Paola Izzo | Università degli Studi di Napoli "Federico II" (original) (raw)

Papers by Paola Izzo

Research paper thumbnail of Alu-Mediated Genomic Deletion of the Serine/Threonine Protein Kinase 11 (STK11) Gene in Peutz–Jeghers Syndrome

Research paper thumbnail of Identification and molecular characterization of the CAMPANIA deletion, a novel α°-thalassemic defect, in two unrelated Italian families

American Journal of Hematology, 2010

Research paper thumbnail of Interleukin10 Receptor Down-Expression in Early-Onset Inflammatory Bowel Disease: A Case-Report

Research paper thumbnail of Cis-acting elements in the promoter region of the human aldolase C gene

We i~vesti~f~d the c&acting sequences involve m the expression of the human Adolph C gene by tran... more We i~vesti~f~d the c&acting sequences involve m the expression of the human Adolph C gene by transient tmnsf~t~ons into b~man neuroblastoma cells (SKNBE). We demonstrate that 420 bp of the Y-flanking DNA dimct at high efficiency the transcription of the CAT reporter gene. A deletion between -420 bp and -164 bp causes a 60% decrease ofCAT activity. Gel shift and DNase I footprmtmg analyses revealed four protected elements: A, B, C and D. Competition analyses indicate that Spl or factors sharing a similar sequence specificity bind to elements A and B, but not to elements C and D. Sequence analysts shows a half palindromic ERE motrf(GGTCA), in elements B and D. Region D binds a transactivatmg factor which appears also essential to stabtlize the initiation complex.

Research paper thumbnail of Lithium chloride induces mesenchymal‑to‑epithelial reverting transition in primary colon cancer cell cultures

International journal of oncology, Jan 2, 2015

Epithelial‑to‑mesenchymal transition (EMT) confers stem cell‑like phenotype and more motile prope... more Epithelial‑to‑mesenchymal transition (EMT) confers stem cell‑like phenotype and more motile properties to carcinoma cells. During EMT, the expression of E‑cadherin decreases, resulting in loss of cell‑cell adhesion and increased migration. Expression of Twist1 and other pleiotropic transcription factors, such as Snail, is known to activate EMT. We established primary colon cancer cell cultures from samples of operated patients and validated cultures by cytogenetic and molecular biology approaches. Western blot assay, quantitative real‑time PCR and immunofluorescence were performed to investigate the expression of E‑cadherin, vimentin, β‑catenin, cytokeratin‑20 and ‑18, Twist1, Snail, CD44, cyclooxygenase‑2 (COX2), Sox2, Oct4 and Nanog. Moreover, cell differentiation was induced by incubation with LiCl‑containing medium for 10 days. We observed that these primary colorectal cancer (CRC) cells lost expression of the E‑cadherin epithelial marker, which was instead expressed in cancer a...

Research paper thumbnail of Differential expression of PTEN gene correlates with phenotypic heterogeneity in three cases of patients showing clinical manifestations of PTEN hamartoma tumour syndrome

Hereditary cancer in clinical practice, 2013

Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS) and proteus syndrome are disorders... more Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS) and proteus syndrome are disorders known as PTEN hamartoma tumour syndrome (PHTS), that can show remarkable clinical overlap and are all caused by germline PTEN mutations. We here present two families, one affected by CS and the other affected by BRRS, both carriers of specific pathogenetic missense mutation in exon 5 of PTEN gene, within the catalitic domain. Both PHTS families exhibited extremely variable phenotypes, showing inter- and intra- familial variability. One of the two characterised mutations, the c.320A- > T; p.107Asp- > Val, identified in the CS family, was not previously described in the literature. Furthermore, the BRRS family, carrier of the c.406 T- > C; p.136Cys- > Arg mutation, shows a substantial alteration of PTEN protein expression that well correlates with intra-familial phenotypic variability. Finally, we describe an apparently sporadic case of an 80-year-old man, with a very low leve...

Research paper thumbnail of “DNA-Guided” Therapy

Multiple Primary Malignancies, 2009

Rapid progress in understanding the biomolecular basis of disease has brought new concepts to the... more Rapid progress in understanding the biomolecular basis of disease has brought new concepts to the diagnosis and treatment of some hereditary tumors. After the genes causing the syndromes were identified, the first step was the adoption of predictive genetic tests to identify within ...

Research paper thumbnail of Synergistic effect of interleukin-10-receptor variants in a case of early-onset ulcerative colitis

World Journal of Gastroenterology, 2013

AIM: To investigated the molecular cause of very early-onset ulcerative colitis (UC) in an 18-mo-... more AIM: To investigated the molecular cause of very early-onset ulcerative colitis (UC) in an 18-mo-old affected child.

Research paper thumbnail of Human aldolase A gene. Structural organization and tissue-specific expression by multiple promoters and alternate mRNA processing

European Journal of Biochemistry, 1988

The complete nucleotide sequence of the human aldolase A isoenzyme gene is reported. The cloned g... more The complete nucleotide sequence of the human aldolase A isoenzyme gene is reported. The cloned gene sequence, spanning 7530 bp, includes twelve exons and occurs as a single copy per haploid human genome. The structural organization of the gene is quite complex: eight exons containing the coding sequence are common to all mRNAs extracted from human and other mammalian sources; four additional exons are present in the 5' untranslated region, of these one is contained in the ubiquitous type of mRNA, the second is in the musclespecific type of mRNA and the third and fourth are in a minor species of mRNA found in human liver tissue. Furthermore, the determined sequence includes 1000 nucleotides upstream from the first exon (exon I) in the 5' flanking region, and 400 nucleotides, which include the polyadenylation signal, downstream from the termination codon.

Research paper thumbnail of Increased mucosal nitric oxide production in ulcerative colitis is mediated in part by the enteroglial-derived S100B protein

Neurogastroenterology & Motility, 2009

In the central nervous system glial-derived S100B protein has been associated with inflammation v... more In the central nervous system glial-derived S100B protein has been associated with inflammation via nitric oxide (NO) production. As the role of enteroglial cells in inflammatory bowel disease has been poorly investigated in humans, we evaluated the association of S100B and NO production in ulcerative colitis (UC). S100B mRNA and protein expression, inducible NO synthase (iNOS) expression, and NO production were evaluated in rectal biopsies from 30 controls and 35 UC patients. To verify the correlation between S100B and NO production, biopsies were exposed to S100B, in the presence or absence of specific receptor for advanced glycation end-products (RAGE) blocking antibody, to measure iNOS expression and nitrite production. S100B and iNOS expression were evaluated after incubation of biopsies with lipopolysaccharides (LPS) + interferon-gamma (IFN-c) in the presence of anti-RAGE or anti-S100B antibodies or budesonide. S100B mRNA and protein expression, iNOS expression and NO production were significantly higher in the rectal mucosa of patients compared to that of controls. Exogenous S100B induced a significant increase in both iNOS expression and NO production in controls and UC patients; this increase was inhibited by specific anti-RAGE blocking antibody. Incubation with LPS + IFN-c induced a signifi-cant increase in S100B mRNA and protein expression, together with increased iNOS expression and NO production. LPS + IFN-c-induced S100B up-regulation was not affected by budesonide, while iNOS expression and NO production were significantly inhibited by both specific anti-RAGE and anti-S100B blocking antibodies. Enteroglial-derived S100B up-regulation in UC participates in NO production, involving RAGE in a steroid insensitive pathway.

Research paper thumbnail of Purification and properties of several transfer RNA methyltransferases fromS. typhimurium

Molecular and Cellular Biochemistry, 1981

A fast method for a single-step fractionation of a number of tRNA methyltransferases from Salmone... more A fast method for a single-step fractionation of a number of tRNA methyltransferases from Salmonella typhimurium is described. The method basically consists of ion-exchange chromatography on a phosphocellulose column and permits the separation of the enzymes forming mt6A, m1G, m5U, m7G. The enzyme fractions appear sufficiently purified to allow the estimation of some molecular and kinetic properties. The apparent KM for adenosylmethionine range between 1.5 to 3.2 X 10(-5) M, whereas KM for undermethylated tRNA range between 3.1 X 10(-5) M to 3.1 X 10(-4) M. Glycerol gradient determination indicates the following Mr for the native proteins: 25 X 10(3), 40 X 10(3), 50 X 10(3) and 65 X 10(3) for m7G-, mt6A-, m1G- and m5U-forming enzymes, respectively. A complete analysis of methylated nucleosides formed in vivo in S. typhimurium has been obtained: it also allowed us to infer the pattern of the various tRNA methyltransferases for this prokaryote. The tRNA methyltransferase forming mt6A has been isolated for the first time from any type of cell.

Research paper thumbnail of An optimized method to separate reticulocytes from peripheral blood for molecular analysis

International Journal of Laboratory Hematology, 2009

Research paper thumbnail of Association of low-risk MSH3 and MSH2 variant alleles with Lynch syndrome: Probability of synergistic effects

International Journal of Cancer, 2011

Mutations in the MLH1 and MSH2 genes account for a majority of cases of families with Lynch Syndr... more Mutations in the MLH1 and MSH2 genes account for a majority of cases of families with Lynch Syndrome. Germ-line mutations in MSH6, PMS2 and MLH3 are responsible for disease in a minority of cases, usually associated with milder and variable phenotypes. No germ-line mutations in MSH3 have so far been associated with Lynch Syndrome, although it is known that impaired MSH3 activity leads to a partial defect in mismatch repair (MMR), with low levels of microsatellite instability at the loci with dinucleotide repeats in colorectal cancer (CRC), thus suggesting a role for MSH3 in carcinogenesis.

Research paper thumbnail of Familial adenomatous polyposis coli: Five novel mutations in exon 15 of the adenomatous polyposis coli (APC) gene in Italian patients

Human Mutation, 1999

Germline mutations within the adenomatous polyposis coli (APC) gene, a tumor suppressor gene, are... more Germline mutations within the adenomatous polyposis coli (APC) gene, a tumor suppressor gene, are responsible for most cases of familial adenomatous polyposis (FAP), an autosomal dominantly inherited predisposition to colorectal cancer. To date, more than 300 germ-line causative mutations within this gene have been described . Of these, about 95% are chain-terminating mutations, and more than 60% have been localized within exon 15 (Nagase and Nakamura, 1993, Beroud and Soussi, 1996). Using polymerase chain reaction-single strand conformation polymorphism, protein truncation test (PTT) and DNA sequencing we have identified five new frameshift mutations (2523insCTTA, 2638delA, 2803insA, 3185delAA, 4145delTCATGT), all occurring within exon 15 and giving rise to truncated protein products. Two of these new mutations are of particular interest because of the unusual phenotypic features shown by probands. The phenotype of the 2 SCARANO ET AL. proband bearing the 2523insCTTA mutation at codon 842 was very aggressive with onset of the symptoms at 12 years, while the patient bearing the 3185delAA mutation at codon 1062 exhibited features of an attenuated form of FAP (AAPC). Our data reiterate the great heterogeneity of the mutational spectrum in FAP that gives rise to an extreme variability of the clinical expression.

Research paper thumbnail of The mutation spectrum of the APC gene in FAP patients from southern Italy: Detection of known and four novel mutations

Human Mutation, 2003

Grant sponsors: MURST (PRIN '99); CNR (target project: P.F. Biotechnology); BIOGEM S.c.a.r.l.; Re... more Grant sponsors: MURST (PRIN '99); CNR (target project: P.F. Biotechnology); BIOGEM S.c.a.r.l.; Regione Campania. Communicated by Mark H. Paalman Familial adenomatous polyposis (FAP), an autosomal dominantly inherited condition accounting for about 1% of all colorectal cancers, results from mutations in the adenomatous polyposis coli (APC) tumor suppressor gene. The clinical spectrum and severity of FAP varies greatly with the mutation site, and both between and within families. Using the protein truncation test, single strand conformation polymorphism analysis and DNA sequencing, we identified 30 (75%) mutant alleles in 40 unrelated FAP families, for a total of 22 different APC mutations. Of these, 18 are known and 4 are novel: c.1797C>A (C599X), c.893_894delAC, (c.3225T>A; c.3226C>A) and c.4526_4527insT. Of the 30 APC gene mutations, 5 (~17%) are nonsense mutations, 17 (~57%) are small deletions, 5 (~17%) are small insertions and 3 (~10%) are complete deletions. All mutations occurred in single pedigrees, except those at codons 1061 and 1062, each found in two unrelated families, and the mutation at codon 1309 in exon 15, found in five unrelated families. About 40% of mutations, mostly small deletions and insertions, are located at repeated sequences; they promote misalignment-mediated errors in DNA replication and could represent a hot spot mutation region. This study enlarges the spectrum of APC gene mutations and sheds light on the correlation between the site of APC germline mutations and clinical manifestations of FAP.

Research paper thumbnail of Detection of four novel mutations in the iduronate‐2‐sulfatase gene

Human Mutation, 1998

Hunter disease (mucopolysaccharidosis type II or MPS II) is an X-linked recessive disorder caused... more Hunter disease (mucopolysaccharidosis type II or MPS II) is an X-linked recessive disorder caused by the deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS) (E.C.3.1.6.13.) involved in the catabolism of mucopolysaccharides dermatan sulfate and heparan sulfate. A large variety of alterations have been detected at the IDS locus. We report here the identification, in 7 unrelated Italian patients, of IDS gene mutations, four of which are novel and have been confirmed by amplification refractory system (ARMS) or restriction analysis. Our findings include: the missense mutation P86L found in a severe phenotype, the splicing mutation G374G and the nonsense mutation W475X, both associated with mild phenotypes. The four novel mutations were: the missense mutations R88P and R88H, associated with severe phenotypes, concerning a position found to be a mutational "hot-spot" for the IDS gene due to a mutation-prone CpG dinucleotide; mutations T1181 and P266H, both in mild patients. Interestingly, four of our mutations are located on exon III of IDS gene, confirming the high mutation frequency of this exon. After this manuscript was submitted, Rathman et al (Am. J.Hum.Genet.59,1202,1996) reported a total of 101 mutations including one R88H which is one of the novel mutations in this report.

Research paper thumbnail of Mucopolysaccharidosis type II: Identification of six novel mutations in Italian patients

Human Mutation, 1997

... Mutations in Brief. Mucopolysaccharidosis type II: Identification of six novel mutations in I... more ... Mutations in Brief. Mucopolysaccharidosis type II: Identification of six novel mutations in Italian patients. Guglielmo RD Villani,; Nicola Balzano,; Michela Grosso,; Francesco Salvatore,; Paola Izzo,; Paola Di Natale. Article first published online: 8 JAN 1999. ...

Research paper thumbnail of Three novel germline mutations in the adenomatous polyposis coli gene

Research paper thumbnail of Familial adenomatous polyposis coli: Five novel mutations in exon 15 of the adenomatous polyposis coli (APC) gene in Italian patients Communicated by: R.G.H. Cotton Online Citation: Human Mutation, Mutation in Brief #225 (1999) Online http://journals.wiley.com/1059-7794/pdf/mutation/225.pdf

Human Mutation, 1999

Germline mutations within the adenomatous polyposis coli (APC) gene, a tumor suppressor gene, are... more Germline mutations within the adenomatous polyposis coli (APC) gene, a tumor suppressor gene, are responsible for most cases of familial adenomatous polyposis (FAP), an autosomal dominantly inherited predisposition to colorectal cancer. To date, more than 300 germ-line causative mutations within this gene have been described (Beroud and Soussi, 1996). Of these, about 95% are chain-terminating mutations, and more than 60% have been localized within exon 15 (Nagase and Nakamura, 1993, Beroud and Soussi, 1996). Using polymerase chain reaction-single strand conformation polymorphism, protein truncation test (PTT) and DNA sequencing we have identified five new frameshift mutations (2523insCTTA, 2638delA, 2803insA, 3185delAA, 4145delTCATGT), all occurring within exon 15 and giving rise to truncated protein products. Two of these new mutations are of particular interest because of the unusual phenotypic features shown by probands. The phenotype of the proband bearing the 2523insCTTA mutation at codon 842 was very aggressive with onset of the symptoms at 12 years, while the patient bearing the 3185delAA mutation at codon 1062 exhibited features of an attenuated form of FAP (AAPC). Our data reiterate the great heterogeneity of the mutational spectrum in FAP that gives rise to an extreme variability of the clinical expression.

Research paper thumbnail of First genotype characterization of Argentinean FAP patients: Identification of 14 novelAPCmutations

Human Mutation, 2004

Using a combination of the protein truncation test, the single strand conformation polymorphism t... more Using a combination of the protein truncation test, the single strand conformation polymorphism technique, DNA sequencing and quantitative PCR analysis, we identified the specific mutation in 39 (average age: 28.4 years) of the 51 probands (detection rate: 76.47%); 13 are novel germline mutations and one is a novel sequence variant. There were 27 small deletions, four small duplications, five nonsense mutations in exon 15, three nonsense mutations in exons 6, 11, and 12, and one sequence variant in exon 3 identified in a patient bearing a truncating mutation in exon 15. The most common mutation (found in 10 cases) was at codon 1309. All patients negative for APC mutations were also negative for the MutY homolog (MYH) gene mutation, as expected because of fully penetrant FAP cases. This study enlarges the spectrum of APC gene mutations, and reinforces the concept of mutation heterogeneity. It also sheds light on correlations between the site of APC germline mutations and the clinical manifestations of FAP. Our data indicate that the genotype/phenotype correlations in Argentinean patients are similar to those observed in other populations.

Research paper thumbnail of Alu-Mediated Genomic Deletion of the Serine/Threonine Protein Kinase 11 (STK11) Gene in Peutz–Jeghers Syndrome

Research paper thumbnail of Identification and molecular characterization of the CAMPANIA deletion, a novel α°-thalassemic defect, in two unrelated Italian families

American Journal of Hematology, 2010

Research paper thumbnail of Interleukin10 Receptor Down-Expression in Early-Onset Inflammatory Bowel Disease: A Case-Report

Research paper thumbnail of Cis-acting elements in the promoter region of the human aldolase C gene

We i~vesti~f~d the c&acting sequences involve m the expression of the human Adolph C gene by tran... more We i~vesti~f~d the c&acting sequences involve m the expression of the human Adolph C gene by transient tmnsf~t~ons into b~man neuroblastoma cells (SKNBE). We demonstrate that 420 bp of the Y-flanking DNA dimct at high efficiency the transcription of the CAT reporter gene. A deletion between -420 bp and -164 bp causes a 60% decrease ofCAT activity. Gel shift and DNase I footprmtmg analyses revealed four protected elements: A, B, C and D. Competition analyses indicate that Spl or factors sharing a similar sequence specificity bind to elements A and B, but not to elements C and D. Sequence analysts shows a half palindromic ERE motrf(GGTCA), in elements B and D. Region D binds a transactivatmg factor which appears also essential to stabtlize the initiation complex.

Research paper thumbnail of Lithium chloride induces mesenchymal‑to‑epithelial reverting transition in primary colon cancer cell cultures

International journal of oncology, Jan 2, 2015

Epithelial‑to‑mesenchymal transition (EMT) confers stem cell‑like phenotype and more motile prope... more Epithelial‑to‑mesenchymal transition (EMT) confers stem cell‑like phenotype and more motile properties to carcinoma cells. During EMT, the expression of E‑cadherin decreases, resulting in loss of cell‑cell adhesion and increased migration. Expression of Twist1 and other pleiotropic transcription factors, such as Snail, is known to activate EMT. We established primary colon cancer cell cultures from samples of operated patients and validated cultures by cytogenetic and molecular biology approaches. Western blot assay, quantitative real‑time PCR and immunofluorescence were performed to investigate the expression of E‑cadherin, vimentin, β‑catenin, cytokeratin‑20 and ‑18, Twist1, Snail, CD44, cyclooxygenase‑2 (COX2), Sox2, Oct4 and Nanog. Moreover, cell differentiation was induced by incubation with LiCl‑containing medium for 10 days. We observed that these primary colorectal cancer (CRC) cells lost expression of the E‑cadherin epithelial marker, which was instead expressed in cancer a...

Research paper thumbnail of Differential expression of PTEN gene correlates with phenotypic heterogeneity in three cases of patients showing clinical manifestations of PTEN hamartoma tumour syndrome

Hereditary cancer in clinical practice, 2013

Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS) and proteus syndrome are disorders... more Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS) and proteus syndrome are disorders known as PTEN hamartoma tumour syndrome (PHTS), that can show remarkable clinical overlap and are all caused by germline PTEN mutations. We here present two families, one affected by CS and the other affected by BRRS, both carriers of specific pathogenetic missense mutation in exon 5 of PTEN gene, within the catalitic domain. Both PHTS families exhibited extremely variable phenotypes, showing inter- and intra- familial variability. One of the two characterised mutations, the c.320A- > T; p.107Asp- > Val, identified in the CS family, was not previously described in the literature. Furthermore, the BRRS family, carrier of the c.406 T- > C; p.136Cys- > Arg mutation, shows a substantial alteration of PTEN protein expression that well correlates with intra-familial phenotypic variability. Finally, we describe an apparently sporadic case of an 80-year-old man, with a very low leve...

Research paper thumbnail of “DNA-Guided” Therapy

Multiple Primary Malignancies, 2009

Rapid progress in understanding the biomolecular basis of disease has brought new concepts to the... more Rapid progress in understanding the biomolecular basis of disease has brought new concepts to the diagnosis and treatment of some hereditary tumors. After the genes causing the syndromes were identified, the first step was the adoption of predictive genetic tests to identify within ...

Research paper thumbnail of Synergistic effect of interleukin-10-receptor variants in a case of early-onset ulcerative colitis

World Journal of Gastroenterology, 2013

AIM: To investigated the molecular cause of very early-onset ulcerative colitis (UC) in an 18-mo-... more AIM: To investigated the molecular cause of very early-onset ulcerative colitis (UC) in an 18-mo-old affected child.

Research paper thumbnail of Human aldolase A gene. Structural organization and tissue-specific expression by multiple promoters and alternate mRNA processing

European Journal of Biochemistry, 1988

The complete nucleotide sequence of the human aldolase A isoenzyme gene is reported. The cloned g... more The complete nucleotide sequence of the human aldolase A isoenzyme gene is reported. The cloned gene sequence, spanning 7530 bp, includes twelve exons and occurs as a single copy per haploid human genome. The structural organization of the gene is quite complex: eight exons containing the coding sequence are common to all mRNAs extracted from human and other mammalian sources; four additional exons are present in the 5' untranslated region, of these one is contained in the ubiquitous type of mRNA, the second is in the musclespecific type of mRNA and the third and fourth are in a minor species of mRNA found in human liver tissue. Furthermore, the determined sequence includes 1000 nucleotides upstream from the first exon (exon I) in the 5' flanking region, and 400 nucleotides, which include the polyadenylation signal, downstream from the termination codon.

Research paper thumbnail of Increased mucosal nitric oxide production in ulcerative colitis is mediated in part by the enteroglial-derived S100B protein

Neurogastroenterology & Motility, 2009

In the central nervous system glial-derived S100B protein has been associated with inflammation v... more In the central nervous system glial-derived S100B protein has been associated with inflammation via nitric oxide (NO) production. As the role of enteroglial cells in inflammatory bowel disease has been poorly investigated in humans, we evaluated the association of S100B and NO production in ulcerative colitis (UC). S100B mRNA and protein expression, inducible NO synthase (iNOS) expression, and NO production were evaluated in rectal biopsies from 30 controls and 35 UC patients. To verify the correlation between S100B and NO production, biopsies were exposed to S100B, in the presence or absence of specific receptor for advanced glycation end-products (RAGE) blocking antibody, to measure iNOS expression and nitrite production. S100B and iNOS expression were evaluated after incubation of biopsies with lipopolysaccharides (LPS) + interferon-gamma (IFN-c) in the presence of anti-RAGE or anti-S100B antibodies or budesonide. S100B mRNA and protein expression, iNOS expression and NO production were significantly higher in the rectal mucosa of patients compared to that of controls. Exogenous S100B induced a significant increase in both iNOS expression and NO production in controls and UC patients; this increase was inhibited by specific anti-RAGE blocking antibody. Incubation with LPS + IFN-c induced a signifi-cant increase in S100B mRNA and protein expression, together with increased iNOS expression and NO production. LPS + IFN-c-induced S100B up-regulation was not affected by budesonide, while iNOS expression and NO production were significantly inhibited by both specific anti-RAGE and anti-S100B blocking antibodies. Enteroglial-derived S100B up-regulation in UC participates in NO production, involving RAGE in a steroid insensitive pathway.

Research paper thumbnail of Purification and properties of several transfer RNA methyltransferases fromS. typhimurium

Molecular and Cellular Biochemistry, 1981

A fast method for a single-step fractionation of a number of tRNA methyltransferases from Salmone... more A fast method for a single-step fractionation of a number of tRNA methyltransferases from Salmonella typhimurium is described. The method basically consists of ion-exchange chromatography on a phosphocellulose column and permits the separation of the enzymes forming mt6A, m1G, m5U, m7G. The enzyme fractions appear sufficiently purified to allow the estimation of some molecular and kinetic properties. The apparent KM for adenosylmethionine range between 1.5 to 3.2 X 10(-5) M, whereas KM for undermethylated tRNA range between 3.1 X 10(-5) M to 3.1 X 10(-4) M. Glycerol gradient determination indicates the following Mr for the native proteins: 25 X 10(3), 40 X 10(3), 50 X 10(3) and 65 X 10(3) for m7G-, mt6A-, m1G- and m5U-forming enzymes, respectively. A complete analysis of methylated nucleosides formed in vivo in S. typhimurium has been obtained: it also allowed us to infer the pattern of the various tRNA methyltransferases for this prokaryote. The tRNA methyltransferase forming mt6A has been isolated for the first time from any type of cell.

Research paper thumbnail of An optimized method to separate reticulocytes from peripheral blood for molecular analysis

International Journal of Laboratory Hematology, 2009

Research paper thumbnail of Association of low-risk MSH3 and MSH2 variant alleles with Lynch syndrome: Probability of synergistic effects

International Journal of Cancer, 2011

Mutations in the MLH1 and MSH2 genes account for a majority of cases of families with Lynch Syndr... more Mutations in the MLH1 and MSH2 genes account for a majority of cases of families with Lynch Syndrome. Germ-line mutations in MSH6, PMS2 and MLH3 are responsible for disease in a minority of cases, usually associated with milder and variable phenotypes. No germ-line mutations in MSH3 have so far been associated with Lynch Syndrome, although it is known that impaired MSH3 activity leads to a partial defect in mismatch repair (MMR), with low levels of microsatellite instability at the loci with dinucleotide repeats in colorectal cancer (CRC), thus suggesting a role for MSH3 in carcinogenesis.

Research paper thumbnail of Familial adenomatous polyposis coli: Five novel mutations in exon 15 of the adenomatous polyposis coli (APC) gene in Italian patients

Human Mutation, 1999

Germline mutations within the adenomatous polyposis coli (APC) gene, a tumor suppressor gene, are... more Germline mutations within the adenomatous polyposis coli (APC) gene, a tumor suppressor gene, are responsible for most cases of familial adenomatous polyposis (FAP), an autosomal dominantly inherited predisposition to colorectal cancer. To date, more than 300 germ-line causative mutations within this gene have been described . Of these, about 95% are chain-terminating mutations, and more than 60% have been localized within exon 15 (Nagase and Nakamura, 1993, Beroud and Soussi, 1996). Using polymerase chain reaction-single strand conformation polymorphism, protein truncation test (PTT) and DNA sequencing we have identified five new frameshift mutations (2523insCTTA, 2638delA, 2803insA, 3185delAA, 4145delTCATGT), all occurring within exon 15 and giving rise to truncated protein products. Two of these new mutations are of particular interest because of the unusual phenotypic features shown by probands. The phenotype of the 2 SCARANO ET AL. proband bearing the 2523insCTTA mutation at codon 842 was very aggressive with onset of the symptoms at 12 years, while the patient bearing the 3185delAA mutation at codon 1062 exhibited features of an attenuated form of FAP (AAPC). Our data reiterate the great heterogeneity of the mutational spectrum in FAP that gives rise to an extreme variability of the clinical expression.

Research paper thumbnail of The mutation spectrum of the APC gene in FAP patients from southern Italy: Detection of known and four novel mutations

Human Mutation, 2003

Grant sponsors: MURST (PRIN '99); CNR (target project: P.F. Biotechnology); BIOGEM S.c.a.r.l.; Re... more Grant sponsors: MURST (PRIN '99); CNR (target project: P.F. Biotechnology); BIOGEM S.c.a.r.l.; Regione Campania. Communicated by Mark H. Paalman Familial adenomatous polyposis (FAP), an autosomal dominantly inherited condition accounting for about 1% of all colorectal cancers, results from mutations in the adenomatous polyposis coli (APC) tumor suppressor gene. The clinical spectrum and severity of FAP varies greatly with the mutation site, and both between and within families. Using the protein truncation test, single strand conformation polymorphism analysis and DNA sequencing, we identified 30 (75%) mutant alleles in 40 unrelated FAP families, for a total of 22 different APC mutations. Of these, 18 are known and 4 are novel: c.1797C>A (C599X), c.893_894delAC, (c.3225T>A; c.3226C>A) and c.4526_4527insT. Of the 30 APC gene mutations, 5 (~17%) are nonsense mutations, 17 (~57%) are small deletions, 5 (~17%) are small insertions and 3 (~10%) are complete deletions. All mutations occurred in single pedigrees, except those at codons 1061 and 1062, each found in two unrelated families, and the mutation at codon 1309 in exon 15, found in five unrelated families. About 40% of mutations, mostly small deletions and insertions, are located at repeated sequences; they promote misalignment-mediated errors in DNA replication and could represent a hot spot mutation region. This study enlarges the spectrum of APC gene mutations and sheds light on the correlation between the site of APC germline mutations and clinical manifestations of FAP.

Research paper thumbnail of Detection of four novel mutations in the iduronate‐2‐sulfatase gene

Human Mutation, 1998

Hunter disease (mucopolysaccharidosis type II or MPS II) is an X-linked recessive disorder caused... more Hunter disease (mucopolysaccharidosis type II or MPS II) is an X-linked recessive disorder caused by the deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS) (E.C.3.1.6.13.) involved in the catabolism of mucopolysaccharides dermatan sulfate and heparan sulfate. A large variety of alterations have been detected at the IDS locus. We report here the identification, in 7 unrelated Italian patients, of IDS gene mutations, four of which are novel and have been confirmed by amplification refractory system (ARMS) or restriction analysis. Our findings include: the missense mutation P86L found in a severe phenotype, the splicing mutation G374G and the nonsense mutation W475X, both associated with mild phenotypes. The four novel mutations were: the missense mutations R88P and R88H, associated with severe phenotypes, concerning a position found to be a mutational "hot-spot" for the IDS gene due to a mutation-prone CpG dinucleotide; mutations T1181 and P266H, both in mild patients. Interestingly, four of our mutations are located on exon III of IDS gene, confirming the high mutation frequency of this exon. After this manuscript was submitted, Rathman et al (Am. J.Hum.Genet.59,1202,1996) reported a total of 101 mutations including one R88H which is one of the novel mutations in this report.

Research paper thumbnail of Mucopolysaccharidosis type II: Identification of six novel mutations in Italian patients

Human Mutation, 1997

... Mutations in Brief. Mucopolysaccharidosis type II: Identification of six novel mutations in I... more ... Mutations in Brief. Mucopolysaccharidosis type II: Identification of six novel mutations in Italian patients. Guglielmo RD Villani,; Nicola Balzano,; Michela Grosso,; Francesco Salvatore,; Paola Izzo,; Paola Di Natale. Article first published online: 8 JAN 1999. ...

Research paper thumbnail of Three novel germline mutations in the adenomatous polyposis coli gene

Research paper thumbnail of Familial adenomatous polyposis coli: Five novel mutations in exon 15 of the adenomatous polyposis coli (APC) gene in Italian patients Communicated by: R.G.H. Cotton Online Citation: Human Mutation, Mutation in Brief #225 (1999) Online http://journals.wiley.com/1059-7794/pdf/mutation/225.pdf

Human Mutation, 1999

Germline mutations within the adenomatous polyposis coli (APC) gene, a tumor suppressor gene, are... more Germline mutations within the adenomatous polyposis coli (APC) gene, a tumor suppressor gene, are responsible for most cases of familial adenomatous polyposis (FAP), an autosomal dominantly inherited predisposition to colorectal cancer. To date, more than 300 germ-line causative mutations within this gene have been described (Beroud and Soussi, 1996). Of these, about 95% are chain-terminating mutations, and more than 60% have been localized within exon 15 (Nagase and Nakamura, 1993, Beroud and Soussi, 1996). Using polymerase chain reaction-single strand conformation polymorphism, protein truncation test (PTT) and DNA sequencing we have identified five new frameshift mutations (2523insCTTA, 2638delA, 2803insA, 3185delAA, 4145delTCATGT), all occurring within exon 15 and giving rise to truncated protein products. Two of these new mutations are of particular interest because of the unusual phenotypic features shown by probands. The phenotype of the proband bearing the 2523insCTTA mutation at codon 842 was very aggressive with onset of the symptoms at 12 years, while the patient bearing the 3185delAA mutation at codon 1062 exhibited features of an attenuated form of FAP (AAPC). Our data reiterate the great heterogeneity of the mutational spectrum in FAP that gives rise to an extreme variability of the clinical expression.

Research paper thumbnail of First genotype characterization of Argentinean FAP patients: Identification of 14 novelAPCmutations

Human Mutation, 2004

Using a combination of the protein truncation test, the single strand conformation polymorphism t... more Using a combination of the protein truncation test, the single strand conformation polymorphism technique, DNA sequencing and quantitative PCR analysis, we identified the specific mutation in 39 (average age: 28.4 years) of the 51 probands (detection rate: 76.47%); 13 are novel germline mutations and one is a novel sequence variant. There were 27 small deletions, four small duplications, five nonsense mutations in exon 15, three nonsense mutations in exons 6, 11, and 12, and one sequence variant in exon 3 identified in a patient bearing a truncating mutation in exon 15. The most common mutation (found in 10 cases) was at codon 1309. All patients negative for APC mutations were also negative for the MutY homolog (MYH) gene mutation, as expected because of fully penetrant FAP cases. This study enlarges the spectrum of APC gene mutations, and reinforces the concept of mutation heterogeneity. It also sheds light on correlations between the site of APC germline mutations and the clinical manifestations of FAP. Our data indicate that the genotype/phenotype correlations in Argentinean patients are similar to those observed in other populations.