hide
Language(s):
eng - English
Dates:
2003
Publication Status:
Accepted / In Press
Pages:
126 pp
Publishing info:
Torino, Italy : Università degli Studi di Torino
Table of Contents:
1. INTRODUCTION..............................................................................................................5
1.1 HISTORY...........................................................................................................................5
1.2 CLINICAL FEATURES...........................................................................................................6
1.2.1 Craniofacial features..............................................................................................6
1.2.2 Cardiovascular anomalies......................................................................................7
1.2.3 Growth....................................................................................................................8
1.2.4 Skeletal findings......................................................................................................8
1.2.5 Genitourinary system..............................................................................................9
1.2.6 Haematology............................................................................................................9
1.2.7 Lymphatics...............................................................................................................9
1.2.8 Development and behaviour..................................................................................10
1.2.9 Hearing anomalies.................................................................................................10
1.2.10 Ocular anomalies...................................................................................................10
1.2.11 Ectoderm................................................................................................................10
1.3 GENETIC STUDIES............................................................................................................12
1.4 GENETIC HETEROGENEITY................................................................................................15
1.4.1 Noonan syndrome 2 (MIM 605275)......................................................................15
1.4.2 Neurofibromatosis-Noonan syndrome (NF-NS) (MIM 601321)...........................16
1.4.3 LEOPARD syndrome (MIM #151100)..................................................................18
1.4.4 Cardio-facio-cutaneous syndrome (CFC)(MIM 115150).....................................18
1.5 CHROMOSOMAL REARRANGEMENTS IN HUMANS.................................................................20
1.5.1 Numerical chromosomal abnormalities................................................................22
1.5.2 Structural chromosomal rearrangements.............................................................21
1.5.3 Disease-associated balanced chromosome rearrangements: a genetic tool for gene identification and cloning..........................................................................................22
1.6 AIMS AND OBJECTIVE.......................................................................................................24
2. MATERIALS AND METHODS....................................................................................25
2.1 MATERIALS......................................................................................................................25
2.1.1 Chemicals and equipment.....................................................................................25
2.1.2 Solutions and media..............................................................................................26
2.1.3 Buffer solutions.....................................................................................................27
2.1.4 Enzymes.................................................................................................................28
2.1.5 Kits........................................................................................................................29
2.1.6 Vectors..................................................................................................................29
2.1.7 Primers..................................................................................................................29
2.1.8 Genomic material..................................................................................................34
2.1.9 Bacterial material.................................................................................................34
2.2 METHODS........................................................................................................................35
2.2.1 Patients' sample collection...................................................................................35
2.2.2 Bioinformatic analysis..........................................................................................35
2.2.3 DNA isolation........................................................................................................36
2.2.4 RNA isolation........................................................................................................36
2.2.5 Fluorescence in Situ Hybridisation (FISH)..........................................................36
2.2.6 Polymerase Chain Reaction (PCR)......................................................................37
2.2.7 Allele-Specific PCR amplification (ASO).............................................................38
2.2.8 RT-PCR.................................................................................................................38
2.2.9 Semiquantitative RT-PCR.....................................................................................38
2.2.10 Purification of PCR products................................................................................39
2.2.11 Recombinant DNA techniques..............................................................................39
2.2.12 Sequencing............................................................................................................39
2.2.13 5'-RACE (Rapid Amplification of cDNA Ends)....................................................39
2.2.14 Labelling of DNA probes for hybridisations.........................................................40
2.2.15 Library screening..................................................................................................41
2.2.16 Southern blot hybridisations.................................................................................42
2.2.17 Northern blot hybridisations.................................................................................42
2.2.18 Mutation screening by Denaturing High Performance Liquid Chromatography (DHPLC)...........................................................................................................................43
3. RESULTS........................................................................................................................44
3.1 MOLECULAR CHARACTERIZATION OF THE BREAKPOINT REGIONS ASSOCIATED
WITH A CONSTITUTIONAL T(2;12)(Q37;Q24)PAT IN A PATIENT WITH NOONAN SYNDROME ..............44
3.1.1 Case report............................................................................................................44
3.1.2 Identification of chromosome-specific BAC clones that spanned the translocation breakpoints........................................................................................................................45
3.1.3 Chromosome 12: cosmid library screening and Southern blot experiments........48
3.1.4 Chromosome 2: cosmid library screening and Southern blot experiments..........49
3.1.5 In silico analysis of the breakpoint-spanning BAC clone on chromosome 12.....51
3.1.6 Molecular analysis of the breakpoint region on chromosome 12.........................52
3.1.7 Identification and characterization of the TRAP240-like gene............................54
3.1.8 Expression analysis of the TRAP240-like gene....................................................58
3.1.9 Expression analysis of the TRAP240-like gene in the patient with t(2;12)(q37;q24)pat...........................................................................................................59
3.1.10 Mutation analysis of the candidate gene TRAP240-like.......................................60
3.1.11 Analysis of the breakpoint region on chromosome 2............................................65
3.2 PTPN11 GENE ANALYSIS....................................................................................................67
3.2.1 Mutation analysis of the PTPN11 gene in 96 NS and 5 CFC patients.................67
3.2.2 Mutation analysis of the PTPN11 gene in patients with LEOPARD syndrome...71
4. DISCUSSION...................................................................................................................74
4.1 ROLE OF PTPN11 GENE IN THE AETIOLOGY OF NOONAN SYNDROME, CARDIO-FACIO-CUTANEOUS SYNDROME, AND LEOPARD SYNDROME..........................................................................................74
4.1.1 Spectrum of PTPN11 mutation in NS patients......................................................77
4.1.2 Genotype-phenotype correlation..........................................................................82
4.1.3 Cosegregation.......................................................................................................86
4.1.4 Cardio-facio-cutaneous syndrome........................................................................86
4.1.5 LEOPARD syndrome............................................................................................87
4.2 ROLE OF TRAP240-LIKE GENE IN THE AETIOLOGY OF NOONAN SYNDROME.............................89
4.3 SELENOCYSTEINE LYASE GENE................................................................................................94
4.4 CONCLUSION........................................................................................................................98
5. REFERENCES...............................................................................................................100
6. SUMMARY....................................................................................................................116
6.1 RIASSUNTO....................................................................................................................116
6.2 SUMMARY......................................................................................................................119
7. APPENDIX.....................................................................................................................124
7.1 PUBLICATIONS...............................................................................................................124
7.2 ACKNOWLEDGMENTS.....................................................................................................126
Rev. Type:
-
Identifiers:
eDoc: 194830
Degree:
PhD
