Tânia Regina Zaccariotto

Polymorphism of human haptoglobin and its clinical importance

Haptoglobin (Hp) is a plasma glycoprotein, the main biological function of which is to bind free hemoglobin (Hb) and prevent the loss of iron and subsequent kidney damage following intravascular hemolysis. Haptoglobin is also a positive acute-phase protein with immunomodulatory properties. In humans, the HP locus is polymorphic, with two codominant alleles (HP1 and HP2) that yield three distinct genotypes/phenotypes (Hp1-1, Hp2-1 and Hp2-2). The corresponding proteins have structural and functional differences that may influence the susceptibility and/or outcome in several diseases. This article summarizes the available data on the structure and functions of Hp and the possible effects of Hp polymorphism in a number of important human disorders.

Genótipos de haptoglobina e hipertensão refratária em pacientes com diabete melito tipo 2

BACKGROUND It has been suggested that haptoglobin polymorphism may influence the pathogenesis of microvascular and macrovascular complications in diabetic patients. OBJECTIVE This cross sectional study was carried out to investigate the existence or not of an association between haptoglobin genotypes and prevalence of ischemic cardiovascular events (stable angina, unstable angina and acute myocardial infarction), systemic arterial hypertension, refractory hypertension, obesity and dyslipidemia in 120 type-2 diabetes mellitus patients followed up at Hospital de Clínicas da UNICAMP in Campinas, São Paulo state, southeastern Brazil. METHODS Haptoglobin genotyping was performed by allele-specific polymerase chain reactions. The frequencies of the haptoglobin genotypes were compared with the presence/absence of cardiovascular disease, systemic arterial hypertension, refractory hypertension, obesity and dyslipidemia; systolic and diastolic blood pressure measurements; plasma levels of glucose, cholesterol (total, high density lipoprotein-HDL and low density lipoprotein-LDL) and triglycerides; and serum creatinine levels. RESULTS Although no association between haptoglobin genotype and the presence of cardiovascular disease could be identified, we found a significant excess of patients with Hp2-1 genotype among those with refractory hypertension, who also had higher systolic and diastolic blood pressure, and total and LDL cholesterol levels. CONCLUSION Our results suggest that type-2 diabetes mellitus patients with the Hp2-1 genotype may have higher chances of developing refractory hypertension. Further studies in other diabetic populations are required to confirm these findings.FUNDAMENTO: Tem sido sugerido que o polimorfismo da haptoglobina pode influenciar na patogenese das complicacoes microvasculares e macrovasculares em pacientes diabeticos. OBJETIVO: O objetivo principal deste estudo transversal foi de realizar uma investigacao da existencia ou nao de uma associacao entre os genotipos de haptoglobina e a prevalencia de eventos isquemicos cardiovasculares (angina estavel, angina instavel e infarto agudo do miocardio), hipertensao arterial sistemica, hipertensao refrataria, obesidade e dislipidemia em 120 pacientes com diabete melito tipo 2, seguidos no Hospital Universitario da Unicamp, em Campinas, Estado de Sao Paulo. METODOS: A genotipagem da haptoglobina foi realizada por reacoes em cadeia da polimerase alelo-especificas. As frequencias dos genotipos de haptoglobina foram comparadas com a presenca/ausencia de doenca cardiovascular, hipertensao arterial sistemica, hipertensao refrataria, obesidade e dislipidemia; medicoes de pressao arterial sistolica e diastolica; glicemia, colesterol (total, lipoproteinas de alta densidade - HDL e lipoproteinas de baixa densidade - LDL) e triglicerideos; assim como niveis de creatinina serica. RESULTADOS: Embora nenhuma associacao entre o genotipo de haptoglobina e a presenca de doenca cardiovascular tenha sido identificada, encontramos um excesso significativo de pacientes com o genotipo Hp2-1 entre as pessoas com hipertensao refrataria, que tambem apresentavam uma maior pressao arterial sistolica e diastolica e niveis de colesterol total e LDL. CONCLUSAO: Nossos resultados sugerem que os pacientes com diabete melito tipo 2 com o genotipo Hp2-1 podem apresentar uma maior chance de desenvolver hipertensao refrataria. Estudos adicionais em populacoes diabeticas sao necessarios para confirmar esses achados.

Haptoglobin polymorphism and diabetic nephropathy in Brazilian diabetic patients

Diabetic nephropathy (DN) has an important impact on morbidity/mortality in diabetic patients. Genetic factors are probably involved in the development of this microvascular complication. Haptoglobin (Hp) is a genetically polymorphic glycoprotein that forms stable complexes with plasma-free hemoglobin (Hb) providing protection against heme-induced oxidative stress and kidney damage. The aim of the present study was to investigate the existence of association between the Hp genotypes and the presence of DN in Brazilian diabetic patients. The Hp genotypes of 265 patients, 95 type 1 diabetes mellitus (DM1) sufferers with at least 10 years of disease and 170 type 2 diabetes mellitus (DM2) sufferers with at least 5 years of disease were determined by allele-specific PCR; both groups included patients with and without DN. Hp allele and genotype frequencies were compared among the patient groups and between the patient groups and a control group of 142 healthy individuals. No association between Hp genotypes and DN could be demonstrated. Additionally, urinary albumin excretion values and the presence or absence of systemic arterial hypertension (SAH) were compared among the patient groups. Again, no significant correlations were found. The Hp polymorphism could not be associated with DN in the population studied here.

Haptoglobin polymorphism in a HIV-1 seropositive Brazilian population.

Background: Haptoglobin (Hp) is a plasma protein with antioxidant and immunomodulatory properties. Three main genotypes/phenotypes (Hp1-1, Hp2-1, Hp2-2) show distinctive efficiencies in their activities and have been related to susceptibility and outcome in different diseases, including HIV infection. Objective: To compare Hp genotype distribution between HIV-1 seropositive patients and healthy controls. Methods: 387 Brazilian HIV-1 seropositive patients, subclassified as A, B, and C according to the Centers for Disease Control, were compared with 142 healthy controls. The influence of the polymorphism on iron status (serum iron, ferritin, transferrin, transferrin saturation), acute phase proteins (Hp, C reactive protein, fibrinogen, albumin), the HIV-1 viral load, and CD4+ T lymphocyte counts was examined. Results: Apart from finding lower Hp concentrations among individuals with genotype Hp2-2, no other significant difference was observed. Conclusions: No association was found between Hp genotype and either HIV status or indices of HIV progression.

Haptoglobin Genotypes in Sickle-Cell Disease

We compared the frequencies of the haptoglobin (Hp) genotypes of 775 Brazilian patients with sickle-cell disease divided into the following age groups: 3 months-5 years, 6-10 years, 11-15 years, 16-20 years, and over 20 years. The last group (>20 years) was also compared with a healthy control group and was further divided into subgroups including only subjects aged 21-30 years (V.a and Control.a) and over 30 years (V.b and Control.b). There was no significant difference in the frequencies of the Hp genotypes between the different patient groups or between the patients and controls. However, the Hp2-2 genotype was always less frequent than the Hp1-1 genotype in the patient groups, whereas the opposite was observed in healthy controls. The frequency of Hp2-2 was 25.0% in patients in the 21-30 years age group and fell to 19.5% in those over 30 years. In the controls, the corresponding frequency was around 28%. Although our results do not allow us to conclude that Hp genotypes on their own confer greater or lesser selective advantage on sickle-cell disease patients in the population studied, this polymorphism may, when combined with other genetic and environmental factors, contribute to the clinical diversity observed in this disease.

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.

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.