Hyperhomocysteinemia, a risk factor for vascular disease, is related to vitamin B-12, vitamin B-6, and especially folate deficiency, or to genetic factors such as mutations in methylenetetrahydrofolate reductase (MTHFR), an enzyme involved in the remethylation pathway of homocysteine to methionine. Recently, a C677 --> T mutation identified in the MTHFR gene was found to be frequently associated with decreased MTHFR activity and an elevated plasma homocysteine concentration. Since hyperhomocysteinemia seems to be determined by both genetic and environmental factors, we studied the interactions between MTHFR (phenotype and genotype) and folate status, including methyltetrahydrofolate (methylTHF), the product of MTHFR, on the homocysteine concentration in 52 healthy subjects, (28 women and 24 men; mean age, 32.7 years). MTHFR activity seems to be dependent on folate status, as shown by a lower activity in folate-deficient subjects and a return to normal values after supplementation with folic acid, and also by a decreased enzymatic activity on phytohemagglutinin (PHA)stimulated lymphocytes grown in a folic acid-deficient medium. Conversely, the C677 --> T mutation seems to influence folate metabolism. Subjects who were homozygous for this mutation (+/+) had significantly higher plasma homocysteine and lower plasma folate and total and methylfolate levels in red blood cells (RBCs) than heterozygous (+/-) and normal (-/-) subjects. The ratio of RBC methylfolate to RBC total folate was, respectively, 0.27 in +/+, 0.66 in +/-, and 0.71 in -/-. This mutation seems to have an impact on methylTHF generation, These data illustrate the interactions between nutritional and genetic factors.

The t (9;22) translocation is present in about 95 per cent of chronic myelogenous leukemia and in a significant subset of acute leukemias, mainly of the lymphoid subtype. This chromosomal rearrangement leads to the fusion of the bcr and c-abl genes and to the transcription of leukemia-specific bcr-abl mRNAs. The accurate identification of the t (9;22) translocation relies on cytogenetics (conventional or Fish) and molecular techniques. The detection of residual Ph positive cells following bone marrow transplant or interferon therapy is critical and has relevant therapeutic implications.

Systemic mast cell disease (SMCD) is a disorder characterized by a mast cell proliferation in various tissues. Mast cells express the c-kit proto-oncogene. A few cases of c-kit mutations have been described in SMCD. We report an aggressive SMCD in a patient who presented with a bone marrow infiltration by abnormal mast cells. Molecular studies of mast cell DNA and RNA revealed a new c-kit heterozygous mutation (Asp820Gly). This mutation leads to a drastic amino-acid change and is located close to the highly oncogenic Asp816Val. These findings suggest that the Asp820Gly has a potential role in c-kit activation.

Mastocytosis are a group of diseases characterized by abnormal proliferation of mast cells. Various forms are observed in respect to the organ system involved, clinical manifestations, and association with hematological disorders. The c-kit proto-oncogene encodes for a receptor tyrosine kinase, which plays a crucial role in hematopoiesis, especially in mast cell growth and differentiation. Mutations in the tyrosine kinase domain of c-kit have been reported in murine and human malignant cell lines, and more recently in some cases of human mast cell diseases. The biochemical and clinical aspects of these mutations are reviewed with special emphasis on the experiments which demonstrate their role in oncogenesis and mast cell proliferation.

Four hundred fifty subjects were screened for the 1691 G --> A mutation in the factor V gene. Two hundred ninety-seven patients were referred to us for unexplained thrombosis, 133 were family members of these patients and 20 were normal subjects. We studied the relationships between the mutation, resistance to APC and thrombosis. Among the 450 subjects tested, 65 belonging to 42 families were found to have the 1691 G --> A mutation in one (n = 61) or both alleles (n = 4). The prevalence of the mutation in the thrombotic patients was 13%. Resistance to APC was tested for in 247 subjects not on anticoagulant treatment (4 homozygous and 44 heterozygous for the mutation, and 199 individuals without the mutation). Incomplete cosegregation of heterozygosity for the 1691 G --> A mutation with APC resistance (APC-SR < 2.4 or n-APC-SR < 0.75) was observed, showing that the functional assay alone is insufficient for a firm diagnosis. In patients carrying the mutation, elevated levels of prothrombin fragment 1 + 2 and D-dimers pointed to increased thrombin generation in vivo. Clinical manifestations in the heterozygous subjects were very similar to those reported in heterozygous PC or PS deficiencies, but the first thrombotic event occurred later than in PC- or PS-deficient patients. Homozygosity for the factor V gene mutation appears to be a far more benign thrombotic disorder than homozygous PC and PS deficiencies.

We report the set-up of a denaturant gradient gel electrophoresis (DGGE) assay to screen for mutations in the whole coding sequence of the p53 gene. These DGGE experimental conditions were applied to the analysis of the p53 gene in acute leukemias. Forty adults with acute myelogenous leukemia (AML) and 21 with acute lymphoid leukemia (ALL) were investigated. Eleven of the AML patients were investigated at the time of the initial diagnosis and at relapse. In contrast with most reports based on amplified fragments analyzed by single-strand conformation electrophoresis and focusing on exons 5 to 8, we analyzed the whole coding sequence of the gene. Two of the 40 AML patients displayed a point mutation in exon 7; it was either an A to G substitution that converted Tyr-234 to Cys, or a G to A change that converted Arg-248 to Gln. The screening procedure led to the discovery of several intronic and exonic polymorphisms. These results confirm the low incidence of p53 mutations in acute leukemias and suggest a limited role of the p53 protein in leukemogenesis. The computerized modeling and electrophoresis parameters presented here provide a powerful tool for the exhaustive characterization of p53 mutants in all kinds of malignancies.

We report a case of mild, clinically asymptomatic, immune thrombocytopenia after allogenic bone marrow transplantation (BMT) for chronic myeloid leukemia (CML) caused by the presence of a recipient-origin Br(a) antibody that recognized the donor platelets. Although the antibody titer decreased, it remained detectable more than 3 years after BMT. Chimerism studies were performed combining cytogenetics, blood cell phenotype studies, and genomic amplification of hypervariable sequences. Cytogenetic studies and molecular analysis of peripheral blood cells, purified B- and T-lymphocyte subpopulations, and bone marrow colonies showed the hematopoiesis to be of donor origin, but absorption-elution experiments with peripheral RBCs showed a small amount of recipient RBCs. The CML chimeric transcript was also detected by means of polymerase chain reaction on samples collected until day +867 post-BMT. This case shows that recipient-origin platelet alloantibodies can cause thrombocytopenia after BMT and that the persistence of small numbers of recipient cells (even leukemic) is not necessarily associated with hematologic relapse.

Extrahematologic manifestations, such as symptoms of rheumatic disease, have been recorded in association with myelodysplastic syndromes, including chronic myelomonocytic leukemia (CMML). We report the case of a 51-year-old woman with tumoral infiltration of the Achilles tendons bilaterally, indicating CMML. This diagnosis was confirmed at postmortem examination.

Anaemia is frequently encountered in daily practice. With full knowledge of its pathophysiology a rational classification is possible allowing a suitable approach for diagnostic investigations. In a first stage, the data provided by blood counts, erythrocyte constants and reticulocyte counts guide the diagnostic rationale. In cases with microcytic and hypochromic anaemia, measurement of ferritin level separates iron deficiency anaemia from the so-called "inflammatory" anaemias. A high number of reticulocytes points to haemolytic anaemia. Among the many causes of haemolysis, one must first look for autoimmune haemolysis. Elsewhere, the clinical context and morphological red cell abnormalities point to a hereditary disease affecting the erythrocyte membrane, enzymes or globin content. Although rare, microangiopathic anemia with schizocytosis and paroxysmal nocturnal haemoglobinuria must not be misdiagnosed. Bone marrow examination remains the clue in non-regenerative normochromic, normo- or macrocytic anaemias. In difficult cases, other investigations, such as cytogenetics, isotopic examination and progenitor culture, may help in characterizing the anaemia. Serum erythroproietin essays and plasma transferrin receptor counts will soon figure among the methods used to explore anaemias.

We looked for mutations of exons 5 to 8 of the P53 gene in 10 patients with acute myeloid leukemia (AML) and 17p monosomy, and 36 patients with AML and no cytogenetic abnormalities of 17p. DNA was analyzed by polymerase chain reaction, single-strand conformation polymorphism analysis, and nucleotide sequencing. Four of the 10 patients with 17p monosomy showed point mutation, single-nucleotide deletion, or insertion in exons 7 or 8. By contrast, only 1 of the 36 patients with AML and no cytogenetic abnormalities of 17p showed a mutation of the P53 gene in exons 5 to 8 (P less than .01). These results suggest that alterations of the P53 gene may have a role in leukemogenesis in some cases of AML. The fact that P53 gene mutations occurred more often in patients with 17p monosomy seems to support the “recessive” model of tumor suppressive activity of the P53 gene rather than the “dominant” model, in which alteration of only one allele is sufficient for the development of malignancy.

We have examined p53 alleles in 151 DNAs from patients with myelodysplastic syndrome using single-strand conformation polymorphism analysis of polymerase chain reaction products. We focused our study on the four highly conserved regions of the p53 gene and detected five patients with aberrantly migrating fragments. We confirmed the putative mutation in each case by direct sequencing analysis. Of these five patients, three had chromosome 17 monosomy associated with p53 mutation, one patient showed one mutated p53 allele and one wild-type allele, and the last patient demonstrated only the mutant allele, suggesting a homozygous state. Unlike many other types of human cancers, point mutations in the p53 tumor-suppressor gene appear to be a rare event in myelodysplastic syndromes.

An 18 year old boy presented with microcytic hypochromic anemia. Erythrocytic abnormalities and family studies suggested congenital sideroblastic anemia (CSA), but atypical features included absence of clinical iron overload, scanty iron deposits in mitochondria of late erythroblasts and reticulocytes, and a high platelet count. An unusual adhesion between bone marrow macrophages and reticulocytes was observed by electron microscopy. Haematological response was seen following pyridoxine administration, thus fending support to the diagnosis of CSA.

The polymerase chain reaction (PCR) allows the detection of minimal amounts of nucleic sequences and has been successfully used to test for the chronic myeloid leukemia-specific bcr/abl transcripts. We studied blood samples from 17 patients who had undergone allogeneic bone marrow transplantation for CML, using a modified polymerase chain reaction-based assay for the detection of leukemic mRNA. This nested PCR technique was found to be highly sensitive, detecting the chimeric bcr/abl transcript in 16 of 17 patients including several long-term survivors. Cytogenetic techniques failed to detect Ph mitoses. The clinical significance of the persisting bcr/abl transcript for long periods following BMT is poorly understood and remains to be elucidated by further studies.