Abstract 2079
Phosphatidylinositol phosphate kinases (PIPKs) are a family of lipid kinase enzymes that produce the second messenger PI4,5P2 (phosphatidylinositol 4,5-biphosphate), which plays an ...important role in the regulation of a variety of cellular activities, including gene expression. PIPKs are classified into 3 subfamilies — PIPK I (a, b, g), PIPK II (a, b, g) and PIPK III — which are functionally distinct and are located in different subcellular compartments. In a recent study in our laboratory, the PIPKIIa gene was differentially expressed in reticulocytes from 2 siblings with hemoglobin (Hb) H disease who had the same genotype (-a3.7/–SEA). Expression of both the PIPKIIa and b-globin genes were higher in the patient with the higher Hb H level, suggesting a possible relationship between PIPKIIa and the production of globins, particularly b-globin. In light of these findings, the aim of this study was to determine the gene expression profiles of PIPKs (I and II - with their isoforms a, b and g - and III) during erythropoiesis in peripheral blood hematopoietic CD34+ cell culture from 11 healthy volunteers and 6 patients with hemoglobinopathies 2 with a-thalassemia (Hb H disease), 2 with b-thalassemia (homozygous for the IVS-I-6-T-C mutation) and 2 with sickle cell anemia using quantitative real time PCR (qRT-PCR) and to compare these profiles with the gene expression profiles of a-, b- and g-globins on the 7th, 10th and 13th days of the erythroid culture. In the cell culture from the normal group, expression of the PIPKIIa and other PIPK genes increased during erythroid differentiation, coinciding with the expression profiles of globin genes and showing in particular that a-globin has a significant effect on PIPKIIa (p<0.0001), as the PIPKIIa on a-globin gene (p=0.0002). In the patients, the expression profile of the PIPKIIa gene also increased during differentiation, whereas the results for the other PIPK genes varied. However, mRNA levels differed between patients, indicating greater complexity in individuals with hemoglobinopathies. PIPKIIa expression level was elevated in the culture from one of the a-thalassemia patients (approximately 12 times higher than in the corresponding control) but was lower than the control in one of the b-thalassemia patients. Expression levels of this gene also varied among sickle cell patients. This is the first study of the gene expression profiles of these kinases during in vitro human erythropoiesis. We identified a standard pattern of gene expression for PIPKs, and PIPKIIa in particular, a gradual increase in expression during erythroid differentiation, similar to the pattern for globin genes. This suggests that PI4,5P2, as an important secondary messenger involved in the regulation of gene expression, may play an important role in the regulation of globin gene expression and the normal process of Hb synthesis in red blood cells. Although our results varied between patients, highlighting the complexity of the regulatory systems involved in Hb production, they reinforce the hypothesis of a relationship between PIPKIIa and globin expression. This work was supported by FAPESP, CNPq and CAPES.
No relevant conflicts of interest to declare.
Abstract 4835
Sickle cell anemia (SCA) is characterized by a chronic inflammatory state in which oxidative stress, particularly in the endothelium, exerts a strong influence on the pathogenesis of ...vaso-occlusion and may be implicated in patients' clinical heterogeneity and survival. It has been suggested that the cytokine production profile of cells involved in the immune response may vary among patients with SCA. Leg ulcers (LU) represent a severe complication in these patients, and this condition has been associated with specific end-organ damage and an increase in morbidity and mortality. Recent studies have shown that venous obstruction, endothelial dysfunction, coagulopathy and infections are implicated in the complex pathogenesis of LU.
To determine IL-1β, IL-6 and IL-8 plasma levels and gene expression rates as well as hematological and coagulation parameters and correlate these with the history of LU in adult SCA patients followed up at HEMOPE, in the state of Pernambuco, northeastern Brazil.
Peripheral blood samples from 92 patients (median age 27 years; 42 female; 52 male; all Afro-descendants) in the steady state who had been diagnosed with SCA (HbSS), had not received a transfusion and were not using hydroxyurea were analyzed. Plasma levels of cytokines were determined by ELISA, and the gene expression rates by qRT-PCR. The patients' clinical and laboratorial characteristics were obtained from their medical charts. Statistical analysis was performed using the SAS System for Windows version 9.2.
Median age was higher in patients with a history of LU than in those without a history (33.1 vs. 28.4; p = 0.04). Although no statistically significant (p = 0.5) differences in IL-8 gene expression rates were observed, IL-8 plasma levels were significantly higher in patients with a history of LU than in patients without a history (23.8 vs. 7.7; p = 0.01) (Figure 1). Thus, patients with high levels of IL-8 had an increased risk for the occurrence of leg ulcers (OR = 1.01; 95% CI = 1.00–1.02). The ROC curve showed that IL-8 levels higher than 8.55 pg/mL could indicate the presence of LU (accuracy = 71.6%; sensitivity = 73.7%; specificity = 68.5%). The laboratory tests revealed reticulocyte counts and activated partial thromboplastin time (aPTT) ratios (R) that were significantly higher in patients with a history of LU than in those without a history (11.8 vs. 8.4, p = 0.01; 1.1 vs. 0.9, p = 0.04, respectively). Both the higher reticulocyte counts and R values were associated with increased risk for the occurrence of leg ulcers in these patients (OR = 1.12, 95% CI = 1.02 – 1.20; OR = 24.28, 95% CI = 1.20 – 486.09, respectively).
In this study, patients who had had LU at some time in their lives showed significantly higher IL-8 levels, reticulocyte counts and R values than patients who had never had LU. Our results therefore suggest a relationship between the parameters described above and LU in patients with SCA. These parameters could perhaps be used, in association with different genetic modulators that may contribute to different clinical phenotypes observed in this disease, as markers of this clinical manifestation of SCA or of a propensity to develop it. Display omitted
Financial Support: CAPES (Brazil)/FAPESP/CNPq/INCTS
No relevant conflicts of interest to declare.
A anemia falciforme caracteriza-se como quadro hemolítico hereditário que evolui cronicamente causando danos físicos e emocionais às pessoas acometidas. Até o presente momento não se dispõe de ...tratamento curativo, a não ser o transplante de medula óssea, que ainda tem sido realizado de maneira experimental. A triagem neonatal de hemoglobinopatias, principalmente da anemia falciforme, tem sido essencial ao diagnóstico precoce e à instituição de medidas preventivas e promotoras de saúde. No entanto, o Ministério da Saúde do Brasil recomenda o exame dos pais a partir da identificação de heterozigotos, mas não faz alusão quanto à ampliação da triagem para outros familiares. Uma família que possua uma criança afetada com estas síndromes passa a ter um marcador para um grupo genético de risco. Neste caso, a triagem ampliada para os familiares mais próximos (avós, pais, irmãos, tios e primos) poderá identificar muitos portadores ou casais em risco, antes do casamento e procriação, além de servir de base a programas de assessoramento genético e de controle epidemiológico das hemoglobinopatias, uma herança genética bastante freqüente em nossa população.
A anemia falciforme caracteriza-se como quadro hemolítico hereditário que evolui cronicamente causando danos físicos e emocionais às pessoas acometidas. Até o presente momento não se dispõe de ...tratamento curativo, a não ser o transplante de medula óssea, que ainda tem sido realizado de maneira experimental. A triagem neonatal de hemoglobinopatias, principalmente da anemia falciforme, tem sido essencial ao diagnóstico precoce e à instituição de medidas preventivas e promotoras de saúde. No entanto, o Ministério da Saúde do Brasil recomenda o exame dos pais a partir da identificação de heterozigotos, mas não faz alusão quanto à ampliação da triagem para outros familiares. Uma família que possua uma criança afetada com estas síndromes passa a ter um marcador para um grupo genético de risco. Neste caso, a triagem ampliada para os familiares mais próximos (avós, pais, irmãos, tios e primos) poderá identificar muitos portadores ou casais em risco, antes do casamento e procriação, além de servir de base a programas de assessoramento genético e de controle epidemiológico das hemoglobinopatias, uma herança genética bastante freqüente em nossa população.Sickle cell anemia is a hereditary condition that evolves to a chronic illness, causing physical and emotional disorders to those involved. As yet there is no cure except for bone marrow transplantation which is still in the experimental stage. Neonatal screening for hemoglobin disorders, particularly sickle cell anemia, has been crucial for ensuring early diagnosis and the application of preventive and health-promoting measures. The Brazilian Health Ministry recommends testing parents thereby identifying heterozygotes, but does not propose extending this screening to other family members. A family that has a child affected by one of these syndromes is a marker for an at-risk group. In this case extending screning to close relatives (grandparents, siblings, aunts and uncles, and cousins) may identify individuals affected by the disease or couples at risk before marriage and reproduction and serve as the basis for programs providing genetic evaluation and epidemiological control of hemoglobin diseases that are relatively common in the Brazilian population.
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