Fabrizio Tosto

In utero exposure to di-(2-ethylhexyl) phthalate affects liver morphology and metabolism in post-natal CD-1 mice

The plasticizer di-(2-ethylhexyl)phthalate (DEHP) affects reproductive development, glycogen and lipid metabolism. Whereas liver is a main DEHP target in adult rodents, the potential impact on metabolic programming is unknown. Effects of in utero DEHP exposure on liver development were investigated upon treatment of pregnant CD-1 mice on gestational days (GD)11-19. F1 mice were examined at post-natal days 21 (weaning) and 35 (start of puberty): parameters included liver histopathological, immunocytochemical and alpha-fetoprotein (AFP) gene expression analyses. In utero DEHP exposure altered post-natal liver development in weanling mice causing significant, dose-related (i) increased hepatosteatosis, (ii) decreased glycogen storage, (iii) increased beta-catenin intracytoplasmic localization (females only). At puberty, significantly decreased glycogen storage was still present in males. A treatment-induced phenotype was identified with lack of glycogen accumulation and intracytoplasmic localization of beta-catenin which was associated with increased AFP gene expression. Our findings suggested that DEHP alters post-natal liver development delaying the programming of glycogen metabolism.

Characterization of HuH6, Hep3B, HepG2 and HLE liver cancer cell lines by WNT/β - catenin pathway, microRNA expression and protein expression profile.

Somatic mutations in the genes members of WNT/β-catenin pathway, especially in CTNNB1 codifying for β-catenin, have been found to play an important role in hepatocarcinogenesis. The purpose of this work is to characterize alterations of the WNT/β-catenin signalling pathway, and to study the expression pattern of a panel of microRNAs and proteins potentially involved in the pathogenesis of liver cancer. In this respect, the molecular characterization of the most used liver cancer cell lines HuH6, Hep3B, HepG2, and HLE, could represent a useful tool to identify novel molecular markers for hepatic tumour. A significant modulation of FZD7, NLK, RHOU, SOX17, TCF7L2, TLE1, SLC9A3R1 and WNT10A transcripts was observed in all the four liver cancer cell lines. The analysis of selected microRNAs showed that miR-122a, miR-125a and miR-150 could be suitable candidates to discriminate tumoural versus normal human primary hepatocytes. Finally, Grb-2 protein expression resulted to be increased more than two-fold in liver cancer cell lines in comparison to normal human primary hepatocytes. These advances in the knowledge of molecular mechanisms involved in the pathogenesis of liver cancer may provide new potential biomarkers and molecular targets for the diagnosis and therapy.

The Italian External Quality Assessment Scheme for Fragile X Syndrome: The Results of a 5-Year Survey

The Italian External Quality Assessment scheme for fragile X syndrome started in 2001 as an activity funded by the National Health System and coordinated by the National Institute of Public Health. The aim of this work is to present the data of 5 years (2001--2004 and 2006) of survey. The External Quality Assessment scheme was designed to cover the following points: (a) genotyping and (b) interpretation and reporting of results. Overall, the scheme covered about 65% of all Italian public laboratories. The average reporting of results was 91.6%, with an overall success rate of 76%. The rate of diagnostic errors observed was on average 5%. Inaccuracy in sizing of CGG repeats of normal and premutated alleles was reported. During the survey the proportion of laboratories using a Southern blotting, polymerase chain reaction, and ABI sizing kit in combination rose from 36.8% to 70.6%. The reports from laboratories showed incompleteness and considerable variations in expected outcomes. For this reason, in 2004 a model for written reports was introduced. In conclusion, these data underscore the need to participate in External Quality Assessment schemes as an educational resource to ensure quality in molecular genetic testing.

The Italian External Quality Control Programme for cystic fibrosis molecular diagnosis: 4 years of activity.

Abstract Background: The Italian External Quality Control Programme for cystic fibrosis molecular diagnosis started in 2001; public laboratories distributed throughout Italy participated on a voluntary basis. Methods: The Italian Public Health Institute (Istituto Superiore di Sanità) sent six validated DNA samples to participating laboratories: technical and clinical information was provided for each sample. Laboratories were required to analyse all six samples. For each sample the laboratories had to provide the results (including raw data) and a report of molecular analysis within 2 months using current methods and nomenclature. Raw data and reports were evaluated by a Steering Committee and their comments were sent to each laboratory. Results: Genotyping results indicated a general good level of quality for all laboratories, i.e., ∼1% of alleles were incorrectly assigned each year due to analytical (45%) and misinterpretation (45%) errors. During the first 2 years, more than 70% of laboratories did not test for some regional Italian mutations. Commercial kits for reverse dot-blot and oligonucleotide ligation assay PCR were used to detect mutations by 52.8% and 29.5%, respectively, of the participating laboratories. Reporting of results was still inadequate; in 2004 a model for the written report was introduced, but not all laboratories used it. Conclusions: Our data show that few genotyping errors were made by laboratories and were principally due to misinterpretation and analytical reasons. However, reports are still inadequate and it will be interesting to evaluate the introduction of the reporting model in future years. Clin Chem Lab Med 2007;45:254–60.

The Italian Scheme of External Quality Assessment for β-Thalassemia: Genotyping and Reporting Results and Testing Strategies in a 5-Year Survey

The Italian scheme of External Quality Assessment for beta-thalassemia started in 2001 as part of a project twice funded by the Italian Ministry of Health and coordinated by the Istituto Superiore di Sanità. To date, five trials have been performed (2001-2004 and 2006). The aim of the Italian scheme is to help public laboratories in improving and reaching a high standard of quality when performing a molecular test. Many laboratories took part in the 5-year project, and their participation was constant during the whole period. The aims of this paper are to describe the genotyping and reporting results as well as focusing on the techniques and the testing strategies adopted to detect mutations. Almost 99% of the alleles analyzed were correctly detected by laboratories, while 0.33% of the analyses gave a wrong result. Reverse dot blot was the most used technique, and it was always used in the strategies adopted by laboratories to detect mutations. The reports sent by laboratories showed incompleteness and heterogeneity; thus, a new model for written reports has been introduced since 2004. It will be interesting to monitor the effects of the reporting model and the output of this educational action in the future.

The Italian External Quality Assessment Scheme in Classical Cytogenetics: Four Years of Activity

Background: The Italian external quality assessment scheme in classical cytogenetics was started in 2001 as an activity funded by the National Health System and coordinated by the Italian Public Institute of Health. Objectives: The aim of our work is to present data from the first 4 years of activity, 2001–2004. Methods: Italian cytogenetics public laboratories were enrolled on a voluntary basis, and this nationwide program covered prenatal, postnatal and oncological diagnosis. The scheme is annual and retrospective; a panel of experts reviewed the quality of images and reports in order to assess technical, analytical and interpretative performance. Results: Over the 4-year period, the number of participating laboratories increased: from 36 in 2001, 46 in 2002, 49 in 2003 to 51 in 2004. The overall technical performance was satisfactory. Inadequacy or lack of information in reporting was the most frequent analytical inaccuracy identified in all parts of the scheme. However, the percentage of complete reports increased significantly during the period: by 36% in postnatal diagnosis between 2001 and 2004 (p < 0.001) and by 42% in oncological diagnosis between 2002 and 2004 (p = 0.003). Conclusions: Our experience reveals that participation in external quality assessment programs has significant advantages, helping to standardize and to assure quality in cytogenetic testing.

The Italian National External Quality Assessment Program in Molecular Genetic Testing: Results of the VII Round (2010-2011)

Since 2001 the Istituto Superiore di Sanità established a quality assurance programme for molecular genetic testing that covers four pathologies: Cystic Fibrosis (CF), Beta Thalassemia (BT), Fragile X Syndrome (FX), and Familial Adenomatous Polyposis Coli (APC). Since 2009 this activity is an institutional activity and participation is open to both public and private laboratories. Seven rounds have been performed until now and the eighth is in progress. Laboratories receive 4 DNA samples with mock clinical indications. They analyze the samples using their routine procedures. A panel of assessors review the raw data and the reports; all data are managed through a web utility. In 2010 the number of participants was 43, 17, 15, 5 for CF, BT, FX, APC schemes respectively. Genotyping results were correct in 96%, 98.5%, 100%, and 100% of CF, BT, FX, and APC samples, respectively. Interpretation was correct in 74%, 91%, 88%, and 60% of CF, BT, FX, and APC reports, respectively; however in most of them it was not complete but a referral to genetic counseling was given. Reports were satisfactory in more than 60% of samples in all schemes. This work presents the 2010 results in detail comparing our data with those from other European schemes.

The Italian External Quality Control Program for Familial Adenomatous Polyposis of the Colon: Five Years of Experience

Familial adenomatous polyposis is a rare autosomal dominant inherited disease (incidence, 1/8000). More than 90% of families affected by familial adenomatous polyposis have a mutation in the tumor suppressor gene adenomatous polyposis coli (APC). Mutations in this gene are characterized by 100% penetrance, although there is a variation in phenotypic expression of the disease. According to a 2004 survey of the Italian Human Genetic Society, about 264 APC gene molecular genetic tests were performed by Italian laboratories per year. The Italian External Quality Assessment (IEQA), financially supported by the Ministry of Health and coordinated by the Istituto Superiore di Sanità, was started in 2000 to improve the quality of molecular genetic tests in Italy. In the frame of the IEQA, about 50% of public laboratories performing APC gene tests have been monitored. The number of responding public laboratories during the 5 years was 6, 7, 7, 7, and 5 from 2001 to 2006, respectively; on average, 96.3% of samples completely analyzed were correctly genotyped. Methods used by laboratories to detect mutation were direct sequencing, single-strand conformation polymorphism, protein truncation test, and denaturing high-performance liquid chromatography. Written reports were not homogeneous among laboratories, although a new form of written report was proposed to laboratories in 2004. It will be interesting to monitor the effects of the reporting model and the output of this educational action in the future.

The Italian National Centre for Rare Diseases: where research and public health translate into action

The Italian National Centre for Rare Diseases (CNMR) is the result of a strategic approach, which the National Institute of Health (ISS) has been developing for more than 10 years, to deal with the public health challenges associated with rare diseases (RDs). The CNMR was formally established within the ISS in 20081. Its mission is to promote and develop experimental research and public health actions, as well as to provide technical expertise and information on RDs and orphan drugs, for the prevention, treatment and surveillance of these diseases. It is also the national focal point for information and communication for patients suffering from one of several thousand RDs, and for their families, collaborating with the national organisations of patients suffering from RDs. The Centre employs a wide range of scientific and technical expertise from various fields (medicine, genetics, molecular biology, epidemiology, public health, psychology, sociology etc.) and holds a network of national and international collaborations, which allow the development of a sound and integrated approach to RDs. The CNMR provides expert advice to the Italian Ministry of Health (MOH), to the National Health Council, to the National Health Service (NHS), and collaborates with the Regions, which are responsible for the provision of health services in the Italian devolved health system. Expert advice on RDs is also provided at EU and at international level. Since its establishment, the Centre has developed into a lively and propulsive hub for experimental research, public health, information, communication and training on RDs in Italy, and for patient empowerment. In addition, it has contributed to networks and scientific boards at national, European and international level and has implemented a number of strategic projects on RDs. The Centre is in continuous evolution in order to follow closely the pace of science and research, the emerging needs of patients, the solicitations of policy makers, and the demands of the health system.