Control of phosphofructokinase by fructose 2,6-bisphosphate in B-lymphocytes and B-chronic lymphocytic leukemia cells.

The levels of fructose 2,6-bisphosphate and glucose 1,6-bisphosphate and the activities of the key glycolytic enzymes have been studied in T- and B-lymphocytes, and in B-chronic lymphocytic leukemia cells (B-CLL). In both kinds of cells these two bisphosphorylated metabolites have been identified and are present at similar concentrations. Their phosphofructokinase, like that of other normal or tumoral cells, is sensitive to these activators. Fructose 2,6-bisphosphate is the most potent stimulator; it displays the properties of a positive effector. It greatly increases the affinity for fructose 6-phosphate and relieves the inhibition by adenosine triphosphate, without changing Vmax. This effect is also synergistic with adenosine monophosphate. Despite few differences in the activity of phosphofructokinase and in the content of its main effectors in B-lymphocytes and in B-CLL cells, the kinetic properties of the enzyme from B-CLL cells were different, the enzyme being more sensitive to fructose 2,6-bisphosphate (Ka 2 orders of magnitude lower) and to glucose 1,6-bisphosphate than the enzyme from normal lymphocytes. The results reported showing that phosphofructokinase from B-CLL lymphocytes is altered in regulatory properties and the observed changes, in comparison to phosphofructokinase from normal B-lymphocytes, fit well with the hypothesis that fructose 2,6-bisphosphate can also assume a regulatory role in these cancer cells characterized by proliferation and accumulation of relatively mature-appearing lymphocytes.

Immunophenotypic characteristics of blast crisis of chronic myeloid leukaemia: correlations with clinico-biological features and survival.

Blast cells from 40 patients with Philadelphia-positive chronic myeloid leukaemia (CML) in blast crisis were analysed by immunophenotypic methods. In 27 cases, BCR gene studies were also performed. By light microscopy morphology and cytochemistry the cases were classified as follows: undifferentiated (n = 7; 17.5%), myeloid (n = 27; 67.5%), and lymphoid (n = 6; 15%). On the basis of the immunological markers, the cases were reclassified as: myeloid (n = 17; 42.5%), megakaryoblastic (n = 17; 42.5%), and lymphoid (n = 6; 15%). The seven cases initially considered as undifferentiated by morphological and conventional cytochemical criteria were classified as myeloid (four cases) and megakaryoblastic (three cases) by marker analysis. The monoclonal antibody anti-myeloperoxidase (anti-MPO) was the most sensitive myeloid associated marker in these cases, being positive in five of them. A significant proportion (27%) of non-lymphoid blast crisis cases were CD7-positive, and myeloid markers were positive in the four lymphoid CML-CB cases studied. Analysis of the clinico-haematological characteristics on the various subgroups of patients showed that patients with lymphoid blast crisis had shorter duration of the chronic phase, more frequent extramedullary blastic involvement, more favourable response to therapy, and longer survival. Finally, a trend for an association between megakaryoblastic involvement of blast crisis and breakpoint localization in the 3' extreme of the M-bcr segment was also noted.

Analysis of the clinical relevance of the breakpoint location within M-BCR and the type of chimeric mRNA in chronic myelogenous leukemia.

It has been suggested that the breakpoint location within the M-BCR segment of chromosome 22 and the type of chimeric mRNA BCR/ABL (b2a2 or b3a2) are associated with differences in the clinical and hematological characteristics of chronic myelogenous leukemia (CML). To assist in clarifying this matter, in a series of Ph-positive CML patients the relationship of both the breakpoint location within M-BCR (n = 71) and the type of chimeric mRNA BCR/ABL (n = 40) with the chronic phase duration, patients' survival, and thrombopoietic activity was analyzed. Median survival for patients with breakpoints in zones 1+2+3 (n = 38) and zones 4+5 (n = 31) was 62 and 75 months, respectively, the difference being not significant; patients with breaks in zones 1+2 (n = 19) and zones 3+4+5 (n = 50) had a median survival of 50 and 67 months, respectively (P also not significant). Moreover, no significant differences were found in the survival of patients with b2a2 (n = 16) and b3a2 (n = 24) mRNA junctions. Finally, no differences were observed in the platelet or megakaryocyte counts between patients with breakpoints in extremes 5' and 3' nor between patients with b2a2 and b3a2 mRNA. The above results are in agreement with those reported in most recent studies, confirming the lack of clinical relevance of molecular pattern in CML.

Evaluation of the blue formazan spot test for screening glucose 6 phosphate dehydrogenase deficiency.

Several screening tests for glucose 6 phosphate dehydrogenase (G6PD) deficiency have been reported thus far, and a standardized method of testing was proposed by the International Council for Standardization in Hematology (ICSH). The screening test used in any particular laboratory depends upon a number of factors such as cost, time required, temperature, humidity, and availability of reagents. In this study, a direct comparison between three different G6PD screening methods has been undertaken. In 71 cases (50 hematologically normal volunteers, 9 hemizygous G6PD-deficient males, and 12 heterozygous deficient females), the blue formazan spot test (BFST) was compared with the conventional methemoglobin reduction test (HiRT) and the ICSH-recommended fluorescent spot test (FST-ICSH). In all cases, the results obtained with the three screening tests were correlated with the enzyme activity assayed spectrophotometrically. In hemizygous G6PD-deficient males, all cases were equally detected with the three methods: BFST (4.7-6.64, controls: 11.1-13.4), BMRT (score +3 in all 9 cases), and FST (no fluorescence in 9 cases). In heterozygous G6PD-deficient females, two methods detected 7 out of 12 cases (BFST: 8.71-11.75, controls: 11.1-13.4; and BMRT: score +3 in 7 cases), whereas the FST-ICSH missed all 12 cases that presented a variable degree of fluorescence. Although the sensitivity for G6PD-deficient carrier detection is the same for the BMRT and the BFST, the latter has the advantage of being semiquantitative and not merely qualitative. Unfortunately, none of the three screening tests compared here allowed the detection of the 100% heterozygote carrier state of G6PD deficiency.

[Molecular genetics in the diagnosis of acute leukemia and chronic lymphoproliferative syndromes in 121 cases]. / Contribución de la genética molecular al diagnoóstico de la leucemia aguda y síndromes linfoproliferativos crónicos a propósito de 121 casos.

BACKGROUND: The introduction of biology and molecular genetics in the hematological laboratory has brought about a new and spectacular advance in the study of cloning and cytological characterization of malignant hemopathies. The principal aim of the present study was to analyze the contribution of this new technology in the diagnosis of acute leukemia (AL) and chronic lymphoproliferative syndromes (CLS) through analysis of lymphoid clonality and genetic rearrangement proper to the lymphoid differentiation of the B and T cells. METHODS: The genetic rearrangement of the heavy chain immunoglobulins (IgH) and the beta (beta) and gamma (gamma) chains of the T receptor (TRC) in 121 patients with the following malignant hemopathies: acute myeloid leukemia (AML), 28 cases; acute lymphoblastic leukemia (ALL), 27 cases; and CLS, 66 cases. The Southern method was used. RESULTS: Clonality analysis: presence of genetic rearrangement (clonality) in all the cases of lymphoblastic AL (ALL) and CLS and in 5 of 28 cases of AML (3 IgH and 2 TRC). Strain analysis: presence of absolute coincidence (exclusive rearrangement of the IgH or TRC genes in proliferations of the B or T strain, respectively) in 18 of 27 cases (67%) of ALL, in 14 of 15 cases (93%) of T-CLS and in all cases (100%) of B-CLS. CONCLUSIONS: In malignant hemopathies the analysis of genetic rearrangement constitutes a method of great practical use for determining the presence of lymphoid clonality and is a good complement to conventional morphological and immunophenotypic procedures for cytological characterization of the same.

[Molecular analysis of chronic Philadelphia chromosome negative myeloid leukemia: study of 6 cases]. / Análisis molecular en la leucemia mieloide crónica cromosoma Filadelfia negativa: estudio de seis casos.

BACKGROUND: Patients with Philadelphia negative (Ph-) chronic myeloid leukemia (CML) constitute a small proportion of the total number of patients with CML. Molecular analysis of these cases has permitted recognition that some cases present a breakpoint in the bcr region of chromosome 22, that is, the alteration constituting the substrate of the Ph chromosome. To date, the number of patients analyzed to this regard is low. METHODS: Six patients with Ph negative CML who constituted part of a series of 96 patients diagnosed with CML over a period of 6 years were studied. Analysis of the BCR gene in the DNA of the leuko-concentrate of peripheral blood was carried out with the Southern Blot technique, using the 3' and 5' probes and Transprobe and the restriction enzymes Bgl II, Eco RI, Hind III and Bam HI. The principal clinical-hematological characteristics of the patients were analyzed. RESULTS: Breakpoints were observed in the bcr region of chromosome 22 in 3 of the 6 patients, all of whom presented typical CML clinical-hematological features. CONCLUSIONS: Half of the patients with Philadelphia negative (Ph-) chronic myeloid leukemia (CML) have a breakpoint in the bcr region of chromosome 22, a similar molecular pattern to the Ph positive CML and their clinical-hematological profile is indistinguishable from those with CML.

[Molecular study of red cell pyruvate kinase deficiency in 15 patients with chronic hemolytic anemia. Group of Erythropathology of Hematology and hemotherapy Association of Spain (HHAS)]. / Estudio molecular del déficit de piruvatocinasa eritrocitaria en 15 pacientes con anemia hemolítica crónica. Grupo de Eritropatología de la Asociación Española de Hematología y Hemoterapia (AEHH).

BACKGROUND: Identification of RBC pyruvate-kinase (PK) gene mutations by polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) followed by PK gene sequencing in positive cases has been assessed and the results obtained with a preliminary study of 15 unrelated patients of Spanish origin are presented. PATIENTS AND METHODS: Patients have been classified into two different groups: group 1, propositus (15 cases), and group 2, relatives of the patients included in group 1 (10 males and 5 females). In group 1, a PCR was followed by SSCP and sequencing, and in group 2, the PCR was followed by digestion with specific restriction endonucleases (PCR-ER). RESULTS: Group 1: from 15 patients included in the study 2 were identified as homozygous, 4 as heterozygous and 9 as compound heterozygous. In this group, were identify 26 affected alleles with 11 different mutations: T1456 10 alleles (38.6%), T721 3 alleles (11.6%), A1010, C514, C1015 and T1223 2 alleles (7.7%), and C1070, A1291, T1508, A1595 y T1675 one allele. Relatives from 8 out of 15 patients from group 1 showed the following pattern: homozygous (one case), heterozygous (10 cases), compound heterozygous (2 cases) and normal (2 cases). CONCLUSIONS: SSCP procedure followed by direct gene sequencing in positive cases is fast and simple enough to allow the identification of PK deficient variants, avoiding the need of biochemical characterisation of semipurified deficient enzyme, which is more cumbersome and time consuming. In addition, the PCR-ER method is a very useful tool for screening of the most frequent molecular variants, as well as, for the detection of the carrier condition of this enzymopathy (family studies).

Heterogeneity of "Mediterranean type" glucose-6-phosphate dehydrogenase (G6PD) deficiency in Spain and description of two new variants associated with favism.

Glucose-6-phosphate dehydrogenase (G6PD); EC 1.1.1.49 from thirty-six unrelated Spanish males was partially purified from blood, and the variants were characterized biochemically and electrophoretically according to the methods recommended by the world Health Organization. Subjects were from multiple geographic regions within Spain, and all suffered from hemolytic anemia, either acute (34 cases) or chronic nonspherocytic (2 cases). Almost all the variants studied presented residual erythrocyte G6PD activity ranging from 0 to 10% of normal, and five different mutants were responsible for the deficient phenotype. Three variants were similar to others previously described: G6PD Mediterranean (11 cases), G6PD Athens-like (3 cases), and G6PD Union (2 cases). The remaining variants were different from the numerous variants already reported and have been considered as new mutants. Provisionally they are called G6PD Betica (19 cases) and G6PD Menorca (1 case). The present study constitutes the first attempt to characterize the deficient G6PD variants found in Spain and supplies new data on the relationship between molecular characteristics of deficient variants and their clinical manifestations. The most important findings can be summarized as follows: (1) The Spanish population is characterized by an important heterogeneity in G6PD deficiency. (2) Although G6PD Mediterranean is very frequent, it presents a relatively high degree of polymorphism. (3) Favism has been observed associated with all kinds of variants described here. (4) G6PD Betica, which is the most frequent variant found in subjects of Southern Spanish origin, has been observed associated with favism in all cases except one.

Nondeletional alpha-thalassemia: first description of alpha Hph alpha and alpha Nco alpha mutations in a Spanish population.

Several different deletions underlie the molecular basis of alpha-thalassemia. The most common alpha-thalassemia determinant in Spain is the rightward deletion (-alpha 3.7). To our knowledge, however, no cases of alpha-thalassemia due to nondeletional mutations have so far been described in this particular Mediterranean area. Here, we report the existence of nondeletional forms of alpha-thalassemia in ten Spanish families. The alpha 2-globin gene was characterized in ten unrelated patients and their relatives only when the presence of deletional alpha-thalassemia was ruled out. The alpha 2-globin gene analysis was performed using the polymerase chain reaction (PCR) followed by restriction enzyme analysis or by allelespecific priming. This allowed the identification of a 5-base pair (bp) deletion at the donor site of IVS I (alpha Hph alpha) in 9 cases and the alpha 2 initiation codon mutation (alpha Nco alpha) in one case. Although these alpha 2-globin gene mutations are found in other mediterranean areas, our results demonstrate their presence in the Spanish population and suggest that the alpha Hph alpha/alpha alpha genotype is probably the most common nondeletional form of alpha-thalassemia in Spain.

Haemoglobin Lleida: a new alpha 2-globin variant (12 bp deletion) with mild thalassaemic phenotype.

Molecular studies of alpha-thalassaemias have revealed defects at different steps in the process of alpha-gene expression. It is not surprising, therefore, that in some cases a single mutation or small deletion can result in a structurally abnormal haemoglobin that produces the alpha-thalassaemia phenotype. In this report we describe a new unstable alpha-globin variant, Hb Lleida, in a Spanish patient with alpha-thalassaemia trait. The mutation was detected by single-strand conformation polymorphism in the third exon of the alpha 2-globin gene. Direct sequence analysis of the alpha-globin gene showed a 12 bp deletion as the only defect of the alpha 2- and alpha 1-globin genes. The propositus was revealed to be a heterozygous carrier, and two alleles were separated by electrophoresis. This deletion causes the loss of four aminoacid residues (from codon 113 to 116) and would be expected to produce an unstable haemoglobin, as a shorter alpha-globin chain variant is created with 137 amino acids instead of 141 amino acids present in a normal alpha-globin chain. However, no abnormal haemoglobin was found by either isoelectric focusing or haemoglobin electrophoresis. Since the deletion affects an aminoacid residue (114 Pro) involved in alpha 1-beta 1-globin chain contacts, the interaction required for efficient Hb assembly is also compromised. The resulting unstable alpha-globin chain is rapidly catabolized and unsuitable for haemoglobin tetramer formation, causing an alpha-thalassaemia trait phenotype in the heterozygous patient.

[Unilateral asterixis associated with anatomic cerebral lesions]. / Asterixis unilateral asociada a lesiones anatómicas cerebrales.

Three patients with unilateral asterixis associated with different vascular lesions of thalamus, basal ganglia and internal and external capsules of contralateral hemisphere are described. Unilateral asterixis is a highly indicative sign of focal cerebral lesion. Its pathophysiology is still unknown. It has been postulated that asterixis is a myoclonic phenomenon resulting from malfunction within neuronal circuits of central nervous system responsible for the active maintenance of posture.

Electrophoretic and kinetic studies of a new mutant red cell pyrimidine 5'-nucleotidase.

Molecular characteristics of a deficient pyrimidine 5'-nucleotidase (P5N) were studied in a partially purified red cell enzyme extract. The results showed a high Michaelis constant for uridine 5'-monophosphate, an acidic shift of the optimum pH and normal heat stability. Enzyme electrophoresis using a starch gel and histidine-citrate buffer pH 7.0 showed a single band with identical mobility to that of the 'minor' band of normal enzyme. This electrophoretic pattern supports the hypothesis that P5N deficiency is, at least in some cases, a consequence of the absence of a 'major' isoenzymatic band characteristically present in normal enzyme.

Relationship between lymphocyte size and enzyme activities in two morphological variants of B-chronic lymphocytic leukaemia.

The activities of the key glycolytic enzymes phosphofructokinase (PFK), pyruvate kinase (PK) and hexokinase in addition to adenosine deaminase, purine nucleoside phosphorylase (PNP) and lactate dehydrogenase (LDH) have been measured in lymphocytes from 39 cases with B-chronic lymphocytic leukaemia (B-CLL). According to the percentage of circulating large non-granular atypical lymphocytes (AL) the B-CLL cases were classified as: typical (less than 10% of AL; 28 cases) and atypical (10-25% AL; 11 cases). In both groups the median lymphocyte volume (MLV) was assessed and correlated with the correspondent enzyme activities. The MLV of B-CLL lymphocytes was significantly (p less than 0.001) decreased (149.9 +/- 19.4 fl) as compared to normal B lymphocytes (175.1 +/- 14.5 fl) and it was significantly (p less than 0.001) lower in typical B-CLL (141.8 +/- 12.2 fl) than in atypical B-CLL (172.0 +/- 17.2 fl). Furthermore, in patients with typical B-CLL, all enzyme activities when expressed as U/10(9) cells were, with the exception of PFK, significantly decreased compared to normal B lymphocytes. However, when the results were expressed as U/ml cells, only PK, PNP and LDH remained significantly low. These findings demonstrate that the determination of MLV in addition to morphology may be a useful tool to distinguish the two previously described morphological B-CLL variants (typical and atypical) and that these two different B-CLL groups are also distinguishable on the basis of three enzyme activities, PK, PNP and LDH which have been shown to be less dependent on cell size than the other enzymes, also studied here.

Erythrocyte fructose 2,6-bisphosphate content in congenital hemolytic anemias.

We have investigated the levels of fructose 2,6-bisphosphate and its synthesizing enzyme 6-phosphofructo-2-kinase in red blood cells from different congenital anemias. Fructose 2,6-bisphosphate concentration and 6-phosphofructo-2-kinase activity are markedly influenced by the number of reticulocytes in all the cases studied with the exception of homozygous pyruvate kinase deficiency, where no correlation was observed with the percentage of reticulocytes.

Heterozygous pyruvate kinase deficiency and severe hemolytic anemia in a pregnant woman with concomitant, glucose-6-phosphate dehydrogenase deficiency.

The aim of this paper is to describe the clinical and hematological characteristics of a 32-year-old woman with concomitant heterozygous pyruvate kinase (PK) and glucose-6-phosphate dehydrogenase (G6PD) deficiencies and severe hemolytic anemia during pregnancy. In 1964, Oski et al. described a family in which a clinically healthy woman was heterozygous for both PK and G6PD deficiencies. To our knowledge, the present case is the first described in which the same condition is associated with hemolysis. A heterozygous condition for both enzymopathies was clearly demonstrated by family study criteria, and all other causes of hemolytic anemia were eliminated. No evidence of genetic relationship between the two disorders was demonstrated. Since late onset of hemolysis in heterozygous PK-deficient women has been observed in association with pregnancy and the molecular characteristics of the concomitant deficient G6PD enzyme were kinetically favorable, partial PK deficiency is suggested as the major cause of hemolysis in this patient.

Combined study of lymphocyte phosphoglucomutase (PGM) and adenylate kinase (AK) isoenzymes in the early characterization of bone marrow engraftment.

A starch-gel electrophoretic study of 2 peripheral blood lymphocyte enzymes, phosphoglucomutase (PGM) and adenylate kinase (AK), was performed in 25 donor-recipient allogenic bone marrow transplantation (BMT) pairs. A different isoenzymatic pattern between donor and recipient for PGM, AK or both, was observed in 11 of these 25 pairs. From these 11 cases, an identical donor isoenzyme pattern was observed in 10 cases between 24 and 85 days (mean 41.8, SD 22.08) after BM infusion confirming engraftment, whereas in the remaining 1 the demonstration could not be substantiated due to early death. The present study demonstrates that, in addition to other cell markers, the simultaneous study of PGM and AK isoenzymes allows confirmation of BM engraftment in about 45% of cases, much earlier than is possible by means of red cell antigen analysis. Moreover, it is a simple, relatively inexpensive and rapid laboratory tool for a better characterization of the established chimera.

Congenital methemoglobin-reductase (cytochrome b5 reductase) deficiency associated with mental retardation in a Spanish girl.

Methemoglobinemia and mental retardation associated with NADH-diaphorase deficiency was found in a 2-year-old girl of Spanish origin. She showed no NADH-diaphorase activity in either erythrocytes or leukocytes, but electrophoretic studies of the hemolysate showed traces of an enzyme with normal mobility. Cytochrome b5 reductase activity was also found to be absent in the leukocytes of the propostius. Intermediate NADH-diaphorase activity was found in erythrocytes and leukocytes in her parents and her sister in accordance with the autosomal recessive mode of inheritance of this enzymopathy. The relationship between a generalized cytochrome b5 reductase deficiency and the progressive neurological involvement in our patient is discussed briefly.

Co-existence of hereditary spherocytosis and a new red cell pyruvate kinase variant: PK mallorca.

BACKGROUND AND OBJECTIVE: A partial red blood cell (RBC) pyruvate-kinase (PK-R) deficiency was found in a patient with concomitant hereditary spherocytosis (HS) and chronic hemolytic anemia. Clinical, biological and molecular studies were performed in the patient, his parents and a brother, in order to characterize the specific PK-R gene mutation and the inheritance mechanism of the transmission of both red cell defects in this particular family. DESIGN AND METHODS: Conventional biological studies were used to identify the PK-LR gene mutation responsible for hereditary transmission of PK-R deficiency and HS. The family study was completed with genotypic and RBC membrane protein analyses in the patient and his family. RESULTS: Molecular study of the PK deficiency was performed in all the family members and demonstrated a heterozygous condition for the 1516 G->A (506Val->Ile) mutation at the PK-LR gene in both the patient and his mother. Since this mutation has not been reported previously, it is provisionally named PK "Mallorca". The study of RBC membrane proteins demonstrated the existence of partial band 3 and protein 4.2 deficiencies in the propositus and his father but not in the mother and brother, who were also studied. These results support the dominant mode of inheritance of HS and PK-LR gene in this family. INTERPRETATION AND CONCLUSIONS: HS and PK deficiency are not exceptional in Spain. The co-existence of both RBC defects in the same patient, however, is very rare; only a few cases have been described to date. Our findings suggest that performing an elementary RBC enzyme survey in all patients with HS would help to determine the real frequency of this apparently rare association.