From mutations to disease mechanism in rett syndrome, breast cancer, and congenital hypothyroidism
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FROM MUTATIONS TO DISEASE MECHANISM IN RETT SYNDROME, BREAST CANCER, AND CONGENITAL HYPOTHYROIDISM by brahim Barış B.S., Molecular Biology and Genetics, Boğaziçi Univesity, 2000 M.S., Molecular Biology and Genetics, Boğaziçi Univesity, 2002 Submitted to the Institute for Graduate Studies in Science and Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy Graduate Program in Molecular Biology and Genetics Boğaziçi University 2008 ii FROM MUTATIONS TO DISEASE MECHANISM IN RETT SYNDROME, BREAST CANCER, AND CONGENITAL HYPOTHYROIDISM APPROVED BY: Assoc. Prof. Esra Battaloğlu…………………………..…. (Thesis Supervisor) Prof. A. Nazlı Başak………...……………………………. Prof. Hande Çağlayan……………………………..……… Prof. Uğur Özbek…………………………………………. Assoc. Prof. Müge Türet Sayar …………………..………. DATE OF APPROVAL: 10.06.2008 iii to my family iv ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my thesis supervisor Assoc. Prof. Esra Battaloğlu for her endless support and intimate encouragement throughout my thesis. My appreciation is also extended to the members of my thesis committee Prof. A. Nazlı Başak, Prof. Hande Çağlayan, Prof. Uğur Özbek, and Assoc. Prof. Müge Türet Sayar for allocating their time to evaluate this work. I would like to extend my gratitude to Boğaziçi University, Scientific Research Projects Fund for funding the expenses of my thesis (Project No: 03S101 and 08B103D). I am grateful to the CMT members Duygu, Çiğdem, rem, Başak, and Dr. Rezan who has provided a warm environment in the lab. I am also thankful to all Retina and plant group members for creating best laboratory environment ever. I am grateful to my dear friends Dr. Birdal Bilir and Dr. Yeşim Özmen for their valuable and wonderful friendship. I would to extend my thanks to all my dear friends (Kader, nanç, Sibel, and Murat) in the department. I wish to thank all of the academic staff, among whom I would like to mention Prof. Kuyaş Buğra, Assoc. Prof. Muge Türet Sayar, and Assoc. Prof. Nesrin Özeren for their kind support and encouragement. I would like to express my great thanks to each of my family member, especially to my parents for standing by my side at all time. I am grateful to my wife Tuğba who has always supported, appreciated and encouraged me with his endless love and patience throughout my life. Additionally, I would not complete my thesis work on time without her help in DNA sequencing and quantitative analyses. v ABSTRACT FROM MUTATIONS TO DISEASE MECHANISM IN RETT SYNDROME, BREAST CANCER, AND CONGENITAL HYPOTHYROIDISM Epidemiological studies provide the correlative data to understand the etiology of human inherited diseases and develop efficient genetic testing assays. Additionally, the accumulated data of genetic and epigenetic findings, expression profiling, and proteomics allows disease diagnosis, to understand the molecular mechanisms leading to the disease pathogenesis, and to develop efficient therapeutic approaches. In the framework of this thesis, we have investigated genetic and epigenetic changes and performed genotype- phenotype correlations to unravel the molecular mechanisms that lead to three different diseases, Rett Syndrome, breast cancer, and congenital hypothyroidism. The genetic basis of Rett Syndrome (RTT) was investigated in a total of 71 RTT patients. A heterogeneous spectrum of disease-causing MECP2 mutations was identified in 68.2 per cent of a clinically well defined group of cases whereas in only 12.5 per cent of the patients referred for differential diagnosis suggesting that this gene does not represent a major cause of the disease among patients with Rett-like features. For the first time, we have identified gene duplications as causative mutations in female atypical RTT cases. Consistent with the animal models, our results support the possibility that duplication of MECP2 that leads to increased expression might underlie some cases of X-linked delayed- onset neurobehavioral disorders including Rett Syndrome. Our results showed that exon rearrangements that could not be detected by standard techniques contribute to 19.3 per cent of these MECP2 mutations, and should be considered in especially RTT variants in order to determine the actual significance of the gene in the etiology of RTT. Genotype/phenotype correlation was performed based on comparison of severity score of patients with the type and location of the mutation and the XCI pattern. The results did not reveal a statistically significant correlation, but, the patients with exon deletions were vi found to be more severely affected than patients with all other types of mutations and patients with exon duplications to present with severe eye contact problems. Additionally, we have developed and validate a novel multiplexed amplification refractory mutation system (ARMS) assay for identification of seven common mutations that accounts for almost 65 per cent of all MECP2 gene mutations. The validation studies revealed that our novel assay is an efficient, reliable, and cost-effective screen for molecular genetic testing of patients with RTT. Furthermore, we tested the effect of DNA concentration on reliablity and reproducibility of SYBR green dye-based Real Time PCR analysis to detect the MECP2 exon rearrangments. The results suggested that Real Time PCR analysis is reliable for determination of the exon copy number if the DNA amount is in the range of 1-50 ng. To our knowledge, there are no known reports investigating the role of methylation of hHR23A and hHR23B genes in the tumor tissues. We have characterized the 5' flanking region of the hHR23A and hHR23B genes using web-based analysis and investigated the involvement of methylation status of putative promoter region of hHR23A and hHR23B genes in breast carcinogenesis. The observations of the hypermethylation of hHR23A gene and the presence of methylated conserved motifs and transcription binding sites in hHR23B gene among the analyzed tumor tissues suggested the involvement of methylation of hHR23 genes in the breast carcinogenesis. Investigation of epigenetic changes in tumor samples of breast cancer patients was a pioneering work since available literature implicates its presence only in cell lines. Since our CH patient was the first case with Bamforth Syndrome and suffered the plasma cholinesterase deficiency, the genetic mechanisms leading to congenital hypothyroidism and prolonged paralysis after mivacurium were investigated. In contrast to other reported two patients with TTF2 gene mutation, the presence of thyroid tissue in our patient suggested further phenotypic heterogeneity associated with human TTF-2 mutations. The functional study with a collaborative work also helped to understand the genetic mechanisms and provided original evidence that implicated differential effects of TTF-2 mutations on downstream target genes required for normal human thyroid organogenesis. vii ÖZET RETT SENDROMU, MEME KANSER , VE KONJEN TAL H POT RO D ZMDE MUTASYONLARDAN HASTALIK MEKAN ZMASINA Epidemiyolojik çalışmalar, insan kalıtsal hastalıklarının etiyolojisinin anlaşılması ve genetik testlerin geliştirilmesi için, karşılaştırmalı veriler sağlar. Bunun yanı sıra, genetik ve epigenetik bulgular, anlatım profilleri ve proteomiksden elde edilen bilgi birikimi, hastalık tanısına, hastalık patogenezine neden olan moleküler mekanizmaların anlaşılmasına ve doğru töropatik yaklaşımların geliştirilmesine ışık tutar. Bu tez çalışması kapsamında, üç farklı hastalığa, Rett Sendromu (RTT), meme kanseri ve Konjenital Hipotiroidizme (CH), neden olabilecek mekanizmaları aydınlatmak amacıyla genetik ve epigenetik değişimler incelendi ve genotip-fenotip karşılaştırması yapıldı. RTT’nin genetik temeli toplam 71 hastada incelendi. Kesin klinik tanı alan hastaların yüzde 68.2’sinde heterojen dağılım gösteren ve hastalığa neden olduğu bilinen MECP2 gen mutasyonları tanımlanırken, ayırımcı tanı amacıyla yönlendirilen hastaların sadece yüzde 12.5’inde mutasyonların belirlenmesi, sözkonusu genin, RTT-benzeri özellikler gösteren hastalar için majör neden olamayacağını düşündürdü. Atipik RTT olgularında, ilk defa, gen duplikasyon mutasyonlarının hastalığa neden olabileceği gösterildi. Hayvan modellerindeki bulgularla örtüşen sonuçlar, duplikasyonların, MECP2 anlatımının artmasına ve Rett Sendromunu da içeren bazı X’e bağlı geç-başlangıçlı nörodavranışsal hastalıklara neden olabileceği olasılığını destekledi. Sonuçlar, standart teknikler ile tanımlanamayan ekson düzenlenme bozukluklarının, mutasyonların yüzde 19.3’ünü kapsadığını ve sözkonusu genin RTT etiyolojisine gerçek katkısının belirlenmesi açısından özellikle RTT varyantlarında araştırılması gerektiğini gösterdi. Hastalık şiddeti skorları ile mutasyonların tipi ve konumlarının, ve XCI paterninin karşılaştırılması temeline dayanan genotip/fenotip korelasyonu gerçekleştirildi. Bulgular istatiksel olarak anlamlı bir korelasyonu desteklemese de ekson delesyonu taşıyan hastaların diğer tüm mutasyon viii tiplerini taşıyan hastalardan daha şiddetli etkilendikleri ve ekson duplikasyonu olan hastalarda şiddetli göz kontağı problemi olduğu bulundu. Ayrıca bilinen tüm MECP2 gen mutasyonlarının yüzde 65’ini oluşturan ve en sık görülen yedi mutasyonun tanımlanması amacıyla yeni bir multipleks amplifikasyon refrakter mutasyon tanımlama sistemi (ARMS) geliştirildi ve geçerliliği sınandı. Yeni metodun RTT hastalarının moleküler genetik analizinde kullanılabilecek elverişli, güvenilir, ve düşük maliyetli bir test olduğu gösterildi. Bunun yanı sıra, DNA konsantrasyonunun, MECP2 ekson düzenleme bozukluklarının belirlenmesinde kullanılan SYBER yeşili boya-bazlı Gerçek Zamanlı PCR analizinin güvenilirliği ve tekrarlanabilirliği üzerindeki etkisi irdelendi. Ayrıca, ekson kopya sayısının belirlenmesi çalışmalarında, DNA miktarının 1-50 ng aralığında olduğu durumlarda Gerçek Zamanlı PCR analizinin güvenilir sonuçlar verdiği gösterildi. Bilgimiz dahilinde, tümör dokularında hHR23A and hHR23B gen metilasyonlarının rolünü araştıran raporlar bulunmamaktadır. Bu konuda katkı sağlamak amacıyla sözkonusu genlerin 5’ uçları web tabanlı analiz kullanılarak karakterize edildi ve olası promotör bölgelerinin metilasyon durumunun meme karsinogenezindeki rolü incelendi. Analiz edilen tümör dokularında hHR23A gen hipermetilasyonunun gözlenmesi ve hHR23B geninde korunmuş metilasyon motifleri ve transkripsiyon bağlanma bölgelerinde metilasyon bulunması hHR23 genlerinin metilasyonunun meme kanseri oluşumunda yer alabileceğini düşündürdü. Literatürde epigenetik değişimlerin sadece hücre hatlarında gösterilmiş olması nedeniyle meme kanseri hastalarının tümör dokularında bu değişimlerin araştırılması öncü bir çalışma niteliğindedir. Tez kapsamında incelenen konjenital hipotiroidizm (CH) hastası ilk Bamford Sendromlu olgu olması ve plazma kolin esteraz yetersizliği göstermesi nedeniyle CH’e ve mivacurim kullanımı sonrası uzun paralize neden olan mekanizmanın aydınlatılması amacıyla incelendi. TTF2 gen mutasyonu olduğu bilinen diğer iki hastanın aksine hastamızda tiroid bezinin varlığının belirlenmesi insanda TTF2 mutasyonları ile ilişkili fenotipik heterojenliğin bilinenden fazla olduğunu ortaya çıkardı. Uluslararası ortak bir çalışma ile gerçekleştirilen işlevsel analizler de genetik mekanizmanın anlaşılmasına yardımcı oldu ve TTF2 mutasyonlarının normal insan tiroid organogenezinde rol alan hedef genler üzerindeki farklı etkilerine işaret eden özgün kanıt sağladı. ix TABLE OF CONTENTS ACKNOWLEDGEMENTS ....................................................................................... iv ABSTRACT .............................................................................................................. v ÖZET ........................................................................................................................ vii LIST OF FIGURES ................................................................................................... xiv LIST OF TABLES .................................................................................................... xix LIST OF ABBREVIATIONS .................................................................................... xxi 1. INTRODUCTION ................................................................................................ 1 1.1. Epigenetics .................................................................................................... 1 1.1.1. Epigenetics ......................................................................................... 1 1.1.2. DNA Packaging .................................................................................. 1 1.1.3. Epigenetic Modifications .................................................................... 3 1.1.3.1. CpG and non-CpG Methylation ............................................. 3 1.1.3.2. Histone Modification ............................................................. 5 1.2. Rett Syndrome .............................................................................................. 6 1.2.1. Historical Background ........................................................................ 6 1.2.2. Clinical and Neuropathological Charecteristics ................................... 6 1.2.3. Phenotypic Variability in RTT ............................................................ 8 1.2.4. Genetic Basis of RTT ......................................................................... 8 1.2.5. MECP2 Gene ..................................................................................... 9 1.2.6. CDKL5 Gene ..................................................................................... 11 1.2.7. MECP2 Mutation Profile .................................................................... 11 1.2.8. MeCP2 Function ................................................................................. 13 1.2.8.1. Transcription Regulation and Chromatin Remodeling ........... 13 1.2.8.2. RNA Splicing ........................................................................ 15 1.2.9. Mouse Models of RTT ........................................................................ 17 1.2.10. MeCP2 Target Genes and Their Relevance with Disease .................. 18 1.3. Breast Cancer ................................................................................................ 20 1.3.1. Breast Tissue ...................................................................................... 20 1.3.2. Breast Cancer Risk ............................................................................. 21 1.3.3. Breast Carcinogenesis ......................................................................... 24 x 1.3.4. DNA Methylation and Genetic Instability ........................................... 27 1.3.5. DNA Repair System ........................................................................... 28 1.3.6. hHR23 (RAD23) Genes ...................................................................... 31 1.4. Congenital Hypothyroidism .......................................................................... 35 1.4.1. The Thyroid Gland ............................................................................. 35 1.4.2. Congenital Hypothyroidism ................................................................ 36 1.4.3. Forkhead Gene Family ....................................................................... 37 1.4.4. Human TTF-2 Gene ............................................................................ 39 1.4.5. Thyroid Development and TTF-2 ....................................................... 42 1.4.6. TTF-2 Gene Mutations ....................................................................... 43 1.4.7. Plasma Cholinesterase Deficiency ...................................................... 44 2. AIM ..................................................................................................................... 45 3. MATERIALS ....................................................................................................... 47 3.1. Subjects and Samples .................................................................................... 47 3.2. Chemicals ..................................................................................................... 47 3.3. Fine Chemicals ............................................................................................. 47 3.3.1. Enzymes ............................................................................................. 47 3.3.2. Oligonucleotide Primers ..................................................................... 48 3.3.3. DNA Size Marker ............................................................................... 51 3.4. Kits ............................................................................................................... 51 3.5. Buffers and Solutions .................................................................................... 51 3.5.1. DNA Extraction from Peripheral Blood .............................................. 51 3.5.2. Polymerase Chain Reaction (PCR) ..................................................... 52 3.5.3. Agarose Gel Electrophoresis ............................................................... 52 3.5.4. Polyacrylamide Gel Electrophoresis ................................................... 53 3.5.5. Silver Staining .................................................................................... 53 3.6. Equipments ................................................................................................... 54 4. METHODS .......................................................................................................... 56 4.1. Molecular Basis of Rett Syndrome (RTT) ..................................................... 56 4.1.1. DNA Extraction from Peripheral Blood .............................................. 56 4.1.2. Quantitative Analysis of the Extracted DNA ....................................... 56 4.1.3. Mutation Analysis of MECP2 Gene .................................................... 57 4.1.3.1. Restriction Endonuclease Analysis ........................................ 57 xi 4.1.3.2. Single Strand Conformation Polymorphism ........................... 57 4.1.3.3. Preparation of SSCP Gels ...................................................... 58 4.1.3.4. SSCP Electrophoresis ............................................................ 59 4.1.3.5. Silver-Staining ...................................................................... 59 4.1.4. DNA Sequence Analysis .................................................................... 59 4.1.5. Quantitative Real Time PCR ............................................................... 59 4.1.6. Quantitative Fluorescent Multiplex PCR Assay .................................. 60 4.1.7. X Chromosome Inactivation ............................................................... 61 4.1.7.1. Preparation of Denaturing Polyacrlamide Gels ...................... 61 4.1.7.2. Electrophoresis of PCR Products ........................................... 61 4.1.8. Clinical Severity Score Analysis ......................................................... 62 4.1.8.1. Clinical Severity Score .......................................................... 62 4.1.8.2. Huppke Scoring .................................................................... 62 4.1.8.3. Statistical Analyses ............................................................... 62 4.1.9. Multiplexed ARMS-PCR Approach for the Detection of Common MECP2 Mutations .............................................................................. 62 4.1.9.1. Primer Design ....................................................................... 62 4.1.9.2. PCR Conditions .................................................................... 64 4.1.9.3. p.R106W Mutation Detection ................................................ 65 4.1.9.4. PCR-RFLP and DNA Sequencing ......................................... 65 4.1.10. The Effect of DNA Concentration on Reliability and Reproducibility of SYBR Green Dye-based Real Time PCR Analysis to Detect the exon Rearrangements Clinical Severity Score Analysis ....................... 65 4.1.10.1. Preparation of DNA Samples .............................................. 65 4.1.10.2. Quantitative Real Time PCR Conditions ............................. 65 4.1.10.3. Quantification ..................................................................... 66 4.1.10.4. Statistical Analysis .............................................................. 66 4.2. Methylation Analyses of the Putative Promoter Region of hHR23 Genes in Breast Tumor Tissues .................................................................................... 67 4.2.1. Non-heating DNA Extraction Protocol ............................................... 67 4.2.2. Promoter Region Analyses and Primer Design .................................... 67 4.2.3. Bisulfite Modification ......................................................................... 68 4.2.4. Amplification and Sequencing of the Bisulfite Modified DNA ........... 68 xii 4.3. Molecular Basis of Congenital Hypothyroidism (CH) ................................... 69 4.3.1. Mutation Analysis of the TTF2 Gene .................................................. 69 4.3.1.1. Direct DNA Sequencing Analysis ......................................... 69 4.3.1.2. AlwNI Digestion .................................................................... 70 4.3.2. Functional Characterization of p.R102C Mutant TTF2 ....................... 70 4.3.3. Mutation Analysis of Butyrylcholinesterase (BChE) Gene .................. 70 5. RESULTS ............................................................................................................ 72 5.1. Molecular Basis of Rett Syndrome ................................................................ 72 5.1.1. Patients ............................................................................................... 72 5.1.2. Mutation Analysis of MECP2 Gene .................................................... 72 5.1.3. Quantitative PCR Analyses ................................................................. 74 5.1.4. X Chromosome Inactivation Status ..................................................... 84 5.1.5. Genotype–Phenotype Correlations ...................................................... 85 5.1.6. Prenatal Diagnosis .............................................................................. 85 5.1.7. Multiplexed ARMS-PCR Approach for the Detection of Common MECP2 Mutations .............................................................................. 88 5.1.7.1. Assay Optimization ............................................................... 88 5.1.7.2. Validation of the Assay ......................................................... 88 5.1.8. The Effect of DNA Concentration on Reliability and Reproducibility of SYBR Green Dye-based Real Time PCR Analysis to Detect the Exon Rearrangements ......................................................................... 90 5.1.8.1. Comparison of Quantification Methods ................................. 91 5.1.8.2. Quantification ....................................................................... 91 5.2. Methylation Analyses of the Putative Promoter Region of Rad23 Genes in Breast Tumor Tissues .................................................................................... 96 5.2.1. Characterization of the 5' flanking region of the hHR23 Genes ........... 96 5.2.1.1. hHR23A Gene ....................................................................... 96 5.2.1.2. hHR23B Gene ....................................................................... 96 5.2.2. Bisulfite Sequencing of hHR23A and hHR23B in paraffin-embedded tissues ................................................................................................. 97 5.2.2.1. hHR23A ................................................................................ 98 5.2.2.2. hHR23B ................................................................................ 103 5.3. Molecular Basis of Congenital Hypothyroidism ............................................ 110 xiii 5.3.1. Clinical Features of the Patient ........................................................... 110 5.3.2. Mutation Analysis of the TTF2 Gene .................................................. 110 5.3.3. Functional Characterization ................................................................ 113 5.3.4. Mutation Analysis of Butyrylcholinesterase (BChE) Gene .................. 113 5.3.4.1. PCR-RFLP ............................................................................ 114 6. DISCUSSION ...................................................................................................... 115 6.1. Molecular Basis of Rett Syndrome ................................................................ 115 6.1.1. Multiplexed ARMS-PCR Approach for the Detection of Common MECP2 Mutations .............................................................................. 120 6.1.2. The Effect of DNA Concentration on Reliability and Reproducibility of SYBR Green Dye-based Real Time PCR Analysis to Detect the Exon Rearrangements ......................................................................... 122 6.2. Methylation Analyses of the Putative Promoter Region of hHR23 Genes in Breast Tumor Tissues .................................................................................... 124 6.3. Molecular Basis of Congenital Hypothyroidism ............................................ 130 6.3.1. Mutation Analysis of Butyrylcholinesterase (BChE) Gene .................. 132 7. CONCLUSION .................................................................................................... 133 REFERENCES .......................................................................................................... 134 Yüklə 4,8 Kb. Dostları ilə paylaş:
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