S.T.O. Saad

alpha-Globin genes: thalassemic and structural alterations in a Brazilian population

Seven unrelated patients with hemoglobin (Hb) H disease and 27 individuals with alpha-chain structural alterations were studied to identify the alpha-globin gene mutations present in the population of Southeast Brazil. The -alpha3.7, --MED and -(alpha)20.5 deletions were investigated by PCR, whereas non-deletional alpha-thalassemia (alphaHphalpha, alphaNcoIalpha, alphaalphaNcoI, alphaIcalpha and alphaTSaudialpha) was screened with restriction enzymes and by nested PCR. Structural alterations were identified by direct DNA sequencing. Of the seven patients with Hb H disease, all of Italian descent, two had the -(alpha)20.5/-alpha3.7 genotype, one had the --MED/-alpha3.7 genotype, one had the --MED/alphaHphalpha genotype and three showed interaction of the -alpha3.7 deletion with an unusual, unidentified form of non-deletional alpha-thalassemia [-alpha3.7/(alphaalpha)T]. Among the 27 patients with structural alterations, 15 (of Italian descent) had Hb Hasharon (alpha47Asp-->His) associated with the -alpha3.7 deletion, 4 (of Italian descent) were heterozygous for Hb J-Rovigo (alpha53Ala-->Asp), 4 (3 Blacks and 1 Caucasian) were heterozygous for Hb Stanleyville-II (alpha78Asn-->Lys) associated with the alpha+-thalassemia, 1 (Black) was heterozygous for Hb G-Pest (alpha74Asp-->Asn), 1 (Caucasian) was heterozygous for Hb Kurosaki (alpha7Lys-->Glu), 1 (Caucasian) was heterozygous for Hb Westmead (alpha122His-->Gln), and 1 (Caucasian) was the carrier of a novel silent variant (Hb Campinas, alpha26Ala-->Val). Most of the mutations found reflected the Mediterranean and African origins of the population. Hbs G-Pest and Kurosaki, very rare, and Hb Westmead, common in southern China, were initially described in individuals of ethnic origin differing from those of the carriers reported in the present study and are the first cases to be reported in the Brazilian population.

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.

Hemochromatosis (HFE) gene mutations in Brazilian chronic hemodialysis patients

Patients with chronic renal insufficiency (CRI) have reduced hemoglobin levels, mostly as a result of decreased kidney production of erythropoietin, but the relation between renal insufficiency and the magnitude of hemoglobin reduction has not been well defined. Hereditary hemochromatosis is an inherited disorder of iron metabolism. The importance of the association of hemochromatosis with treatment for anemia among patients with CRI has not been well described. We analyzed the frequency of the C282Y and H63D mutations in the HFE gene in 201 Brazilian individuals with CRI undergoing hemodialysis. The analysis of the effects of HFE mutations on iron metabolism and anemia with biochemical parameters was possible in 118 patients of this study (hemoglobin, hematocrit, ferritin levels, transferrin saturation, and serum iron). A C282Y heterozygous mutation was found in 7/201 (3.4%) and H63D homozygous and heterozygous mutation were found in 2/201 (1.0%) and 46/201 (22.9%), respectively. The allelic frequencies of the HFE mutations (0.017 for C282Y mutation and 0.124 for H63D mutation) did not differ between patients with CRI and healthy controls. Regarding the biochemical parameters, no differences were observed between HFE heterozygous and mutation-negative patients, although ferritin levels were not higher among patients with the H63D mutation (P = 0.08). From what we observed in our study, C282Y/H63D HFE gene mutations are not related to degrees of anemia or iron stores in CRI patients receiving intravenous iron supplementation (P > 0.10). Nevertheless, the present data suggest that the H63D mutation may have an important function as a modulating factor of iron overload in these patients.

IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES INDUCED BY HYDROXYUREA IN RETICULOCYTES FROM SICKLE CELL ANAEMIA PATIENTS

1 The major effect associated with hydroxyurea (HU) treatment of sickle cell anaemia (SCA) patients is an increase in fetal haemoglobin (HbF) synthesis, which inhibits the polymerization of haemoglobin S. 2 Hydroxyurea improves clinical symptoms by reducing the frequency of pain and vaso‐occlusive crises, acute chest syndrome, transfusion requirements and hospitalization. 3 The molecular mechanisms responsible for HU‐mediated induction of fetal globin transcription are not completely understood. Therefore, the aim of the present study was to identify differentially expressed genes participating in these mechanisms. 4 We established two suppression subtractive hybridization (SSH) libraries from reticulocytes obtained from SCA patients either not on or on HU treatment. The gene expression of some of the genes identified was subsequently evaluated by real‐time polymerase chain reaction (PCR). 5 Genes identified with altered expression included SUDS3, FZD5 and PHC3, which may be associated with the regulation of globin expression. 6 This is the first demonstration of an association between HU treatment and the expression of genes identified in erythroid cells.

Molecular identification of Sicilian (deltaß)º-thalassemia associated with ß-thalassemia and hemoglobin S in Brazil

We describe the clinical and molecular characteristics of two unrelated Brazilian families with an association of the Sicilian form of (ds)o-thalassemia with hemoglobin S and s-thalassemia. Direct sequencing of the s-globin gene showed only the hemoglobin S mutation in patient 1 and the s-thalassemia IVS1-110 in patient 2. The other allele was deleted in both patients and PCR of DNA samples of the breakpoint region of both patients showed a band of approximately 1,150 bp, expected to be observed in the DNA of carriers of Sicilian (ds)o-thalassemia. The nucleotide sequence of this fragment confirmed the Sicilian deletion. There are few reports concerning the Hb S/(ds)o-thalassemia association and patient 2 is the first reported case of Sicilian type of (ds)o-thalassemia in association with s-thalassemia documented at the molecular level.

Hemoglobin H disease resulting from the association of the – α3.7 rightward deletion and the (αα)ΜΜ deletion in a Brazilian patient

Abstract:u2002 A patient with Hb H disease resulting from the association of the –u200aα3.7 rightward deletion with the rare (αα)ΜΜ deletion, which removes the entire α‐major regulatory element (MRE), is reported. This is the first description of an α‐thalassemic mutation resulting from deletion of the locus‐controlling sequences in the South‐American population.

Identification of Hb Zürich [α2β263(E7)His→Arg] by DNA analysis in a brazilian family

Hb Zurich, in which the β63(E7) histidine is replaced by arginine, was the first unstable hemoglobin (Hb) variant to be analyzed structurally (1-3). The structural alteration in Hb Zurich leads to particularly interesting functional and clinical consequences. The substitution of arginine for the distal histidine at helix E7 causes a marked change in the space where ligand binding occurs (4). The positively charged arginine attaches to the propionate of the heme, leaving the heme pocket wide open, and allowing ready access to the heme iron. This explains the propensity for individuals with this variant to have hemolytic episodes following ingestion of sulfa derivatives (1,5).

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).

Early circulating erythroid progenitor cells and expression of erythropoietin receptors in sickle cell disease.

Abstract: The ability of circulating progenitor cells from 22 patients with sickle cell disease (SCD) to develop erythroid colonies was studied in vitro in the presence or absence of growth factors (5637‐CM and erythropoietin). In both conditions, SCD patients presented significantly higher numbers of circulating burst‐forming unit‐erythroid (BFU–E/5×105MNC) when compared to control subjects. The study of the expression of erythropoietin receptors revealed an increased level in SCD patients. Moreover, there was a correlation between both stimulated and autocrine (without stimulus) BFU–E and the expression of erythropoietin receptors. These results are of particular interest since they indicate that the phenomenon of spontaneous BFU–E‐derived colonies observed in SCD patients may be due to an increased expression of erythropoietin receptors.