Acute promyelocytic leukemia: the study of t(15; 17) translocation by fluorescent in situ hybridization, reverse transcriptase-polymerase chain reaction and cytogenetic techniques

Acute promyelocytic leukemia (AML M3) is a well-defined subtype of leukemia with specific and peculiar characteristics. Immediate identification of t(15;17) or the PML/RARA gene rearrangement is fundamental for treatment. The objective of the present study was to compare fluorescent in situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR) and karyotyping in 18 samples (12 at diagnosis and 6 after treatment) from 13 AML M3 patients. Bone marrow samples were submitted to karyotype G-banding, FISH and RT-PCR. At diagnosis, cytogenetics was successful in 10 of 12 samples, 8 with t(15;17) and 2 without. FISH was positive in 11/12 cases (one had no cells for analysis) and positivity varied from 25 to 93 per cent (mean: 56 per cent). RT-PCR was done in 6/12 cases and all were positive. Four of 8 patients with t(15;17) presented positive RT-PCR as well as 2 without metaphases. The lack of RT-PCR results in the other samples was due to poor quality RNA. When the three tests were compared at diagnosis, karyotyping presented the translocation in 80 per cent of the tested samples while FISH and RT-PCR showed the PML/RARA rearrangement in 100 per cent of them. Of 6 samples evaluated after treatment, 3 showed a normal karyotype, 1 persistence of an abnormal clone and 2 no metaphases. FISH was negative in 4 samples studied and 2 had no material for analysis. RT-PCR was positive in 4 (2 of which showed negative FISH, indicating residual disease) and negative in 2. When the three tests were compared after treatment, they showed concordance in 2 of 6 samples or, when there were not enough cells for all tests, concordance between karyotype and RT-PCR in one. At remission, RT-PCR was the most sensitive test in detecting residual disease, as expected (positive in 4/6 samples). An incidence of about 40 per cent of 5' breaks and 60 per cent of 3' breaks, i.e., bcr3 and bcr1/bcr2, respectively, was observed.

The Lebanese mutation as an important cause of familial hypercholesterolemia in Brazil

Familial hypercholesterolemia (FH) is a common autosomal disorder that affects about one in 500 individuals in most Western populations and is caused by a defect in the low-density-lipoprotein receptor (LDLr) gene. In this report we determined the molecular basis of FH in 59 patients from 31 unrelated Brazilian families. All patients were screened for the Lebanese mutation, gross abnormalities of the LDLr gene, and the point mutation in the codon 3500 of the apolipoprotein B-100 gene. None of the 59 patients presented the apoB-3500 mutation, suggesting that familial defective ApoB-100 (FDB) is not a major cause of inherited hypercholesterolemia in Brazil. A novel 4-kb deletion in the LDLr gene, spanning from intron 12 to intron 14, was characterized in one family. Both 5' and 3' breakpoint regions were located within Alu repetitive sequences, which are probably involved in the crossing over that generated this rearrangement. The Lebanese mutation was detected in 9 of the 31 families, always associated with Arab ancestry. Two different LDLr gene haplotypes were demonstrated in association with the Lebanese mutation. Our results suggest the importance of the Lebanese mutation as a cause of FH in Brazil and by analogy the same feature may be expected in other countries with a large Arab population, such as North American and Western European countries.

Renal dysfunction in patients with sickle cell anemia or sickle cell trait

Patients with sickle cell anemia (Hb SS) or sickle cell trait (Hb AS) may present several types of renal dysfunction; however, comparison of the prevalence of these abnormalities between these two groups and correlation with the duration of disease in a large number of patients have not been thoroughly investigated. In a cross-sectional study using immunoenzymometric assays to measure tubular proteinuria, microalbuminuria, measurement of creatinine clearance, urinary osmolality and analysis of urine sediment, we evaluated glomerular and tubular renal function in 106 adults and children with Hb SS (N = 66) or Hb AS (N = 40) with no renal failure (glomerular filtration rate (GFR)>85 ml/min). The percentage of individuals with microalbuminuria was higher among Hb SS than among Hb AS patients (30 vs 8 per cent, P<0.0001). The prevalence of microhematuria was similar in both groups (26 vs 30 per cent, respectively). Increased urinary levels of retinol-binding protein or ß2-microglobulin were detected in only 3 Hb SS and 2 Hb AS patients. Urinary osmolality was reduced in patients with Hb SS or with Hb AS; however, it was particularly evident in Hb SS patients older than 15 years (median = 393 mOsm/kg, range = 366-469) compared with Hb AS patients (median = 541 mOsm/kg, range = 406-722). Thus, in addition to the frequently reported early reduction of urinary osmolality and increased GFR, nondysmorphic hematuria was found in 26 and 30 per cent of patients with Hb SS or Hb AS, respectively. Microalbuminuria is an important marker of glomerular injury in patients with Hb SS and may also be demonstrated in some Hb AS individuals. Significant proximal tubular dysfunction is not a common feature in Hb SS and Hb AS population at this stage of the disease (i.e., GFR>85 ml/min)

Necrólise epidérmica tóxica secundária ao uso da citosina-arabinosídeo em dose intermediária / Toxic epidermal necrolysis after the use of intermediate dose of cytosine arabinoside

A necrólise epidérmica tóxica é afecçao dermatológica secundária ao uso de drogas e corresponde à síndrome de Lyell, relacionada ao eritema multiforme e à síndrome de Stevens-Johnson. Objetivos. Relatar um caso de necrólise epidérmica fatal secundária à citosina-arabinosídeo (Ara-C) em dose intermediária. Relato de Caso. Paciente do sexo feminino, com 16 anos de idade, portadora de leucemia linfóide aguda - LLA-L1. Iniciou tratamento segundo o protocolo do Grupo Brasileiro de Tratamento da Leucemia Infantil/85, alto risco. Na fase II da induçäo, após o uso de Ara-C na dose de 1,5g/m2, intravenoso, 12/12h x três dias, desenvolveu múltiplas lesöes cutâneas bolhosas, que aumentaram rapidamente por progressäo das bordas. As bolhas continham secreçäo serosa, evoluíram para ulceraçäo superficial central, com infecçäo secundária múltipla. Faleceu por septicemia, no 13 dia após o início do quadro dermatológico. Conclusäo. O Ara-C tem sido relacionado a diversas manifestaçöes de toxicidade dermatológica; no entanto, até o momento, näo há relato de necrólise epidérmica tóxica, sendo este o primeiro caso da literatura.

Fetal hemoglobin in patients with chronic renal disease

To investigate whether hemoglobin (Hb) synthesis is affected by different treatment protocols used for end-stage renal disease, we analyzed the electrophoretic pattern of hemoglobin in 136 adult patients with chronic renal failure. Forty-seven patients were not in a dialysis program (ND), 29 individuals were on continuous ambulatory peritoneal dialysis (CAPD), 33 patients were on hemodialysis(HD), and 27 subjects had received a kidney transplant (KT). We found 3.6 per cent hemoglobin C, 1.4 per cent hemoglobin S and 3.6 per cent beta-thalassemia minor as reported in other studies of Brazilian patients. In addition, we found increased fetal hemoglobin (Hb F) levels in 7.4 per cent of the patients which contrasts with the reported 0.01 per cent prevalence rate of hereditary persistence of Hb F in Brazil. Seven out of ten patients with elevated Hb F belonged to either the CAPD or the KT group. We postulate that stress erythropoiesis is probably the mechanism responsible for the Hb F increase in these patients. However, properly designed clinical studies are still necessary to clarify these questions.

The importance of molecular biology in hematologic disorders

The advances in molecular biology and recombinant DNA technology have permitted the understanding of numerous diseases. Some of the earliest advances have occurred in the field of hematology when these approaches revealed genetic alterations in the hemoglobin gene of patients with sickle-cell anemia and thalassemias. In the field of onco-hematology, these powerful tools allowed to identify the chemical and biological changes that happen during the transformation of normal to malignant cells. Futhermore, it has led to the development of new agents for cancer therapy, such as interleukins and interferons. We describe here basic concepts of molecular biology, and the effect this technology had on the diagnostic capabilities for defining, diagnosing, and predicting the natural history of hematologic diseases.

Location and rapid analysis of the intragenic BamHI polymorphic site of the factor IX gene

We report the precise location and a polymerase chain reaction (PCR)-based method for the analysis of the intragenic BamHI polymorphism of the factor IX (FIX) gene. After screening DNA samples from the Brazilian Black population by a selective amplification of a segment of the FIX gene containing entire exon 2, intron 2, and exon 3 followed by digestion of PCR products with BamHI, we were able to identify individuals presenting the polymorphic BamHI site. By DNA sequencing of selected samples, the dimorphic base was located at nucleotide number 6575 (intron 2). The PCR method outlined here allows rapid and easy analysis of this polymorphism. Its application may be particularly useful for carrier detection and prenatal diagnosis of hemophilia B in the Black population, thus far the only one that has been shown to be polymorphic for this site. Because of its apparent restrictive pattern it may also be used in combination with other markers for estimates of racial admixture in mixed populations in which the Black population is present (as is the case for most countries in the American continent).

Molecular analysis of hemophilia B in Brazilian patients

1. The molecular basis of hemophilia B was investigated in 19 Brazilian patients from 14 unrelated families. 2. Southern blotting of TaqI-digested DNA samples was employed for the screening of gene lesions followed by polymerase chain reaction and DNA sequencing for the characterization of mutations. 3. Three different gene mutations were characterized: a nonsense mutation at nucleotide 30875 (codon 252, CGA-->TGA), a partial deletion comprising exons 1-3 (at least 7.4 kb of extension), and a complete deletion (at least 42 kb of extension). These patients are now referred to as Ribeir ao Preto 1, Ribeir ao Preto 2 and Ribeir ao Preto 3, respectively. 4. The factor IX haplotype (composed of 7 polymorphic sites) associated with each mutation was determined. Comparisons with previous studies confirmed an independent origin for the nonsense mutation. 5. This study represents the first survey of gene lesions associated with hemophilia B in South America. The results indicate the presence of heterogeneous mutations, as observed in other populations. These results also contribute to the understanding of the mechanisms involved in the mutations affecting the FIX gene