Brigida Coppola

Analysis of cyclin-dependent kinase inhibitor genes (CDKN2A, CDKN2B, and CDKN2C) in childhood rhabdomyosarcoma.

p16INK4A, p15INK4B, and p18 proteins are highly specific inhibitors of cyclin‐dependent serine/threonine kinase (CDK) activities required for G1‐S transition in the eukaryotic cell division cycle. Mutations, mainly homozygous deletions, of the CDKN2A (p16INK4A/MTSI) gene have been recently found in tumor cell lines and in many primary tumors. We looked for homozygous deletions of CDKN2A, CDKN2B (p15INK4B), and CDKN2C (p18) in 12 primary rhabdomyosarcoma (RMS) specimens and in five cell lines established from this cancer type. By means of polymerase chain reaction (PCR) and PCR‐single strand conformation polymorphism (PCR‐SSCP), we analyzed the presence of biallelic gene deletion or point mutation causing gene function loss. All the examined tumor cell lines (100%) and three of 12 (25%) primary tumors showed homozygous deletion of CDKN2A. Furthermore, no aberrant bands in primary tumors were detected via SSCP, suggesting the absence of mutations in the coding region. In all cases the deleted area at 9p21 also involved the CDKN2B gene. Conversely, no homozygous deletion or point mutations were detected when CDKN2C was analyzed. Our results strongly indicate that the p16INK4A (and/or p15INK4B) protein plays a key role in the development and/or progression of childhood rhabdomyosarcoma and suggest that this CDK‐inhibitor protein might control proliferation and/or differentiation of human muscle cells. Moreover, alteration of CDKN2C does not appear to be involved in the genesis of rhabdomyosarcoma. Genes Chromosom Cancer 15:217–222 (1996).

THROMBIN GENERATION IN CHILDREN WITH ACUTE LYMPHOBLASTIC LEUKEMIA: Effect of Leukemia Immunophenotypic Subgroups

Elevated plasma concentrations of endogenous thrombin generation markers and thrombotic events have been reported in children with leukemia. The aim of this study was to evaluate the effects of cancer and its treatment on thrombin generation (TAT levels) in children with acute lymphoblastic leukemia (ALL). The authors evaluated 32 children (23 M, 9 F) aged between 1 and 15 years (mean 6) affected by ALL (immunophenotypic subgroups: 16 common, 7 T, and 9 pre-B type). In all patients TAT levels at onset and after 5-6 doses of L-asparaginase were evaluated. TAT levels were higher in patients both at onset (13.04 +/- 10.90 ng/L) and after the 5-6 doses of L-asp (19.41 +/- 11.05 ng/L) with respect to controls (4 +/- 1 ng/L) (p < .001 and p < .001). TAT levels after 5-6 doses of L-asp were higher than those at onset (p < .001). Factorial ANOVA showed that at onset there was a significant effect of leukemia immunophenotypic subgroups upon TAT levels (p < .05) and no effect of inherited thrombotic risk factors. These results indicate that in children with ALL an important role is played by acquired thrombotic risk factors, among which the indirect cancer procoagulant activity has its importance.

Crigler-Najjar syndrome type II resulting from three different mutations in the bilirubin uridine 5′-diphosphate-glucuronosyltransferase (UGT1A1) gene

Editor—Crigler-Najjar syndromes (CN, MIM 218800) are inborn errors of metabolism characterised by unconjugated hyperbilirubinaemia resulting from the defective activity of the hepatic enzyme bilirubin uridine 5′-diphosphate-glucuronosyltransferase (B-UGT). CN syndrome has been classified into two types according to the degree of hyperbilirubinaemia and to the response to phenobarbital administration. The more severe CN type I is characterised by severe chronic non-haemolytic unconjugated hyperbilirubinaemia with high levels of serum bilirubin owing to the absence of bilirubin UGT activity. In the milder CN type II, bilirubin UGT activity is only decreased and a consistently significant reduction is obtained with phenobarbital treatment, which does not occur in CN I. Like other members of the UGT isozyme family, the two human liver bilirubin UGT isozymes, UGT1A1 and UGT1D, are encoded by the UGT1 gene complex through a mechanism of alternative splicing. Each gene has a unique promoter and a unique exon 1, while exons 2-5 are common to both genes.1 Most of the enzymatic activity results from the expression of the UGT1A1 gene.2 At the molecular level, CN I results from a number of different defects; nonsense (or frameshift) and missense mutations are represented in almost the same amounts both in homozygosity and in the compound heterozygous state.3 4 The milder phenotype in CN II patients seems to be mainly the result of homozygosity for missense mutations5 and more …

Homozygous Prothrombin Gene Mutationand Ischemic Cerebrovascular Disease

We report the case of a 31-year-old woman who, at the age of 26 suffered from an episode of superficial thrombophlebitis in the left leg, experienced two episodes of transient ischemic attacks at the age of 30 and had an ischemic stroke with left-sided hemiparesis at the age of 31 years. A cerebral CT scan showed an ischemic lesion in the right sylvian area involving the opercular and nucleocapsular regions. Her father had had an ischemic stroke at the age of 54 years and died at the age of 58; her mother had had a myocardial infarction at the age of 48 years and died at 51 years from breast cancer. Laboratory investigation of the patient demontrated high levels of fibrinogen, F II, F VII, F 1 + 2, FPA and ACA-IgG with low levels of HDL cholesterol associated with homozygosity for the 20210 A genotype. There were no other genetic or acquired prothrombotic defects. In conclusion, this case strongly suggests a clinically significant role ot the prothrombin gene mutation in both arterial and venous thrombosis.

High frequency of homozygous deletions of CDK4I gene in childhood acute lymphoblastic leukaemia

Summary. To determine the incidence of homozygous deletions of the newly identified tumour suppressor gene, CDK4I, molecular genomic DNA analyses by PCR technique were performed on primary neoplastic cells from 22 childhood acute leukaemias obtained at presentation. The blast cells derived in all the analysed cases from bone marrow. We found that none of acute myeloblastic leukaemias (four cases) showed the CDK4I alteration, whereas 6/13 (46%) common acute lymphoblastic leukaemias (ALLs) displayed homozygous deletions. Moreover, and even more important, all the blasts purified from ALLs derived from early lymphoid precursors (three early‐T ALLs and two pre‐B ALLs) showed the absence of CDK4I gene. When the entire coding sequence of the CDK4I gene from samples without homozygous deletions was analysed by the single‐strand conformational polymorphism method, no point mutations were identified. These results demonstrate that CDK4I gene deletions are very frequent and probably early events in childhood acute leukaemias of lymphoid origin and especially in early‐T and pre‐B ALLs. Moreover, the molecular mechanism of the loss of function of the gene is correlated, at least in childhood ALLs, almost exclusively to deletions and not to point mutations.

Resistance to activated protein C in thalassaemic patients: an underlying cause of thrombosis

Abstract: We evaluated 81 thalassaemia major and 4 thalassaemia intermedia patients (48 M, 37 F), median age 17 years; 62/85 patients were HCV‐positive, 3/85 HIV‐positive, 19/85 were splenectomized. Forty normal healthy children were recruited as the control group. The number of thrombotic events was studied retrospectively. Platelet poor plasma was filtered and quick‐frozen at ‐70°C until time of assay. APC resistance was measured in an activated thromboplastin time and results were expressed as normalized ratio. All tests were done with diluted 1 in 5 (v/v) factor V deficient plasma and with undiluted plasma. Molecular genetic investigation of factor V gene was performed with polymerase chain reaction, followed by digestion of amplified products with restriction enzyme Mnl I. Data obtained with molecular investigation revealed the presence of 4 heterozygous subjects for factor V Leiden (4.7%). Functional tests were able to detect all heterozygotes for factor V Leiden both with undiluted and with diluted plasma, and there were no false negative subjects. However, undiluted plasma revealed a greater number of false positive subjects (n = 15) than did diluted plasma. Therefore, tests done with undiluted and diluted plasma revealed a 100% sensitivity, while specificity was 81 % for undiluted plasma and 97% for diluted plasma. Only one thrombotic event was observed in one of the 85 studied patients, as a case of stroke in a thalassaemia intermedia patient with APC resistance. In the same patient an additional thrombogenic risk factor was represented by a pronounced haematocrit increase at the beginning of her tranfusion regimen.

p16INK4A gene homozygous deletions in human acute leukaemias with alterations of chromosome 9

Acute leukaemias are characterized by non‐random chromosomal aberrations which are often strictly related to the inactivation of tumour suppressor genes (TSGs). Alterations at the short arm of chromosome 9 have been reported in a remarkable percentage of acute lymphoblastic leukaemias (ALL) and have been suggested to cause the loss of activity of the putative TSG, p16INK4A (MTS1/CDKN2) gene. In order to evaluate the correlation between this gene inactivation and visible cytogenetic abnormalities, we have investigated p16INK4A homozygous gene deletions in 10 paediatric acute leukaemias of different cell lineages which demonstrated karyotype aberrations involving chromosome 9. Moreover, the dimension of the genetic alteration was evaluated by studying the loss of heterozygosity of two highly polymorphic markers of chromosome 9p, namely α‐interferon (IFNA) and D9S104, and the deletion of 5′‐methylthioadenosine phosphorylase (MTAPase) gene. Finally, the deletion of a gene belonging to p16INK4A family, the p18 gene, was analysed in these acute leukaemias. Our results demonstrated that: (i) the biallelic loss of p16INK4A gene is strictly related to a specific immunophenotype, namely ALL of T‐cell lineage; (ii) no significant correlation exists between alterations at chromosome 9p level and the homozygous deletions of p16INK4A gene; and (iii) p18 gene was not deleted in the examined cases. These findings suggest a possible correlation between the T‐lymphocyte phenotype and the expression of p16INK4A gene. Moreover, the absence of MTAPase activity seems to be a valuable marker of p16INK4A gene inactivation, thus indicating that the deleted chromosomal area on 9p21 very frequently involves the MTAPase gene.

Lactate dehydrogenase isoenzyme patterns in blood cells from histiocytosis X children

To find a clinical assay for histiocytosis X (HX) diagnosis, measurements were made of both activity and isoenzyme distribution of lactate dehydrogenase (LDH; EC 1.1.1.27) from the blood cells of 6 acute phase and 9 remission patients. A significant increase in the LDH activity measured in the monocytes and lymphocytes isolated from the blood of the acute phase patients was found. The increased activity was due to an enhancement of the normal pattern of LDH isoenzymes in these cells and not to a change in isoenzyme distribution. No increase was found in monocyte LDH isoenzymes from the patients in remission.

Alteration of cell division cycle regulation in human cancers: The role of CDKN2A gene

Cancer might be thought of as a disease characterized by a deregulated cellular growth. Thus, it is not surprising that some of the molecular components of cell division cycle machinery are altered in human tumors. The cell cycle of all post-embryonic eukaryotic cells (including malignant cells) is divided into four phases, namely: G1 phase (period prior to DNA synthesis), S phase (period of DNA synthesis), G2 phase (period between DNA synthesis and mitosis) and M phase (mitosis). Collectively, G1, S and G2 are called interphase, the cell cycle period distinct from division of the nucleus (mitosis) and cytoplasm (cytokinesis) (Pardee, 1989; Desai et al., 1992). The length of the S, G2 and M phases is remarkably similar in many different cells, while the greatest variation is seen in the length of G1. At some point late in G1, called restriction or R point, a cell becomes committed to traverse the remainder of the cell cycle. Thus, variations in cell cycle time are mostly due to variations in the length of G1 up to the R point (Pardee, 1989; Desai et al., 1992).