Low expression of APAF-1XL in acute myeloid leukemia may be associated with the failure of remission induction therapy

Apoptotic protease activating factor 1 (APAF-1) has a critical role in the regulation of apoptosis. In the present study, the mRNA expression analysis of different APAF-1 transcripts (APAF-1S, APAF-1LC, APAF-1LN, and APAF-1XL) was analyzed in bone marrow samples from 37 patients with acute myeloid leukemia (newly diagnosed, with no previous treatment). APAF-1XL and APAF-1LN transcripts (with and without an extra WD-40 repeat region, respectively) were detected in all samples, although the major form expressed was APAF-1XL in 65 percent of the samples (group 1), while 35 percent of the samples expressed primarily APAF-1LN (group 2). Only 46 percent of the patients presented complete remission in response to remission induction therapy (represented by less than 5 percent marrow blasts and hematological recovery), all but 2 cases being from group 1, 21.6 percent did not attain complete remission (only 1 case from group 1), and 32.4 percent of the patients died early. Lower expression of APAF-1XL (APAF-1XL/APAF-1LN ratio <1.2) was associated with a poor response to therapy (P = 0.0005, Fisher exact test). Both groups showed similar characteristics regarding white blood cell counts, cytogenetic data or presence of gene rearrangements associated with good prognosis as AML1-ETO, CBFB-MYH11 and PML/RARA. Since it has been shown that only the isoforms with the extra WD-40 repeat region activate procaspase-9, we suggest that low procaspase-9 activation may also be involved in the deregulation of apoptosis and chemotherapy resistance in acute myeloid leukemia.

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 percent) and H63D homozygous and heterozygous mutation were found in 2/201 (1.0 percent) and 46/201 (22.9 percent), 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.

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 (deltaß)º-thalassemia with hemoglobin S and ß-thalassemia. Direct sequencing of the ß-globin gene showed only the hemoglobin S mutation in patient 1 and the ß-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 (deltaß)º-thalassemia. The nucleotide sequence of this fragment confirmed the Sicilian deletion. There are few reports concerning the Hb S/(deltaß)º-thalassemia association and patient 2 is the first reported case of Sicilian type of (deltaß)º-thalassemia in association with ß-thalassemia documented at the molecular level

ß-Spectrin Säo PauloII, a novel frameshift mutation of the ß-spectrin gene associated with hereditary spherocytosis and instability of the mutant mRNA

Hereditary spherocytosis (HS) is a common inherited anemia characterized by the presence of spherocytic red cells. Defects in several membrane protein genes have been involved in the pathogenesis of HS. ß-Spectrin-related HS seems to be common. We report here a new mutation in the ß-spectrin gene coding region in a patient with hereditary spherocytosis. The patient presented acanthocytosis and spectrin deficiency and, at the DNA level, a novel frameshift mutation leading to HS, i.e., a C deletion at codon 1392 (ß-spectrin Säo PauloII), exon 20. The mRNA encoding ß-spectrin Säo PauloII was very unstable and the mutant protein was not detected in the membrane or in other cellular compartments. It is interesting to note that frameshift mutations of the ß-spectrin gene at the 3' end allow the insertion of the mutant protein in the red cell membrane, leading to a defect in the auto-association of the spectrin dimers and consequent elliptocytosis. On the other hand, ß-spectrin Säo PauloII protein was absent in the red cell membrane, leading to spectrin deficiency, HS and the presence of acanthocytes

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.

The Agamma-195 (C->G) mutation in hereditary persistence of fetal hemoglobin is not associated with activation of a reporter gene in vitro

Hereditary persistence of fetal hemoglobin is an uncommon, benign disorder in which the expression of gamma-globin genes persists into adult life. Several point mutations have been associated with the increased gamma-globin gene promoter activity. We evaluated the -195 (C->G) mutation by a functional in vitro assay based on the luciferase reporter gene system. The results indicated that the increased promoter activity observed in vivo could not be reproduced in vitro under the conditions employed, suggesting that other factors may be involved in the overexpression of the gamma-globin gene containing the -195 (C->G) mutation. Furthermore, this is the first time that the -195 (C->G) mutation of the Agamma-globin gene has been evaluated by in vitro gene expression

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, aaNcoI, 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/(aa)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

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.

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 per cent 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-a2 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 chaim 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 per cent vs 79.3 per cent). The frequencies of b2-a2 and b3-a2 rearrangements were 28.1 per cent and 65.7 per cent, 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.

Microalbuminuria in sickle cell disease

The kidney is involved in virtually all individuals who inherit the suckle cell form of hemoglobin. though asymptomatic and relatively common, proteinuria in patients with sickle cell anemia (SS) over 40 years old is associated with reduced creatinine clearance. The subclinical incrase in urinary albumin is termed microalbuminuria and is a marker of preclinical glomerular damage. The aim of the present study was to determine the presence of microalbuminuria measured by radioimmunoassay in patients with sickle cell disease. The study inclused 41 patients with SS, 11 patients with hemoglobin SC disease, 4 subjects with Sß-thalassemia and 10 normal controls. All subjects were teenagers or adults. Sixteen SS patients (40%) and 1 SC (9%) and 1 Sß (25%) patient presented mean urinary albumin excretion (UAE) above normal values (30 mg/l). No correlation was observed between UAE and age, creatinine clearance, hemoglobin level or %HbF. These parameters, as well as the presence of ulcers, were not significantly differente between SS patients with and without UAE above 30 mg/dl. The high prevalence of microalbuminuria in patient with sickle cell anemia indicates that glomerular damage is common. The connection between microalbuminuria and clinical neplhropathy has been demonstrated in diabetes and may indicate a sign of early disease rather than a marker for susceptibility. Thus, microalbuminuria may be an early indicator of flomerular damage for patients with sickle cell disease

Differences in first-phase insulin release between normal blacks and whites

This study was designed to compare insuslin release during the intravenous glucose tolerance test (GTT) in normal blacks and whites. The study included 14 normal blacks (9 males and 5 females) and 15 normal whites (10 males and 5 females). The index of first phase insulin release (sum of 1 + 3 min insulin levels and total area under the curve for the first 10 min of the GTT) was significantly higher in blacks. The difference is discussed in terms of genetic and acquired trats