested that the CCR5Δ32 allele, which is responsible for the production of truncated C-C chemokine receptor type 5 (CCR5), could confer a selective advantage on patients with SCD because it leads to a less efficient Th1 response. We determined the frequency of the CCR5Δ32 polymorphism in 795 Afro-Brazilian SCD patients followed up at the Pernambuco Hematology and Hemotherapy Center, in Northeastern Brazil, divided into a pediatric group (3 months–17 years, n = 483) and an adult group (18–70 years, n = 312). The adult patients were also compared to a healthy control group (blood donors, 18–61 years, n = 247). Methods. The CCR5/CCR5Δ32 polymorphism was determined by allele-specific PCR. Results. No homozygous patient for the CCR5Δ32 allele was detected. The frequency of heterozygotes in the study population (patients and controls) was 5.8%, in the total SCD patients 5.1%, in the children 5.4%, in the adults with SCD 4.8%, and in the adult controls 8.1%. These differences did not reach statistical significance. Conclusions. Our findings failed to demonstrate an important role of the CCR5Δ32 allele in the population sample studied here.

Influence of the polymorphisms of the alpha-major regulatory element HS-40 on in vitro gene expression

The alpha-MRE is the major regulatory element responsible for the expression of human alpha-like globin genes. It is genetically polymorphic, and six different haplotypes, named A to F, have been identified in some population groups from Europe, Africa and Asia and in native Indians from two Brazilian Indian tribes. Most of the mutations that constitute the alpha-MRE haplotypes are located in flanking sequences of binding sites for nuclear factors. To our knowledge, there are no experimental studies evaluating whether such variability may influence the alpha-MRE enhancer activity. We analyzed and compared the expression of luciferase of nine constructs containing different alpha-MRE elements as enhancers. Genomic DNA samples from controls with A (wild-type alpha-MRE) and B haplotypes were used to generate C-F haplotypes by site-directed mutagenesis. In addition, three other elements containing only the G-->A polymorphism at positions +130, +199, and +209, separately, were also tested. The different alpha-MRE elements were amplified and cloned into a plasmid containing the luciferase reporter gene and the SV40 promoter and used to transiently transfect K562 cells. A noticeable reduction in luciferase expression was observed with all constructs compared with the A haplotype. The greatest reductions occurred with the F haplotype (+96, C-->A) and the isolated polymorphism +209, both located near the SP1 protein-binding sites believed not to be active in vivo. These are the first analyses of alpha-MRE polymorphisms on gene expression and demonstrate that these single nucleotide polymorphisms, although outside the binding sites for nuclear factors, are able to influence in vitro gene expression.

A novel α0‐thalassemia deletion in a Brazilian child with Hb H disease [‐‐(Braz)]

Sir, Alpha-thalassemias are among the most common genetic diseases in the world and are characterized by impaired synthesis of the a-globin chains of hemoglobin. Deletions involving the a cluster on chromosome 16p13.3 are the main cause of this condition [1], and although the most common deletions can be detected by PCR, rare or new deletions require more elaborate techniques. Multiplex ligation-dependent probe amplification (MLPA) has been used for this purpose [2–4] and enabled a new, extensive a deletion in trans with the a deletion to be detected in a Brazilian patient with Hb H disease (--/-a). A 3-month-old Brazilian girl of mixed ethnic origin (European and African) was referred to us for investigation of microcytic and hypochromic anemia. Her parents and two sisters were also analyzed. Although they did not present with any clinical complaints, they had hematological changes suggestive of thalassemia. Hematological analysis of the proband and her 2-year-old sister revealed Hb H. The hematological and molecular data for the family studied here are shown in Table 1. A Sysmex hematology analyzer (Sysmex XE-2100, Sysmex, Kobe, Japan) was used for cell counts and hematological indices, and electrophoresis in cellulose acetate in neutral and alkaline pHs and cation-exchange high-performance liquid chromatography (HPLC) (VariantTM, Bio-Rad Laboratories, Hercules, CA, USA) were used in the hemoglobin analysis. Genomic DNA from each member of the family was extracted from peripheral blood leukocytes using a commercial kit (QIAamp DNA Blood Mini Kit, Qiagen, Hilden, Germany). Screening for the seven most common a-thalassemic deletions [-a, -a, -(a), --, --, --, --] was performed by multiplex-PCR [5], and the -a deletion was confirmed by a gap-PCR [6]; a

Differential profile of PIP4K2A expression in hematological malignancies

PIP4K2A is a lipid kinase that phosphorylates PtdIns5P, generating PtdIns4,5P2. Recently, PIP4K2A was identified as a potential target in acute myeloid leukemia cells. The objective of the present study was to investigate the PIP4K2A expression in hematological malignancies and verify the effects of PIP4K2A silencing on proliferation and survival of leukemia cell lines. PIP4K2A was found to be a cytoplasmic and nuclear protein with reduced levels in leukemia cell lines compared to normal leukocytes. PIP4K2A mRNA levels were significantly reduced in bone marrow cells from acute lymphoid leukemia (ALL) patients compared with healthy donors and in myelodysplastic syndromes (MDS) with ≥5% compared with <5% bone marrow blasts. Low PIP4K2A expression (lowest tertile versus 2 higher tertiles) negatively impacted overall survival of MDS patients by univariate analysis. PIP4K2A silencing did not modulate cell proliferation, clonogenicity and apoptosis of HEL and Namalwa leukemia cells. In summary, we characterized the expression of PIP4K2A in a cohort of patients with hematological malignancies and we found that PIP4K2A mRNA expression is downregulated in RAEB-1/RAEB-2 MDS and ALL cells, and PIP4K2A silencing does not modulate cell survival in HEL and Namalwa leukemia cells.

PIPKIIα is widely expressed in hematopoietic-derived cells and may play a role in the expression of alpha- and gamma-globins in K562 cells

Characterized for the first time in erythrocytes, phosphatidylinositol phosphate kinases (PIP kinases) belong to a family of enzymes that generate various lipid messengers and participate in several cellular processes, including gene expression regulation. Recently, the PIPKIIα gene was found to be differentially expressed in reticulocytes from two siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIα and the production of globins. Here, we investigated PIPKIIα gene and protein expression and protein localization in hematopoietic-derived cells during their differentiation, and the effects of PIPKIIα silencing on K562 cells. PIPKIIα silencing resulted in an increase in α and γ globins and a decrease in the proliferation of K562 cells without affecting cell cycle progression and apoptosis. In conclusion, using a cell line model, we showed that PIPKIIα is widely expressed in hematopoietic-derived cells, is localized in their cytoplasm and nucleus, and is upregulated during erythroid differentiation. We also showed that PIPKIIα silencing can induce α and γ globin expression and decrease cell proliferation in K562 cells.

Estudo da expressão dos genes de globinas em células HEL mediante o silenciamento do gene PIP4KIIα

Resumo Diversos estudos têm demonstrado a possível relação existente entre a expressão gênica das globinas e as fosfatidilinositol-fosfato-quinases (PIPKs), dentre as quais a PIPK da família 4 do tipo II, pertencente ao subtipo α (PIP4KIIα) se destaca por apresentar os resultados mais significativos e reprodutivos em cultura de células de pacientes com doença de hemoglobina H e de células eritroleucêmicas imortalizadas. A fim de elucidar a interação entre tais proteínas, este trabalho visou mensurar os níveis de expressão gênica e de síntese proteica das globinas α e γ em células em que o gene PIP4KIIα foi silenciado. Os resultados obtidos revelaram que a expressão gênica da globina α apresentou tendência de elevação mediante o silenciamento do gene PIP4KIIα, ocorrendo o oposto em relação à globina γ. As análises a nível protéico não confirmaram as de expressão gênica, o que evidencia a necessidade de repetição dos experimentos. No entanto, ainda assim os dados obtidos corroboram a hipótese de relação entre a expressão do gene PIP4KIIα e a dos genes das globinas α e γ, abrindo horizontes para a descoberta de novas medidas terapêuticas para hemoglobinopatias.