Rossella Tomaiuolo - Academia.edu (original) (raw)

Papers by Rossella Tomaiuolo

Research paper thumbnail of Molecular diagnostics: between chips and customized medicine

Clinical Chemistry and Laboratory Medicine, May 1, 2010

Recently there has been an explosive increase in molecular diagnostics. This is due to translatio... more Recently there has been an explosive increase in molecular diagnostics. This is due to translational research on the molecular basis of human diseases, and to technological developments that have resulted in efficient procedures for extensive analysis of the human genome. However, a large body of data on the genome is still difficult to interpret at the clinical level. For many monogenic diseases, "modifier" genes, inherited independently of the disease gene interact, thereby resulting in a distinct phenotype for each patient. Multigenic diseases depend on complex interactions between genes and the environment. Response to drugs and side effects are modulated by gene variants. The same is true for the response to nutrients. All these interactions, which vary from patient to patient, led to the concept of "personalized medicine". Our genome consists of 25,000 genes, a surprisingly low number when compared to other species. Therefore, the complex phenotype of humans depends on a number of mechanisms that regulate gene expression, which, in turn, may be altered resulting in disease. For example, DNA methylation modulates the level of gene expression, and altered methylation of some genes is related to human neoplasias. MicroRNAs regulate the expression of a myriad of genes, and mounting evidence indicates that this mechanism may be impaired in human diseases. Finally, the relationships between genetics and human behavior are starting to be elucidated. For example, suicide may be related to alterations of methylation of specific genes. To conclude: the chip-wide analysis of human genomes is becoming easier, but the understanding of molecular genetics that confirmed the real "uniqueness" of each genome is an excellent opportunity for laboratory medicine to reposition the patient at the heart of the medical process.

Research paper thumbnail of Lung cancer metastatic cells detected in blood by reverse transcriptase-polymerase chain reaction and dot-blot analysis

Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 1997

We analyzed the blood of patients with lung cancer at different stages of presentation for the pr... more We analyzed the blood of patients with lung cancer at different stages of presentation for the presence of carcinoembryonic antigen (CEA) mRNA detected by reverse transcriptase-polymerase chain reaction (RT-PCR) combined with the dot-blot procedure as an indicator of micrometastatic malignant cells. We studied 24 lung cancer patients (10 with distant metastases and 14 with no evidence of distant metastases), eight age- and sex-matched patients affected by nonneoplastic respiratory diseases (four smokers), and eight healthy subjects. We used immunohistochemistry and RT-PCR dot-blot analysis to evaluate CEA expression in the neoplastic tissue, and the RT-PCR dot-blot procedure to analyze CEA mRNA in circulating cells. The RT-PCR dot-blot procedure was highly sensitive aspecific: it detected CEA mRNA in samples of RNA from lung cancer diluted 10(6)-fold with RNA extracted from normal blood cells, and sequence analysis confirmed that the amplified product was CEA. CEA mRNA was found in ...

Research paper thumbnail of Nasal polyposis in atypical cystic fibrosis: A case report

International Journal of Pediatric Otorhinolaryngology Extra, 2010

Cystic fibrosis (CF) is one of the most common inherited life-shortening diseases with an inciden... more Cystic fibrosis (CF) is one of the most common inherited life-shortening diseases with an incidence of 1:2.500–3500 and a carrier frequency of 4–5% [1]. It is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a protein expressed in the apical membrane of exocrine epithelial cells, resulting in changes to the fluid and electrolytes on cell

Research paper thumbnail of Congenital diarrheal disorders: An updated diagnostic approach

International Journal of Molecular Sciences, 2012

Congenital diarrheal disorders (CDDs) are a group of inherited enteropathies with a typical onset... more Congenital diarrheal disorders (CDDs) are a group of inherited enteropathies with a typical onset early in the life. Infants with these disorders have frequently chronic diarrhea of sufficient severity to require parenteral nutrition. For most CDDs the disease-gene is known and molecular analysis may contribute to an unequivocal diagnosis. We review CDDs on the basis of the genetic defect, focusing on the significant contribution of molecular analysis in the complex, multistep diagnostic work-up.

Research paper thumbnail of Tropomyosin-related kinase B receptor polymorphisms and isoforms expression in suicide victims

Psychiatry Research, 2014

. TrkB isoforms expression. TrkB isoforms expression/HPRT1-GAPDH geometric average in control and... more . TrkB isoforms expression. TrkB isoforms expression/HPRT1-GAPDH geometric average in control and suicide subjects. The values are represented as mean and standard deviation of the mean, and the P value is conducted by t-test or Mann-Whitney-test (*P¼ 0.031).

Research paper thumbnail of Exploitation of a very small peptide nucleic acid as a new inhibitor of miR-509-3p involved in the regulation of cystic fibrosis disease-gene expression

BioMed research international, 2014

Computational techniques, and in particular molecular dynamics (MD) simulations, have been succes... more Computational techniques, and in particular molecular dynamics (MD) simulations, have been successfully used as a complementary technique to predict and analyse the structural behaviour of nucleic acids, including peptide nucleic acid- (PNA-) RNA hybrids. This study shows that a 7-base long PNA complementary to the seed region of miR-509-3p, one of the miRNAs involved in the posttranscriptional regulation of the CFTR disease-gene of Cystic Fibrosis, and bearing suitable functionalization at its N- and C-ends aimed at improving its resistance to nucleases and cellular uptake, is able to revert the expression of the luciferase gene containing the 3'UTR of the gene in A549 human lung cancer cells, in agreement with the MD results that pointed at the formation of a stable RNA/PNA heteroduplex notwithstanding the short sequence of the latter. The here reported results widen the interest towards the use of small PNAs as effective anti-miRNA agents.

Research paper thumbnail of A Novel DHPLC-Based Procedure for the Analysis of COL1A1 and COL1A2 Mutations in Osteogenesis Imperfecta

The Journal of Molecular Diagnostics, 2011

Approximately 90% of patients with osteogenesis imperfecta (OI) exhibit dominant COL1A1 or COL1A2... more Approximately 90% of patients with osteogenesis imperfecta (OI) exhibit dominant COL1A1 or COL1A2 mutations; however, molecular analysis is difficult because these genes span 51 and 52 exons, respectively. We devised a PCR-denaturing high-performance liquid chromatography (DHPLC) procedure to analyze the COL1A1 or COL1A2 coding regions and validated it using 130 DNA samples from individuals without OI, 25 DNA samples from two cells to investigate the procedure's potential for preimplantation diagnosis, and DNA samples from 10 patients with OI. Three novel intronic variants in vitro were expressed using a minigene assay to assess their effects on splicing. The procedure is rapid, inexpensive, and reproducible. Analysis of samples from individuals without OI revealed six novel and some known polymorphisms useful for linkage diagnosis because of high heterozygosity. Analysis of two-cell samples confirmed the known genotype in 24 of 25 experiments; DNA failed to amplify in only one case. No incidence of allele dropout was recorded. DHPLC revealed six novel mutations, three of which were intronic, in all patients with OI, and these results were confirmed by means of COL1A1 and COL1A2 direct sequencing. Expression of intronic mutations demonstrated that variant 804 ؉ 2_804 ؉ 3delTG in intron 11 disrupts normal splicing, thereby leading to formation of two alternative products. Variants c.3046-4_3046-5dupCT (COL1A1) and c.891 ؉ 77A>T (COL1A2) did not affect splicing. The described DHPLC protocol combined with the minigene assay may contribute to molecular diagnosis in OI. Moreover, this protocol will aid in counseling about prenatal and preimplantation diagnosis.

Research paper thumbnail of Extensive Molecular Analysis of Patients Bearing CFTR-Related Disorders

The Journal of Molecular Diagnostics, 2012

Cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders (CFTR-RDs) may prese... more Cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders (CFTR-RDs) may present with pancreatic sufficiency, normal sweat test results, and better outcome. The detection rate of mutations is lower in CFTR-RD than in classic CF: mutations may be located in genes encoding proteins that interact with CFTR or support channel activity. We tested the whole CFTR coding regions in 99 CFTR-RD patients, looking for gene mutations in solute carrier (SLC) 26A and in epithelial Na channel (ENaC) in 33 patients who had unidentified mutations. CFTR analysis revealed 28 mutations, some of which are rare. Of these mutations, RT-PCR demonstrated that the novel 1525-1delG impairs exon 10 splicing; by using minigene analysis, we excluded the splicing effect of three other novel intronic variants. Analysis of SLC26A genes revealed several variants, some of which are novel, that did not affect mRNA expression. Other mutations occurred in the ENaC genes encoding the ENaC subunits, but their frequency did not significantly differ between patients and controls. Our data, although obtained on a preliminary cohort of CFTR-RD patients, exclude a role of mutations in SLC26A and in SCNN genes in the pathogenesis of such disease; we confirm that CFTR analysis has a relevant role in CFTR-RD patients; and it appears mandatory to use CFTR scanning techniques and approaches to reveal the effect of novel mutations.

Research paper thumbnail of Molecular and Functional Analysis of the Large 5′ Promoter Region of CFTR Gene Revealed Pathogenic Mutations in CF and CFTR-Related Disorders

The Journal of Molecular Diagnostics, 2013

The ASCP designates this journal-based CME activity ("JMD 2013 CME Program in Molecular Diagnosti... more The ASCP designates this journal-based CME activity ("JMD 2013 CME Program in Molecular Diagnostics") for a maximum of 48 AMA PRA Category 1 Credit(s) TM . Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. 2 0 1 3 J M D C M E P r o g r a m

Research paper thumbnail of Gene Mutation in MicroRNA Target Sites of CFTR Gene: A Novel Pathogenetic Mechanism in Cystic Fibrosis?

PLoS ONE, 2013

Cystic fibrosis (CF) is the most frequent lethal genetic disorder among Caucasians. It depends on... more Cystic fibrosis (CF) is the most frequent lethal genetic disorder among Caucasians. It depends on alterations of a chloride channel expressed by most epithelial cells and encoded by CFTR gene. Also using scanning techniques to analyze the whole coding regions of CFTR gene, mutations are not identified in up to 10% of CF alleles, and such figure increases in CFTRrelated disorders (CFTR-RD). Other gene regions may be the site of causing-disease mutations. We searched for genetic variants in the 1500 bp of CFTR 39 untranslated region, typical target of microRNA (miRNA) posttranscriptional gene regulation, in either CF patients with the F508del homozygous genotype and different clinical expression (n = 20), CF (n = 32) and CFTR-RD (n = 43) patients with one or none mutation after CFTR scanning and in controls (n = 50). We identified three SNPs, one of which, the c.*1043A.C, was located in a region predicted to bind miR-433 and miR-509-3p. Such mutation was peculiar of a CFTR-RD patient that had Congenital Bilateral Absence of Vas Deferens (CBAVD), diffuse bronchiectasis, a borderline sweat chloride test and the heterozygous severe F508del mutation on the other allele. The expression analysis demonstrated that the c.*1043A.C increases the affinity for miR-509-3p and slightly decreases that for the miR-433. Both miRNAs cause in vitro a reduced expression of CFTR protein. Thus, the c.*1043A.C may act as a mild CFTR mutation enhancing the affinity for inhibitory miRNAs as a novel pathogenetic mechanism in CF.

Research paper thumbnail of Design, synthesis and biochemical investigation, by in vitro luciferase reporter system, of peptide nucleic acids as new inhibitors of miR-509-3p involved in the regulation of cystic fibrosis disease-gene expression

Research paper thumbnail of Prothrombotic gene variants as risk factors of acute myocardial infarction in young women

Journal of Translational Medicine, 2012

Background: Acute myocardial infarction (AMI) in young women represent an extreme phenotype assoc... more Background: Acute myocardial infarction (AMI) in young women represent an extreme phenotype associated with a higher mortality compared with similarly aged men. Prothrombotic gene variants could play a role as risk factors for AMI at young age. Methods: We studied Factor V Leiden, FII G20210A, MTHFR C677T and beta-fibrinogen -455G>A variants by real-time PCR in 955 young AMI (362 females) and in 698 AMI (245 females) patients. The data were compared to those obtained in 909 unrelated subjects (458 females) from the general population of the same geographical area (southern Italy).

Research paper thumbnail of Congenital Diarrheal Disorders: Improved Understanding of Gene Defects Is Leading to Advances in Intestinal Physiology and Clinical Management

Journal of Pediatric Gastroenterology and Nutrition, 2010

Congenital diarrheal disorders (CDD, Online Mendelian Inheritance in Man [OMIM] 251850) represent... more Congenital diarrheal disorders (CDD, Online Mendelian Inheritance in Man [OMIM] 251850) represent one of the most challenging clinical conditions for pediatric gastroenterologists because of the severity of the clinical picture and the broad range of disorders in its differential diagnosis. The number of conditions included within CDD has gradually increased. Recent advances made in the pathophysiology of these conditions have led to a better understanding of the more common diarrheal diseases. Based on the body of data accumulated in recent years, we suggest that CDD be classified in 4 categories depending on the alteration in absorption and transport of nutrients and electrolytes, enterocyte differentiation and polarization, enteroendocrine cell differentiation, and modulation of the intestinal immune response. Our knowledge of the genes responsible for CDD is also rapidly increasing, thanks to linkage studies based on genome-wide analysis of polymorphisms. In this context, the identification of disease genes is a step forward in the diagnostic approach to a patient in whom CDD is strongly suspected. However, it is conceivable that faster, less expensive molecular procedures will, in the near future, become available. This approach could spare the patient invasive procedures and limit complications associated with a delay in diagnosis. Furthermore, carrier and prenatal molecular diagnosis may help pediatricians better manage the condition in the early stages of life.

Research paper thumbnail of Atypcal Cystic Fibrosis (Acf): An Experience of a Cystic Fibrosis Center

Journal of Cystic Fibrosis, 2008

Research paper thumbnail of Molecular Analysis of Genes Encoding CFTR Interactors of SLC26 Family in CF Patients: Preliminary Results

Journal of Cystic Fibrosis, 2008

Research paper thumbnail of DIRECT SEQUENCING OF CSTs IN CYSTIC FIBROSIS PATIENTS BEARING UNDEFINITE GENOTYPE

Journal of Cystic Fibrosis, 2008

Research paper thumbnail of Cystic fibrosis presenting as metabolic alkalosis in a boy with the rare D579G mutation

Journal of Cystic Fibrosis, 2004

We report on a 10-month-old boy with hypotonic dehydration and metabolic alkalosis. Sweat test wa... more We report on a 10-month-old boy with hypotonic dehydration and metabolic alkalosis. Sweat test was borderline and genetic analysis was negative for common mutations. Analysis of the whole coding regions of the CFTR gene revealed the rare mutation D579G in homozygosity. D

Research paper thumbnail of Phenotypic discordance in three siblings affected by atypical cystic fibrosis with the F508del/D614G genotype

Journal of Cystic Fibrosis, 2006

We report an example of atypical CF, i.e., a family in which three siblings were affected by late... more We report an example of atypical CF, i.e., a family in which three siblings were affected by late-diagnosed mild CF, and showed discordant pulmonary and pancreatic phenotypes. Sibling no. 1 (male), showed a severe pulmonary involvement and pancreatic sufficiency; sibling no. 2 (female) showed a mild pulmonary disease with pancreatic sufficiency; sibling no. 3 (male) had a very mild pulmonary expression and pancreatic insufficiency. The sweat test was altered in all three siblings, and all had intestinal occlusion in young age. The whole scanning of CFTR revealed the rare F508del/D614G genotype. The discordance of clinical expression within the same family reinforces the putative role of modifier genes of CF phenotype.

Research paper thumbnail of Epidemiology and a novel procedure for large scale analysis of CFTR rearrangements in classic and atypical CF patients: A multicentric Italian study

Journal of Cystic Fibrosis, 2008

Background: Mutation epidemiology in each ethnic group is a crucial step of strategies for cystic... more Background: Mutation epidemiology in each ethnic group is a crucial step of strategies for cystic fibrosis (CF) diagnosis and counselling. To date, the scanning of the whole coding region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene permits to identify about 90% of alleles from patients bearing CF and a lower percentage in patients bearing atypical CF. CFTR rearrangements in heterozygosis elude current techniques for molecular analysis, and some of them have been reported with a frequency up to 6% in various ethnic groups. Methods: Using quantitative PCR analysis of all coding regions, we assessed the occurrence of CFTR rearrangements in 130 alleles from classic CF patients and in 198 alleles from atypical CF patients (all unrelated and from Italian descent) bearing unidentified mutations after the scanning of CFTR. Results: Seven rearrangements (i.e., dele1, dele2, dele2_3, dele 14b_17b, dele17a_18, dele22_23, and dele22_24) were identified in 34/131 (26.0%) CF alleles bearing undetected mutations (which means about 2.5% of all CF alleles) and in none of the 198 alleles from atypical CF. The CFTR haplotype and the sequence analysis of the breakpoints confirmed the common origin of all the rearrangements. Thus, we set up a novel duplex PCR assay for the large-scale analysis of the seven rearrangements. The procedure was rapid (all PCR amplifications were obtained under the same conditions), costless and repeatable. Conclusions: It is useful to select the CFTR rearrangements more frequent in specific ethnic groups and to set up procedures for large-scale analysis. Their study can be performed in cases in which a high detection rate is required (i.e., partners of CF carriers/patients). On the contrary, the analysis of rearrangement is useless in atypical CF patients.

Research paper thumbnail of Prediction of acute pancreatitis risk based on PIP score in children with cystic fibrosis

Journal of Cystic Fibrosis, 2014

Background: Currently no tools to predict risk of acute (AP) and recurrent pancreatitis (ARP) in ... more Background: Currently no tools to predict risk of acute (AP) and recurrent pancreatitis (ARP) in children with cystic fibrosis (CF) are available. We assessed the prevalence of AP/ARP and tested the potential role of Pancreatic Insufficiency Prevalence (PIP) score in a cohort of children with CF. Methods: We identified two groups of children, on the basis of presence/absence of AP/ARP, who were compared for age at diagnosis, clinical features, genotypes and sweat chloride level. PIP score was calculated for each patient. Results: 10/167 (5.9%) experienced at least one episode of AP during follow up; 10/10 were pancreatic sufficient (PS). Patients with AP/ARP showed a PIP score ≤0.25 more frequently (6/10) than patients without AP/ARP. The odds ratio (95% CI) of developing pancreatitis was 4.54 (1.22-16.92) for patients with PIP b 0.25 when compared with those who have a PIP score N 0.25 (p 0.0151). PIP score was correlated with sweat chloride test (p b 0.01). Conclusion: PIP score, PS status and normal/borderline sweat chloride levels could be applied to predict pancreatitis development in children with CF. ARP could lead to pancreatic insufficiency.

Research paper thumbnail of Molecular diagnostics: between chips and customized medicine

Clinical Chemistry and Laboratory Medicine, May 1, 2010

Recently there has been an explosive increase in molecular diagnostics. This is due to translatio... more Recently there has been an explosive increase in molecular diagnostics. This is due to translational research on the molecular basis of human diseases, and to technological developments that have resulted in efficient procedures for extensive analysis of the human genome. However, a large body of data on the genome is still difficult to interpret at the clinical level. For many monogenic diseases, "modifier" genes, inherited independently of the disease gene interact, thereby resulting in a distinct phenotype for each patient. Multigenic diseases depend on complex interactions between genes and the environment. Response to drugs and side effects are modulated by gene variants. The same is true for the response to nutrients. All these interactions, which vary from patient to patient, led to the concept of "personalized medicine". Our genome consists of 25,000 genes, a surprisingly low number when compared to other species. Therefore, the complex phenotype of humans depends on a number of mechanisms that regulate gene expression, which, in turn, may be altered resulting in disease. For example, DNA methylation modulates the level of gene expression, and altered methylation of some genes is related to human neoplasias. MicroRNAs regulate the expression of a myriad of genes, and mounting evidence indicates that this mechanism may be impaired in human diseases. Finally, the relationships between genetics and human behavior are starting to be elucidated. For example, suicide may be related to alterations of methylation of specific genes. To conclude: the chip-wide analysis of human genomes is becoming easier, but the understanding of molecular genetics that confirmed the real "uniqueness" of each genome is an excellent opportunity for laboratory medicine to reposition the patient at the heart of the medical process.

Research paper thumbnail of Lung cancer metastatic cells detected in blood by reverse transcriptase-polymerase chain reaction and dot-blot analysis

Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 1997

We analyzed the blood of patients with lung cancer at different stages of presentation for the pr... more We analyzed the blood of patients with lung cancer at different stages of presentation for the presence of carcinoembryonic antigen (CEA) mRNA detected by reverse transcriptase-polymerase chain reaction (RT-PCR) combined with the dot-blot procedure as an indicator of micrometastatic malignant cells. We studied 24 lung cancer patients (10 with distant metastases and 14 with no evidence of distant metastases), eight age- and sex-matched patients affected by nonneoplastic respiratory diseases (four smokers), and eight healthy subjects. We used immunohistochemistry and RT-PCR dot-blot analysis to evaluate CEA expression in the neoplastic tissue, and the RT-PCR dot-blot procedure to analyze CEA mRNA in circulating cells. The RT-PCR dot-blot procedure was highly sensitive aspecific: it detected CEA mRNA in samples of RNA from lung cancer diluted 10(6)-fold with RNA extracted from normal blood cells, and sequence analysis confirmed that the amplified product was CEA. CEA mRNA was found in ...

Research paper thumbnail of Nasal polyposis in atypical cystic fibrosis: A case report

International Journal of Pediatric Otorhinolaryngology Extra, 2010

Cystic fibrosis (CF) is one of the most common inherited life-shortening diseases with an inciden... more Cystic fibrosis (CF) is one of the most common inherited life-shortening diseases with an incidence of 1:2.500–3500 and a carrier frequency of 4–5% [1]. It is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a protein expressed in the apical membrane of exocrine epithelial cells, resulting in changes to the fluid and electrolytes on cell

Research paper thumbnail of Congenital diarrheal disorders: An updated diagnostic approach

International Journal of Molecular Sciences, 2012

Congenital diarrheal disorders (CDDs) are a group of inherited enteropathies with a typical onset... more Congenital diarrheal disorders (CDDs) are a group of inherited enteropathies with a typical onset early in the life. Infants with these disorders have frequently chronic diarrhea of sufficient severity to require parenteral nutrition. For most CDDs the disease-gene is known and molecular analysis may contribute to an unequivocal diagnosis. We review CDDs on the basis of the genetic defect, focusing on the significant contribution of molecular analysis in the complex, multistep diagnostic work-up.

Research paper thumbnail of Tropomyosin-related kinase B receptor polymorphisms and isoforms expression in suicide victims

Psychiatry Research, 2014

. TrkB isoforms expression. TrkB isoforms expression/HPRT1-GAPDH geometric average in control and... more . TrkB isoforms expression. TrkB isoforms expression/HPRT1-GAPDH geometric average in control and suicide subjects. The values are represented as mean and standard deviation of the mean, and the P value is conducted by t-test or Mann-Whitney-test (*P¼ 0.031).

Research paper thumbnail of Exploitation of a very small peptide nucleic acid as a new inhibitor of miR-509-3p involved in the regulation of cystic fibrosis disease-gene expression

BioMed research international, 2014

Computational techniques, and in particular molecular dynamics (MD) simulations, have been succes... more Computational techniques, and in particular molecular dynamics (MD) simulations, have been successfully used as a complementary technique to predict and analyse the structural behaviour of nucleic acids, including peptide nucleic acid- (PNA-) RNA hybrids. This study shows that a 7-base long PNA complementary to the seed region of miR-509-3p, one of the miRNAs involved in the posttranscriptional regulation of the CFTR disease-gene of Cystic Fibrosis, and bearing suitable functionalization at its N- and C-ends aimed at improving its resistance to nucleases and cellular uptake, is able to revert the expression of the luciferase gene containing the 3'UTR of the gene in A549 human lung cancer cells, in agreement with the MD results that pointed at the formation of a stable RNA/PNA heteroduplex notwithstanding the short sequence of the latter. The here reported results widen the interest towards the use of small PNAs as effective anti-miRNA agents.

Research paper thumbnail of A Novel DHPLC-Based Procedure for the Analysis of COL1A1 and COL1A2 Mutations in Osteogenesis Imperfecta

The Journal of Molecular Diagnostics, 2011

Approximately 90% of patients with osteogenesis imperfecta (OI) exhibit dominant COL1A1 or COL1A2... more Approximately 90% of patients with osteogenesis imperfecta (OI) exhibit dominant COL1A1 or COL1A2 mutations; however, molecular analysis is difficult because these genes span 51 and 52 exons, respectively. We devised a PCR-denaturing high-performance liquid chromatography (DHPLC) procedure to analyze the COL1A1 or COL1A2 coding regions and validated it using 130 DNA samples from individuals without OI, 25 DNA samples from two cells to investigate the procedure's potential for preimplantation diagnosis, and DNA samples from 10 patients with OI. Three novel intronic variants in vitro were expressed using a minigene assay to assess their effects on splicing. The procedure is rapid, inexpensive, and reproducible. Analysis of samples from individuals without OI revealed six novel and some known polymorphisms useful for linkage diagnosis because of high heterozygosity. Analysis of two-cell samples confirmed the known genotype in 24 of 25 experiments; DNA failed to amplify in only one case. No incidence of allele dropout was recorded. DHPLC revealed six novel mutations, three of which were intronic, in all patients with OI, and these results were confirmed by means of COL1A1 and COL1A2 direct sequencing. Expression of intronic mutations demonstrated that variant 804 ؉ 2_804 ؉ 3delTG in intron 11 disrupts normal splicing, thereby leading to formation of two alternative products. Variants c.3046-4_3046-5dupCT (COL1A1) and c.891 ؉ 77A>T (COL1A2) did not affect splicing. The described DHPLC protocol combined with the minigene assay may contribute to molecular diagnosis in OI. Moreover, this protocol will aid in counseling about prenatal and preimplantation diagnosis.

Research paper thumbnail of Extensive Molecular Analysis of Patients Bearing CFTR-Related Disorders

The Journal of Molecular Diagnostics, 2012

Cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders (CFTR-RDs) may prese... more Cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders (CFTR-RDs) may present with pancreatic sufficiency, normal sweat test results, and better outcome. The detection rate of mutations is lower in CFTR-RD than in classic CF: mutations may be located in genes encoding proteins that interact with CFTR or support channel activity. We tested the whole CFTR coding regions in 99 CFTR-RD patients, looking for gene mutations in solute carrier (SLC) 26A and in epithelial Na channel (ENaC) in 33 patients who had unidentified mutations. CFTR analysis revealed 28 mutations, some of which are rare. Of these mutations, RT-PCR demonstrated that the novel 1525-1delG impairs exon 10 splicing; by using minigene analysis, we excluded the splicing effect of three other novel intronic variants. Analysis of SLC26A genes revealed several variants, some of which are novel, that did not affect mRNA expression. Other mutations occurred in the ENaC genes encoding the ENaC subunits, but their frequency did not significantly differ between patients and controls. Our data, although obtained on a preliminary cohort of CFTR-RD patients, exclude a role of mutations in SLC26A and in SCNN genes in the pathogenesis of such disease; we confirm that CFTR analysis has a relevant role in CFTR-RD patients; and it appears mandatory to use CFTR scanning techniques and approaches to reveal the effect of novel mutations.

Research paper thumbnail of Molecular and Functional Analysis of the Large 5′ Promoter Region of CFTR Gene Revealed Pathogenic Mutations in CF and CFTR-Related Disorders

The Journal of Molecular Diagnostics, 2013

The ASCP designates this journal-based CME activity ("JMD 2013 CME Program in Molecular Diagnosti... more The ASCP designates this journal-based CME activity ("JMD 2013 CME Program in Molecular Diagnostics") for a maximum of 48 AMA PRA Category 1 Credit(s) TM . Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. 2 0 1 3 J M D C M E P r o g r a m

Research paper thumbnail of Gene Mutation in MicroRNA Target Sites of CFTR Gene: A Novel Pathogenetic Mechanism in Cystic Fibrosis?

PLoS ONE, 2013

Cystic fibrosis (CF) is the most frequent lethal genetic disorder among Caucasians. It depends on... more Cystic fibrosis (CF) is the most frequent lethal genetic disorder among Caucasians. It depends on alterations of a chloride channel expressed by most epithelial cells and encoded by CFTR gene. Also using scanning techniques to analyze the whole coding regions of CFTR gene, mutations are not identified in up to 10% of CF alleles, and such figure increases in CFTRrelated disorders (CFTR-RD). Other gene regions may be the site of causing-disease mutations. We searched for genetic variants in the 1500 bp of CFTR 39 untranslated region, typical target of microRNA (miRNA) posttranscriptional gene regulation, in either CF patients with the F508del homozygous genotype and different clinical expression (n = 20), CF (n = 32) and CFTR-RD (n = 43) patients with one or none mutation after CFTR scanning and in controls (n = 50). We identified three SNPs, one of which, the c.*1043A.C, was located in a region predicted to bind miR-433 and miR-509-3p. Such mutation was peculiar of a CFTR-RD patient that had Congenital Bilateral Absence of Vas Deferens (CBAVD), diffuse bronchiectasis, a borderline sweat chloride test and the heterozygous severe F508del mutation on the other allele. The expression analysis demonstrated that the c.*1043A.C increases the affinity for miR-509-3p and slightly decreases that for the miR-433. Both miRNAs cause in vitro a reduced expression of CFTR protein. Thus, the c.*1043A.C may act as a mild CFTR mutation enhancing the affinity for inhibitory miRNAs as a novel pathogenetic mechanism in CF.

Research paper thumbnail of Design, synthesis and biochemical investigation, by in vitro luciferase reporter system, of peptide nucleic acids as new inhibitors of miR-509-3p involved in the regulation of cystic fibrosis disease-gene expression

Research paper thumbnail of Prothrombotic gene variants as risk factors of acute myocardial infarction in young women

Journal of Translational Medicine, 2012

Background: Acute myocardial infarction (AMI) in young women represent an extreme phenotype assoc... more Background: Acute myocardial infarction (AMI) in young women represent an extreme phenotype associated with a higher mortality compared with similarly aged men. Prothrombotic gene variants could play a role as risk factors for AMI at young age. Methods: We studied Factor V Leiden, FII G20210A, MTHFR C677T and beta-fibrinogen -455G>A variants by real-time PCR in 955 young AMI (362 females) and in 698 AMI (245 females) patients. The data were compared to those obtained in 909 unrelated subjects (458 females) from the general population of the same geographical area (southern Italy).

Research paper thumbnail of Congenital Diarrheal Disorders: Improved Understanding of Gene Defects Is Leading to Advances in Intestinal Physiology and Clinical Management

Journal of Pediatric Gastroenterology and Nutrition, 2010

Congenital diarrheal disorders (CDD, Online Mendelian Inheritance in Man [OMIM] 251850) represent... more Congenital diarrheal disorders (CDD, Online Mendelian Inheritance in Man [OMIM] 251850) represent one of the most challenging clinical conditions for pediatric gastroenterologists because of the severity of the clinical picture and the broad range of disorders in its differential diagnosis. The number of conditions included within CDD has gradually increased. Recent advances made in the pathophysiology of these conditions have led to a better understanding of the more common diarrheal diseases. Based on the body of data accumulated in recent years, we suggest that CDD be classified in 4 categories depending on the alteration in absorption and transport of nutrients and electrolytes, enterocyte differentiation and polarization, enteroendocrine cell differentiation, and modulation of the intestinal immune response. Our knowledge of the genes responsible for CDD is also rapidly increasing, thanks to linkage studies based on genome-wide analysis of polymorphisms. In this context, the identification of disease genes is a step forward in the diagnostic approach to a patient in whom CDD is strongly suspected. However, it is conceivable that faster, less expensive molecular procedures will, in the near future, become available. This approach could spare the patient invasive procedures and limit complications associated with a delay in diagnosis. Furthermore, carrier and prenatal molecular diagnosis may help pediatricians better manage the condition in the early stages of life.

Research paper thumbnail of Atypcal Cystic Fibrosis (Acf): An Experience of a Cystic Fibrosis Center

Journal of Cystic Fibrosis, 2008

Research paper thumbnail of Molecular Analysis of Genes Encoding CFTR Interactors of SLC26 Family in CF Patients: Preliminary Results

Journal of Cystic Fibrosis, 2008

Research paper thumbnail of DIRECT SEQUENCING OF CSTs IN CYSTIC FIBROSIS PATIENTS BEARING UNDEFINITE GENOTYPE

Journal of Cystic Fibrosis, 2008

Research paper thumbnail of Cystic fibrosis presenting as metabolic alkalosis in a boy with the rare D579G mutation

Journal of Cystic Fibrosis, 2004

We report on a 10-month-old boy with hypotonic dehydration and metabolic alkalosis. Sweat test wa... more We report on a 10-month-old boy with hypotonic dehydration and metabolic alkalosis. Sweat test was borderline and genetic analysis was negative for common mutations. Analysis of the whole coding regions of the CFTR gene revealed the rare mutation D579G in homozygosity. D

Research paper thumbnail of Phenotypic discordance in three siblings affected by atypical cystic fibrosis with the F508del/D614G genotype

Journal of Cystic Fibrosis, 2006

We report an example of atypical CF, i.e., a family in which three siblings were affected by late... more We report an example of atypical CF, i.e., a family in which three siblings were affected by late-diagnosed mild CF, and showed discordant pulmonary and pancreatic phenotypes. Sibling no. 1 (male), showed a severe pulmonary involvement and pancreatic sufficiency; sibling no. 2 (female) showed a mild pulmonary disease with pancreatic sufficiency; sibling no. 3 (male) had a very mild pulmonary expression and pancreatic insufficiency. The sweat test was altered in all three siblings, and all had intestinal occlusion in young age. The whole scanning of CFTR revealed the rare F508del/D614G genotype. The discordance of clinical expression within the same family reinforces the putative role of modifier genes of CF phenotype.

Research paper thumbnail of Epidemiology and a novel procedure for large scale analysis of CFTR rearrangements in classic and atypical CF patients: A multicentric Italian study

Journal of Cystic Fibrosis, 2008

Background: Mutation epidemiology in each ethnic group is a crucial step of strategies for cystic... more Background: Mutation epidemiology in each ethnic group is a crucial step of strategies for cystic fibrosis (CF) diagnosis and counselling. To date, the scanning of the whole coding region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene permits to identify about 90% of alleles from patients bearing CF and a lower percentage in patients bearing atypical CF. CFTR rearrangements in heterozygosis elude current techniques for molecular analysis, and some of them have been reported with a frequency up to 6% in various ethnic groups. Methods: Using quantitative PCR analysis of all coding regions, we assessed the occurrence of CFTR rearrangements in 130 alleles from classic CF patients and in 198 alleles from atypical CF patients (all unrelated and from Italian descent) bearing unidentified mutations after the scanning of CFTR. Results: Seven rearrangements (i.e., dele1, dele2, dele2_3, dele 14b_17b, dele17a_18, dele22_23, and dele22_24) were identified in 34/131 (26.0%) CF alleles bearing undetected mutations (which means about 2.5% of all CF alleles) and in none of the 198 alleles from atypical CF. The CFTR haplotype and the sequence analysis of the breakpoints confirmed the common origin of all the rearrangements. Thus, we set up a novel duplex PCR assay for the large-scale analysis of the seven rearrangements. The procedure was rapid (all PCR amplifications were obtained under the same conditions), costless and repeatable. Conclusions: It is useful to select the CFTR rearrangements more frequent in specific ethnic groups and to set up procedures for large-scale analysis. Their study can be performed in cases in which a high detection rate is required (i.e., partners of CF carriers/patients). On the contrary, the analysis of rearrangement is useless in atypical CF patients.

Research paper thumbnail of Prediction of acute pancreatitis risk based on PIP score in children with cystic fibrosis

Journal of Cystic Fibrosis, 2014

Background: Currently no tools to predict risk of acute (AP) and recurrent pancreatitis (ARP) in ... more Background: Currently no tools to predict risk of acute (AP) and recurrent pancreatitis (ARP) in children with cystic fibrosis (CF) are available. We assessed the prevalence of AP/ARP and tested the potential role of Pancreatic Insufficiency Prevalence (PIP) score in a cohort of children with CF. Methods: We identified two groups of children, on the basis of presence/absence of AP/ARP, who were compared for age at diagnosis, clinical features, genotypes and sweat chloride level. PIP score was calculated for each patient. Results: 10/167 (5.9%) experienced at least one episode of AP during follow up; 10/10 were pancreatic sufficient (PS). Patients with AP/ARP showed a PIP score ≤0.25 more frequently (6/10) than patients without AP/ARP. The odds ratio (95% CI) of developing pancreatitis was 4.54 (1.22-16.92) for patients with PIP b 0.25 when compared with those who have a PIP score N 0.25 (p 0.0151). PIP score was correlated with sweat chloride test (p b 0.01). Conclusion: PIP score, PS status and normal/borderline sweat chloride levels could be applied to predict pancreatitis development in children with CF. ARP could lead to pancreatic insufficiency.