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Biomedical subjects

L Luzzatto

Publications and source records attributed to L Luzzatto.

At least 91 records · Page 5Linked to original sources
PubMed1994

Cloning of the glucose 6-phosphate dehydrogenase gene from Plasmodium falciparum.

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is one of the human genetic traits that confer relative resistance against malaria caused by Plasmodium falciparum. It has been previously shown that this organism, during its intraerythrocytic development, produces its own G6PD, which has properties different from those of human G6PD. In order to investigate the role of this enzyme in parasite-host cell interactions, we have isolated the G6PD gene from Plasmodium falciparum as a set of overlapping lambda gt11 clones. By sequence analysis we have found a single open reading frame, uninterrupted by introns, coding for a protein of 910 amino acids, almost twice as long as any previously sequenced G6PD molecule. The P. falciparum G6PD mRNA is 5.1 kb in size and has an exceptionally long 5' untranslated region of some 1000 nucleotides. We have mapped the G6PD gene to chromosome 14. The C-terminal portion of the predicted protein, from amino acid 310-910 (except for an 'insert' of 62 amino acids), has 39% homology to human G6PD, with a number of characteristic, fully conserved peptides. The N-terminal portion of the predicted protein has no homology to G6PD, but it contains a peptide in which 7 out of 12 amino acids are identical to the putative glutathione binding site of human glutathione S-transferase.

Amino Acid Sequence↗
PubMed1994

Genomic organization of the X-linked gene (PIG-A) that is mutated in paroxysmal nocturnal haemoglobinuria and of a related autosomal pseudogene mapped to 12q21.

The PIG-A gene, whose product is involved in one of the early steps in the synthesis of glycan phosphatidylinositol (GPI) anchors, has been recently found to be defective in all cases of paroxysmal nocturnal haemoglobinuria (PNH). By isolating genomic clones from a human phage library we now show that the PIG-A gene consists of six exons (the first of which is non-coding) spanning 17 kb of DNA, and we have mapped the gene to chromosomal position Xp22.1. The PIG-A promoter has features of a housekeeping gene. We have also isolated additional clones which cross-hybridize to PIG-A cDNA, and we have thus identified an intronless PIG-A pseudogene (psi PIG-A), which we have mapped to chromosomal position 12q21. psi PIG-A cannot be functional because it contains several stop codons and a frameshift. These data make it possible to design primers for amplification of the entire PIG-A coding region, with exclusion of psi PIG-A sequences, which will facilitate characterization of PIG-A mutations in patients with PNH. Database searches revealed that PIG-A contains homologies with a number of glycosyl transferases and is highly homologous (45%) to the protein encoded by the yeast SPT14 gene.

Amino Acid Sequence↗
PubMed1994

Myelodysplasia in a patient with pre-existing paroxysmal nocturnal haemoglobinuria: a clonal disease originating from within a clonal disease.

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemolytic anaemia, clonal in nature, due to somatic mutation. PNH may evolve to aplastic anaemia; more rarely to a myelodysplastic syndrome (MDS) or to acute myeloid leukaemia (AML). We have studied a patient who suffered from PNH and later developed refractory anaemia with ringed sideroblasts (RARS) associated with trisomy 8. By testing peripheral blood cells with appropriate antibodies we have shown that all of the red cells, neutrophils and monocytes, as well as 20% of the lymphocytes, belonged to the PNH clone; in contrast, only 43% of neutrophils and 22% of monocytes belonged to the MDS clone. We infer that the MDS must have arisen from within the PNS clone.

Adult↗
PubMed1994

Management of pregnancy when maternal blood has a very high level of fetal haemoglobin.

Fetal blood normally has a higher oxygen affinity than maternal blood because of the predominance of haemoglobin (Hb) F in the former and of Hb A in the latter; this predominance facilitates the transfer of oxygen from maternal to fetal blood. We report two patients who had exclusively or predominantly Hb F in their blood and were managed differently. When patient 1 became pregnant she had regular exchange blood transfusions in order to reduce her Hb F from 80% to below 50%; patient 2, who had 100% Hb F, was not transfused before, during or after her pregnancy. Each patient delivered a normal healthy baby. We conclude that the differential oxygen affinity produced by the combination of Hb A in the maternal blood and Hb F in the fetal blood is not indispensable to ensure an oxygen supply adequate for normal fetal development and growth.

Adult↗
PubMed1994

Mutations in the PIG-A gene causing partial deficiency of GPI-linked surface proteins (PNH II) in patients with paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria (PNH) is due to the absence or marked reduction of glycan phosphatidylinositol (GPI)-anchored proteins on the surface of blood cells. Affected patients may have a population of red blood cells that are completely deficient (PNH III) or partially deficient (PNH II) in these proteins, or they may have both. PNH III has recently been shown to be due, in all cases examined, to a somatic mutation in the PIG-A gene, whose product is required for an early step in GPI anchor synthesis. We now show that two patients with PNH II cells also have somatic mutations of the same gene: these produce a partial rather than a total loss of PIG-A function.

Adult↗
PubMed1994

Tissue plasminogen activator for hepatic vein thrombosis in paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder thought to arise in a multipotent haemopoietic stem cell. A distinct clinical feature is a tendency to thrombosis, with a particular predilection for the hepatic veins (Budd-Chiari syndrome). We report here on two patients with PNH who developed hepatic vein thrombosis (HVT) and who were treated with tissue plasminogen activator (t-PA). Both patients had a marked clinical and radiological improvement following the t-PA treatment and remain well over 2 years and 6 years after the treatment. This method of thrombolysis for HVT occurring in PNH has only been reported in two previous patients with limited follow-up. We suggest that this therapy is a useful first-line treatment for PNH patients who develop HVT.

Adult↗
PubMed1994

Dyskeratosis congenita is a chromosomal instability disorder.

Dyskeratosis congenita (DC) is a rare inherited disorder characterized by dystrophic changes in the skin and mucous membranes, bone marrow failure, and a predisposition to malignancy. In the majority of families the pattern of inheritance of DC has been compatible with X-linkage, the most likely location being Xq28. The primary defect responsible for this disease remains unknown. As DC shares many features (congenital abnormalities, bone marrow failure) with the chromosomal instability disorder. Fanconi's anaemia (FA), several studies have focused on cytogenetic features in DC. Unlike in FA, cytogenetic studies on peripheral blood lymphocytes have shown no significant difference between DC and normal lymphocytes with or without prior incubation with clastogens (bleomycin, diepoxybutane, mitomycin-c, 4-nitroquinoline-1-oxide). However, studies on DC fibroblasts have shown abnormalities in both morphology (polygonal cell shape, ballooning, dendritic-like projections) and growth rate (doubling time about twice normal), as well as numerous unbalanced chromosomal rearrangements (dicentrics, tricentrics, translocations) in the absence of any clastogenic agents. Bone marrow metaphases from one out of three patients studied (the eldest of the three) also showed unbalanced chromosomal rearrangements in the absence of any clastogens. Cell-specific difference and a higher rate of chromosomal rearrangements in the older patients appear to correlate with the clinical evolution of the disease. These findings suggest that the DC defect predisposes DC cells to developing chromosomal rearrangements.

Adolescent↗
PubMed1994

High resolution of proteins by double-inverted gradient polyacrylamide gel electrophoresis (DG-PAGE).

We have designed a new method for high resolution electrophoretic separation of proteins that have similar molecular weights. The proteins migrate first through a conventional gradient gel, in which molecular friction increases as pore size decreases. The proteins then enter an inverted sodium dodecyl sulfate (SDS) gradient gel in which friction decreases; thus, smaller molecules gradually migrate faster and achieve improved separation from larger molecules, which remain near the border between the two gels. We therefore call this technique double-inverted gradient polyacrylamide gel electrophoresis (DG-PAGE). This technique was used to resolve mixtures of aldolase, horseradish peroxidase precursors, glucose 6-phosphate dehydrogenase and pyruvate kinase. By comparison with other established methods, we show that DG-PAGE has a higher resolving power, which achieves clear separation of proteins differing as little as 0.5 kDa in molecular weight.

Animals↗
PubMed1994

Retroviral-mediated gene transfer of a mutant H-ras gene into normal human bone marrow alters myeloid cell proliferation and differentiation.

To investigate the effects of mutant ras expression on the growth and differentiation of normal human bone marrow, we used retrovirus-mediated gene transfer. A retrovirus (HR-1) containing a mutant ras gene (H12-ras) in addition to the selectable neo gene was transferred by cocultivation of a packaging cell line with long-term cultures of normal human bone marrow. Controls were established by cocultivating aliquots of the same bone marrow with a retrovirus (VSN-2) containing only neo. The efficiency of gene transfer, as determined by the percentage of G418-resistant colony-forming units-granulocyte/macrophage (CFU-GM) immediately after termination of cocultivation, was similar: 8 +/- 4% with HR-1 and 5 +/- 3% with VSN-2. After a further week in long-term culture, there was an increase in the number and percentage of G418-resistant CFU-GM in both the HR-1-infected and VSN-2-infected marrows. Thereafter, the numbers of G418-resistant CFU-GM declined, becoming undetectable at 4 weeks. The time course of the production of G418-resistant colonies was not significantly different in HR-1- and VSN-2-infected marrows, indicating that H12-ras did not alter the proliferation of normal CFU-GM. However, the total cellularity of HR-1-infected marrow cultures was significantly greater than that of VSN-2-infected marrow cultures. This was due to increased cellular proliferation of HR-1-infected cultured cells, since differential counts showed a significant increase in myeloid blast cells together with a slight reduction in mature myeloid cells in HR-1-infected marrow compared to baseline and to VSN-2-infected marrow. No leukemic blast cell colonies were grown from HR-1-infected marrows or control marrows, and HR-1 infection did not confer serum independence. These data show successful retroviral infection of normal bone marrow progenitor cells and suggest that expression of mutant H12-ras in such cells results in enhanced proliferation of early myeloid cells at the expense of differentiation.

Animals↗
PubMed1993

Specific defect in N-acetylglucosamine incorporation in the biosynthesis of the glycosylphosphatidylinositol anchor in cloned cell lines from patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal disorder arising in a multipotent hemopoietic stem cell. PNH manifests clinically with intravascular hemolysis resulting from an increased sensitivity of the red cells belonging to the PNH clone to complement-mediated lysis. Numerous studies have shown that surface proteins anchored to the membrane via a glycosylphosphatidylinositol (GPI) anchor (including proteins protecting the cell from complement) are deficient on the cells of the PNH clone, leading to the notion that GPI-anchor biosynthesis may be abnormal in these cells. To investigate the biochemical defect underlying PNH we have used lymphoblastoid cell lines (LCLs) with the PNH phenotype obtained by Epstein-Barr virus immortalization of lymphocytes from nine patients with PNH. By labeling cells with myo-[3H]inositol we have found that PNH LCLs produce phosphatidylinositol normally. By contrast, PNH LCLs fail to incorporate [3H]mannose into GPI anchor precursors. When cell-free extracts of PNH LCLs and normal LCLs obtained from the same patients (and expected therefore to be isogeneic except for the PNH mutation) were incubated with uridine diphospho-N-acetyl[3H]glucosamine (UDP-[3H]GlcNAc), we observed complete failure or marked reduction in the production of N-acetylglucosaminyl(alpha-1,6)phosphatidylinositol and glucosaminyl(alpha-1,6)phosphatidylinositol by the PNH LCLs in all cases. These findings pinpoint the block in PNH at an early stage in the biosynthesis of the GPI anchor, suggesting that the defective enzyme is UDP-GlcNAc:phosphatidylinositol-alpha-1,6-N- acetylglucosaminyltransferase. The existence of PNH type III cells and type II cells is probably explained by the transferase deficiency being total or partial, respectively.

Acetylglucosamine↗
PubMed1993

Production and characterization of lymphoblastoid cell lines with the paroxysmal nocturnal hemoglobinuria phenotype.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic disorder caused by a somatic mutation in a hematopoietic stem cell. The fact that, in some cases, not only myeloid but also lymphoid cells are affected suggests that the mutation has occurred in a multipotent stem cell. By studying the expression of CD59 antigen (membrane inhibitor of reactive lysis) and of decay accelerating factor (DAF) on the lymphocytes of 16 patients with PNH, we found an abnormal population of lymphocytes (with absent CD59 and DAF) in 10 cases. From 4 of these patients we were able to produce Epstein-Barr virus-immortalized lymphoblastoid cell lines (LCLs) that have a PNH phenotype (absent CD59, DAF, and CD48). PNH LCL cells have apparently normal DAF messenger RNA despite not having DAF on their surface. These cell lines will be a valuable resource for further investigation of the defect or defects underlying PNH.

Adult↗
PubMed1993

V kappa gene segments rearranged in chronic lymphocytic leukemia are distributed over a large portion of the V kappa locus and do not show somatic mutation.

The structure of the human V kappa locus has been thoroughly investigated, but how the germ-line V kappa gene segment repertoire is actually sampled in kappa chain gene rearrangements is not known. In order to begin to answer this question we have polymerase chain reaction (PCR) amplified the rearranged V kappa genes from 26 kappa-expressing cases of chronic lymphocytic leukemia (CLL), followed by cloning and sequencing of the PCR product. All four V kappa gene families were represented amongst rearranged genes. In 25 out of 32 cases, the sequence of the rearranged gene matches perfectly that of 1 of 11 different known germ-line V kappa genes, indicating that no somatic mutation has occurred. Of the remaining 7 rearranged V kappa genes, 4 differ from known germ-line genes by only one or two amino acid residues; and 3 differ from each other and from all known sequences by 5 or more residues, suggesting that somatic mutation has occurred in these 3 cases. We conclude that: (a) in at least three-quarters of cases the rearranged genes are unmutated; (b) there is preferential usage of individual V kappa genes but not of V kappa gene families; and (c) the V kappa genes used are widely dispersed in the V kappa locus.

Aged↗
PubMed1993

Variants of glucose-6-phosphate dehydrogenase are due to missense mutations spread throughout the coding region of the gene.

Glucose-6-phosphate dehydrogenase (G6PD) is remarkable for its genetic diversity in humans. Many variants of G6PD have been described with wide ranging levels of enzyme activity and associated clinical symptoms. Fifty-eight different mutations have now been identified and these account for 97 named G6PD variants. The mutations are almost exclusively missense mutations, causing single amino acid substitutions. They are spread throughout the coding region of the gene, although there appears to be a cluster of mutations that cause a more severe clinical phenotype towards the 3' end of the gene. The absence of large deletions, frameshift mutations and nonsense mutations is consistent with the notion that a total lack of G6PD activity would be lethal.

Amino Acid Sequence↗
PubMed1993

G6PD haplotypes spanning Xq28 from F8C to red/green color vision.

The most telomeric region of the human X chromosome within band Xq28 consists of a gene-rich region of about 3 Mb which contains the genes for coagulation factor VIIIc, glucose-6-phosphate dehydrogenase (G6PD), and red/green color vision. We have studied five polymorphic sites from this region, in a sample of normal people from the Cosenza province of Southern Italy. These sites, which span a distance of some 350 kb, are in strong linkage disequilibrium. Of the 32 possible haplotypes only 10 were found, and 4 of these account for 80% of all X chromosomes analyzed. In addition, we found that all G6PD-deficient people with the G6PD Mediterranean mutation belong to only two haplotypes. One of these (Med 1) is found only within a small subregion of the area investigated, west of the Appennine mountain range. Most remarkably, all Med 1 G6PD-deficient individuals also had red/green color blindness. The more frequent haplotype (Med 2) is the same in Calabria and in Sardinia, where it accounts for about 90% of the G6PD Mediterranean mutations, despite the fact that gene flow between the populations of Sardinia and Southern Italy must have been limited. These data do not enable us to determine whether the two types of G6PD Mediterranean have arisen through two separate identical mutational events or through a single mutational event followed by recombination. However, the data indicate relatively little recombination over an extended region of the X chromosome and they suggest that the G6PD Mediterranean mutation is recent by comparison to the other polymorphisms investigated.

Adult↗
PubMed1993

G6PD Mediterranean accounts for the high prevalence of G6PD deficiency in Kurdish Jews.

The Jews of Kurdistan are a small inbred population with a high incidence of beta-thalassaemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Recently, it was reported that the beta-thalassaemia in this population shows an unusual mutational diversity; 13 different mutations were identified, of which 4 had not previously been observed in any other population. In contrast, we now report that the G6PD deficiency, which has the highest known incidence in the world, and which affects about 70% of males, is almost entirely attributable to a single widespread mutation, G6PD Mediterranean.

Female↗