V. R. Arruda

Sickle Cell Disease in a Brazilian Population from Sao Paulo: A Study of the βs Haplotypes

In this study we have determined the frequency of βs haplotypes in a Brazilian sickle cell disease population from Sao Paulo, Brazil, by analyzing sequence variations in the immediate 5’ fl

High-dose dexamethasone therapy in chronic idiopathic thrombocytopenic purpura

Abstract A high-dose pulse of dexamethasone has been described as a current option for the treatment of refractory idiopathic thrombocytopenic purpura (ITP), but the results are controversial. Here we describe the use of a high dose of dexamethasone (40 mg per day for 4 days every month) in 18 patients with chronic ITP. The median age of the patients was 42.5 years (range, 16–77 years); 13 were female and five male. The duration of the disease ranged from 5 to 480 months, and splenectomy was carried out in six of the 18 patients.The overall results obtained revealed a satisfactory response (platelet counts higher than 50×109/l) in eight of the 18 patients. However, a long-term remisson was achieved in only three of the eight patients with a follow up of 7–16 months. We were not able to identify any clinical or laboratory prognostic parameters or previous treatment which would allow one to predict a successful outcome of this treatment. These results suggest that a high dose of dexamethasone may provide an alternative, be it a poor one, for the treatment of refractory IPT, in which the use of a low-cost drug with limited side effects is an important consideration.

Genetic analysis of beta-thalassemia major and beta-thalassemia intermedia in Brazil.

The development of methodologies to identify the molecular lesions responsible for different types of beta-thalassemia has made it possible to correlate these data with clinical and hematological severity. We examined DNA from 35 patients with beta-thalassemia, residents of the State of São Paulo, Brazil, for some types of genetic modifying factors: beta-thalassemia mutations, the upstream Xmnl GY-globin gene polymorphisms, and alpha-globin gene deletions. Additionally, the beta-like gene cluster haplotypes and the presence of the AYT variant were studied. The following mutations were present in the 70 chromosomes studied: 54.3% codon 39 (C-->T) (beta degree); 18.6% IVS-I-6 (T-->C) (beta+); 18.6% IVS-I-110 (G-->A) (beta+), and 4.3% IVS-I-1 (G-->T) (beta degree). Haplotype II was associated with the nonsense mutation at codon 39, haplotype I with the IVS-I-110 and codon 39 mutations, and haplotypes VI and VII with the IVS-I-6 mutation. The Xmnl polymorphism was detected in three out of 31 patients studied. No alpha-thalassemia was detected among the thalassemia intermedia patients. The AYT variant was present in 87.1% of 31 thalassemia patients and was associated with the codon 39/haplotype II and IVS-I-6/haplotype VI mutations. This is the first study of the Brazilian population that has analyzed the beta-thalassemia mutations and other molecular variants, and has correlated them with the clinical manifestations.

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 S beta thalassemia (8SS, 2S beta), 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 higher 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%) 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.

β0‐Thalassemia resulting from a novel mutation: β66/u→stop codon

To the Editor: We describe here a novel point mutation which affects the 6th codon of the b-globin gene (GAGpTAG), creating a stop codon instead of a glutamic acid codon. This nonsense mutation was found in a 11-year-old White Brazilian girl with Hb C-b thalassemia. The patients mother was heterozygous for Hb C and the father was heterozygous for the b-thalassemia mutation. This is the ®rst nonsense mutation described in codon 6 of the b-globin gene. b-Thalassemia (b-thal) occurs at high frequencies in individuals of Mediterranean, Middle Eastern, East Indian, African and Southeast Asian descent (1). The b-thal syndromes were brought into Brazil mainly by Italian immigration during the end of the 19th century and the beginning of the 20th century (2). They are usually due to point mutations in the b-globin gene, and 160 different mutations have been described (3). Four of them are responsible for the majority of the b-thal cases in the Brazilian population (b 39, b IVS-I-nt 110, b IVS-I-nt 6 and b IVS-I-nt 1) (4). In this paper we describe a novel point mutation found in a 11-year-old White Brazilian girl with haemoglobin C-b-thal (C-b-thal). Haematological data were determined electronically (Cell Dyn 3.500, Abbott). Hb electrophoresis was performed with standard techniques (5). Hb F was determined by alkali denaturation (6). The b-globin gene was ampli®ed using the polymerase chain reaction (PCR) with primers described elsewhere (7), and direct-sequenced using Thermo Sequenase Cycle Sequencing (Amersham). The results are shown in Table 1. Familial analysis revealed that the mother was heterozygous for Hb C and the father, probably an Italian descendent, was heterozygous for b-thal. In this case, the b-thal resulted from a GpT substitution at the ®rst nucleotide position of the 6th codon (Fig. 1), and created a stop codon in replacement of the glutamic acid codon (GAGpTAG). Premature termination of the globin chain synthesis due to interruption of translation can be caused by either nonsense or frameshift mutations. They usually originate an unstable product which is quickly degraded in the cell (8, 9). Only 14 (8.8%) of the 160 b-thal mutations are nonsense mutations. Only 5 molecular alterations in codon 6 are known (Hb S, Hb C, Hb G-Makassar, Hb Machida and a frameshift ±A from GAG) (3). This is the ®rst thalassemic nonsense mutation described in this codon. Fig. 1. Nucleotide sequence of the PCR-ampli®ed b-globin gene of the studied patient: Lane A, normal control: codon 6 ± GAG (Glu); Lane B, patient: codon 6; there are two mutations GpT, TAG (stop codon) and GpA, AAG (Lys).

A Novel Splice Site Mutation in a Brazilian Patient with Hereditary Antithrombin Deficiency Type I

Source of Material. The proband is a 30-year-old male patient who had deep vein thrombosis and pulmonary embolism, in March 1994. His father and aunt (father’s sister), who are deceased, had deep vein thrombosis at young age. In the proband, AT deficiency was diagnosed on the basis of reduced levels of activity (54%) and antigen (57%) in plasma. Of the family members, one brother and a nephew, who were asymptomatic, had decreased AT activity, with levels of 63 and 59%, respectively. Methods. Genomic DNA was isolated from peripheral blood leukocytes according to standard procedures. Each of the 7 exons and all the exon/intron junctions of the AT gene were amplified by PCR, using allele-specific primers. The amplified fragments were submitted to nonradioactive single strand conformation polymorphism (SSCP). Independent of results obtained from SSCP, the amplified fragments were submitted to another screening method, conformational sensitive gel electrophoresis (CSGE). Direct sequencing was performed by the Sanger method, using the Thermo-Sequenase Cycle Sequencing Kit (United States Biochemical, Cleveland, Ohio, USA) with the same sense primer as used for PCR amplification. Fig. 1. Patient direct sequencing of the amplified exon 1 AT gene, showing the mutation in intron 1 at nucleotide position +5 (G → A).

Molecular identification of hereditary persistence of fetal hemoglobin type 2 (HPFH type 2) in patients from Brazil

The HPFH deletion type 2 was first described in a patient from Ghana and is characterized by a large deletion of approximately 105 kb extension. We report here the results obtained in studying a black Brazilian patient who presented an association ofβ -thalassemia and HPFH type 2, using a PCR strategy for detection of the breakpoint region. This procedure allows a rapid molecular identification of this condition and is a reliable procedure for screening patients with a hematological picture of HPFH deletion types.

Determination of the Allele Frequencies of Three Polymorphisms in the Promoter Region of the Human Protein C Gene in Three Brazilian Ethnic Groups

The frequencies of three polymorphisms in the promoter region of the human protein C gene have been determined in three ethnic groups of the Brazilian population. The allele frequencies observed in south-eastern Brazilian Caucasians and Blacks were similar to the values obtained for the Dutch population and were different from those observed in Amazonian Indians living in the north of the country. The most frequent genotypic combination found in this latter group was the CC/GG/AA genotype, which is very rare among Caucasians. The complete heterozygous genotype CT/AG/At̲ was the most frequent both in Brazilian Caucasians and Blacks. Among 27 possible genotypic combinations, 21 were found in the Caucasian group and 15 in Blacks, revealing a high degree of genetic diversity in south-eastern Brazilian populations.