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A Iolascon

Publications and source records attributed to A Iolascon.

At least 91 records · Page 5Linked to original sources
PubMed1997

Frequent de novo mutations of the ANK1 gene mimic a recessive mode of transmission in hereditary spherocytosis: three new ANK1 variants: ankyrins Bari, Napoli II and Anzio.

A subset of spherocytosis cases associated with mutations of the ANK1 gene present an apparently recessive inheritance pattern on a clinical and haematological basis. We identified three novel out-of-frame deletions in the ANK1 gene: allele Bari (1361delG), Napoli II (2883delC) and Anzio (3032delCA) in three Italian patients, two of whom have been splenectomized. Analysis of the cDNA showed small or trace amounts of ankyrin mRNAs in Bari, Napoli II and Anzio. The parents were normal clinically and haematologically and did not carry the mutations exhibited by their children. We confirmed the de novo character of the HS mutations based on paternity testing. Recessive HS associated with the ANK1 gene is probably rarer than initially thought, and spherocytosis may often be due to de novo mutations.

Ankyrins↗
PubMed1997

Localization of the congenital dyserythropoietic anemia II locus to chromosome 20q11.2 by genomewide search.

Congenital dyserythropoietic anemias (CDA) are genetic disorders characterized by anemia and ineffective erythropoiesis. Three main types of CDA have been distinguished: CDA I and CDA III, whose loci have been already mapped, and CDA II (MIM 224100), the most frequent among CDAs, which is transmitted as an autosomal recessive trait and is known also as "HEMPAS" (hereditary erythroblast multinuclearity with positive acidified serum). We have recruited a panel of well-characterized CDA II families and have used them to search for the CDA II gene by linkage analysis. After the exclusion of three candidate genes, we ob-tained conclusive evidence for linkage of CDA II to microsatellite markers on the long arm of chromosome 20 (20q11.2). A maximum two-point LOD score of 5.4 at a recombination fraction of .00 was obtained with marker D20S863. Strong evidence of allelic association with the disease was detected with the same marker. Some recombinational events established a maximum candidate interval of approximately 5 cM.

Anemia, Dyserythropoietic, Congenital↗
PubMed1997

Rapid detection of neonatal sepsis using polymerase chain reaction.

Clinical diagnosis of sepsis in newborn infants is not easy and there is no laboratory test with 100% specificity and sensitivity, with the exception of blood culture, the results of which are not available for at least 48-72 h. Polymerase chain reaction methodology has been used to diagnose different bacterial, viral and protozoal infections, and the possibility of amplifying the DNA region common to all bacteria could represent an optimal method for the diagnosis of sepsis. The authors have performed PCR in a group of 33 neonates at risk for early-onset sepsis, correlating molecular data with blood culture results. The presence of bacterial DNA in blood samples was evaluated, amplifying the DNA region encoding the 16S rRNA. There were no false negative results (four positive blood cultures and four positive PCR), with competitive costs and time. This method also allows the diagnosis of sepsis due to uncommon species and also, using a second PCR with specific primers, an aetiological diagnosis.

DNA, Bacterial↗
PubMed1997

Characterisation of CAH alleles with non-radioactive DNA single strand conformation polymorphism analysis of the CYP21 gene.

The major cause of congenital adrenal hyperplasia (CAH), a common recessive genetic disease, is the deficiency of steroid 21-hydroxylase (21OH), a microsomal enzyme encoded by the CYP21 gene. Although several CAH causing mutations have been identified in the CYP21 gene of patients with 21OH deficiency, genotyping of the 21OH locus is quite complex because of the high frequency of gene conversion and the presence of multiple mutations on single CAH alleles. In order to perform the complete characterisation of the CYP21 gene coding region more simply, we developed a highly sensitive, non-radioactive method allowing DNA single strand conformation polymorphism (DNA-SSCP) analysis. This method was applied to the characterisation of all the exons and intron-exon junctions of the CYP21 gene in five patients affected by the simple virilising form and one affected by the salt wasting form. In all samples showing SSCP signals, direct sequence analysis showed the presence of more than one single sequence variant. In particular, four mutations which are already known to cause the disease, 16 polymorphisms, and one newly identified C to T transition at position 849 were detected. A random sequence analysis, performed on 31 out of 81 exons showing a normal SSCP pattern, shows the method to be highly sensitive: no sequence variant was detected, thus confirming the validity of this non-radioactive DNA-SSCP analysis in characterising the CYP21 gene in patients with steroid 21OH deficiency. Notwithstanding the complete characterisation of all exons and exon/intron junctions of the CYP21 gene, no complete genotype/phenotype correlation was found in the panel of patients analysed, thus suggesting that characterisation of CAH alleles must be extended to outside the coding region of the CYP21 gene, most probably into the promoter region.

Adrenal Hyperplasia, Congenital↗
PubMed1997

MYCN oncogene amplification in neuroblastoma is associated with worse prognosis, except in stage 4s: the Italian experience with 295 children.

PURPOSE: To evaluate the prognostic role of MYCN oncogene amplification in children with neuroblastoma. PATIENTS AND METHODS: Of 694 children (age, 0 to 15 years) with previously untreated neuroblastoma, 295 (42%) were evaluated at diagnosis for MYCN gene amplification. RESULTS: Clinical characteristics and survival results of 295 patients studied and 399 not studied for MYCN were comparable. In 48 of 295 patients studied for MYCN (16%), the gene was amplified (> or = three gene copies). Amplification was more frequent in children older than 1 year, with abdominal tumor (18% v 7%), advanced disease, normal vanillylmandelic (VMA) urinary excretion, and high lactate dehydrogenase (LDH), ferritin, and neuron-specific enolase (NSE) serum levels. In patients studied for MYCN, the 5-year overall survival (OS) rate was higher for children aged less than 1 year (90% v 44%), with extraabdominal tumor, stage 1 or 2 versus 3 versus 4, and normal NSE, LDH, and ferritin serum levels. Patients with amplified MYCN had a worse OS (odds ratio [OR], 3.38; confidence interval [CI], 2.22 to 5.16). This association held after adjustment for other characteristics. The impact of MYCN amplification was greater in patients with favorable characteristics, in particular age (OR, 10.28 for infants; 2.08 for older children) and stage (OR, 35.3 for stage 1 to 2; 8.41 for stage 3; 1.76 for stage 4). However, of 29 children with stage 4s, all three with amplified MYCN survive. In a multivariate analysis, the prognostic role of MYCN amplification, age, and stage was confirmed, but the size of the effect of MYCN was dependent on age and stage. CONCLUSION: MYCN amplification is associated with a worse prognosis in children with neuroblastoma at all ages and stages except 4s. This association is most pronounced in children with otherwise favorable prognostic indicators, and in these children should be considered as an indication for more intensive intervention.

Adolescent↗
PubMed1997

Inactivation of cyclin-dependent kinase inhibitor genes and development of human acute leukemias.

A large body of evidence has definitely demonstrated that cancer development and/or progression is strictly linked to alterations of molecular mechanisms controlling the cell division cycle. In particular, those aberrations which cause a shortening of G1 phase length and a deregulated S phase entry seem to be very important. Two main tumor suppressor loci, involved in the cell cycle regulation, are frequently altered in human tumors. One is located on 13q14 chromosome and includes the gene coding pRb protein while the other is located on 9p21 chromosome and involves two genes, namely p16INK4A and p15INK4B which belong to the same gene family. While RB1 gene is scarcely altered in hematological tumors, the putative tumor suppressor gene(s) on 9p21 appear(s) to be frequently inactivated in some subtypes of cancers derived from hematopoietic tissues. This manuscript will review the main biochemical aspects of the cell division cycle with major emphasis devoted to the findings regarding the recently characterized small proteic mitotic inhibitors and to their possible role in cancer formation. Particular attention will be paid to the data concerning the incidence of p16INK4A (and p15INK4B) gene(s) inactivation in human acute lymphoblastic leukemias. Indeed, such gene(s) seems to be the main, and until now the unique, tumor suppressor gene consistently altered in this acute hematological cancer diseases. Finally, future directions in studies on the connection between cell cycle control and leukemogenesis will be analyzed.

Carrier Proteins↗
PubMed1996

Study of the molecular defects in glucose phosphate isomerase-deficient patients affected by chronic hemolytic anemia.

We have studied four unrelated Italian patients with chronic hemolytic anemia associated with glucose phosphate isomerase (GPI) deficiency. Using intronic primers, we were able to detect the gene alterations on the genomic DNA of the patients. Five different mutations were identified among the eight mutated alleles found: three missense mutations (301A,584T,1028G), one nonsense mutation (286T), and a four nucleotides deletion [Del 1473-IVS16(+2)]. All of these were new except for mutation 1028G, which was previously identified in a Japanese variant (GPI Narita). Two patients were homozygotes (301A/301A and 1028G/1028G), whereas the other two were compound heterozygotes sharing a common mutation [286T/584T and Del 1473-IVS16(+2)/584T]. The missense mutations were found to involve highly conserved amino acids, suggesting that these residues are crucial for the maintenance of the enzyme function. The mutation 286T results in a truncated protein of 95 amino acids in comparison with the 558 of the normal one. The four nucleotides deletion located at the junction of exon/intron 16(5'-TTGGTCGgtgagt-3') is the first GPI mutation affecting a splice site. Moreover one difference from the published sequence (473T-->G) was found in exon five in all of the eight alleles studied and in 30 normal subjects. Correlation was made between mutations, biochemical characteristics of the enzyme, and clinical course of the disease.

Adult↗
PubMed1996

The cisternae decorating the red blood cell membrane in congenital dyserythropoietic anemia (type II) originate from the endoplasmic reticulum.

We studied 20 individuals from 17 unrelated families with congenital dyserythropoietic anemia (type II; CDAII). The clinical phenotype was mild to moderate. The inheritance pattern was invariably recessive. Coomassie blue stained gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) show that band 3 was thinner and migrated slightly faster than usual. In addition, staining showed two unknown minor bands (in the patients), but not in normal controls, the obligate carrier parents, or in patients with other anemic syndromes. These minor proteins were studied using partial digestion, amino acid sequencing, Western blotting, immunofluorescence, and immunogold electron microscopy. They were identified as the glucose-regulated protein GRP78 and calreticulin that are resident proteins of the endoplasmic reticulum (ER). Using specific antibody, we showed that protein disulfide isomerase (PDI), a third major protein of the ER, was also present on the SDS-PAGE of red blood cell (RBC) ghosts. Immunofluorescence colocalized PDI with the dense discontinuous ring decorating the RBC membrane. Immunogold electron microscopy showed that PDI was localized in the lumen of the cisternae, confirming that these originate from the smooth ER. From a practical point of view, screening the above minor proteins in RBC membranes appears to be a straightforward and reliable diagnostic test for CDAII.

Amino Acid Sequence↗
PubMed1996

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 GI-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.

Adolescent↗
PubMed1996

Peculiar allelotype associated with susceptibility to neuroblastoma.

Human neuroblastoma (NB) is characterized genetically by deletions of the short arm of chromosome I and by MYCN amplification. Loss of heterozygosity (LOH) has been found frequently for region 1p36. We have studied restriction fragment length polymorphisms (RFLPs) by using anonymous and hypervariable region (HVR) sequences to demonstrate LOH for 1p loci in 50 Italian neuroblastoma patients. Twelve cases (25%) showed LOH at one or more loci. Locus D1S94 was the most frequently involved (8/12 cases with deletion; 67%). MYCN amplification was observed in 20% of the patients. We also studied the allelic distribution in the constitutional DNA of neuroblastoma patients and of healthy Italian subjects for loci D1S112 and D1S94. A significantly (P = 0.01) different allele frequency was detected in the two groups at locus D1S94, but not at D1S112. Furthermore, the NB population was not in Hardy-Weinberg equilibrium at the former locus. This new observation suggests the existence of an allelotype associated with the susceptibility to neuroblastoma.

Alleles↗
PubMed1996

Cytoskeletal behaviour in spectrin and in band 3 deficient spherocytic red cells: evidence for differentiated splenic conditioning role.

Based on quantitative analysis of red cell membrane proteins, hereditary spherocytosis (HS) can be divided into two main groups including isolated or ankyrin combined spectrin deficiency and band 3 reduction. Protein methyl esterification catalysed by protein carboxyl methyl-transferase (PCMT type II; EC 2.1.1.77) is a post-biosynthetic modification which is involved in the metabolism of damaged membrane proteins. We utilized the evaluation of erythrocyte membrane protein methyl esterification as a marker of cytoskeletal disarray in seven HS subjects with spectrin reduction and in seven patients with HS due to band 3 deficiency. Our results support the notion that band 3 deficient erythrocytes are not affected by an extensive cytoskeletal derangement. On the contrary, we found a remarkable increase of membrane methylation in the unsplenectomized, spectrin-deficient. HS patients, suggesting a striking membrane skeleton disarray. This phenomenon was not observed in the spectrin-deficient red cells of splenectomized patients. Therefore in spectrin deficient erythrocytes the induction of cytoskeletal damage, specifically recognized by PCMT type II, could be one of the splenic steps producing conditioned spherocytes.

Adolescent↗
PubMed1996

P16INK4A gene homozygous deletions in human acute leukaemias with alterations of chromosome 9.

Acute leukaemias are characterized by nonrandom 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 alpha-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: (1) 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.

Base Sequence↗
PubMed1996

Ankyrin Napoli: a de novo deletional frameshift mutation in exon 16 of ankyrin gene (ANK1) associated with spherocytosis.

We report a case of apparently recessive hereditary spherocytosis in an Italian child. The proband exhibited a reduction of overall ankyrin in the red cell membrane. The parents were free of any haematological manifestations. The VNDR associated with the ankyrin gene (ANK1) were consistent with the following diplotypes: AC11/ AC14 (father), AC14/AC14 (mother) and AC11/AC14 (child). The cDNA of the patient disclosed the expression of the AC11 allele only. As a consequence, we put forward the hypothesis of a de novo inactivation affecting the ankyrin allele of maternal origin (AC14) and accounting for the disease. PCR amplification of exons, SSCP analysis and nucleotide sequencing disclosed a polymorphism: GAC --> AAC; Asp --> Asn in codon 328 of exon 10, and a one-nucleotide deletion : CTG --> CG in codon 573 of the exon 16. This frameshift mutation placed in phase the TGA triplet that normally overlaps codons 636 and 637. Termination of translation near the middle of ankyrin mRNA coding sequence resulted, presumably, in its premature degadation. The present allele has been designated allele Napoli.

Amino Acid Sequence↗
PubMed1996

Homozygous deletions of cyclin-dependent kinase inhibitor genes, p16(INK4A) and p18, in childhood T cell lineage acute lymphoblastic leukemias.

p16(INK4A) and p18 proteins are highly specific inhibitors of cyclin-dependent serine/threonine kinase activities required for the overcoming of the G1 checkpoint in the eukaryotic cell division cycle. The frequent cytogenetic aberrations occurring in several human neoplasms at the level of their codifying genes along with their molecular function strongly suggest that they might be important tumor suppressor genes. We looked for homozygous deletions of p16(INK4A) and p18 genes in 21 cases of childhood T cell lineage acute lymphoblastic leukemia (ALL). Twenty of 21 patients (95%) had homozygous deletions of p16(INK4A) gene while three out of 21 (14%) showed p18 gene biallelic deletion. Loss of heterozygosity studies were performed in 18 of the T cell ALL investigated by means of two highly polymorphic 9p21 markers. The results obtained demonstrated that genetic deletions of different extension occur on the short arms of the 9 chromosome pair. Karyotypic analyses, performed in 13 cases, failed to demonstrate 9p alterations in 12 samples, (92%) thus demonstrating that p16(INK4A) gene homozygous deletions are not restricted to cases with cytogenetically detectable 9p aberrations. The high incidence of p16(INK4A) gene deletions in pediatric T cell lineage ALL suggests that this genetic alteration could represent an early and key event in the development of such a malignancy but it should not have any prognostic value. Conversely, the inactivation of p18 gene, observed in a lower but significant number of cases, could participate in the progression of acute leukemias towards a more aggressive disease. Finally, our results may suggest that p16(INK4A) protein plays a key role in the control of proliferation and/or differentiation of human T lymphocytes.

Adolescent↗
PubMed1995

Increased membrane-protein methylation in hereditary spherocytosis. A marker of cytoskeletal disarray.

Protein carboxyl methyltransferase of type II selectively recognizes L-isoaspartyl and D-aspartyl residues spontaneously occurring in proteins and peptide substrates. Membrane protein methylation levels increase with erythrocyte aging in circulation, in parallel with the spontaneous formation of abnormal aspartyl sites, due to protein intrinsic instability. We found that enzymic methyl esterification of erythrocyte membrane proteins in hereditary spherocytosis, a model of cytoskeletal disarray, is significantly increased compared to normal red blood cells. This cannot be explained by an increase in mean age of spherocytes, which are on the contrary significantly younger than control cells. No differences in cytosolic methyltransferase specific activity, as well as in the intracellular concentrations of the methyl donor adenosylmethionine and/or of the methylation inhibitor adenosylhomocysteine were observed. We identified bands 2.1, 4.1 and 4.2 as the main targets for increased methylation, whose levels were correlated with the degree of spectrin deficiency associated with this anemia. Our findings indicate that membrane-protein methyl esterification represents a marker of membrane structural alteration in vivo in spherocytosis. We hypothesize that either an increased accessibility of methylation sites normally not available to the methyltransferase, or accelerated formation of methyl-accepting sites in membrane proteins are present in spherocytosis.

Cytoskeleton↗
PubMed1995

5'-Deoxy-5'-methylthioadenosine phosphorylase and p16INK4 deficiency in multiple tumor cell lines.

5'-Deoxy-5'methylthioadenosine phosphorylase (MTA-Pase) gene is localized at the 9p21 region linked to the recently identified putative tumor suppressor gene, p16INK4, which appears implicated in the control of cell division cycle. The phosphorylase is a housekeeping enzyme involved in the purine and amino acid metabolism whose activity is evidentiable in all the normal tissues. Chromosomal deletions encompassing both MTAPase and p16INK4 genes cause the total absence of the enzymatic activity only in malignant cells, thus resulting in defined metabolic differences between malignant and normal cells. MTAPase deficiency was investigated by direct radiochemical assay method and by immunochemical techniques in 35 different human malignant cell lines established from several tumor types. The enzyme-deficient cells derived from breast, lung, ovary and liver cancer, malignant melanomas, malignant gliomas and liposarcomas. Two of the MTAPase-deficient cell preparations (from a liver carcinoma and from a melanoma) are primary cultures thus directly representing the original cancer genotypes. Several of the MTAPase-negative cells were studied for p16INK4 gene deletions and for p16INK4 protein deficiency. In all the examined samples a full correlation exists between the lack of MTAPase and that of p16INK4. A similar result was obtained analysing extracts of Vero cell line, which is a fibroblast MTAPase-negative cell line established from the kidney of a normal adult monkey. Conversely, Cos cells, which also are fibroblasts derived from monkey kidney, show both MTAPase and p16INK4 protein. These results: (i) demonstrate that the phosphorylase deficiency is distributed among almost all the most important human cancers; (ii) confirm and extend the tumor types were p16INK4 gene inactivation is observable and (iii) suggest that deletions at 9p21 (in humans) or at syntenic chromosomes (in other species) might represent a general mechanism of p16INK4 gene loss of function and possibly, in turn, of cancer development and/or progression.

Animals↗
PubMed1995

Involvement of the cyclin-dependent kinase-4 inhibitor (CDKN2) gene in the pathogenesis of lymphoid blast crisis of chronic myelogenous leukaemia.

Recent data suggest that homozygous deletion of the cyclin-dependent kinase 4 inhibitor gene (CDKN2), a putative tumour suppressor gene located on chromosome 9p21, represents a common genetic event in human cancer. As the molecular basis of the evolution of chronic myelogenous leukaemia (CML) into blast crisis remains largely unknown, we decided to investigate if the occurrence of similar deletions could represent one of the mechanisms underlying the disease progression. Whereas none of 22 chronic phase CML cases examined showed alterations, we found that 3/17 total blast crisis examined (18%) showed a homozygous deletion of the CDKN2 gene. The deletions were restricted to cases of lymphoid blast crisis, being present in 3/8 (40%) of the lymphoid and in none of the nine myeloid cases examined. The fact that the chronic phase DNA obtained at diagnosis in one of the cases lacks the homozygous deletion observed in blast crisis, suggests that the final deletion event took place concomitantly with the progression of the disease. Furthermore, the analysis of polymorphic regions on chromosome 9p21 flanking at both sides the CDKN2 gene, showed that deletions at 9p21 differ between cases and are characterized by a wide range of extensions. A concomitant search for a possible involvement of the p53 tumour suppressor gene in the same series of patients showed mutations of the gene and loss of heterozygosity at 17p only in myeloid blast crisis, suggesting the presence of distinct molecular pathways in the pathogenesis of lymphoid and myeloid blast crisis.

Base Sequence↗