phd students’ works annual progress report 2007-2008 ... · on the basis of research activity the...

Oncology and Genetics

PhD Students ’ works

Annual Progress Report 2007-2008

University of Siena

Doctoral School

Molecular Biology Department

and

Human Pathology and Oncology Department

Information Engineering Department

Pediatrics, Obstetrics and Reproduction Medicine Department

Surgery Department

Surgery and Bioengineering Department

and

S.H.R.O. Sbarro Health Research Organization

Fiorgen Onlus

Oncology and GeneticsDoctoral School

PhD Students ’ works

Annual Progress Report 2007-2008

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Oncology and Genetics Doctoral School

This initiative is aimed to spread the information on the research activities of PhDstudents in our academic community.

The pamphlet is in English in order to promote Doctoral Schools of our University atinternational level, with particular attention to those foreign institutions with which wehave signed international cooperation agreements. Moreover, it could also be use-ful to foster new agreements with foreign partners.

The RectorProf. Silvano Focardi

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This pamphlet was created to regroup and present together the research activities of thestudents of the Doctoral School in Oncology and Genetics in order to spread informationabout the work of the students and to promote the collaboration on research projects.

The first pages illustrate the activity of the “annual progress report day”. This event takesplace at the end of each academic year and is dedicated to the presentation of both theresearch projects proposed by the new entered students and the annual progress reportsof the older students.The pamphlet continues with the presentation of the research abstracts of the 36 PhD stu-dents. Finally, the last pages are dedicated to the “thesis discussion days”. In the last ses-sion of thesis discussion, the qualification of “Doctor Europaeus” was conferred to two stu-dents.

I wish to dedicate this pamphlet to the PhD students who represent the “mainstay” of theInstitution that we call University with their continuous daily work, their perseverance andmotivation.

The director of the SchoolProf. Alessandra Renieri

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The Doctoral School in Oncology and Genetics is constituted of 4 sections or “educationtrainings”:1) Medical Genetics coordinated by Alessandra Renieri2) Oncological Genetics coordinated by Antonio Giordano3) Colorectal and Gastroesophageal Diseases coordinated by Gabriello Tanzini4) Hepatobiliopancreatic Diseases and Multitumoral Syndromes coordinated by FrancescoCetta

In addition to the four above mentioned coordinators, the Faculty Board is composed byteachers from the University of Siena: Antonio Acquaviva, Alfio Andronico, MonicaBianchini, Alessandro Cappelli, Anton Ferdinando Carli, Maddalena Cioni, GiuseppePasquale Cito, Serenella Civitelli, Antonio De Martino, Paolo Frezzotti, TheodoraHadjistilianou, Marco Lorenzi, Sergio Mancini, Giuseppe Marzocca, Clelia Daniela AnnaMiracco, Roberto Ponchietti, Francesco Salvestrini, Francesco Tani, Walter Testi, PaoloToti, Luigi Verre; and by teachers from other Universities: Maurizio Genuardi from theUniversity of Florence, Pier Paolo Pandolfi from the Cornell University, New York, Hans vanBokhoven from the University of Nijmegen, The Netherlands.

On the basis of research activity the School has signed 7 International CooperationAgreements with the following Universities:Bilkent University, Ankara, Turkey;Duisburg-Essen University , Germany;Freiburg University, GermanyGreenwood Genetic Center, Greenwood, South Carolina, USA;Kentucky University, Lexington, USA;Radboud University of Nijmegen, The Netherlands;St. Kliment Ochridski University, Sofia, Bulgaria.

The Doctoral School in Oncology and Genetics at the University of Siena trains students tocarry out research in Medical Genetics and in Clinical and Molecular Oncology over a fouryears program. The aim of this Doctoral School is to train researchers who will be able toplan and develop competitive research proposals. The School has a dedicated web site atthe following address: http://www.unisi.it/ricerca/dottorationweb/genetica_medica/. In thissite it is possible to find general information on the School, seminar activities, research pro-jects, and PhD students scientific “identity card”.

Index

Annual progress report day

Students Project Abstracts

• Giovanni Abbadessa Gene Therapy with oncosuppressor genes in Primary Liver Cancer xenograft

• Mariangela Amenduni Searching The Gene Responsible For Ter-Haar Syndrome

• Rosangela ArtusoAlport Syndrome With Diffuse Leiomyomatosis

• Annamaria AzzaràOxidative Stress In The Pathogenesis Of Environment Related Health DamageIn The City Of Milan.

• Leonardo Barellini Genetic predisposition in patients with inherited multitumoral syndromes and environmental factors (ionizing radiations, toxic agents) in the pathogenesis of inflammatory and neoplastic disease of the thyroid.

• Simona Benoni Identification Of Four New Germinal Mutations In Pten Gene

• Maria Giovanna BoianoNovel biomarkers in prostate cancer

• Rossella Caselli Multiple congenital anomalies and mental retardation: analysis by oligo array-CGH

• Vincenza Causarano Analysis of fetal nucleic acids in maternal plasma

• Antonella Chessa Quality of life in colorectal cancer patients after surgery and a related counselling program

• Filomena CisterninoPolyciclic Aromatic Hydrocarbons (Pahs) As A Measure Of Pollution Related Health Risk. Possible Use As A Marker Of Road Traffic Emission.

• Roberta De Filippis Expression analysis of the mental retardation gene FACL4

• Armand DhamoFamilial Adenomatous Polyposis (FAP)

• Eleonora FerrariSignal-Dependent control of Autophagy

• Irene GrassiFamiliar risk in colorectal cancer

• Valeria Guarnaccia The prolyl-isomerases PIN1 regulates the cyto-differentiating activity of retinoids in myeloid leukemia cells

• Eleni KatzakiClinical and molecular characterization of Italian patients affected by Cohen syndrome

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• Khadang Baharak Denaturing High Performance Liquid Chromatography(DHPLC) in X-Linked Alport Syndrome Diagnosis

• Elvana KolaPollution related Inflammatory and Malignant Diseases in subjects living in Milan, in particular, concerning the possible role of infection and airway pH.

• Raffaele La MontagnaProline-Rich Antibactirial Peptides: Inhibition DnaK Chaperone Activiy

• Paolo Laviano Pollution related cardiovascular, cutaneous and osteoarticular diseases in patients living in the city of Milan

• Giuliana Malagnino Familial adenomatous polyposis

• Mario MancinoMechanisms regulating Cripto 1 gene expression in cancer cells

• Elena Marcocci Autosomal Alport syndrome

• Federico MarianiPredicting Invasivity Of Intraductal Papillary Mucinous Tumours Of The Pancreas

• Monica MischitelliHuman Polyomavirus BK and Prostate Cancer: evaluation of BKV variants,host immune response and tumor host cytokines in cancer development

• Filomena Tiziana Papa Rett syndrome: MECP2 genotype-phenotype correlation

• Veronica Parri Functional characterization of FACL4 protein involved in X-linked mental retardation

• Flavio RizzolioThe role of pRb2/p130 in epigenetic modulation of Estrogen Receptor- in breast cancer

• Annalisa RobertiAberrant methylation in the Gal1 sporadic Burkitt lymphoma-derived cell line

• Dalila RondinellaNew HPRT mutation in an Italian family affected by Lesch-Nyhan syndrome

• Ariele Spanhol RossetoGenetic modifier factors in Rett syndrome

• Katia SampieriGenomic differences between retinoma and retinoblastoma

• Elisa Scala Molecular bases of the clinical variability in Rett syndrome

• Tiziana SquillaroMesenchymal stem cells: a model for Rett syndrome analysis

• Marina Vignoli Comprehensive analysis of CDKN2A gene mutations in familial melanoma patients

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Annual Progress ReportOncology and Genetics Doctoral SchoolOctober 1, 2007 Centro Didattico S Maria alle Scotte

Morning session

8.45 Introduction Alessandra Renieri

9.00 Final report of the XIX cycle (15 minutes for each student) Chairman Mario De Marchi

Rossella Caselli (A. Renieri) Multiple congenital anomalies and mental retardation: analysis by oligo array-CGH

Armand Dhamo (F. Cetta) Extracolonic manifestations of Adenomatous Polyposis of the Colon

Federico Mariani (F. Cetta-E. Pinto) Predicting Invasivity Of Intraductal Papillary Mucinous Tumours Of The Pancreas

Elisa Scala (A. Renieri) Molecular bases of the clinical variability in Rett syndrome

10.00 Progress report of the 3rd year, XX cycle (10 minutes for each student)Chairman Antonio Giordano

Leonardo Barellini (F. Cetta) Genetic predisposition related to inherited multitumoral syndromes and environmental polluttans (ionizing radiations, toxic substance) in the occurrence of inflammatory and neoplastic diseases of the thyroid.

Giulia Calamati (F. Cetta) Pollution related chronic inflammation in the occurrence of inflammatory or neoplastic diseases of abdominal organs, in particular concerning the role of p53, as an early event in the ouset of the disease.

Vincenza Causarano (A.Renieri-M. Ferrari) postponed at 6.30 pm (videoconference)Analysis of fetal nucleic acids in maternal plasma

Annalisa Roberti (A. Giordano) Aberrant methylation in the Gal1 sporadic Burkitt lymphoma-derived cell line.

Katia Sampieri (A. Renieri) Genetic differences between retinoma and retinoblastoma

11.00 Progress report of the 2nd year, XXI cycle (10 minutes for each one)Chairman Gabriello Tanzini

Rosangela Artuso (A. Renieri) Molecular basis of Alport syndrome and leiomiomatosis (ATS-DL)

Antonella Chessa (G. Tanzini) Quality of life in colorectal cancer patients after surgery and a related counselling program

Irene Grassi (G. Tanzini) Familial risk in colorectal cancer patients

Eleni Katzaki (A. Renieri) ) Clinical and molecular heterogeneity in Italian patients affected by Cohen syndrome.

Giuliana Malagnino (F. Cetta) Physicochemical and toxicological analysis of PM

Mario Mancino (A. Giordano) postponed at 6.40 pm (videoconference)Mechanisms regulating Cripto 1 gene expression in cancer cells

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Tiziana Squillaro (A. Renieri- U. Galderisi) Mesenchymal stem cells: a model for Rett syndrome analysis

Marina Vignoli (M. Genuardi) Comprehensive analysis of CDKN2A gene mutations in familial melanoma patients

12.30 Finger social lunch and poster viewing

Afternoon session:

14.30 Progress report of the 1st year, XXII cycle (5 minutes for each student) Chairman Antonio Giordano

Giovanni Abbadessa (A. Giordano) postponed at 6.50 pm (videoconference) Gene Therapy with oncosuppressor genes in Primary Liver Cancer xenograft.

Simona Benoni (F. Cetta - M. De Marchi)Identification of four new germinal mutations in PTEN gene

Filomena Cisternino (F. Cetta) "In vitro" and in humans toxicity and teratogenicity of polycyclic aromatic hydrocarbons, transitional metals and organic component of PM in subjects living in Milan.

Eleonora Ferrari (A. Giordano - G. Guanti)) Signal-Dependent control of Autophagy

Valeria Guarnaccia (A. Renieri - E. Garattini)The prolyl-isomerases PIN1 regulates the cyto-differentiating activity of retinoids in myeloid leukemia cells

Baharak Khadang (A. Renieri)X-linked Alport syndrome

Elena Marcocci (A. Renieri)Autosomal Alport syndrome due to COL4A4 mutation

Filomena Papa (A. Renieri)Oligo array-CGH analysis: comparison of two different platforms, identification of atypical phenotype in known syndromes and novel microdeletion syndromes.

Ariele Rosseto (A. Renieri)Genotype-phenotype correlation in 159 Rett patients: the combined effect of MECP2 mutation type, CDKL5 and BDNF polymorphisms.

Flavio Rizzolio (A. Giordano)The role of pRb2/p130 in epigenetic modulation of Estrogen Receptor- in breast cancer

16.30 Presentation of the PhD students program of the XXIII cycle (3 minutes for each student) Chairman Francesco Cetta

Mariangela Amenduni (A. Renieri - H. Van Bokhoven)Searching the gene responsible for the Ter-Haar syndrome

Annamaria Azzarà (F. Cetta)Oxidative stress, ROS production, in vitro and in vivo alterations due to PM in residents in the city of Milan.

Maria Giovanna Boiano (A. Giordano)Novel biomarkers in prostate cancer

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Roberta De Filippis (A. Renieri)Expression analysis of the mental retardation gene FACL4.

Elvana Kola (F. Cetta)Pollution related inflammatory and malignant diseases in subjects living in Milan, in particular concerning the possible role of infection and airway pH.

Raffaele La Montagna (A. Giordano)Proline-Rich Antibactirial Peptides: Inhibition DnaK Chaperone Activiy.

Paolo Angelo Maria Laviano (F. Cetta)Pollution related cardiovascular, allergic, cutaneous and osteoarticular diseases in residents of the city of Milan.

Monica Mischitelli (A. Giordano- VA. Pietropaolo)Human Polyomavirus BK and Prostate Cancer: evaluation of BKV variants, host immune response and tumor host cytokines in cancer development

Veronica Parri (A. Renieri)Functional characterization of FACL4 protein involved in X-linked mental retardation.

Dalila Rondinella (A. Renieri)New HPRT mutation in an Italian family affected by Lesch-Nyhan

6.30 pm Videoconference

Vincenza Causarano (A.Renieri-M. Ferrari) Analysis of fetal nucleic acids in maternal plasma

Mario Mancino (A. Giordano) Mechanisms regulating Cripto 1 gene expression in cancer cells

Giovanni Abbadessa (A. Giordano)Gene Therapy with oncosuppressor genes in Primary Liver Cancer xenograft.

7.00 pm Closing session and attribution of credits by the faculty board

A copy of the minutes is available at http://www.unisi.it/ricerca/dottorationweb/genetica_medica/ accessing the "Minutes" link

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Gene Therapy with oncosuppressor genes in Primary Liver Cancer xenograft

Medical therapy for hepatocellular carcinoma (HCC) is currently inefficient. Clinical trials are ongoing to testthe efficacy of new molecules, but definitive results are not available yet. Rb2 monitors cell cycle progres-sion mostly by interacting with E2F family members of transcription factors and with D cyclins. Its expres-sion levels correlate inversely with HCC prognosis and VEGF production. Since angiogenesis is fundamen-tal for the development of this particular kind of cancer, tumor transfection with Rb2 should stop the tumorgrowth and possibly its related angiogenesis. The addion of other oncosuppressor genes working on diffe-rent pathways may even cause tumor regression.

METHODS: 2.5 X 106 of Hep3b human liver carcinoma cells have been injected subcutaneously in theflanks of nude mice. When the tumors reached a volume of approximately 200 mm3, 5 X 1012 particles ofadenoviruses containing various genes have been injected intratumorally, in different treatment and controlgroups of mice. Each group was formed by 8 animals, and the experiment was repeated twice. Resultshave been evaluated by measuring tumor volumes and weight, by pathological assessment, and are beingevaluated by dosing the expression of the gene products, of markers of angiogenesis and of cyclins.

Results were interesting but cannot be disclosed yet due to ongoing patent application.

Oncology and Genetics Doctoral SchoolOncological GeneticsXXII cycle

Giovanni Abbadessa, MD

Tutor A. Giordano


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Oncology and Genetics Doctoral SchoolMedical Genetics

XXIII cycleMariangela Amenduni, MS

Tutor A. Renieri

Searching The Gene Responsible For Ter-Haar Syndrome

Homozygosity mapping is a highly efficient method for the discovery of autosomal recessive disease loci. Thismethodology maps homozygous regions in patients from related parents. The greater the number of affectedindividuals who have a shared homozygous region defined by a stretch of consecutive homozygous markersmapping to the region, the more likely the region is to harbour the disease-causing mutation. In this projectwe applied homozygosity mapping to identify a disease locus for Ter-Haar syndrome. Fourteen affected indi-viduals from 10 families where subject to whole-genome homozygosity mapping using a 250K Single-Nucleotide-Polymorphism (SNP) microarray. Analysis of the SNP microarray data using an excel homozy-gosity template revealed evidence for genetic heterogeneity, i.e. there was no single locus for Ter-Haarsyndrome matching all families. The most promising homozygous locus is on chromosome 5. We selectedthree candidate genes, STC-2, LOC153222 and BNIP1 from this region, according to their predicted biologi-cal function or tissue-expression. No mutations were identified in these genes, suggesting that another genein the homozygous locus may carry the disease mutations. If the causative gene from this locus will be resol-ved it may guide the identification of other genes in Ter-Haar families not mapping to chromosome 5.

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Oncology and Genetics Doctoral SchoolMedical GeneticsXXI cycleRosangela Artuso, MS

Tutor A. Renieri

Alport Syndrome With Diffuse Leiomyomatosis

Alport syndrome (AS) is a hereditary glomerulonephritis progressing to end-stage renal failure and usuallyaccompanied by high-tone sensorineural deafness and ocular abnormalities (anterior lenticonus and macu-lar flecks). Inheritance is X-linked in about 85% of cases, caused by different mutations in the COL4A5 gene.Rarely AS is associated with diffuse leiomyomatosis (DL). DL is a benign nodular smooth muscle tumor ofthe digestive tract, mainly of the esophagus , of the tracheobronchial wall, of the lower intestinal tract and thefemale genitalia. The COL4A5 and COL4A6 genes are located head to head on chromosome Xq22. It'sknown that patients with intragenic deletion of COL4A5 or deletion of COL4A5 and COL4A6 extendingbeyond exon 3 of COL4A6 develop only AS. Patients with deletion of COL4A5 and only exon 1 and 2 ofCOL4A6 develop ATS-DL (Figure). In our previous study, we have performed a detailed characterization ofa submicroscopic deletion in a patient with AS and DL. The mechanisms of smooth muscle overgrowth inATS-DL are unknown. We have hypothesize that the partial deletion of COL4A6 may cause a rearrangementof the gene eventually leading to overexpression of an alternative transcript in an inappropriate tissue leadingto tumor development. To confirm this hypothesis I performed Race-PCR of COL4A5 and COL4A6 genes in36 tissues. This technique allows the amplification of an unknown 5' or 3' of a transcript using known sequen-ce. Race-PCR products with an unexpected length were analysed by direct sequencing. I have analysed 8out of 16 selected tissues but until now no variations have been found.

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Oncology and Genetics Doctoral School Hepatobiliopancreatic Disease and Multitumoral Syndromes

XXIII cycle Annamaria Azzarà, MS

Tutor F. Cetta

Oxidative Stress In The Pathogenesis Of Environment Related Health Damage In

The City Of Milan

Airborne particulate matter (PM) increases morbidity and mortality from cardiopulmonary diseases includingcancer, as well as the occurrence of permanent damage to the development of respiratory tract in children. One possible mechanism is that PM is genotoxic to alveolar ephitelial cells (AEC) by causing DNA damageand apoptosis, in a manner similar to other toxic particulates such as silica and asbestos.Mutagenicity of airborne PM is caused by at least 500 mutagenic components of varying chemical classes,PM causes dose dependent AEC DNA strand break formation, reductions in mitochondrial membrane poten-tial, caspase 9, and caspase 3 activation, and apoptosis. These effects are prevented by iron chelators andfree radical scavengers. PM causes AEC DNA damage and apoptosis by mechanism involving the mito-chondrial regulated death pathway and the generation of free radicals. Oxidative stress contributes to thepathophysiology of both asthma and interstitial lung diseases (fibrosing alveolitis, sarcoidosis) as well as car-diovascular diseases. An imbalance between oxidants and antioxidants is a major determinant of many path-ways leading to clinical diseases, even if individual susceptibility and vulnerability is also responsible for thewide variety of final results in terms of health damage. When cultured in vitro alveolar macrophages isolatedfrom patients with a wide range of pollution related diseases produce increased amounts of superoxideanions . Moreover indicators of free radical activity are increased in both serum and bronchoalveolar lavage(BAL gluid).The aim of the present research has been to try to elucidate the possible role of oxidative stress in the patho-genesis of environment related health damage in the city of Milan.In particular, evaluation of oxidative stress performed in macrophages and alveolar cells obtained either byBAL or by biopsies or surgical speciemens in patients with COBD, asthma, lung cancer, and other major dis-eases of the tract and componed with a population without evident pulmonary diseases living since birth ormore than 40 years in the city of Milan

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Surgery Doctoral School Hepatobiliopancreatic Disease and Multitumoral Syndromes XX cycle Leonardo Barellini, MD

Tutor F. Cetta

Genetic predisposition in patients with inherited multitumoral syndromes and envi-

ronmental factors (ionizing radiations, toxic agents) in the pathogenesis of inflam-

matory and neoplastic disease of the thyroid

Thyroid gland completes its full development during childhood. Therefore thyroid cells are higly vul-nerable to environmental pollutants (ionizing radiations, toxic substances) during this specialsusceptibility window. An increased number of thyroid carcinomas occurred after the Chernobylnuclear disaster, not only in children living in Bielorus, but also in frail subjects, such as those withinherited multitumoral syndromes, namely familial adenomatous polyposis, (FAP) even if living at adistance of hundreds to thousands km from the primary site of emission. In particular in about onethird of FAP patients a very unusual variant of papillary thyroid carcinoma, was found, the so calledcribiform, that is very rare in sporadic patients (0.1%). Aim of the study: in order to further evaluatethe possible role of environmental pollutants in the occurrence of both inflammatory and neoplasticdisease of the thyroid, 2 groups of patients have been evaluated.• 18 patients with thyroid papillary carcinomas associated with FAP.• 300 consecutive patients younger than 30 years, who underwent to thyroidectomy for papil-lary thyroid carcinoma.

The following outcomes will be expected:1) To demonstrate that in a consecutive series of patients with thyroid carcinoma younger

than 30, when the cribriform morular variant was present, there is a significant proportion of patients with previously undiagnosed FAP.

2) Not only big nuclear disaster, but also longlasting environmental toxic agents, could play a role in the occurrence of thyroid diseases, namely in predisposed or frail subjects, with greater susceptibility to health damage from airborne pollutants.

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Oncology and Genetics Doctoral School Hepatobiliopancreatic Diseases and Multitumoral Syndromes

XXII cycle Simona Benoni, MS

Tutor F. Cetta - Co-tutor M. De Marchi

Identification Of Four New Germinal Mutations In PTEN Gene

Germline mutations of PTEN gene (Phosphatase, TEnsin homologue, deleted on chromosome TEN,10q23.3) are responsible for at least four syndromes: Cowden syndrome (CS), Bannayan-Riley-Ruvalcabasyndrome (BRRS), Proteus syndrome (PS) and Proteus-like syndrome (PSL). These syndromes are charac-terized by macrocephaly, hamartomatous intestinal polyps. Moreover, patients are at risk of developingbreast, thyroid, colon, uterus and endometrium carcinomas.Mutations in PTEN gene are distributed in allexons and in the promoter region too. Untill now we have received 31 samples for PTEN molecular analysis,of which we have analyzed all the 9 exons and the promoter region by DHPLC and direct sequencing. This approach has permitted to identify 4 new mutations:

1) c. 686 C>G (p. Ser229X) in exon 7, (by DHPLC and confirmed by direct sequencing) in a 6 years old girlwith hamartomatous fibrolipomas of the wrist and back region;2) c. 634+5 G>A in intron 6; a de novo mutation (identified by DHPLC and direct sequencing) probably a spli-cing mutation as predicted by in silico analysis. This has been found in a 7 years old boy with macrocrania, angiomas, autistic behaviour and developmentaldelay;3) c. 492 +1 G>C with loss of the donor-site in a 10 years old boy with clinical suspect of BRRS (overgrowth,mental retardation, macrocrania and cafË au lait spots). The same mutation was found in his mother too, with macrocrania,4) c. delA20 (p. Glu7Gly fs X23) in exon 1 (identified by DHPLC and direct sequencing), a de novo mutationcausing a stop codon in a 11 years old boy, with clinical diagnosis of BRRS.Conclusions:This project was carried on using two distinct techniques: DHPLC and direct sequencing.- DHPLC wasn't been able to demonstrate the presence of all the mutations above described- Sequencing is a reliable technique able to identify all the mutations and intragenic SNPs.In our experience for patients negative for PTEN mutations and homozigotes for known PTEN SNPs, sequen-ce is not sufficient to exclude the presence of large deletions and so we are going to consider the MLPA analy-sis.

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Oncology and Genetics Doctoral School Oncological Genetics XXIII cycle Maria Giovanna Boiano, MS

Tutor A. Giordano

Novel biomarkers in prostate cancer

Prostate cancer is the second leading cause of cancer deaths in men. It is not invariably lethal and is a hete-rogeneous disease ranging from asymptomatic to a rapidly fatal systemic malignancy. The prevalence of pro-state cancer is so high that it could be considered a normal age related phenomenon. The difficulty of earlydiagnosis of PC is in distinguishing between clinically indolent prostate cancers, which will be asymptomatic,and aggressive prostate cancers with the potential to kill the patient. In spite of the availability of some bio-markers for prostate cancer, the basic molecular mechanisms regulating its development and progression arestill very poorly understood. Prostate cancer can be divided epidemiologically into hereditary and sporadicforms, but it is not possible to distinguish these two groups at a molecular level. However, most prostate can-cers are sporadic. The molecular biology of prostate cancer is still in its relative infancy. These observationshave led researchers to find for novel diagnostic and prognostic tools for prostate cancer. MicroRNAs(miRNAs, miRs) are a growing class of short non-coding RNAs that negatively regulate the expression ofgenes involved in proliferation, apoptosis, development, differentiation, antiviral defense and cancer. MaturemiRs derive from longer transcripts (pri-miRNAs) which are processed to shorter hairpin precursors (pre-miRNAs) by the action of Drosha enzyme. The 70- nucleotides precursors are exported to the cytoplasmwhere they are cleaved by Dicer to mature 19-22 nucleotides miRs, which enter RNA-induced silencing com-plex. Complementary mRNA sequences are inactivated by cleavage in a fashion similar to RNAi, while pai-ring with partially complementary sequences in the 3' UTR of target mRNAs can either repress translationalefficiency or induce transcript decay. Present estimates suggest that nearly a third of all cellular transcriptsmay be regulated by the few hundred human miRNAs currently known to exist. Accumulating evidence, suchas aberrant expression of miRNAs, suggests that they are involved in the development of cancer. They havebeen identified in various tumor types, showing that different sets of miRNAs are usually deregulated in dif-ferent cancers. In this project, I propose to: (1) characterize miRNA expression profiles in prostate cancer; (2)characterize miRNA gene target pathways in prostate cancer. The discovery of new prognostic or predictivemolecular markers (miRNAs) and miRNA target gene network analysis could help in distinguishing indolentfrom aggressive prostate tumors. A clinical application of this knowledge would be that radical treatment andits associated morbidity could be avoided in prostate cancers that are unlikely to progress. This project willaddress this need.

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Doctorate in Medical GeneticsXIX cycle

Rossella Caselli, MS

Tutor A. Renieri

Multiple congenital anomalies and mental retardation: analysis by oligo array-CGH

Chromosomal abnormalities are a major cause of mental retardation and congenital malformations. It isknown that a considerable fraction of patients with multiple congenital anomalies and mental retardation havesubmicroscopic chromosomal imbalances. This study has been focused on the oligo array-CGH analysis inorder to investigate for the presence of the submicroscopic imbalances in patients with mental retardation andmultiple congenital anomalies, normal analysis of G-banded karyotype and subtelomeric regions. By oligoarray with an average resolution of about 75 kb method (44K, Agilent) we have analyzed a cohort of 32patients. We have identified a clear pathogenic rearrangement in 9 cases (28%). This percentage is likelyhigher than those previously reported. In five cases we identified four known deletion syndromes in atypicalcases (15q11, 17p11, 22q11 in two patients) and one duplication reciprocal of the deletion responsible forSmith-Magenis difficult to recognize on clinical ground. In these cases, the diagnosis was quite unexpected.Array-CGH analysis demonstrated the presence of four novel de novo chromosomal deletions (2q24, 2q32,6q25, 7q36). We compared the clinical features of patients with new deletions with other rare cases descri-bed in the literature in order to define clinical diagnostic criteria useful in the clinical practice. The emergingphenotype of 6q25 deletion is characterized by short palpebral fissures, smooth philtrum, thin upper lip, asym-metric dysplastic protruding ears, heart septal defect, and slightly delayed psychomotor development. Thesecharacteristics should be taken into account in order to identify other patients. In two cases we have identi-fied 2 novel inherited rearrangements (Xq25, 17q12). The inherited 17q12dup (reciprocal of renal cyst-dia-betes del syndrome) was identified in a sex reversal male with MR-Peters' anomaly and in his healthy father.Since the duplication includes TCF2 and it is located 4Mb apart to HSD17B1 (a key enzyme of testosteronebiosynthesis) low penetrance may be considered. In addition to the screening of a cohort of 32 patients withunknown diagnosis, we have revaluated by oligo aCGH cases with clinical and molecular diagnosis to bet-ter define the deletion breakpoints. We performed in silico analysis of the gene content of the deleted regionto select genes potentially involved in generating the phenotype. We characterised two patients with 13q dele-tion syndrome and another one with isolated retinoblastoma in order to narrow the critical region responsiblefor mental retardation and to identify the causative gene(s). This study confirmed the power of the array-CGHmethod to clarify the molecular basis of these difficult cases. Through the employment of this innovativeapproach, several families finally received a definitive diagnosis and a correct recurrence risk.

Part of this work was published in: Caselli R et al. A 2.6 Mb deletion of 6q24.3-25.1 in a patient with growth failure, cardiac septal defect, thin lip and asymmetric dysmor-phic ears Eur J Med Genet 2007 Jul-Aug;50(4):315-21.

Caselli R et al., Retinoblastoma and mental retardation microdeletion syndrome: clinical characterization and molecular dissection usingarray CGH. J Hum Genet. 2007;52(6):535-42.

Mencarelli MA et al. Private inherited microdeletion/microduplications: Implications in clinical practice. Eur J Med Genet. 2008 Jul 9.

Uliana V et al. Expanding the phenotype of 22q11 deletion syndrome: the MURCS association. Clin Dysmorphol. 2008 Jan;17(1):13-7

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Medical Genetics Doctoral SchoolXX cycleVincenza Causarano, MS

Tutor: A. RenieriCo-tutor: M. Ferrari

Analysis of fetal nucleic acids in maternal plasma

The first report concerning the identification of fetal DNA in maternal plasma appeared in 1997 (Lo et al.,1997). Fetal DNA circulating in plasma of pregnant women may represent an accessible source of fetal gene-tic material that can be obtained nonivasively. It opens up new prenatal diagnostic applications and can impro-ve our understanding of the fetomaternal relationship. The main problem is that the fetal DNA in maternal pla-sma is circulating in an excess background of maternal DNA (1:1000) and this is a great hurdle in distinguingbetween maternal and fetal sequences. Fetal DNA was found in maternal plasma as early as 6 weeks' gesta-tion (Galbiati et al., 2005) and it was possible to identify fetal sequences at 5 weeks in in-vitro fertilization(Guibert et al., 2003). It represents 3.4% and 6.2% of total maternal plasma DNA in early and late pregnancy,respectively (Lo et al., 1998). It has been shown that fetal and maternal DNA fragments in plasma differ insize, where the length of most fetal DNA fragments was found to be < 300 bp (Chan et al., 2004). It has beenalso demonstrated the complete clearance of fetal DNA from maternal plasma after delivery, which is crucialto avoid false positive results from previous pregnancies (Lo et al., 1999; Smid et al., 2003). The presence offetal DNA in maternal plasma has been assessed by real-time quantitative PCR of the SRY gene in male-bea-ring pregnancies as the simplest way to distinguish between fetal and maternal DNA (Galbiati et al., 2005).Female pregnancies were identified by exclusion, as SRY gene was absent in these pregnancies as well asin non pregnant controls. These tests are close to 100% accurate and are useful for prenatal diagnosis of pre-gnancies that involve mothers who carry mutations for sex-linked diseases such as Haemophilia A andCongenital Adrenal Hyperplasia (Costa et al., 2002; Rijnders et al., 2001). At the genetic level, the most easilyand clinically applicable procedure is, perhaps, the detection of the RHD gene from a RHD-positive fetus inplasma of RHD-negative mothers (15% of the Caucasian population) (Lo et al., 1998). The early detection offetal RhD status in negative Rh mothers is of great importance in defining the need of gestational immuno-prophylaxis. The use of fetal DNA can be applied to noninvasive prenatal diagnosis of genetic diseases, suchas -thalassemia or cystic fibrosis, in pregnancies where the fetus bears a mutant allele different from the onecarried by its mother. We have developed a strategy based on PNA-clamping (Galbiati et al., 2006) of thematernal allele coupled with Microelectronic Microchip (Foglieni et al., 2004). Also the presence of fetal RNAin maternal plasma was first demonstrated using transcripts from the Y chromosome (Poon et al., 2000). Thisstudy opened up new perspectives and suggested that plasma RNA analysis may provide information regar-ding the gene expression pattern of feto-placental tissues. Thus, maternal plasma RNA analysis may allowthe noninvasive monitoring of feto-placental gene expression in physiological and pathological conditions(preeclampsia and intrauterine growth restriction, IUGR). IUGR is defined as the presence of an ultrasono-graphically estimated fetal weight below the fifth percentile confirmed postnatally, in the absence of chromo-somal and structural abnormalities. Preeclampsia is a hypertensive disorder affecting approximately 5% ofpregnancies and is one of the main causes of mortality of both fetus and mother. Typical symptoms are mater-nal hypertension and proteinuria, which usually develop in the late second or third trimester of pregnancy. Nget al. (2003) first detected as placental markers human Placental Lactogen (hPL) and beta subunit of humanchorionic gonadotropin (ßhCG) mRNAs in maternal plasma. Circulating levels of Corticotropin-releasing hor-mone (CRH) mRNA have been found to be increased in maternal plasma in preeclampsia (Ng et al., 2003).The main advantages of the plasma RNA approach include its potential disease specificity and broad popu-lation coverage, irrespective of fetal gender and polymorphisms. We are presently evaluating whether CRHmRNA and other mRNAs can be markers for preeclampsia and/or IUGR in a population at high risk (IUGR,IDDM and previous preeclampsia).

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Oncology and Genetics Doctoral School Colorectal and Gastroesophageal Diseases

XXI CycleAntonella Chessa, MD

Tutor G. Tanzini

Quality of life in colorectal cancer patients after surgery and a related counsellingprogram

An evaluation of quality of life has been conducted by the use of specific questionnaires-European Organization for Research and Treatment of Cancer QLQ C30 QLQ CR38 modules-during thestandard program of clinical and instrumental follow up for colorectal cancer patients. In the present study allrectal cancer patients operated on between November 2005 and June 2007 have been enrolled and specifi-cally studied. Six out of twelve subjects have completed the questionnaires. Functional scales and symptomitems have been scored at 3,6,9 and 12 months. All scales range from 0 to 100 with a higher scale scorerepresenting a higher level of functioning or better symptomatology. Preliminary scores suggest that rectalcancer surgery has specific variables involved with quality of life as patient age, radiotherapy, surgical tech-nique in relation with local cancer diffusion and need of a colostomy. Age in male subjects influences func-tional outcome concerning sexual items: patients under 65 years have a more efficient communication aboutthese problems and appears more interested in solving them. Pelvic floor rehabilitation may have a role infunctional items. Further data are under collection considering in general colon cancer patient quality of life.Quality of life needs in particular a correct information for the patients and their relatives, with the possibilityfor a genetical counselling in selected families in order to assess inherited cancer forms and multiple cancersyndromes expecially in juvenile expressions. This survey can drive to a better multidisciplinary managementin order to improve short and middle time results in terms of quality of life not only as disease free survivalrates but also as overall wellbeing in people who underwent surgery and to prevent from cancer their fami-lies.

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Oncology and Genetics Doctoral School Hepatobiliopancreatic Disease and Multitumoral Syndromes XXII cycle Filomena Cisternino, MS

Tutor F. Cetta

Polyciclic Aromatic Hydrocarbons (Pahs) As A Measure Of Pollution Related Health

Risk. Possible Use As A Marker Of Road Traffic Emission

Interactions between air pollution and human health is complex, because of the complexity of airborne pollu-tants and that of host response, which greatly varies, according to individual susceptibility. Recent studieshave challenged 2 long lasting postulates 1) health damage is due exclusively to the toxic activity of pollu-tants, as single substances, or as a whole; 2)differences in pollutant concentration within the same city isnegligible. In particular road traffic has emerged as an autonomosly dangerous emission source, which isresponsible for specific and well documented adverse health effects. Therefore, to try to distinguish the traf-fic related particulate material (PM) component as a separate entity, is noteworthy. Polycyclic aromatic hydro-carbons (PAHs), on the basis of their mutagenicity when matched in vitro with human cells, are consideredas main responsibles of the mutagenic power of particulate material (PM), and classified as probable or pos-sible carcinogens. Physicochemical analysis of PM in the city of Milan, will be performed using high volumesample with 6 stage dimensional impactor. Samples will be obtained from different sites, with different proxi-mity to major roads, in different hours of the day and seasons of the year . In addition, in order to analyseremote transportation of pollutant, values obtained in Milan will also be compared with values obtained in aremote alpine site. One major outcome of the present research will be the possible use of PAHs as a risk mar-ker of traffic related pollution. However, their possible use as a parameter of potential health damage doesnot imply that clinical adverse effects in individuals is determined by or related to their intrinsic mutagenicityor carcinogenicity. In fact, the pathophysiological consequences of a substance such as benzo(A)pyrenewhich is diluted 1:100000 in the athmosphere and is even more diluted when interferes with air tract cells arelikely very little On the contrary, there are other substances which are present in fuels, as well as in tire debrisor brake linings (which are part of road emission) which could play a major role in traffic related health effects.Further studies are warranted, in particular to stress from one hand the specificity of PAHs as traffic relatedpollutants and, on the other hand the risk of oversimplification, while extrapolating the actual role of PAHs inthe occurrence of health damage in single individuals.

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Medical Genetics Doctoral SchoolOncological Genetics

XXIII cycleDe Filippis Roberta, MS

Tutor A.Renieri

Expression analysis of the mental retardation gene FACL4

In 2002, the Medical Genetics laboratory of Siena demonstrated that mutations in FACL4 gene cause non-syndromic X-linked mental retardation. FACL4 protein belongs to an enzyme family (the Acyl CoA syntetases)that catalyze the formation of acyl-CoA esters from fatty acids, ATP and coenzyme A. The favourite substra-te of FACL4 is arachidonic acid, a polyunsaturated fatty acid that is an important component of membranephospholipids and is particularly abundant in brain. The FACL4 gene codes for two different isoforms of theprotein: a brain specific isoform of 711 aminoacids and an ubiquitous one of 670 amino acids. The brain spe-cific isoform is encoded by two distinct splicing variants, differing for the presence/absence of exon 2 (5'-UTR). Since patients with FACL4 mutations show only a cognitive defect, it's probably the absence of the pro-tein in brain that has pathogenic relevance. Previous studies revealed that FACL4 mRNA levels are higher inboth newborn and adult brain respect to other tissues, both in human and mouse. However, the relativeexpression levels of the two isoforms in brain and the eventual low expression of the brain-specific isoform inother tissues have not been investigated. In order to characterize FACL4 expression, my project will focus onquantitative expression analysis of the different splicing variants through Real Time RT-PCR in mouse. In par-ticular, we will evaluate mRNA levels during embryonic brain development in order to investigate the poten-tial role of FACL4 in this process. In addition, expression in adult brain and in other tissues will be characte-rized

Part of this work is included in:Meloni I et al. The XLMR gene ACSL4 plays a role in dendritic spine architecture". Submitted to Neuroscience.

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Doctorate in Hepatobiliopancreatic Surgery andMultitumoral Syndromes XIX cycle Armand Dhamo, MD

Tutor F. Cetta

Extracolonic maifestation of adenmatous polypsis of the colon

Familial Adenomatous Polyposis (FAP) is an autosomal dominant hereditary syndrome characterized by thepresence of hundreds to thousands of colorectal adenomatous polyps, which if not surgically treated, deve-lop into colorectal cancer (CRC) in 100%. FAP is determined by germline mutations of the AdenomatousPolyposis Coli (APC) gene, mapped at chromosome 5q21. Because of the accumulation of mutations withinthe 5' end of exon 15, between codons 1250 to 1513, this region is termed the "Mutation Cluster Region"(MCR). Over 70% of FAP patients develop extracolonic manifestations. Extracolonic manifestations of FAPinclude: congenital hypertrophy of the retinal pigment epithelium (CHRPE), epidermoid cysts and lipomas,dental and bone abnormalities, primary central nervous system (CNS) tumors, osteomas, desmoid tumors,ovarian and adrenocortical tumors, duodenal and periampullary adenomatous polyps, gastric fundic glandpolyps, duodenal carcinoma, gastric carcinoma, thyroid carcinomas and primary liver tumors. Several corre-lations have been observed between the FAP and extracolonic manifestations, leading to some historicallyrecognized syndromes and FAP variants, such as Gardner's Syndrome, Turcot's Syndrome and HereditaryDesmoid Disease. In particular, 3 peculiar even if unusual extracolonic manifestations, have been extensivelyanalyzed: 1) thyroid carcinoma (PTC); 2) hepatoblastoma (HB) and primary liver tumors; 3) brain tumors(BTP). The present study reports on a personal series of 16 patients with FAP associated HB, 18 FAP patientswith associated PTC and 3 with FAP associated BTP. Genetic analysis included: 1) analysis of germline andsomatic mutations of the APC gene; 2) detection of p53 mutations; 3) detection of ret/PTC activation inpatients with FAP associated PTC, in particular, when they occurred in the kindred of a subject with HB and/orBT. HB in FAP patients is associated with germline APC mutations in the genomic area between codons 141-1230, whereas mutations located downstream could be more frequently associated with tumors deriving fromthe mature hepatocyte. FAP associated PTC and BTP usually occurs in patients with germline APC mutationsin the genomic area between codons 140-1220, (outside of the MCR). HB, PTC and BTP in FAP patientsusually have a very low incidence (< 2%). PTCs usually develop in the absence of LOH for APC. GermlineAPC mutations cooperate with ret/PTC for occurrence of PTC. One third of FAP associated PTC show cri-briform-morular variant (CMV/PTC), the remaining are typical or conventional PTC. PTC occurs almost exclu-sive in females (F:M ratio > 17:1 vs 2,5:1 in sporadic tumors).The main finding of the present study is that BTP co-segregate with PTC and HB in patients with FAP.HB, PTC and BTP, like other extracolonic manifestations, may precede clinical diagnosis of FAP.

Signal-Dependent control of Autophagy

Autophagy is a process that emerges as a critical response of normal and cancer cell to a variety of externalcues. This process has an important role in all cells during adaptation to changing environmental conditions,cellular and tissue remodelling and cell death. Moreover autophagy allows to preserve the integrity of thesignalling network that protects genome stability, such as cell death, during the cell cycle by a vacuolar pro-cess leading to the degradation of long-live proteins and cytoplasmic organelles in eukaryotes. To date, seve-ral signaling cascades have been described to regulate autophagy in a cell type-specific and signal-depen-dent manner. It has been recently shown that pharmacological blockade of the p38 pathway in colorectal can-cer cells (CRCs), either by the inhibitor SB202190,specific for p38 kinases,or by genetic inactivation indu-ced growth arrest, autophagy and cell death. Combination of SB and LY reduced the number and size ofvacuoles without significant decrease in the percentage of vacuolated cells, wehreas cell death was aug-mented. These effects could be dependent on the presence of three different classes of PI3K kinasesDeficiency of p38 activity induces a tissue-restricted upregulation of the GABARAP gene, an essential com-ponent of autophagic vacuoles and autophagosomes,whereas simultaneous inhibition of autophagy signifi-cantly increases cell death by triggering apoptosis. Moreover, SB addiction to logarithmically growing cultu-res caused cell cycle arrest by a significant downregulation of cyclin E and A within the first 24 and 72 hours.This growth inhibitory effect was not accompanied by the accumulation of the cdk inhibitor p21, as it com-monly happens in the case of G1 or G2 arrest imposed by PIK3/Akt, MEK/ERK or mTOR pathways inhibition,using inhibitors specific for PI3Ks (LY,3MA),MEK1 (PD),mTOR (rapamycin). Specific inhibition of MEK/ERKcomplex give rise to an increase of both the number and size of vacuoles and cell death, suggesting that thepro-autiphagic role sustained cell survival in the presence of p38 blockade. Inhibition of mTOR induced onlya slight increase in SB-mediated autophagic vacuole formation and failed to effect cell growth. These suggestthat mTOR activity is neither essential for CRCs survival nor involved in the p38-dependent autophagicresponse. Combination of SB and LY reduced the number and size of vacuoles without significant decreasein the percentage of vacuolated cells, whereas cell death was augmented. These effects could be dependenton the presence of three different classes of PI3K kinases, where, class I and III are involved in autophagycontrol. Class I enzymes have an inhibitory role. Given the number of drugs that target the p38 pathway, itstands to reason that elucidating the molecular mechanisms that link p38 and autophagy might have animpact on the clinical translation of these drugs.

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Medical Genetics Doctoral SchoolOncological Genetics

XXII cycleEleonora Ferrari, DVM

Tutor A. Giordano

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Ocology and Genetic Doctoral SchoolColorectal and Gastroesophageal DiseasesXXI cycleIrene Grassi, MD

Tutor G.Tanzini

Familiar risk in colorectal cancer

Colorectal cancer is the second leading cause of death for neoplastic diseases in Western World and its inci-dence is increasing. Cumulative cancer-related deaths are high (about 50%), mostly because a large amountof cancers are diagnosed in advanced stages.Prevention and early diagnosis are the most useful tools forabating these mortality rates since the large bowel cancerogenesis is an evolving process (so-called adeno-ma-carcinoma sequence) that may be interrupted by removing the precancerous lesions. Familiarity is awell-known risk factor both for adenomas and cancer and can be used to optimize cost-benefit programs ofscreening. Furthermore, the familial risk may be more elevated if potential-related characteristics of heredi-tary colorectal cancer are present (juvenile age of onset, multiple primary tumours, several affected in thesame family, histologic and molecular features..). All these characteristics have been evaluated in a conse-cutive cohort of patients operated for colorectal cancer at our Insitution. The following are the preliminaryresults. From 2005 to september 2006, we traced the perdigrees of 175 patients: at least one first-degreerelative with the same cancer was reported by 29 of them (16,11 %). In 5 patients a hereditary form couldbe hypothesized and a DNA testing has been offered. A FAP was ascertained in a family (APC mutation posi-tive). In another family an attenuated form (AAPC) was suspected and biomolecular analysis is in progress.

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Oncology and Genetics Doctoral School Medical Genetics

XXII cycle Valeria Guarnaccia, MS

Tutor A. Renieri - Co-tutor E. Garattini

The prolyl-isomerases PIN1 regulates the cyto-differentiating activity of retinoids in

myeloid leukemia cells

Retinoids are natural and synthetic derivates of vitamin A and are used in the treatment of certain types ofsolid tumor and leukaemia. All-trans retinoic acid (ATRA) is used in the treatment of acute promyelocytic leu-kemia (APL), a rare form of acute myelogenous leukemia (AML) characterized by a balanced chromosomaltranslocation t(15;17) leading to expression of PML-RAR, an oncogenic form of the retinoic acid receptorRAR . Retinoic acid (RA) influences the proliferation, differentiation and apoptosis of a variety of cell typesthrough modifications in the expression of subsets of RA-target genes. The RA response is mediated by twoclasses of nuclear receptors, the RA receptors (RARs) and the RXRs , which function as ligand-dependentheterodimeric RAR/RXR transcription factors. The activity of these nuclear retinoic acid receptors is control-led not only by the ligand but also by a number of signals, including phosphorylation and proteosomal-depen-dent degradation. Prolyl-isomerases are a family of enzymes that recognize phosphorylated Ser(Thr)/Promotifs in target proteins and isomerize the bond from a cis- to trans- conformation. This leads to alterationsin the secondary and tertiary structure of the target protein. PIN-1, a member of the prolyl-isomerases family,is implicated in the activation and inactivation of various intra-cellular transcription factors; in particular PIN-1 induces conformational changes that mediate the targeting of certain proteins along the proteosomal path-way. The aims of my study are to establish whether PIN-1 interacts with RAR and/or PML-RAR , the moda-lities of this interaction, and in particular if the expression of PIN-1 exerts any functional effects on the RARand PML-RAR receptor complexes in terms of trans-activation and degradation. We have seen that over-expression of PIN-1 inhibits the ligand-dependent transactivation of RAR and PML-RAR , and inhibition ofprolyl-isomerase activity by pharmacological agents such as PiB (PIN-1 inhibitor) restores the ligand-depen-dent transactivation of RAR suppressed by PIN-1 over-expression in a dose-dependent manner. The resultsindicate that PIN-1 interacts with RAR and PML-RAR , causing inhibition of the transcriptional activity ofthese two receptors. Pharmacological inhibition of PIN by PiB potentiates the growth inhibitory and cyto-dif-ferentiating activity of ATRA in retinoid-responsive AML cells. Thus, we will analyze the effects of PIN-1 inhi-bitors on the activity of RA receptors. By gene expression microarray analysis, we will investigate the effectsof PIN-1 modulation on the differentiation or ATRA mediated growth inhibition in AML cells. Finally the targetgenes responding to ATRA and PIN-1 will be studied, regardless of the mechanistic aspects. So far, ourresults demonstrate that targeting of PIN-1 represents a viable strategy to increase the therapeutic efficacyof retinoids.

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Oncology and Genetics Doctoral School Medical GeneticsXXI CYCLEEleni Katzaki, MS

Tutor A. Renieri

Clinical and molecular characterization of Italian patients affected by Cohen

syndrome

Cohen syndrome is an autosomal recessive disorder with variability in the clinical manifestations, characteri-zed by developmental delay, visual disability, facial dysmorphisms and intermittent neutropenia. We descri-bed a cohort of 10 patients affected by Cohen syndrome from 9 Italian families and ranging from 5 to 52 yearsat assessment. Characteristic age related facial changes were well documented. Visual anomalies, namelyretinopathy and myopia, were present in 9/10 patients (retinopathy in 9/10 and myopia in 8/10). Truncal obe-sity has been described in all patients older than 6 years (8/8). DNA samples from all patients were analy-zed for mutations in COH1 by DHPLC. We detected fifteen COH1 alterations most of them were truncatingmutations, only 1 being a missense change. Partial gene deletions have been found in two families. Mostmutations were private. Two were already reported in the literature just once. A single base deletion leadingto p.T3708fs3769, never reported before, was found in three apparently unrelated families deriving from arestricted area of the Veneto's lowland, between Padova town and Tagliamento river, in heterozygous state.Given the geographical conformation of this region, which is neither geographically or culturally isolated, arecent origin of the mutation could be hypothesized.

Fig1: Schematic description of the predicted COH1 protein with representation of the transmembrane domains andintracellular and extracellular loops. Mutations are indicated along the protein. An arrow indicates a splice-site mutationnamed at the DNA level. The others mutations, which are named at the protein level, are indicated with straight lines. The p.Q3772X mutation (cases 1, 2 and 3) creates a stop codon in position 3772, cutting off the last transmembranedomain. The frameshift mutation p.Q721fs744X and the nonsense mutation p.R2707X, both leading to premature pro-tein interruption were found in case 7. The frameshift mutation p.Y3855fs3877X was identified in case 8, in which a par-tial gene deletion in the other allele has been found.

Part of this work was published in:Katzaki E., et al. Clinical and molecular characterization of Italian patients affected by Cohen syndrome.J Hum Genet. 2007; 52:1011-7

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XXII cycleBaharak Khadang, MD

Tutor A.Renieri

Denaturing High Performance Liquid Chromatography(DHPLC) in X-Linked Alport

Syndrome Diagnosis

Alport Syndrome (ATS) is a progressive glomerulonephritis characterized by irregular thinning, thickening andsplitting of the glomerular basement membrane usually associated with hearing loss and ocular lesions. About80% of Alport cases are X-linked inherited with mutations in COL4A5 gene. Among remaining cases, 15% isautosomal recessive, and about 5% is autosomal dominant due to mutations in either COL4A3 or COL4A4genes. A wide allelic heterogeneity exists in X-linked ATS, because a large majority of patients bear a diffe-rent COL4A5 mutation. In my study 30 patients ,out of which 14 were with strong evidence of X-linked pedi-gree, were analysed. Three abnormal profiles have been revealed. Automated sequencing was done and 3missense mutations were found. DHPLC analyses PCR products previously denatured and reannealed inorder to obtain homoduplex and heteroduplex molecules. Mismatches are revealed by appearance of one ormore early eluting peaks in the chromatographic profile representing heteroduplex species. The heteroduplexspecies can be a representative of nucleotide variation in the gene. To ensure the proper formation of homoand heteroduplices of a gene on X chromosome ,it is necessary to mix the male probands with a normal con-trol male. In picture , the difference of DHPLC profile in a male with known mutation ,with and without mixingwith normal control, is shown. My research project is focused on analysis of COL4A5 gene in a large numberof patients with strong family history of X-linked disorder. Denaturing High Performance LiquidChromatography (DHPLC) and automated sequencing methods are used for the analysis in this project.

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Oncology and Genetics Doctoral School Hepatobiliopancreatic Disease and Multitumoral syndromes XXIII cycle Elvana Kola, MD

Tutor F. Cetta

Pollution related Inflammatory and Malignant Diseases in subjects living in Milan, in

particular, concerning the possible role of infection and airway pH

Environmental pollution and in particular air pollution, from traffic and toxic products, are the principal causeof human morbidity and mortality in last years, in the city of Milan. Particulate material (PM) is able to causeenvironmental related health damage, but the precise cellular and molecular mechanism undergoing thistoxic effect of PM is not yet fully established. There is a great variability in host response in subjects living inthe same area, as we see in the city of Milan, that has a big problem with environmental pollution, also fromthe major traffic and without green areas.

Therefore, 2 different studies starting this year have been planned regarding the pollution of the city of Milan:

1.In vitro study to evaluate the toxic and damaging effect on cell lines or isolated alveolar cells on PM, obtai ned by special filters from detectors placed in various areas of the city, with variable PM concentration, and in particular with different incidence of traffic related pollution.

2.Correlation between the clinical picture and the variable exposure to pollutants (major number of exposition, working place or residence place, etc.)

The aim of the present study is to evaluate the basal function of the respiratory system in Milan residents,affected by respiratory diseases after a long exposure to traffic and environmental pollution. For this, it will beanalysed through laboratory and specialized procedures (Functional Respiratory Proves, DCLO,Fibrobronchoscopy, BAL) the pulmonary function of the hospitalyzed patients with signs and symptoms of pul-monary and in particular to evaluate the difference of pH on the Exhaled Breath Condensate (EBC),Cytokines, Thiol status redox, NO measurement, bacterial population. Another line of this study is also themicroscopic and necroptic study of the sudden deaths by Myocardial Infarction without coronary obstruction.

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Oncology and Genetics Doctoral SchoolOncological Genetics

XXIII cycleRaffaele La Montagna, MS

Tutor A. Giordano

Proline-Rich Antibactirial Peptides: Inhibition DnaK Chaperone Activiy

Peptides, as processed end-products, or as fragments of larger proteins, are the natural ligands for most pro-teins, and even for nucleic acid, lipidic or other bacterial components. Due to a large possible variability andpotentially high binding specificity, synthetic peptides are the reagents of choice to initially characterize tar-get/ligand interactions. Naturally occurring antimicrobial peptides (AMPs) are an excellent example of natu-re's ability to produce a diverse array of ligands suitable to selectively inhibit bacterial processes. Althoughthese can be quite effective molecules in antimicrobial biochemistry, they are not necessarily optimal.Appropriate amino acid modifications can lead to increased specificity and selectivity for the targets, and willhelp the detailed analysis of the intimate biochemical anteractions between bacterial targets and their pepti-de ligands. The principal objective of my program is to improve the binding of the lead A3-APO praline-richpeptide to the bacterial target protein DnaK, characterize the efficacy of target inhibition in vitro and mass pro-duce the optimal ligands for in vivo efficacy assays. We incorporated both basic and applied research objec-tives, namely: a) optimal binding to target proteins, b) effective penetration into bacterial cells, and c) increa-sed stability in a biological environment. Thus, my additional goals include improving the serum stability of theproline-rich antimicrobial peptides and their membrane penetration, as well as fulfill direct drug developmentrequirements such as synergy with conventional antibiotics, acceptable level of pharmacokinetics and biodi-stribution. Ideally, this process will pave the road to peptide-based antibacterial drug candidates for control-led preclinical development.

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Oncology and Genetics Doctoral School Hepatobiliopancreatic Disease and Multitumoral syndromesXXIII cyclePaolo Laviano, MD

Tutor: F. Cetta

Pollution related cardiovascular, cutaneous and osteoarticular diseases in patients

living in the city of Milan

The city of Milan is in the center of a large metropolitan area which, because of meteorological and climaticcondition, high population density and industrial concentration, is one of the most polluted sites in Europe.Recent epidemiologic have shown that airborne particular material (PM) and in particular diesel exhaust andtraffic related pollution, is able to cause evident health damage, not only to the respiratory system, but alsoto the cardiovascular system. These adverse effects are more evident in predisposed patients, i.e. subjectwith previous coronary disease or myocardial infarction. In addition cutaneous (atopia) and osteoarticularside-effects, possibly facilitated by PM exposure, have been reported, as well as allergic rhinitis, otitis media,etc.It has been suggested that PM affects vasomotility, atherosclerotic plaque formation and endogenousfibrinolytic capacity. However, despite the large amount of epidemiologic and laboratory studies, there is notpathological evidence showing how PM interferes with the cardiovascular system in subjects currently dyingor suffering from acute heart attacks.The pathophysiologic mechanisms responsible for cardiovascular imba-lance are not well elucidated. The aim of the present research will be to follow in various Milan Hospital(Niguarda, Policlinico, San Paolo Hospitals) a panel of patients with previous documented coronary disease,in order to assess whether episodes of high pollution exacerbated myocardic ischemia, either at rest or afterphysical exercise. Pulmonary function using both analysis of exhalate breath condensate and respiratoryparameters, as assessed by spirometry will be concomitantly evaluated. In particular, pathological and mole-cular biology analysis will also be performed in surgical specimens or necroscopy specimens in patients dyingbecause of acute heart attacks, during episodes of high pollution in the absence of cardiac level and at bron-chioloalveolar level will be useful for better knowledge of pathophysiologic mechanism.

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Oncology and Genetics Doctoral SchoolHepatobiliopancreatic Disease and Multitumoral syndromes

XXI cycleGiuliana Malagninio, PCS

Tutor: F. Cetta

Physicochemical and toxicological analysis of PM. Relevance of aggregation of ultra-

fine and fine PM within the respiratory tract in the presence of mucus and bacteria

Environment health damage is a major problem in metropolitan areas. Despite the large amount of epidemiolo-gical studies and in vitro studies, suggesting that airbone pollutans can determine a wide variety of cardiorespi-ratory alterations, no clear-cut cause and effect relationship has been documented between PM and clinicallyevident disease in humans. In particular, pathological and pathophysiological evidence of what actually occursin humans when particulate material (PM) interacts with host defenses is lacking. One major problem is to knowwhat actually happens when PM reaches the bronchiolo-alveolar level of old patients who are those at higherrisk for healt effects. These frail subjects, in addition to coronary diseases, also have chronic broncopulmonaryinfections, with mucus hyperproduction, bacteria, cellular debris and an acid pH in their air tract.Two classic postulates establish that: 1) damage to human health mainly depends on toxicity (i.e. direct dama-ge) of each pollutant; 2) fine and ultrafine PM (<2,5?) are more dangerous, because they reach the most distalareas of the respiratory tract, i.e. because they are fine. The aim of the present research is to challenge this twostatements, giving evidence to the view that: 1) fine particles may be more dangerous because they become "big" and form plug within the airway;2) mechanic obstruction due to these plugs can play a major role in the pathogenesis, maybe greater than the"pure toxic" effect.In particular, plugs could block conspicuous proportions of the respiratory tract, determining not only sudden dis-tension and vagal nerve stimulation, but also alteration in the distribution of pulmonary blood flow.In order to support this hypothesis both "in vitro" study and clinical syudies in patients with asthma, COPD, lungcancer and/or cardiovascular diseases will be performed. In particular: 1) the possibility will be investigated thattypical "toxic" compounds such as transitional elements or PAHs, could exert their activity not only by their intrin-sic toxic capacity (genotoxicity, cancerogenicity, teratogenicity, etc.), but also because they could facilitate aggre-gation of carbon particles; 2) specimens by affected individuals (BAL, pulmonary biopsies, surgical specimens)will be examined by pathological methods, including molecular biology and scanning and transmission electronmicroscopy, to document the actual occurrence of "plugs" at the bronchiolo-alveolar level.

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Oncology and Genetics Doctoral School Oncological Genetics XXI cycle Mario Mancino, MS

Tutor A. Giordano

Mechanisms regulating Cripto 1 gene expression in cancer cells

Human Cripto-1 (CR-1) is a cell membrane protein that is overexpressed in several different types of humancarcinomas. In the present study we investigated the mechanisms that regulate the expression of CR-1 genein cancer cells. We cloned a 2481 bp 5' flanking region of the human CR-1 gene into a luciferase reportervector and transfected NTERA-2 human embryonal carcinoma cells and LS174-T colon cancer cells to testfor promoter activity. Activity of CR-1 promoter in both cell lines was modulated by two TGF- family members,TGF- ß1 and BMP-4. In particular, TGF- ß1 significantly up-regulated CR-1 promoter activity, whereas a dra-matic reduction in CR-1 promoter activity was observed with BMP-4 in NTERA-2 and LS174-T cells. Changesin the CR-1 promoter activity following TGF- ß1 and BMP-4 treatments correlated with changes in CR-1mRNA and protein expression in NTERA-2 and LS174-T cells. We also identified three Smad binding ele-ments (SBEs) within the CR-1 promoter and point mutation of SBE1 (-2197/-2189) significantly reducedresponse of the CR-1 promoter to both TGF- ß1 and BMP-4 in NTERA-2 and LS174-T cells. Chromatin immu-noprecipitation assay also demonstrated binding of Smad-4 to a CR-1 promoter DNA sequence containingSBE1 in LS174-T cells. Finally, BMP-4 inhibited migration of LS174-T cells and F9 mouse embryonal carci-noma cells by down-regulation of CR-1 protein. In conclusion, these results suggest a differential modulationof CR-1 gene expression in embryonal and colon cancer cells by two different members of the TGF-ß family.

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XXII cycleElena Marcocci, MS

Tutor A. Renieri

Autosomal dominant Alport syndrome due to COL4A4 mutations

Alport syndrome is a clinically and genetically heterogeneous nephropathy characterized by irregular thinning,thickening, and splitting of the glomerular basement membrane often associated with hearing loss and ocularsymptoms. While the X-linked and the autosomal recessive forms are well known, the autosomal dominant formis not well acknowledged. We have investigated 26 individuals with a clinical and molecular diagnosis of auto-somal ATS belonging to 8 different families. The mean age of patients was 35.6 ranging from 8 to 76 years. Only5 out of 26 (19.2%) individuals reached the ESRD. The mean age of ESRD was 52.5 years. Two patients (7.6%)have hearing loss and none of them had ocular changes with the exception of one patient who has retinophatyprobably non correlated with ATS. The DHPLC analysis (Denaturing High Performance Liquid Chromatography)revealed 8 novel private COL4A4 gene mutations: 3 frameshift mutations, 3 missense mutations and 2 splicesite mutations. This data indicate that autosomal Alport syndrome is a distinct disease with a very low risk ofocular and ear anomalies but with a clear risk to develop renal insufficiency although in at later stage that auto-somal recessive and X-linked forms.

Part of this work is included in: Elena Marcocci, Vera Uliana, et al. Autosomal dominant Alport syndrome due to COL4A4 mutations.Nephrology Dialysis Transplantation - Submitted

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Oncology and Genetics Doctoral SchoolHepatobiliopancreatic Disease and Multitumoral syndromesXIX cycleFederico Mariani, MD

Tutor: F. Cetta - Co-tutor: E. Pinto

Predicting Invasivity Of Intraductal Papillary Mucinous Tumours Of The Pancreas

Cystic lesions of the pancreas has been steadily increasing with use of CT and US, accounting for 5-15 % ofcystic pancreatic masses. First four cases of IPMT were recognized by Ohhashi in 1982 and differentiated inIPMA (intraductal papillary-mucinous adenomas), IPMB (intraductal papillary-mucinous borderline), IPMC (intra-ductal papillary-mucinous carcinoma), nIPMC (non-invasive type) and IPMCs (muc vs. Tub) in 1997 by WHO.IPMT (Intraductal papillary mucinous tumours) and MCT (Mucinous cystic tumours) share rare incidence, mucinproduction, cystic mass and better prognosis vs. DC (ductal carcinoma) but IPMT have unique clinicopatholo-gic findings. IPMT due to its diffuse involvement and multifocality may recur after partial pancreatic resections.Different CT\US\RNM features seem to be present in IPMT and the role of ERCP is mandatory. e-US, pan-creatic-duodenoscopy and intraduttal-us have been also used.The presence of the major apomucin and cell-cycle regulatory proteins and immunohistochemical mucins (MUC1, MUC2, MUC5AC) and p53, p16 and ki-67expression seems to be related with invasivity, but at present there isn't a sensitive and specific marker of inva-sive carcinoma. New scores has been proposed in order to predict malignancy of IPMN. Aberrant promotermethylation of tumor suppressor genes is associated with a loss of gene function. The finding of p16 methyla-tion in pancreatic tissue and in pancreatic juice collected in ERCP has been strongly proposed as invasivity mar-ker. Treatment criteria is not yet well defined. Different procedures have been proposed (DCP, total pancrea-tectomy, pancreatic resections guided by frozen section, conservative approach, follow-up). 54 patients withpancreatic carcinoma who underwent surgical resections between January 2001 and May 2007 at HumanPatology and Oncology Department, Surgical Oncology Unit, Università degli Studi di Siena were retrospecti-vely reviewed. We performed 12 distal pancreatectomies, 3 total pancreatectomy and 39 Whipple's procedu-res. We found five cases of IPMT, 4 males and 1 female, mean age 65 years (range: 42-83). We collected twofurther cases from Surgery Division of S. Donato Hospital, Arezzo. Of all pts with pancreatic cancer, fresh spe-cimen and blood samples were obtained. Normal, neoplastic tissues and patients serum were obtained from 5IPMN resected were stored at - 70° C. Data were stored with TNM OS DFS LS and median follow-up.The methylation status of 5' CpG islands by bisulfite modification of DNA and methylation specific PCR (MS-PCR) will be performed according to the method of Herman et al., carried out for 16 h at 50° on 1 ? of genomicDNA at Surgical Oncology Unit Laboratory in order to define p16 and e-cadherin methylation status. Intraductalgrowth and secondary chronic pancreatitis were associated with IPMT. Larger median cyst size, tumor diame-ter and dilated main pancreatic duct, Ca 19.9, mucin expression profile, and promoter methylation of P16 andE-cadherin found to be predictive factors of malignancy.

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Oncology and Genetics Doctoral SchoolOncological Genetics

XXIII cycleMonica Mischitelli, MS

Tutor A. Giordano - Co-tutor V.A. Pietropaolo

Human Polyomavirus BK and Prostate Cancer: evaluation of BKV variants,host immu-

ne response and tumor host cytokines in cancer development

Prostate cancer (PC) remains the most commonly diagnosed malignancy and the second leading cause of can-cer related death in men of Western countries. Screening programs have been implemented to diagnose menwith PC in its early stages and, although only a subset of men diagnosed with localized PC will end up with meta-stasis, more specific biological criteria should be addressed to better clinically differentiate patients with moreaggressive versus indolent prostate carcinoma. Moreover the molecular pathology of PC is complex and in itsrelative infancy. Multiple factors contribute to the development and progression of PC: infectious agents, hostimmune response and tumor host cytokines might play a role in its pathogenesis and/or progression. HumanPolyomavirus BK (BKV) is a good candidate because it naturally infects humans, is usually acquired early in lifeand almost 90% of adults are seroconvert. BKV resides in the kidneys in a latent or persistent state, but can bereactivated upon immunosuppression of the host, it oncogenically transforms rodent cells in culture, causes kid-ney tumours in transgenic mice, and transforms primary human cells in culture when coexpressed with an acti-vated ras oncogene. The oncogenic potential of BK is linked especially to large T-Antigen. In fact T-Ag displaysmultiple functions that alter the normal physiological metabolism of cells, leading to immortalization and neopla-stic transformation. T-Ag binds and blocks the functions of tumor suppressor proteins, in particular p53.Moreover it, preparing the cellular metabolism to support optimal viral replication, deregulates cell's control cyclepathways inducing cell proliferation. BKV has been reported to be detected in a number of human tumours, inparticular those in the urinary tract, since BKV is an ureteliotropic virus. It is well known that human cytokinesplay a determinant role in cancer development, hence BKV infection could stimulate the secretion of vascularendothelial growth factor (VEGF) and pro-inflammatory cytokines promoting cancer onset or development.VEGF, in particular, is regulated by androgen, suggesting a potential pathway by which androgen regulates pro-state cancer growth. Finally BKV genome contains a transcriptional control region (TCR) that binds transcriptio-nal cell factors (i.e. p53 oncosuppressor) and undergoes to deletion and enhancement process. It generatesvariants that might impair the replication ability of the virus, leading to an increased transformation potential.On these bases I will focus my attention on -Search the presence of BKV sequences and proteins in prostate tumor samples to clarify the role of the BKVin PC onset or progression. It will be used a specific PCR for BKV genome and Immunoistochemistry for BKVproteins;-Screening BKV TCR variants in prostate tumor samples to understand if TCR rearrangements could rise viraloncogenic potential. It will be used a specific PCR for BKV TCR and PCR products will be sequenced and analy-ze for transcriptional cell factors;-Screening p53 localization and expression in BKV infected cells;-Evaluation of VEGF and pro-inflammatory cytokines gene expression following BKV infection or reactivationusing a semiquantitative RT-PCR and Immunoistochemistry.

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Oncology and Genetics Doctoral SchoolMedical GeneticsXXII cycleFilomena Tiziana Papa, MS

Tutor A. Renieri

Oligo array-CGH analysis: comparison of two platform with different resolution,

identification of novel microdeletion syndromes and atypical phenotype in known

syndromes

A cohort of 110 patients with MCA/MR have been analyzed by 44K Agilent oligo array-CGH. A first group(67) was selected for having MCA/MR not recognizable on clinical ground and normal karyotype. In thisgroup we identified 5 novel de novo deletions (2q24, 2q32, 6q25, 7q36 14q12), 6 known deletions in atypi-cal cases (4p16, 15q11, 17p11, 22q11x3), 3 known rearrangements difficult to recognize on clinical ground(22q13del, 17p11dup, 6q27del) and 4 novel imbalances inherited from healthy parent (del7q11, dup17q12,delXq25, dupXq28). Excluding the last 4, mutation detection rate was 21%(14/67). A recognizable phenoty-pe for novel deletions could be traced: long and broad alluces, untreatable seizures for 2q24; sleep distur-bance, behavioral problems, bifid nasal tip, micrognatia for 2q32; septal heart defect, Williams-like upperface, dysmorphic ears, short stature for 6q25; fetal phenytoin-like-face, renal hypoplasia, long QT for 7q36;cortical displasy, epilepsy and microcefaly for 14q12. A second group (43) was selected for having definedclinical diagnosis and array-CGH was used to clarify disease mechanism. Deletion mapping in 3 retinobla-stoma patients with or without MR pinpointed the MR critical region on 13q14. Analysis of 10 Rett patientsruled out a 16p11dup polymorphism, probably responsible for phenotype modulation. Analysis of a patientwith Alport and leiomyomatosis (ATS-DL) and Xq23del allowed to confirm that only smaller del cause DL inATS. Among the above 110 patients, 44 were analyzed by 105K oligo array and 48 known polymorphisms,3 novel inherited rearrangements by healthy parent and any pathogenetic alteration was identificated. Inconclusion, the employment of a platform with higher resolution (25 Kb vs 75 Kb) does not increase the dia-gnostic sensitivity.

Part of this work is published in:Caselli R., et al. Delineation of the phenotype associa-ted with 7q36.1q36.2 deletion: long QT syndrome,renal hypoplasia and mental retardation. Am J MedGenet A 2008; 146A(9):1195-9;

Mencarelli MA et al. Private inheritedmicrodeletion/microduplications: Implications in clinicalpractice. Eur J Med Genet. 2008 Jul 9.

Papa FT et al. A 3 Mb deletion in 14q12 causes severemental retardation, mild facial dysmorphisms and Rett-like features. Am J Med Genet A. 2008 Aug1;146A(15):1994-8

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XXIII cycleVeronica Parri, MS

Tutor A. Renieri

Functional characterization of FACL4 protein involved in X-linked mental retardation

Mutations in FACL4 gene are responsible for non-syndromic X-linked mental retardation. The pathogenicmechanism leading from FACL4 absence to mental retardation is not known, since the exact function of theprotein within the cell has not been clarified. FACL4 gene encodes for an acyl-CoA synthetase, an enzymethat adds coenzyme A to long-chain fatty acids with formation of acyl-CoA, the active form necessary for sub-sequent reactions. The favourite substrate of FACL4 is arachidonic acid, a fatty acid abundant in brain lipidsand involved in many different processes. FACL4 protein is composed by 670 aminoacids with a predictedmolecular weight of about 74 KDa and it contains two domains essential for catalytic activity (Figure A). Asecond, apparently brain-specific, isoform of the protein resulting from alternative splicing is present; this iso-form contains 41 additional highly hydrophobic aminoacids at the N-terminus (Figure B - red circle). The sub-cellular localization of the two isoforms is not known, but it is possible that the hydrophobic N-terminus maycause a re-localization of the brain-specific form. My research project is aimed at the functional characteri-zation of the protein in order to elucidate its role in brain function and/or development. For this purpose, thesubcellular localization of FACL4 will be investigated by double immunofluorescence with the use of a rabbitantiserum which specifically recognizes both protein isoforms. In addition, their expression levels will be eva-luated by Western blot in brain and other tissues. Immunoprecipitation with antibodies against modified resi-dues will be also employed to verify whether FACL4 protein is subjected to post-translational modifications.

Part of this work is included in:Meloni I et al. The XLMR gene ACSL4 plays a role in dendritic spine architecture. Submitted to Neuroscience

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Oncology and Genetics Doctoral School Oncological Genetics XXII cycle Flavio Rizzolio, MS

Tutor A. Giordano

The role of pRb2/p130 in epigenetic modulation of Estrogen Receptor- in breast

cancer

Breast cancer is a highly prevalent and morbid disease, afflicting approximately 1 in 9 women in the UnitedStates. The death rate from breast cancer in the United States has recently declined for most age groups,although it remains a major killer with 45,000 deaths annually. The clonal genetic model of cancer suggeststhat different genetic alterations in a single cell are responsible of tumour proliferation, invasion, metastasisand drug resistance. Despite the fact that in leukaemia and solid tumours clonal genetic changes are com-mon, evidences for pathological epigenetics changes are increasingly also DNA methylation, chromatinalteration and loss of imprinting. ER- , actively involved in hormone and chemotherapeutic response, is animportant actor in normal and tumour breast development. However, ER- status can change during thecourse of disease, and consequently, a resistance to therapy can occur. A fraction of breast cancer losesER- expression and it is associated with the epigenetic process regulating gene silencing. Several recentevidences suggest that the pRb pocket proteins (pRb1/p105, p107, and pRb2/p130), regulators of themammalian cell cycle progression and suppressors of cellular growth and proliferation, might be involved inthis process through interaction with chromatin remodelling enzymes. In our laboratory, we demonstratedthat ER- positive and negative breast cancer cell showed different pRb2/p130 protein complexes bound toER- promoter (see figure). Here, we suggest that in MDA-MB231 ER- negative cells, 5-aza-2'-deoxycytidi-ne demethylating agent treatment can change the protein complex bound to the promoter like in untreatedMCF7 ER- positive cells. In addition, we have demonstrated that ICBP90 (Inverted CCAAT box bindingprotein of 90 kDa), a transcriptional regulator of many genes (e.g Rb1/p105), is overexpressed in severalcancer cell lines and it is bound to pRb2/p130 and cooperates with DNMT in maintaining a specific methy-lation pattern for the ER- promoter. However, pRb1/p105 and p107 can bind the ER- promoter suggestinga possible mechanism of cooperation with pRb2/p130 in ER- specific gene expression. Future experiments will be addressed to elucidate the role of pRb protein family, ICBP90 and DNMT duringER- gene expression. Our working model highlights the central role of pRb2/p130 multimolecular comple-xes in the ER- promoter specific DNA methylation with obvious implication for the therapeutic outcom.

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Oncology and Genetics Doctoral School Oncological Genetics

XX cycleAnnalisa Roberti, MS

Tutor A. Giordano

Aberrant methylation in the Gal1 sporadic Burkitt lymphoma-derived cell line

Burkitt lymphoma (BL) is an aggressive B-cell tumor characterized by high growth rate and a large fractionof cycling cells. All Burkitt lymphomas (BLs) carry reciprocal chromosomal translocations that activate the c-myc oncogene through juxtaposition to one of the immunoglobulin (Ig) loci. This genetic anomaly is the criti-cal event in BL development, but subsequent tumor progression have to involve the selection for additionalgenetic and epigenetic changes. The aim of my research is to investigate if epigenetic alterations have a rolein sporadic Burkitt lymphoma progression and if the reactivation of TSGs by demethylation may play a rolein sporadic Burkitt lymphoma cancer therapy in the future.Because the dysregulation of the pRb pathway byp16INK4a methylation could facilitate c-Myc-mediated growth stimulation and the inactivation of componentsin the p53 pathway should protect the BL cell from apoptosis induced by c-Myc, we have analyzed the expres-sion of genes involved in these patways. Western blotting and immunocytochemistry assay have revealedthe lack of p16/INK4a protein in the Gal1 cell line. It has been reported that the p16/INK4a gene is inactiva-ted by DNA methylation of the promoter region in a variety of human cancers. We analyzed the methylationstatus of this gene using methylation-specific PCR (MSP). After sodium bisulphite modification only methyledband, using p16/INK4a specific set of primers, was found, this suggests that loss of p16/INK4a is due toimproper methylation of the promoter region. We treated the Gal1 cell line with 5-Aza-2 deoxycytidine (5-Aza-CdR), a demethylating agent, to assay if it is possible to reactivate p16/INK4a expression and the effect ofthis treatment on cell cycle regulating genes. Tumor cell growth of treated cells was significantly inhibitedcompared with the untreated groups, demostrating that 5-Aza-CdR has a profund effect on the rate of celldivision. We performed MSP-PCR on treated and untreated cell lines. After 5-Aza-CdR treatment, bothmethyled and unmethyled bands were observed , whereas in the control only methyled band appeared,demonstrating that 5-Aza-CdR is able to inhibit partially the p16/INK4a promoter methylation. Real Time q-PCR was performed on cDNA derived from 5-Aza-CdR-treated and untreated cell lines, to assay if thedemethylation of p16/INK4a promoter region is followed by gene re-expression. Expression of p16/INK4awas increased in the Gal1 cell line following treatment, confirming that its expression is epigenetically silen-ced in this cell line. At the same time, we analyzed the expression changes of some cell cycle regulatinggenes after demethylating tratment. 5-Aza-CdR treatment significantly increased the level of p21WAF1/CIP1without changing the amount of its direct activator p53. This therefore suggests that also p21WAF1/CIP1 maybe silenced by aberrant promoter methylation in Gal1 cells. Still remains to be proven if the increase of genetranscription may be translated in protein re-expression and if the reactivation of TSGs including p16/INK4aand p21WAF1/CIP1 by demethylation could provide the input to develope new approaches for sporadicBurkitt lymphoma therapy in the future.

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Oncology and Genetics Doctoral SchoolMedical GeneticsXXIII cycleDalila Rondinella, MS

Tutor A. Renieri

New HPRT mutation in an Italian family affected by Lesch-Nyhan syndrome

The Lesch-Nyhan syndrome (LNS) is an inherited X-linked disorder of purine metabolism caused by analmost complete lack of hypoxanthine phosphoribosyltransferase (HPRT) activity. The disease is characteri-zed by hyperuricemia, neurologic dysfunction, including severe spasticity and choreoathetoid movements,mental retardation and a typical self-injurious behavior. Partial deficiency of HPRT has been described in avariety of patients with phenotypic differences ranging from isolated hyperuricemia to varying degrees ofneurologic disfunction. Notably, in HPRT deficiency there is an inverse correlation between residual HPRTenzyme activity and the severity of the neurologic disease. Is note that gross deletions and truncating muta-tions that cause complete enzyme deficiency lead to typical LNS phenotype, while point mutations maycause either partial or complete deficiency and therefore atypical or milder cases. We analyzed an Italianfamily with biochemical diagnosis of Lesch-Nyhan syndrome, and we found a new frame-shift mutationc.89_96dupAGGATTTG leading to a truncated protein. The mutation was found in the proband and in thecarrier mother. Genetic analysis provides a valuable tool for the confirmation of the diagnosis in the pro-band and in particular for the identification of potential carriers. The mutation identification could be a toolfor predicting the disease phenotype but a large cohort of Italian patients are required.

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Oncology and Genetics Doctoral school Medical Genetics

XXII cycleAriele Spanhol Rosseto, MS

Tutor A. Renieri

Genetic modifier factors in Rett syndrome.

Rett syndrome (RTT), caused by mutations in MECP2, represents the second cause of mental retardation ingirls, with an estimated prevalence of 1:10000-1:15000. Affected patients develop normally for the firstmonths of life but subsequently present regression, with loss of speech and purposeful hand use, growthretardation, acquired microcephaly, ataxia and breathing problems. The clinical presentation of patients isvariable and a classic form and many variants can be distinguished. In order to explain such heterogeneity,we decided to study the effect of MECP2 mutation type on the phenotype. A severity score (0-2) has beenassigned to 22 different clinical signs in a cohort of 158 MECP2 mutated RTT patients. MECP2 mutationshave been classified in three different groups: A) early truncating mutations and large deletions; B) missen-se mutations; C) late truncating mutations. The non parametric test of Kruskal-Wallis revealed a statisticallysignificant association (p=0.001) between mutation type and the total severity score (0-44): group A has amean severity score of 26.5 7.4, group B of 22.6 7.9, and group C of 19.6 7.6. The mutation type corre-lated well also with some specific clinical signs: presence of speech, level of speech, height, hand use, auto-nomic dysfunction, age of achievement of psychomotor milestone, sphinter control, hypotonia. In order toinvestigate whether other relevant interacting/target genes may have a role in modulating the phenotype wehave analyzed the CDKL5 p.Q791P and the BDNF p.V66M polymorphisms. While significant correlation wasnot found between the total severity score, interesting correlations were found with specific clinical signs.None of the drug-resistant epilepsy patients had a QP or PP genotype of the CDKL5 gene, suggesting a pro-tective effect of the P allele (p=0.025). Analysis of the BDNF p.V66M polymorphism revealed correlation withcold extremities (p=0.026). None of the patients with the MM genotype had severe cold extremities sugge-sting that this polymorphism, especially at the homozygous state, prevents the impairment of autonomicsystem.The identification of genetic modifiers of RTT will have a broad impact not only on the elucidation ofthe pathogenic mechanisms underlying the syndrome but also on the design of future therapeutic strategies.By acting on those specific modifier genes/proteins affected RTT girls may improve their condition.

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Oncology and Genetics Doctoral SchoolMedical GeneticsXX cycleKatia Sampieri, MS

Tutor A. Renieri

Genomic differences between retinoma and retinoblastoma

Genomic copy number gains or losses are involved in the multi-step process transforming normal retina inretinoblastoma after RB1 mutational events. Previous studies on large number of retinoblastoma sampleslead to a multi-step model in which after two successive RB1 mutations (M1 and M2), further genomicchanges accompany malignancy: 1q32.1 gain (M3), 6p22 gain (M4), 16q22 loss (M5a) and 2p24.1 gain(M5b). Genetic studies on retinoma tissue have never been performed. Retinoma is a rare benign retinallesion, considered a pre-tumoral lesion by some authors and a tumor regression by others. In order to cla-rify retinoma-retinoblastoma relationship, we investigated by Real-Time qPCR, copy number changes ofMDM4 (1q32.1), MYCN (2p24.1), E2F3 (6p22) and CDH11 (16q22) genes in retina, retinoma and retinobla-stoma tissues from two different patients. Our results demonstrated that some genomic gains thought tobelong exclusively to retinoblastoma (1q32.1, 6p22 and 2p24) are already present in retinoma, at the samenumber of copies (1q32.1) or at lower number of copies (6p22 and 2p24) respect to retinoblastoma. The16q22 region is not altered in the two retinoma samples. However, in one case, we found 16q22 gain inretinoblastoma tissue, suggesting that copy number variations in this region must be considered a laterevent in the multi-step process. All these results are in favors of the pre-tumoral nature of retinoma.

Part of this work is published in:Sampieri K et al. Genomic differences between retinoma and retinoblastoma. Acta Oncologica In press.

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Doctorate in Medical GeneticsXIX cycle

Scala Elisa, MS

Tutor A. Renieri

Molecular bases of the clinical variability in Rett syndrome

Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder, mostly affecting females. It is cau-sed by MECP2 mutations in 90% of classic patients and in a lower percentage of variant cases. Among RTTpatients, there is a high level of phenotypic variability in type and age of onset, severity of impairment andprofile of clinical course. In order to identify the molecular bases causing this heterogeneity, in our cohort ofRTT patients (http://www.biobank.unisi.it) we searched for: i) the genetic defect in variant RTT patientswithout MECP2 mutations, and ii) modifier factors involved in phenotype modulation of MECP2 mutated RTTpatients. In order to explain the fraction of MECP2 mutation negative RTT variant cases, we hypothesized theexistence of an additional RTT locus. We found mutations in another gene, CDKL5, in patients with a phe-notype overlapping with the infantile seizure onset variant of RTT. We hypothesized that the two genes,MECP2 and CDKL5, belong to the same molecular pathway. We showed that CDKL5 is a nuclear proteinwhose expression in the nervous system overlaps with that of MeCP2, during neural maturation and synap-togenesis. Importantly, we demonstrate that the two genes interact both in vivo and in vitro and that CDKL5is a kinase which ia able to phosphorylate itself and to mediate MeCP2 phosphorylation. We also hypothesi-zed that a fraction of mutation-negative RTT cases could be due to the limited sensitivity of the methodologyused for MECP2 mutations detection. The identification of MECP2 large deletions by MLPA in a subset ofRTT cases suggested that these rearrangements are a common cause of classic form of the syndrome, whilethey are rarely implicated in variant phenotype. In order to understand the genetic differences between theclassic RTT and the milder phenotype described in preserved speech variant (PSV), we searched for diffe-rences in segmental genomic variations in two RTT sisters with the same MECP2 deletion but discordantphenotype. Molecular investigation of the entire family by Real Time quantitative PCR revealed that the dele-tion is absent in parents' DNA. X-inactivation status was balanced in blood of both sisters. We identified byarray-CGH analysis a duplication of about 370 Kb on chromosome 16p11.2 in the classic RTT patient inhe-rited from the healthy father and absent in the mother and PSV sister. The duplicated segment includes 10known genes. Among them, ATP2A1 encodes for a calcium-transporting ATPase found in different membra-nes whose loss of function cause Brody myophaty. In addition another duplicated gene, ATXN2L encoding forataxin type 2 related protein, seems quite interesting. Its function is presently unknown; however, it is a mem-ber of the spinocerebellar ataxia (SCAs) family, which is associated with the SCA group of neurodegenerati-ve disorders. This study allowed to determine the genetic causes in a subset of "unsolved" RTT variantcases and to identify a molecular link between MECP2 and CDKL5 genes. Interestingly, this work representsan initial step towards the identification of genetic modifier factors of RTT.

Part of this work is published in:Scala E et al. CDKL5/STK9 is mutated in Rett syndrome variant with infantile spasms. J Med Genet. 2005Feb;42(2):103-7.Mari F et al. CDKL5 belongs to the same molecular pathway of MeCP2 and it is responsible for the early-onset seizurevariant of Rett syndrome. Hum Mol Genet. 2005 Jul 15;14(14):1935-46. Epub 2005 May 25.

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Oncology and Genetics Doctoral SchoolMedical GeneticsXXI cycleTiziana Squillaro, MS

Tutor A RenieriCo-Tutor U. Galderisi

Mesenchymal stem cells: a model for Rett syndrome analysis

Rett syndrome (RTT; OMIM 312750) is a severe neurodevelopmental disorder affecting almost exclusivelygirls. It has an estimated prevalence of 1:10000-1:15000 femals, and it is thought to be one of the mostcommon genetic causes of mental retardation in girls, second only to Down syndrome. RTT presents withregression, loss of speech, purposeful hand movements, acquired microcephaly, ataxia, stereotypic hand-washing activities and growth retardation.Mutations in the MeCP2 gene were idendified in the great majorityof RTT patients. The MeCP2 protein binds to methylated DNA and mediates gene silencing by causingchanges in chromatin structure through the interaction with co-repressor such as Sin3A and the histonedeacetylase complex. MeCP2 seems to have an important role in development processes of nervoussystem and in osteogenesis. Nevertheless there are not enough data to identify the molecular pathways lin-ked to this protein. The identification of this pathways is of fundamental importance to understand the originof the alterations in RTT patients.In the context of the RTT research, I have focused my attention on thestudy of the mesenchymal stem cells (MSCs) because they may represent a useful experimental model toanalyse the MeCP2 role in cellular ontogenesis. These cells offer several advantages: i) MSCs are easilyisolated from bone marrow cells with a procedure that causes limited uncomfortable pain to patients; ii)MSCs have been shown to possess great somatic plasticity since they can differentiate into non-mesenchy-mal lineages. In fact, it has been demonstated that MSCs are capable of differentiating into neurons.According to this observations it is clear that this experimental model results to be useful to understandwhether if MeCP2 mutations alter the correct neural development. The MSCs great somatic plasticity willallow to verify if also others differential pathways are altered in RTT patients. In particular, MSCs are capa-ble to differentiate in osteocytes and it been demonstrated that several RTT patients show osteoporosis.

Several experimental protocols have been tested to induce neuronal differentiation of MSCs obtainedfrom healthy children. Some of these protocols have included a chemical treatment to elevate endocellularc-AMP levels. These conditions have generate an incompleate and reversible neural differentiaton. Otherprotocols based on a retinoic acid treatment or the cell growth leading to "neurosphere" did not peovideuseful results. The 10-15 days differentiation throught an hormonal cocktail compsed by EGF, PDGF, FGF-2 risulted in several neural-like cells. RT-PCR analysis have shown an expression level increase of seve-ral neuronal markers.I set up an experimental method to silence MeCP2 gene in MSCs by RNA -interferen-ce. For this purpose MSCs have been transducted by adenoviral vectors expressing shRNA directed vsMeCP2 mRNA. This method will allow to evaluate the molecular pathways alteration in absence of MeCP2mRNA.

Part of this work is published in:Squillaro T et al. A case report: bone marrowmesenchymal stem cells from a Rett syndromepatient are prone to senescence and show alower degree of apoptosis. J Cell Biochem.2008 Apr 15;103(6):1877-85.

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XXI cycleMarina Vignoli, MS

Tutor M. Genuardi

Comprehensive analysis of CDKN2A gene mutations in familial melanoma patients

Familial cutaneous melanoma accounts for about 10% of all melanoma cases and is inherited as an autoso-mal dominant trait, with incomplete penetrance and variable expressivity. Genetic linkage analyses in largefamilial melanoma kindreds have identified a melanoma susceptibility locus on human chromosome band9p21. This region harbors the INK4 gene cluster consisting of the CDKN2A and CDKN2B genes. CDKN2Aencodes two distinct proteins translated, in alternate reading frame, from alternatively spliced transcripts, thealpha transcript, comprising exons 1 , 2, and 3, encodes p16 INK4a, while the smaller beta transcript, com-prising exons 1 , 2, and 3, specifies the alternative product p14ARF. The p16 protein inhibits the activity ofthe cyclin D1-cyclin-dependent kinase 4 (CDK4) or 6 (CDK6) complexes that phosphorylate the retinoblasto-ma protein, thus p16 acts as a tumor suppressor and negatively regulates cell growth by arresting cells atG1. p14ARF acts via the p53 pathway to induce cell cycle arrest or apoptosis. Germline mutations in INK4locus are associated with melanoma susceptibility in 20-40% of multiple case melanoma families. Althoughthe CDKN2A gene has been shown to be the major genetic determinant governing high-penetrance suscep-tibility to melanoma, there remains a significant proportion of melanoma pedigrees in which germline muta-tions of CDKN2A have not been identified. To determine the contribution of these candidate genes to familialmelanoma genetic predisposition in italian population, we performed the screening of 115 melanoma patients.Firstly we studied the presence of germline mutations in the CDKN2A locus by direct sequencing of exons 1, 1 2, and 3 and then we performed quantitative analysis of this locus to investigate the possibility thatCDKN2A is affected by germline deletions in patients who do not show point mutation. For the quantitativeanalysis we used two independent methods, based on MLPA (multiplex ligation-dependent probe amplifica-tion) and real-time quantitative PCR using sybr green. The former investigates multiple sequences clusteredon chromosome 9p21, including the CDKN2A promoter regions and the CDKN2B locus, while for the latterwe initially set up analytical conditions limited to CDKN2A coding regions. We have identified a 5bp deletionin the promoter region of ARF gene that probably has not a pathogenic significance. No germline alterationsin any of the tested genes were detected among the families under study. The results revealed a paucity ofmutations in CDKN2A/ARF, suggesting that in the Italian population these genes alone do not contributesignificantly to FMM. Recently, several studies have also demonstrated an increased risk of pancreatic can-cer among CDKN2A melanoma-prone families. We have therefore extended our analysis to familial melano-ma patients with a positive family history of pancreatic cancer. We have found a 6 bp deletion in the codingsequence of exon 1 of CDKN2A, that lead to the loss of two aminoacids. The mutation has been detected intwo patients of the same family, both affected from melanoma and one of them from pancreatic cancer. It hasbeen considered to be a pathogenic mutation that could be responsible for the predisposition to pancreaticcancer in this family.

Part of this work is publis-hed in:Gensini F et al. Thep.G23S CDKN2A foundermutation in high-risk mela-noma families from CentralItaly. Melanoma Res. 2007Dec;17(6):387-92.

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13.00 Entering of the PhD dissertation board composed by

- Prof. Francesco Cetta, (President)Professor of Surgery, Univesity of Siena, Italy

- Prof. Corradino Campisi, (Member)Professor of Surgery, University of Genova, Italy

- Prof. Luigi Maria Pernice, (Secretary)Professor of Surgery, University of Florence, Italy

13.15 Thesis discussion in English language

- Extracolonic manifestation of the Familial Adenomatous PolyposisArmand Dhamo XIX cycle

14.15 Awarding of the PhD degree in Hepatobiliopancreatic Surgery

14.30 End of session

A copy of the thesis is available athttp://www.unisi.it/ricerca/dottorationweb/genetica_medica/accessing the PhD student link and then PhD student in Medical Genetics.

Thesis discussionDoctorate in Hepatobiliopancreatic SurgerySeptember 19, 2007 room 7 Centro Didattico S. Maria alle Scotte, 14.30



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12.30 Entering of the PhD dissertation board composed by:

- Prof. Maurizio Genuardi (President)Professor of Medical Genetics, University of Firenze, Italy- Prof. Alessandra Renieri (Secretary)Professor of Medical Genetics, University of Siena, Italy- Prof. Barbara Pasini (Member)Professor of Medical Genetics, University of Torino, Italy- Prof. Hugh JM Brady (Member)Immunology, Imperial College, London, UK

Thesis discussion in English language

- “Constitutive and somatic genetic events in retinoma and retinoblastoma”Katia SampieriXX cycle

13.30 Awarding of the PhD degree in Medical Genetics and qualification of Doctor

Europaeus

Thesis discussionDecember 15, 2008 Centro Didattico S Maria alle Scotte, room 13

From left to right: Prof. Alessandra Renieri Prof. Maurizio Genuardi Prof. Barbara Pasini Prof. Hugh JM Brady Dr. Katia Sampieri

phd students’ works annual progress report 2007-2008 ... · on the basis of research activity the...

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