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Biomedical subjects

L Luzzatto

Publications and source records attributed to L Luzzatto.

At least 55 records · Page 3Linked to original sources
PubMed1998

Clinical and haematological consequences of recurrent G6PD mutations and a single new mutation causing chronic nonspherocytic haemolytic anaemia.

We have determined the causative mutation in 12 cases of glucose-6-phosphate dehydrogenase deficiency associated with chronic non-spherocytic haemolytic anaemia. In 11 of them the mutation we found had been previously reported in unrelated individuals. These mutations comprise seven different missense mutations and a 24 base pair deletion. G6PD Nara, previously found in a Japanese boy. Repeated findings of the same mutations suggests that a limited number of amino acid changes can produce the CNSHA phenotype and be compatible with normal development. The one new mutation we have found, G6PD Serres, is 1082 C-->T causing a 361 Ala-->Val substitution in the dimer interface where most other severe G6PD mutations are found. Now that several patients with the same mutation have been reported we can compare the resulting clinical phenotypes. For each mutation we find a reasonably consistent clinical picture, ranging from mild (G6PD Clinic) through moderate (G6PD Nashville) to severe (G6PD Beverly Hills and G6PD Nara).

Adolescent↗
PubMed1997

In vivo telomere dynamics of human hematopoietic stem cells.

Aging in vivo and cell division in vitro are associated with telomere shortening. Several lines of evidence suggest that telomere length may be a good predictor of the long term replicative capacity of cells. To investigate the natural fate of chromosome telomeres of hematopoietic stem cells in vivo, we measured the telomere length of peripheral blood granulocytes from 11 fully engrafted bone marrow transplant recipients and from their respective donors. In 10 of 11 donor-recipient pairs, the telomere length was significantly reduced in the recipient and the extent of reduction correlated inversely with the number of nucleated cells infused. These data provide internally controlled in vivo evidence that, concomitantly with their proliferation, hematopoietic stem cells lose telomere length; it is possible that, as a result, their proliferative potential is reduced. These findings must be taken into account when developing new protocols in which few stem cells are used for bone marrow transplantation or for gene therapy.

Adolescent↗
PubMed1997

Somatic mutation in paroxysmal nocturnal hemoglobinuria.

The mutation, occurring in a hematopoietic stem cell, creates an erythrocyte clone deficient in proteins capable of blocking complement-mediated lysis. The precise defect lies, however, in the biosynthesis of anchors to tether the proteins. Future research may explain how the clone gains a growth advantage (perhaps shedding light on aplastic anemia). Meanwhile, there remains the challenge of optimal diagnosis and management.

Erythrocytes↗
PubMed1997

Murine embryonic stem cells without pig-a gene activity are competent for hematopoiesis with the PNH phenotype but not for clonal expansion.

Paroxysmal nocturnal hemoglobinuria (PNH) develops in patients who have had a somatic mutation in the X-linked PIG-A gene in a hematopoietic stem cell; as a result, a proportion of blood cells are deficient in all glycosyl phosphatidylinositol (GPI)-anchored proteins. Although the PIG-A mutation explains the phenotype of PNH cells, the mechanism enabling the PNH stem cell to expand is not clear. To examine this growth behavior, and to investigate the role of GPI-linked proteins in hematopoietic differentiation, we have inactivated the pig-a gene by homologous recombination in mouse embryonic stem (ES) cells. In mouse chimeras, pig-a- ES cells were able to contribute to hematopoiesis and to differentiate into mature red cells, granulocytes, and lymphocytes with the PNH phenotype. The proportion of PNH red cells was substantial in the fetus, but decreased rapidly after birth. Likewise, PNH granulocytes could only be demonstrated in the young mouse. In contrast, the percentage of lymphocytes deficient in GPI-linked proteins was more stable. In vitro, pig-a- ES cells were able to form pig-a- embryoid bodies and to undergo hematopoietic (erythroid and myeloid) differentiation. The number and the percentage of pig-a- embryoid bodies with hematopoietic differentiation, however, were significantly lower when compared with wild-type embryoid bodies. Our findings demonstrate that murine ES cells with a nonfunctional pig-a gene are competent for hematopoiesis, and give rise to blood cells with the PNH phenotype. pig-a inactivation on its own, however, does not confer a proliferative advantage to the hematopoietic stem cell. This provides direct evidence for the notion that some additional factor(s) are needed for the expansion of the mutant clone in patients with PNH.

Animals↗
PubMed1996

High-level regulated expression of the human G6PD gene in transgenic mice.

The glucose-6-phosphate dehydrogenase-encoding gene (G6PD) belongs to a group with constitutive expression in all tissues. The regulation of these housekeeping genes is poorly understood, as compared to what is known about many genes whose expression is restricted to a particular tissue or stage of development, and which are often regulated by locus control regions (LCR) able to act over wide distances. In order to identify sequences in human G6PD which are necessary for its expression, we have generated transgenic mice carrying a 20-kb G6PD construct, including only 2.5 kb of upstream and 2.0 kb of downstream flanking sequence. All mice which carried the transgene (TG) expressed it, and the levels of expression detected in a range of tissues from three independent lines of mice were comparable to that of the endogenous murine G6PD. The variation in enzyme activity from tissue to tissue was remarkably similar for both the TG and the endogenous gene, and was shown to be due in both cases to variations in the steady-state mRNA levels.

Animals↗
PubMed1996

Incidence of second primary malignancies after a malignant tumor in childhood: a population-based survey in Piedmont (Italy).

We have studied the frequency of second primary malignancies (SPM) among the 2,328 children registered in 1967-1969 at the Childhood Cancer Registry of Piedmont, the largest population-based childhood cancer registry in Southern Europe. Since the population of Piedmont is not served by a conventional cancer registry covering all ages, SPMs were identified through a number of ad hoc surveys within a variety of sources. Eighteen SPM (all histologically diagnosed) were observed after a thorough survey conducted in the ontological departments in Piedmont and after a postal questionnaire addressed to general practitioners. Death certificates were also examined. The crude incidence rate was 116.5 per 100,000 person-years. Risk was higher among children whose first malignancy was diagnosed more recently (SIR = 9.8 for diagnoses in 1983-1989 vs. 4.5 for diagnoses in 1967-1974). The same tendency was confirmed in analyses restricted to children in whom leukemia was diagnosed as the first cancer. Clinical data regarding the treatment of the first malignancy were available for 16 children out of 18: 15 had received chemotherapy and 12 radiotherapy (9 SPM originated in the irradiation field). The interest of measuring the risk of SPM on a population basis (and not only in clinical series) and the advantage of close cooperation between epidemiologists and clinical oncologists are underlined.

Adolescent↗
PubMed1996

Glucose 6-phosphate dehydrogenase mutations causing enzyme deficiency in a model of the tertiary structure of the human enzyme.

Human glucose 6-phosphate dehydrogenase (G6PD) has a particularly large number of variants resulting from point mutations; some 60 mutations have been sequenced to date. Many variants, some polymorphic, are associated with enzyme deficiency. Certain variants have severe clinical manifestations; for such variants, the mutant enzyme almost always displays a reduced thermal stability. A homology model of human G6PD has been built, based on the three-dimensional structure of the enzyme from Leuconostoc mesenteroides. The model has suggested structural reasons for the diminished enzyme stability and hence for deficiency. It has shown that a cluster of mutations in exon 10, resulting in severe clinical symptoms, occurs at or near the dimer interface of the enzyme, that the eight-residue deletion in the variant Nara is at a surface loop, and that the two mutations in the A- variant are close together in the three-dimensional structure.

Amino Acid Sequence↗
PubMed1996

Independent origin of single and double mutations in the human glucose 6-phosphate dehydrogenase gene.

The vast majority of both polymorphic and sporadic G6PD variants are due to single missense mutations. In the four polymorphic variants that have two point mutations, one of the mutations is always 376 A-->G (126 Asn-->Asp), which on its own gives rise to the nondeficient polymorphic variant, G6PD A. In a study of G6PD deficient patients who presented with clinical favism in Spain, we have found a new polymorphic variant that we have called G6PD Malaga, whose only abnormality is a 542 A-->T (181 Asp-->Val) mutation. This is the same mutation as previously found in association with the mutation of G6PD A in the double mutant, G6PD Santamaria. G6PD Malaga is associated with enzyme deficiency (class III), and the enzymic properties of G6PD Malaga and G6PD Santamaria are quite similar, indicating that in this case the effects of the two mutations are additive rather than synergistic. G6PD Santamaria might have been produced by recombination between G6PD A and G6PD Malaga; however haplotype analysis, including the use of a new silent polymorphism, suggests that the same 542 A-->T mutation has taken place independently in a G6PD B gene to give G6PD Malaga and in a G6PD A gene to give G6PD Santamaria. These findings help to outline the relationship and evolution of mutations in the human G6PD locus.

Amino Acid Sequence↗
PubMed1996

Serum erythropoietin levels in paroxysmal nocturnal haemoglobinuria: implications for therapy.

In order to assess the rationale and possible indications for the use of recombinant erythropoietin in paroxysmal nocturnal haemoglobinuria (PNH), we have measured endogenous erythropoietin (Epo) levels in 18 patients with PNH and in 44 patients with iron deficiency anaemia (IDA). In both groups of patients we found a significant inverse correlation between Epo and haemoglobin (Hb). However, the mean Epo level was significantly higher in the PNH group (385 mU/ml) than in the IDA group (136 mU/ml). The range of Epo levels at any given Hb was greater in the PNH group than in the IDA group. There was a significant positive correlation between Epo and absolute reticulocyte count. Since Epo administration is unlikely to benefit patients with high levels of endogenous Epo, we conclude that in the majority of patients with PNH there is no indication for treatment with Epo.

Erythropoietin↗
PubMed1996

The dual pathogenesis of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired blood disease with distinct and rather peculiar characteristics that have puzzled hematologists for more than a century. PNH cells are deficient in a set of membrane proteins that have in common a glycolipid anchor. We refer to this combination of deficiencies as the PNH abnormality or the PNH phenotype. Biochemical analysis has recently made it possible to pinpoint the metabolic block in PNH cells to an early step in the biosynthesis of the glycolipid anchor. This block is due in turn to the deficiency of a protein, called PIG-A, which is encoded by an X-linked gene. Expression cloning of the PIG-A gene has been followed by the identification in patients with PNH of somatic mutations in this gene that inactivate or impair the function of the PIG-A protein. These findings explain in full the molecular basis of the PNH abnormality, but they do not explain how the PNH clone, which is biochemically defective, can expand to the extent of contributing a substantial proportion of the patient's hematopoiesis. Thus a second factor is required to explain the pathogenesis of PNH. This is most likely the coexistence of an element of bone marrow failure that produces, paradoxically, a survival or growth advantage for the PNH clone. The notion that the injury causing failure of normal stem cells spares selectively cells with the PNH phenotype is supported by a number of observations, including the finding of multiple independently arisen PNH clones in patients with PNH.

Bone Marrow↗
PubMed1996

Somatic-cell selection is a major determinant of the blood-cell phenotype in heterozygotes for glucose-6-phosphate dehydrogenase mutations causing severe enzyme deficiency.

X-chromosome inactivation in mammals is regarded as an essentially random process, but the resulting somatic-cell mosaicism creates the opportunity for cell selection. In most people with red-blood-cell glucose-6-phosphate dehydrogenase (G6PD) deficiency, the enzyme-deficient phenotype is only moderately expressed in nucleated cells. However, in a small subset of hemizygous males who suffer from chronic nonspherocytic hemolytic anemia, the underlying mutations (designated class I) cause more-severe G6PD deficiency, and this might provide an opportunity for selection in heterozygous females during development. In order to test this possibility we have analyzed four heterozygotes for class I G6PD mutations: two with G6PD Portici (1178G-->A) and two with G6PD Bari (1187C-->T). We found that in fractionated blood cell types (including erythroid, myeloid, and lymphoid cell lineages) there was a significant excess of G6PD-normal cells. The significant concordance that we have observed in the degree of imbalance in the different blood-cell lineages indicates that a selective mechanism is likely to operate at the level of pluripotent blood stem cells. Thus, it appears that severe G6PD deficiency affects adversely the proliferation or the survival of nucleated blood cells and that this phenotypic characteristic is critical during hematopoiesis.

Autoradiography↗