Detection of somatic mutations of the PIG-A gene in Brazilian patients with paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal syndrome characterized by intravascular hemolysis mediated by complement, thrombotic events and alterations in hematopoiesis. Basically, the molecular events which underlie the complexity of the syndrome consist of the absence of the glycosylphosphatidylinositol (GPI) anchor as a consequence of somatic mutations in the PIG-A gene, located on the X chromosome. The GPI group is responsible for the attachment of many proteins to the cytoplasmic membrane. Two of them, CD55 and CD59, have a major role in the inhibition of the action of complement on the cellular membrane of blood cells. The absence of GPI biosynthesis can lead to PNH. Since mutations in the PIG-A gene are always present in patients with PNH, the aim of this study was to characterize the mutations in the PIG-A gene in Brazilian patients. The analysis of the PIG-A gene was performed using DNA samples derived from bone marrow and peripheral blood. Conformation-sensitive gel electrophoresis was used for screening the mutation and sequencing methods were used to identify the mutations. Molecular analysis permitted the identification of three point mutations in three patients: one G->A transition in the 5' portion of the second intron, one T->A substitution in the second base of codon 430 (Leu430->stop), and one deletion deltaA in the third base of codon 63. This study represents the first description of mutations in the PIG-A gene in a Brazilian population.

Minimal doses of hydroxyurea for sickle cell disease

The use of hydroxyurea (HU) can improve the clinical course of sickle cell disease. However, several features of HU treatment remain unclear, including the predictability of drug response and determination of adequate doses, considering positive responses and minimal side effects. In order to identify adequate doses of HU for treatment of sickle cell disease, 10 patients, 8 with sickle cell anemia and 2 with Sbeta thalassemia (8SS, 2SBeta), were studied for a period of 6 to 19 months in an open label dose escalation trial (10 to 20 mg kg(-1) day(-1)). Hemoglobin (Hb), fetal hemoglobin (Hb F) and mean corpuscular volume (MCV) values and reticulocyte, neutrophil and platelet counts were performed every two weeks during the increase of the HU dose and every 4 weeks when the maximum HU dose was established. Reduction in the number of vasoocclusive episodes was also considered in order to evaluate the efficiency of the treatment. The final Hb and Hb F concentrations, and MCV values were significantly higer than the initial values, while the final reticulocyte and neutrophil counts were significantly lower. There was an improvement in the concentration of Hb (range: 0.7-2.0 g/dl) at 15 mg HU kg(-1) day(-1), but this concentration did not increase significantly when the HU dose was raised to 20 mg kg(-1) day(-1). The concentration of Hb F increased significantly (range: 1.0-18.1 per cent) when 15 mg HU was used, and continued to increase when the dose was raised to 20 mg kg(-1) day (-1). The final MCV values increased 11-28 fl (femtoliters). However, reticulocyte (range: 51-205 x 10(9)/l) and neutrophil counts (range: 9.5-1.3 x 10(9)/l) obtained at this dose were significantly lower than those obtained with 15 mg kg(-1) day(-1). All patients reported a decrease in frequency or severity of vasoocclusive episodes. These results suggest that a hydroxyurea dose of 15 mg kg(-1) day(-1) seems to be adequate for treatment of sickle cell disease in view of the minimal side effects observed and the improvement in laboratory and clinical parameters.

Detecçäo de portadoras de gene da hemofilia A em uma populaçäo brasileira / Carriers detection of hemophilia A gene in a Brazilian population

A identificaçäo de portadores do gene para a hemofilia A foi levada a efeito em 20 famílias de pacientes que apresentavam essa condiçäo. A detecçäo foi realizada com base no estudo dos polimorfismos de restriçäo (RLFP) do gene do fator VIII para as enzimas BcII e HindIII. Os métodos utilizados incluíram a amplificaçäo de fragmentos do gene em análise pela reaçäo em cadeia de polimerase e posterior digestäo com as enzimas específicas. Foram estudados 84 indivíduos pertencentes a 20 famílias de pacientes. Foi possível identificar ou excluir portadoras do gene anormal em 76// das famílias estudadas. Esse traballho evidencia a possibilidade de detecçäo de portadoras na maioria das famílias de pacientes com hemofilia A na populaçäo brasileira com o emprego de um número restrito de polimorfismo de restriçäo