Umberto Mazza

Significance of a new type of human fetal hemoglobin carrying a replacement isoleucine → threonine at position 75 (E 19) of the γ chain

SummaryA new type of hemoglobin F, in which isoleucine in position 75 (E 19) of the γ chain is replaced by a threonine residue, has been found in 29 out of 32 homozygotes for β thalassemia. The amount of this hemoglobin ranges from traces to 40% of the total Hb F. The same γ75 Thr chain is also present in the Hb F of 40% of normal newborns and premature infants examined, of one 14-week-old fetus and in one out of 3 patients with aplastic anemia and raised levels of Hb F. Our results strongly suggest that the synthesis of this new chain is under the control of a γ gene nonallelic with those coding for Aγ and Gγ chains.

Distribution of Kaposi's sarcoma herpesvirus sequences among lymphoid malignancies in Italy and Spain

Summary. In this study we have tested the distribution of Kaposis sarcoma herpesvirus (KSHV) DNA sequences throughout the spectrum of lymphoid neoplasia in Italy and Spain. 180 cases of lymphoid malignancies representative of the major histologic and immunophenotypic categories of B‐ and T‐cell tumours were analysed by means of a polymerase chain reaction‐based assay. KSHV sequences were consistently absent in all categories of lymphoid malignancies studied, with the exception of a subset of B‐cell non‐Hodgkins lymphomas localizing in the pleural, pericardial or peritoneal cavities, and fulfilling the diagnostic criteria of body‐cavity‐based lymphoma. The selective and consistent association of KSHV sequences with cases of body‐cavity‐based lymphoma throughout the spectrum of lymphoid neoplasms suggests that KSHV may be involved in the pathogenesis of this peculiar type of lymphoid malignancy.

Molecular heterogeneity of B-lineage diffuse large cell lymphoma

B‐lineage diffuse large cell lymphoma (B‐DLCL) arising de novo is characterized by a marked degree of clinical heterogeneity. To determine whether or not the clinical heterogeneity of de novo B‐DLCL is reflected by heterogeneity in the molecular features of these tumors, we investigated the pattern of distribution of several genetic lesions in 70 cases of de novo B‐DLCL at diagnosis. The panel of genetic lesions tested comprised the molecular alterations most frequently detected in B‐DLCL, including rearrangements of BCL2, BCL6, and MYC as well as deletions of 6q and mutations of TP53. One or more genetic lesions were detected in 39/70 cases of B‐DLCL. Isolated structural alterations of BCL2, BCL6, 6q or TP53 were detected in 8/70, 10/70, 11/70, and 3/70 cases, respectively. No isolated MYC lesions were detected. Six cases carried different combinations of two genetic lesions, including lesions of BCL2 + BCL6 (1 case), BCL2 + MYC (1 case), BCL2 + 6q (2 cases), or BCL6 + 6q (2 cases). One case had accumulated three genetic lesions, namely a rearrangement of BCL2 and BCL6 and a mutation of TP53. Overall, these data show that multiple distinct patterns of genetic lesions may associate with de novo B‐DLCL, indicating that the molecular pathogenesis of this group of lymphomas is characterized by a high degree of molecular heterogeneity. Genes Chromosom Cancer 16:21–30 (1996).

Analysis of microsatellite instability in chronic lymphoproliferative disorders.

Microsatellite instability (MSI) represents one specific pattern of genomic instability and is one of the genetic lesions most frequently detected in human neoplasia. Although MSI has been found to be associated with a wide variety of solid cancers, its involvement in lymphoid malignancies is virtually unexplored. In this study, we have investigated the presence of MSI in chronic lymphoproliferative disorders by comparing the pattern of nine microsatellite repeats (two tetranucleotides, two trinucleotides, and five dinucleotides) on autologous germline and tumor DNA of 23 patients, including 17 with B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL), four with hairy cell leukemia, one with lymphoplasmacytoid lymphoma, and one with T-cell chronic lymphocytic leukemia. All samples at diagnosis displayed a germline pattern of the microsatellites examined, thus suggesting that MSI is not involved in the pathogenesis of these lymphoproliferations. Also, no microsatellite alterations were observed in consecutive samples of B-CLL/SLL obtained from the same patient at various stages of the disease both before and after chemotherapy. Conversely, alterations in 3/9 microsatellite repeats were detected in one case of Richters syndrome which had evolved from a pre-existent B-CLL/SLL phase. Overall, the low frequency of MSI among chronic lymphoproliferative disorders adds further weight to the common view that the mechanisms and patterns of genomic instability in lymphoid neoplasia differ markedly from those commonly observed in solid cancers.

A benign form of thalassaemia intermedia may be determined by the interaction of triplicated α locus and heterozygous β‐thalassaemia

In this paper we report that the combination of a triplicated α globin locus with heterozygous β‐thalassaemia produces a clinical phenotype of thalassaemia intermedia in five Italian subjects from four unrelated families, while in two other cases the phenotype was thalassaemia minor.

Clinical and Haematological Data in 254 Cases of Beta-Thalassaemia Trait in Italy

The haematological and clinical data in 254 Italian subjects with β‐thalassaemia trait are reported. 46% of the patients were anaemic, 40% complained of weakness, 19% showed enlargement of the spleen and 10% enlargement of the liver. The haemoglobin levels ranged from 8 to 15.5 g/dl with a normal distribution and a mean of 12.73 for males, 10.93 for females and 11.34 for children (4–15 years). Reticulocyte counts and serum bilirubin levels were slightly increased and both showed a statistically significant relationship with haemoglobin levels. The serum iron level was increased in 27% and decreased in 6% of the cases. Haemoglobin A2 concentrations ranged from 3.5 to 8% with a normal distribution and a mean of 5.37; Hb F values were less than 1% in 36% and varied from 1 to 14% in the remainder. Red cell osmotic fragility was decreased in all but 6% of the subjects: low MCV, MCH and MCHC values were observed in 75%, 86% and 10% respectively. A comparison is made between the data and those obtained by other workers.

Genetic analysis of p53 and RB1 tumor-suppressor genes in blast crisis of chronic myeloid leukemia.

SummaryWe have investigated the involvement of the p53 and RB1 tumor-suppressor genes in 26 cases of chronic myeloid leukemia (CML) blast crisis, including 17 myeloid, eight lymphoid, and one megakaryoblastic crisis. The presence of p53 mutations in exons 5 through 9 was tested by the PCR-single-strand conformation polymorphism (SSCP) assay, followed by PCR-direct sequencing; in addition, loss of heterozygosity (LOH) at 17p13, the site of the p53 gene, was assayed by Southern blot. Given the variability of the mechanisms of inactivation of the RB1 gene in human tumors, a combination of Southern blot and mutational analysis by PCR-SSCP was used. p53 mutations were restricted to one case of myeloid blast crisis, showing a CGC→TGC (Arg→Cys) mutation at codon 283; two additional cases displayed LOH at 17p13 in the absence of p53 mutations. No molecular lesions of the RB1 gene were detected in any of the cases analyzed. These data indicate that inactivation of p53 and RB1 is a rare event in the molecular pathogenesis of CML acute transformation.

Molecular Defects Associated with the Acute Phase CML

Parts of the Bcr/Abl hybrid transcript supposed to be important for its transforming ability were sequenced in a series of CML blast crises, in order to evaluate the possible presence of alterations responsible for the disease transition from the chronic to the acute phase. In addition, the N- and Ki-ras as well as the p53 involvement was investigated by exploring their structure and expression in the same patients. We used traditional types of molecular analysis including Southern and Northern blot, together with methods that allow a rapid detection of point mutations and microdeletions, such as SSCP, single strand conformation polymorphism and direct sequencing. The results obtained may be summarized as follows: no alterations were found in the parts of the Bcr/Abl transcripts investigated in the present study (SH2, SH3 and the region surrounding codon 832); p53 alterations were observed in 5% and N- and Ki-RAS mutations in 5% of the cases examined. These molecular defects are therefore responsible for the clinical progression of the Ph1-positive CML only in a minority of cases.

Molecular pathology of AIDS-related lymphomas. Biologic aspects and clinicopathologic heterogeneity.

SummaryA high frequency of lymphoma in human immunodeficiency virus-infected individuals has been reported since the outbreak of the acquired immunodeficiency syndrome (AIDS) epidemic in 1982. AIDS-associated non-Hodgkins lymphoma (AIDS-NHL) is almost invariably derived from B cells and is classified as high- or intermediate-grade NHL, according to the working formulation. Two main histologic types are recognized, including small noncleaved cell lymphoma (SNCCL) and diffuse large cell lymphoma (DLCL). Pre-existing host factors putatively involved in lymphoma development include disrupted immunosurveillance, deregulated cytokine production, chronic antigen stimulation, and infection by Epstein-Barr virus (EBV). These alterations are associated with the development of multiple oligoclonal expansions which correspond to the clinical phase known as persistent generalized lymphadenopathy (PGL). The appearance of a true AIDS-NHL is characterized by the presence of a monoclonal B-cell population displaying several genetic lesions, including monoclonal EBV infection, c-MYC and BCL-6 rearrangements, RAS mutations, p53 inactivation, and 6q deletions. These genetic lesions cluster into two distinct molecular pathways, which specifically associate with the different histologic subtypes of AIDS-NHL, i.e., AIDS-SNCCL and AIDS-DLCL. The presence of distinct genetic pathways for AIDS-SNCCL and AIDS-DLCL correlate with a number of clinical features which distinguish these two groups of tumors, including differences in the age of onset, CD4 counts at the time of presentation, time elapsed since HIV infection, and clinical outcome.

Red Cell Glycolysis in a Case of 3‐Phosphoglycerate Kinase Deficiency

Abstract. A severe deficiency in red cell 3‐phosphoglycerate kinase was observed in a 62‐year‐old woman with haemolytic anaemia. Compared with a normal “young” red cell population with the same degree of reticulocytosis (6–7%) the 3‐phosphoglycerate kinase activity was reduced to 27%. A concomitant decrease of hexokinase and pyruvate kinase (both reduced by about 30%) was observed. The activities of glucoses‐phosphate dehydrogenase, phosphofructokinase, fructose‐1,6‐di‐phosphate aldolase, gIyceraIdehyde‐3‐phosphate dehydrogenase and lactate dehydrogenase were slightly increased (7 to 15%). The total glycolytic output of the deficient cells was decreased by 28% at pH 7.0, by 36% at pH 7.4 and by 34% at pH 7.6. Compared with a normal “adult” red cell population the 3‐phosphoglycerate kinase activity was reduced to 42% of the control values. Hexokinase, glyceraldehyde‐3‐phosphate dehydrogenase and lactate dehydrogenase activities were increased by approximately 20–50%. Phosphofructokinase activity was unchanged and pyruvate kinase only slightly increased. The steady state levels of the intermediates preceding the 3‐phosphoglycerate kinase step were increased 2–3 fold. The subsequent metabolites were decreased or practically unmodified. ATP, ADP, NAD+, and NADH were not affected. The reduced glutathione level was increased and the ratio of reduced to oxidized glutathione was doubled. The glycolytic output and its pH‐dependency were normal. The metabolic significance of the enzyme defect was assessed by the in vitro creation of cell stressing conditions, i.e. low pH and high pyruvate levels. In both cases, the 3‐phosphoglycerate kinase activity became limiting at low pH, glucose‐6‐phosphate accumulated at a faster rate and fructose‐ 1,6‐diphosphate and dihydroxyacetone phosphate disappeared more slowly in the deficient cells. After pyruvate loading these cells showed: a faster, more pronounced rise in 1,3‐diphosphoglycerate and a decrease in 2,3‐diphosphoglycerate (slightly increased in the controls): a drop in reduced glutathione (constant in the controls): constant ATP and slightly increased 3‐phosphoglycerate concentrations (both strongly increased in the controls): a slight increase in NADH (dropped to nil in the controls). Steady state glycolysis under normal conditions seemed to be affected by the enzyme deficiency. 3‐phosphoglycerate kinase however, became more severely limiting a low pH or after the addition of pyruvate. In these conditions, the flow was diverted to the 2,3‐diphosphoglycerate bypass, less ATP was produced and the concentration of reduced glutathione decreased. This may be assumed to have led to impairment of the ionic pump and may thus explain the increased haemolysis.