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Biomedical subjects

S T Saad

Publications and source records attributed to S T Saad.

At least 55 records · Page 3Linked to original sources
PubMed1998

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.

Adolescent↗
PubMed1998

The relationship of bone marrow histology with the molecular pattern in chronic myeloid leukemia.

The chromosomal abnormality in chronic myeloid leukemia (CML) results from a reciprocal translocation between chromosomes 9 and 22 transferring the c-abl proto-oncogene from chromosome 9 to the restricted breakpoint region on chromosome 22 M (bcr). In this study the breakpoint was determined within the M-bcr in 35 CML patients in the chronic phase, by Southern blotting analysis, and it was then correlated with bone marrow Granulocytic-Megakaryocytic (GRAN-MEG) and Granulocytic (GRAN) histological subgroups, as well as with the clinical findings and laboratory parameters. In the 35 patients analyzed, 46% were grouped as 5' and 54% as 3'. There was an increase in bone marrow basophils in 5' breakpoint patients compared to 3' breakpoint (p = 0.042) but the M-bcr breakpoint site did not differ significantly in the subgroup GRAN or GRAN-MEG (p = 0. 12). In conclusion, the patient population had a higher frequency of M-bcr breakpoint in zone 4 and 3' position; there was no correlation between 5' and 3' positions and clinical or haematological features, except a significant increase in bone marrow basophil cells in 5' breakpoint patients compared to 3' breakpoint. Although a higher frequency of the 3' breakpoint was found in patients with a low number of megakaryocytes compared to the cases with a granulocytic-megakaryocytic proliferation, this difference was not statistically significant.

Bone Marrow↗
PubMed1997

Band 3 Campinas: a novel splicing mutation in the band 3 gene (AE1) associated with hereditary spherocytosis, hyperactivity of Na+/Li+ countertransport and an abnormal renal bicarbonate handling.

We have studied the molecular defect underlying band 3 deficiency in one family with hereditary spherocytosis using nonradioactive single strand conformation polymorphism of polymerase chain reaction (PCR) amplified genomic DNA of the AE1 gene. By direct sequencing, a single base substitution in the splicing donor site of intron 8 (position + 1G --> T) was identified. The study of the cDNA showed a skipping of exon 8. This exon skipping event is responsible for a frameshift leading to a premature stop codon 13 amino acids downstream. The distal urinary acidification test by furosemide was performed to verify the consequences of the band 3 deficiency in alpha intercalated cortical collecting duct cells (alphaICCDC). We found an increased basal urinary bicarbonate excretion, associated with an increased basal urinary pH and an efficient distal urinary acidification. We also tested the consequences of band 3 deficiency on the Na+/H+ exchanger, by the measurement of Na+/Li+ countertransport activity in red blood cells. The Na+/Li+ countertransport activity was increased threefold to sixfold in the patients compared with the controls. It is possible that band 3 deficiency in the kidney leads to a decrease in the reabsorption of HCO3- in alphaICCDC and anion loss, which might be associated with an increased sodium-lithium countertransport activity.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1997

Beta-spectrin Campinas: a novel shortened beta-chain variant associated with skipping of exon 30 and hereditary elliptocytosis.

Beta-Spectrin Campinas is a novel spectrin variant associated with a shortened beta-chain in a kindred with hereditary elliptocytosis (HE). The propositus and her mother exhibited increased amounts of spectrin dimers and an increase in the alphaI 74 kD fragment from the alpha-chain after partial tryptic digestion of spectrin. The shortened beta-chain appeared as an additional band of approximately 200 kD on SDS-PAGE. In order to delineate the molecular defect of this abnormality at the gene level, reticulocyte mRNA was transcribed into cDNA and the last four exons of the beta-spectrin gene were amplified. Agarose gel of the amplification product of the propositus revealed the expected band of 487 bp as well as a shortened band of approximately 300 bp (size determined on gel). This shortened cDNA amplification product was cloned and nucleotide sequencing revealed the absence of the entire exon 30. In order to determine the underlying mutation responsible for this abnormal splicing, a genomic DNA fragment containing exons 30 and 31 was amplified and nucleotide sequencing revealed a G-->A substitution at the 5' donor splice site consensus sequence of intron 30 (nt + 1 IVS30). The skip splicing observed in this study results in a frameshift, creating a new stop codon and causing a deletion of 129 amino acids at the very COOH-terminus of the protein, thus impairing spectrin dimers self-association. We classified this HE as spherocytic HE because the propositus presented a few spherocytes in addition to many elliptocytes in the blood smear, whereas her mother, who was splenectomized, showed many schizocytes, poikilocytes and spherocytes.

Adult↗
PubMed1997

cDNA-derived amino-acid sequence of a land turtle (Geochelone carbonaria) beta-chain hemoglobin.

The cDNA sequence encoding the turtle Geochelone carbonaria beta-chain was determinated. The isolation of hemoglobin mRNA was based on degenerate primers' PCR in combination with 5'- and 3'-RACE protocol. The full length cDNA is 615 bp with the ATG start codon at position 53 and TGA stop codon at position 495; The AATAAA polyadenylation signal is found at position 599. The deduced polypeptyde contains 146 amino-acid residues. The predicted amino acid sequence shares 83% identity with the beta-globin of a related specie, the aquatic turtle C. p. belli. Otherwise, identity is higher when compared with chicken beta-Hb (80%) than with other reptilian orders (Squamata, 69%, and Crocodilia, 61%). Compared with human HbA, there is 67% identity, and at least three amino acid substitutions could be of some functional significance (Glu43 beta-->Ser, His116 beta-->Thr and His143 beta-->Leu). To our knowledge this represents the first cDNA sequence of a reptile globin gene described.

Amino Acid Sequence↗
PubMed1997

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.

Adolescent↗
PubMed1997

Relationship between the type of BCR-ABL rearrangement and bone marrow histopathological features in chronic myeloid leukemia.

The aim of the present study was to characterize the type of BCR-ABL transcript and to correlate the molecular feature with bone marrow histology. For this purpose, we analysed the BCR-ABL rearrangement in 26 patients with chronic myeloid leukemia (CML) in the chronic phase by RT-PCR, and we also classified the bone marrow histology according to the predominance of granulocylic (GRAN) or granulocytic and megakaryocytic (GRAM/MEG) proliferation, after analysis of two independent observers. We did not find any significant difference in survival of patients presenting b2-a2 and b3-a2 transcripts or GRAN and GRAN/MEG bone marrow types, nor did we find any significant correlation of the type of BCR-ABL transcript with the bone marrow histological subgroups GRAN and GRAN-MEG (Fisher's test = 0.31). Thus, we conclude that the presence of exon b3 is not correlated to bone marrow histology in CML.

Adolescent↗
PubMed1997

N-ras gene point mutations in Brazilian acute myelogenous leukemia patients correlate with a poor prognosis.

The frequency of ras gene mutations varies from 11 to 27% in AML populations from the United States and Europe but it seems that there is no study regarding the frequency of mutated N-ras gene in patients with AML in South America. In order to study the frequency of N-ras gene mutations (exons 1 and 2) in Brazilian patients with AML and to evaluate the possible correlation between the presence of the mutation and clinical features, 40 patients were analyzed. N-ras mutations were identified in DNA samples from eight of 40 AML patients (20%). No significant correlation was found between N-ras mutation and age, sex, race, response to therapy, FAB subtype or occupational exposure. However, the overall survival and AML-free survival were significantly shorter in patients with N-ras mutations than in those without these abnormalities.

Acute Disease↗
PubMed1996

Effect of alpha-thalassemia and beta-globin gene cluster haplotypes on the hematological and clinical features of sickle-cell anemia in Brazil.

To compare the features of sickle-cell anemia in Brazil with those in other locales, we studied the effects of the beta-globin-like gene cluster haplotype and alpha-thalassemia upon the clinical and hematological features in 85 patients. The distribution of haplotypes differed from that in the United States and Jamaica. The Central African Republic (CAR) haplotype predominated; 34% of patients were CAR haplotype homozygotes, 45% CAR/Benin homozygotes, and 11% Benin homozygotes. No Senegal haplotype chromosomes were observed. Alpha-thalassemia was present in 17.5% of patients. HbF levels were higher in Benin homozygotes, compared with the other two groups (P < 0.05). Nearly half the patients with a CAR haplotype had leg ulcers, compared to 12.5% of the Benin homozygote group; stroke did not occur in alpha-thalassemia carriers, but neither result was statistically significant. As in other studies, our results indicate that the CAR haplotype may be associated with more severe disease.

Adolescent↗
PubMed1996

The release of nitric oxide and superoxide anion by neutrophils and mononuclear cells from patients with sickle cell anaemia.

The aim of this work was to investigate the release of nitric oxide and superoxide by neutrophils and mononuclear cells from patients with sickle cell anaemia. Nitric oxide release was assayed by the ability of leucocytes to inhibit thrombin-induced washed platelet aggregation. Superoxide release was assessed by a cytochrome c reduction assay. Neutrophils from sickle cell anaemia patients released nitric oxide in a similar manner to those from healthy controls, because inhibition of platelet aggregation by neutrophils from sickle cell anaemia or from healthy controls was blocked by the inhibitor of nitric oxide synthesis N(omega)-nitro-L-arginine methyl ester (300 microM), but not by N(omega)-nitro-D-arginine methyl ester (300 microM) and was reversed by L-arginine (1 mM). Additionally, a similar number of neutrophils from sickle cell anaemia patients and from healthy controls was required to inhibit platelet aggregation. Mononuclear cells from sickle cell anaemia patients inhibited platelet aggregation only in the presence of superoxide dismutase (60 U ml(-1)). Phorbol 12-myristate 13-acetate (PMA, 30 nM)- or zymosan (100 particles/cell)-induced release of superoxide by mononuclear cells from sickle cell anaemia patients was significantly higher than that observed in mononuclear cells from healthy controls (P<0.001 and P<0.01 respectively, Mann-Whitney test). The levels of superoxide released by neutrophils from sickle cell anaemia patients were similar to those from healthy controls. We conclude that mononuclear cells from sickle cell anaemia patients release more superoxide than those from healthy controls, when stimulated with PMA or zymosan in vitro. Considering that superoxide inactivates nitric oxide, that nitric oxide is an important endogenous vasodilator, and that superoxide produces oxidant damage, this greater production of superoxide by mononuclear cells from sickle cell anaemia patients may represent an additional risk factor for the obstruction of the microcirculation and tissue damage in these patients.

Adolescent↗
PubMed1996

Expression of spectrin alpha I/65 hereditary elliptocytosis in patients from Brazil.

We report the clinical and laboratory findings in three unrelated families from southeastern Brazil with Sp alpha I/65 hereditary elliptocytosis (HE), including one homozygote and a patient presenting an elongated beta-spectrin. In family 1, three patients presented the allele alpha-Lely in trans to the elliptocytogenic allele. In these three patients the blood smear showed pronounced elliptocytosis, poikilocytosis and a few small red cell fragments instead of the mild elliptocytosis observed in their father, who did not present the polymorphism. In family 2 we describe one homozygote, with consanguineous parents presenting with anaemia, splenomegaly, severe poikilocytosis and elliptocytosis, budding, microspherocytes and numerous fragments in the blood smear. In family 3 we found an elongated beta Sp in a patient with Sp alpha I/65. The cause of the HE was the Sp alpha I/65 since the elongated beta Sp was not found in his brother, who also presented with HE and Sp alpha I/65. Apparently the abnormal beta Sp did not aggravate the HE, because both individuals had the same clinical and laboratory findings. However, the propositus presented a few more elliptocytes and poikilocytes than his brother, probably because the elongated beta-spectrin may have disturbed the spectrin self-association. In fact, in the propositus an abnormal band was observed in the nondenaturing gels, just above the Sp dimer, probably as a result of the association of the abnormal beta Sp with the normal spectrin chains. In the family studied here, both brothers presented the allele alpha Lely, but as their mother was dead, it was not possible to determine the polymorphism transmission. However, the high number of poikilocytes observed in the blood smear of both cases suggests an association in trans with the Sp alpha I/65. Thus, taken together, the data in this report indicate that HE secondary to Sp alpha I/65 abnormality is frequent in Brazil, and in one case it was associated with an apparently novel abnormal large beta-spectrin.

Adult↗
PubMed1996

Glutathione peroxidase, reduced glutathione, superoxide dismutase and catalase in red cells of patients with hairy cell leukemia.

Red cell antioxidant enzymes have been recently studied in malignant lymphomas and the results are controversial. Hairy cell leukemia is a rare chronic lymphoproliferative disorder originating probably in a pluripotent stem cell. In the present study, glutathione peroxidase (Gpx), reduced glutathione (GSH), superoxide dismutase (SOD), and catalase were measured in a homogeneous group of patients with untreated hairy cell leukemia and normal controls. Glutathione peroxidase, catalase and SOD activities were significantly lower in patients than in normals. GSH was not significantly different in patients compared to controls. There was no correlation between Gpx, GSH, SOD, and catalase and hemoglobin; reticulocytes, leukocytes, hairy cells, platelets number or splenomegaly. Taken together these data suggest a decreased activity of red cell antioxidant enzymes in hairy cell leukemia and support a pluripotent stem cell defect of these abnormalities.

Adult↗
PubMed1996

Prognostic significance of BCR-ABL rearrangement in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the presence of a reciprocal translocation between chromosomes 9 and 22 in at least 95% of cases. At the molecular level, this translocation results in the activation of the ABL oncogene of chromosome 9, which becomes contiguous with the 5' end of the BCR gene on chromosome 22. The breakpoint usually occurs between exons 2 and 3 (b2-a2 rearrangement), or 3 and 4 (b3-12 rearrangement) of the major breakpoint cluster region (M-BCR) of the BCR gene. The aim of the present study was to characterize the type of BCR-ABL transcript in 32 patients with CML using the reverse transcriptase-polymerase chain reaction (RT-PCR) and to determine if this type of rearrangement is related to the survival of the patients. Our results confirmed that RT-PCR is more sensitive than cytogenetic analysis for identifying the Philadelphia (Ph1) chromosome (96.9% vs 79.3%). The frequencies of b2-a2 and b3-a2 rearrangements were 28.1% and 65.7%, respectively. The survival of patients presenting the b2-a2 or the b3-a2 rearrangement was not significantly different (P = 0.27750). The data suggest that the type of transcript has no prognostic value for CML patients.

Adolescent↗