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UNIVERSITI MALAYA

ORIGINAL LITERARY WORK DECLARATION

Name of Candidate: KAVIMALAR A/P RENGANATHAN

I/C/Passport No: 800513-01-5378 Regisration/Matric No.: SGF110011

Name of Degree: MASTER OF BIOTECHNOLOGY

Title of Project Paper/Research Report/Dissertation/Thesis (“this Work”):

“IDENTIFICATION OF RB1 GERM-LINE MUTATIONS IN PATIENTS WITH RETINOBLASTOMA”

Field of Study: MOLECULAR GENETICS

I do solemnly and sincerely declare that:

(1) I am the sole author/writer of this Work, (2) This Work is original,

(3) Any use of any work in which copyright exists was done by way of fair dealing and for permitted purposes and any excerpt or extract from, or reference to or reproduction of any copyright work has been disclosed expressly and sufficiently and the title of the Work and its authorship have been acknowledged in this Work,

(4) I do not have any actual knowledge nor do I ought reasonably to know that the making of this work constitutes an infringement of any copyright work,

(5) I hereby assign all and every rights in the copyright to this Work to the University of Malaya (“UM”), who henceforth shall be owner of the copyright in this Work and that any reproduction or use in any form or by any means whatsoever is prohibited without the written consent of UM having been first had and obtained,

(6) I am fully aware that if in the course of making this Work I have infringed any copyright whether intentionally or otherwise, I may be subject to legal action or any other action as may be determined by UM.

(Candidate Signature) Date:

Subscribed and solemnly declared before,

Witness‟s Signature Date:

Name PROFESSOR DR HANY MOHD ARIFFIN

Designation

Witness‟s Signature Date:

Name PROFESSOR DR ZULQARNAIN MOHAMED

Designation

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ABSTRACT

Background: Retinoblastoma (RB1; OMIM#180200) is the most common intraocular tumor in early childhood. The malignancy mainly affects children under five years of age, with a prevalence of 1:15,000 to 1:20,000. RB1 or retinoblastoma susceptibility gene is the gene associated with retinoblastoma and is located on chromosome 13q14.2.

The whole coding region of RB1 has 27 exons. The gene encodes a tumor suppressor, the retinoblastoma protein (pRB). Retinoblastoma develops when both alleles of the tumor suppressor gene are inactivated by loss-of-function mutations. The hereditary predisposition to the disease is caused by germ-line mutations in RB1. Since RB1 is a tumor suppressor gene, the survivors of hereditary retinoblastoma also possess high risk of developing non-ocular cancers in their subsequent life. The identification of oncogenic germ-line mutations in the RB1 gene of patients with retinoblastoma is significant for genetic counselling. There are very few reports on RB1 mutations identified in Malaysian patients with retinoblastoma although several studies have been published on the prevalence and clinical features of the disease.

Aims: This was a hospital-based retrospective study to screen the entire coding region of RB1 for de novo germ-line mutations in patients with no reported family history of retinoblastoma.

Methods: This study involved a cohort of six unrelated cases of sporadic retinoblastoma with different laterality of disease: three unilateral and three bilateral cases. Genomic DNA was extracted from peripheral blood of healthy control and patients with retinoblastoma. Polymerase chain reaction (PCR) was carried out to amplify the 27 exons of RB1. The purified PCR amplicons were then subjected to bidirectional DNA sequencing. Sequence data was compared with RB1 DNA sequence

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of two healthy controls of Chinese and Malay ethnicity and RB1 reference sequence (GenBank accession no. L11910.1) to identify mutations. This exon-by-exon mutation analysis was conducted in silico.

Results: Analysis of DNA from blood leukocytes revealed heterozygous and heritable mutations in the RB1 gene of two patients with bilateral retinoblastoma. The mutations were identical single base substitutions which caused a C to T transition: g.162237C>T.

The nonsense mutation which was found in exon 23 changed codon 787 from wild-type CGA to mutant TGA (p.Arg787X), resulting in a premature stop codon. All three patients with unilateral retinoblastoma did not show any mutation in the RB1 gene.

Conclusions: The findings proved that the mutations detected in DNA extracted from blood of patients were constitutional. The identified mutations were de novo mutations as both parents of respective patients lacked the abovementioned mutation in their RB1 gene. The experiment results were consistent with past reports that patients with bilateral retinoblastoma usually harbour germ-line mutations in the RB1 gene. Based on the study result, exon 23 and CGA codons are apparently more frequent mutational targets and should be initially screened in Malaysian patients with retinoblastoma though no mutation hot spots have been reported in RB1 gene thus far. However, further studies with extended sample size would be needed to evaluate and establish a protocol for routine RB1 mutation analysis.

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ABSTRAK

Latar belakang: Retinoblastoma (RB1; OMIM#180200) adalah tumor intraokular yang paling kerap berlaku di peringkat awal kanak-kanak. Kanser ini menjejaskan terutamanya kanak-kanak di bawah umur lima tahun, dengan kelaziman 1:15 000 hingga 1:20 000. RB1 atau gen kecenderungan retinoblastoma adalah gen yang dikaitkan dengan retinoblastoma dan terletak pada kromosom 13q14.2. Seluruh rantau pengekodan RB1 mempunyai 27 ekson. Gen ini mengekod sejenis penindas tumor iaitu protein retinoblastoma (pRB). Retinoblastoma terjadi apabila kedua-dua alel gen penindas tumor dijadikan tidak aktif oleh mutasi hilang-fungsi. Predisposisi terhadap penyakit ini adalah melalui keturunan yang diakibatkan oleh mutasi titisan germa di RB1. Oleh sebab RB1 merupakan sejenis gen penindas tumor, pesakit yang terselamat

daripada retinoblastoma keturunan juga mempunyai risiko tinggi menghidap kanser bukan okular dalam kehidupan berikutnya. Identifikasi mutasi titisan germa onkogenik di dalam gen RB1 pesakit retinoblastoma adalah penting untuk kaunseling genetik.

Setakat ini, amat kurang laporan mengenai mutasi RB1 yang ditemui pada pesakit retinoblastoma di Malaysia walaupun beberapa kajian telah diterbitkan berkaitan kelaziman dan ciri-ciri klinikal penyakit ini.

Matlamat: Ini adalah satu kajian retrospektif yang berasaskan hospital untuk menyaring seluruh rantau pengekodan RB1 bagi mutasi titisan germa baharu di kalangan pesakit yang ada atau tidak mempunyai sejarah keluarga retinoblastoma.

Kaedah: Kajian ini telah melibatkan sebuah kohort yang terdiri daripada enam kes retinoblastoma sporadik yang tiada kaitan antara satu sama lain, dengan kesisian penyakit yang berbeza: tiga kes yang melibatkan satu sisi mata dan tiga kes yang melibatkan kedua-dua sisi mata. DNA genom telah diekstrak daripada darah periferi

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kontrol sihat dan pesakit retinoblastoma. Reaksi rantai polimerase (PCR) telah dijalankan untuk mengamplifikasi 27 ekson RB1. Seterusnya, amplikon PCR tulen telah dilalui penjujukan DNA dwiarah. Data jujukan telah dibandingkan dengan urutan DNA RB1 dua kontrol sihat dari etnisiti Melayu dan Cina dan jujukan rujukan RB1 (no. induk

GenBank L11910.1) untuk mengesan mutasi. Analisis mutasi ekson demi ekson ini telah dijalankan menggunakan perisian komputer.

Keputusan: Analisis DNA daripada leukosit darah mendedahkan mutasi-mutasi heterozigus dan yang diwarisi di dalam gen RB1 dua pesakit retinoblastoma dua sisi mata. Mutasi-mutasi yang dikenal pasti merupakan penggantian bes tunggal yang serupa dan telah menyebabkan suatu transisi, iaitu C kepada T: g.162237C> T. Mutasi tak bererti itu ditemui di ekson 23 dan didapati mengubah kodon 787 daripada jenis liar CGA kepada mutan TGA (p.Arg787X), mengakibatkan suatu kodon terminasi pra- matang. Ketiga-tiga pesakit retinoblastoma satu sisi mata tidak menunjukkan sebarang mutasi di dalam gen RB1.

Kesimpulan: Penemuan membuktikan bahawa mutasi yang dikesan di dalam DNA yang diekstrak daripada darah pesakit adalah konstituen. Mutasi yang ditemui adalah mutasi baru kerana kedua-dua ibu bapa pesakit berkenaan tidak menunjukkan sebarang mutasi di dalam gen RB1. Keputusan eksperimen adalah konsisten dengan laporan sebelumnya bahawa pesakit retinoblastoma dua sisi mata biasanya mempunyai mutasi titisan germa di dalam gen RB1. Berdasarkan hasil kajian ini, ekson 23 dan kodon- kodon CGA merupakan sasaran mutasi dan perlu disaring dahulu pada pesakit retinoblastoma walaupun tiada kawasan khas mutasi telah dilaporkan di dalam gen RB1 setakat ini. Walau bagaimanapun, kajian lanjut dengan saiz sampel yang lebih besar diperlukan untuk menilai dan mewujudkan protokol untuk analisis mutasi yang rutin di dalam RB1.

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ACKNOWLEDGEMENTS

This manuscript would not have been possible without the help of many kind hearts in various ways. I am pleased to acknowledge their contributions.

So right up front, I would like to express my sincere appreciation to my project supervisor, Professor Dr. Hany Ariffin. I am indebted to her beyond measure for her remarkable guidance, incredible patience and constructive feedback which never failed to encourage me during hard times. Likewise, I would also like to thank Professor Dr.

Zulqarnain Mohamed, co-supervisor of the project, for rendering DNA sequencing facility in his lab. Not forgetting his research colleague, Dr. Sally, who carried out DNA sequencing and delivered the results promptly. Thousand thanks to Dr. Saharuddin of Department of Bioinformatics and Miss Ili of CEBAR laboratory who sincerely taught me the in silico protocols with some useful tips on sequence analysis and mutation identification. I am fortunate to have benefitted from interactions with many talented research colleagues in Paediatric Oncology Lab – Jannah, Soo Sin, Leon, Mawar and Adeline. During my study, I had wonderful friends who went through thick and thin with me and therefore deserve mention, namely, Kasthuri, Neelaveni and Vanitha.

Together their influences and support over my study and research period are inestimable. My loving family, through their presence and patience, their tender care and comforting words, has made this sometimes trying effort much more palatable.

To anyone I have inadvertently omitted: my apologies. It‟s the people in one‟s life that count – my heartfelt thanks go out to all.

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TABLE OF CONTENTS

Page

ABSTRACT iii

ABSTRAK v

ACKNOWLEDGEMENTS vii

TABLE OF CONTENTS viii

LIST OF FIGURES xi

LIST OF TABLES xii

LIST OF SYMBOLS AND ABBREVIATIONS xiii

1.0 INTRODUCTION 1

1.1 Significance of the Study 5

1.2 Research Questions 6

1.3 Objectives of the Study 6

2.0 LITERATURE REVIEW 7

2.1 Overview of Retinoblastoma 7

2.1.1 Epidemiology 11

2.1.2 Clinical Presentation 11

2.1.3 Diagnosis 13

2.1.4 Treatment 14

2.1.5 Risk of Secondary Malignancies 16

2.2 Molecular Genetics of Retinoblastoma 21

2.2.1 Brief History 21

2.2.2 Knudson‟s Two-Hit Hypothesis 23

2.2.2.1 Mode of inheritance 26

2.2.3 Retinoblastoma Susceptibility Gene (RB1) 27

2.2.3.1 Structure of RB1 32

2.2.3.2 Function of RB1 32

2.2.4 Retinoblastoma Protein 35

2.2.4.1 Effect of mutation on retinoblastoma protein 39 2.2.5 Development of Retinoblastoma (Tumorigenesis) 40 2.2.6 Frequencies of Various Types of Retinoblastoma 42

2.2.7 Types of Retinoblastoma 43

2.2.7.1 Non-hereditary retinoblastoma 44

2.2.7.2 Hereditary retinoblastoma 45

2.2.8 Laterality of the Disease 47

2.2.8.1 Unilateral retinoblastoma 47

2.2.8.2 Bilateral retinoblastoma 47

2.2.9 Genotype-Phenotype Correlation of Retinoblastoma 48

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2.2.10 Germ-Line Mutation and Retinoblastoma 51 2.2.11 Spectrum of Mutations in RB1 Gene 55 2.2.12 Clinical Specimen for Germ-Line RB1 Mutation Screening 60 2.2.13 Molecular Screening for Identification of RB1 Gene Mutations 62

2.2.13.1 Advantages of molecular screening in identification of

RB1 mutations 66

3.0 METHODOLOGY 70

3.1 Study Design 70

3.2 Sampling 72

3.2.1 Patients 72

3.2.2 Clinical Specimen Collection 73

3.3 DNA Extraction 74

3.3.1 Peripheral Blood DNA Extraction 74

3.3.1.1 Cell lysis 75

3.3.1.2 Washing 75

3.3.1.3 Protein precipitation and organic extraction of DNA 76 3.3.1.4 Ethanol precipitation of DNA 77

3.3.2 Buccal Cell DNA Extraction 77

3.4 DNA Quantification 78

3.5 Storage of Genomic DNA 79

3.6 PCR Amplification of Exonic Regions of RB1 Gene 79

3.7 Selection of Healthy Control 80

3.8 Analysis of PCR Amplicons 82

3.8.1 Preparation of Agarose Gel 82

3.8.2 Gel Electrophoresis 82

3.9 Purification of PCR Amplicons 83

3.10 DNA Sequencing 83

3.11 In-silico Analysis of DNA Sequence 84

4.0 RESULTS 85

4.1 Patients and Control 85

4.2 DNA Extraction 87

4.3 Primer Map and PCR Amplification of RB1 Gene 89

4.4 Mutation Analysis by DNA Sequencing 92

5.0 DISCUSSION 101

5.1 Detection of an Identical Germ-line Mutation in the RB1 Gene

of Two Patients with Bilateral Retinoblastoma 102

5.2 Parental Origin of Mutations 104

5.3 Association of Identified Germ-line Mutation with Laterality of

the Disease 104

5.4 Consequence of Mutation g.162237C>T Found in RB1 Gene 105 5.5 Possible Causes for Absence of Germ-line Mutations in RB-I, RB-II,

RB-IV and RB-VI 107

5.6 Molecular Testing by PCR-Sequencing 108

5.7 Significance of Mutation Screening for Genetic Counselling

and Patients Management 109

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6.0 CONCLUSION 111

6.1 Suggestions for Future Work 112

BIBLIOGRAPHY 115

APPENDICES 130

Appendix I 130

Appendix II 131

Appendix III 132

Appendix IV 133

Appendix V 144

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LIST OF FIGURES

Page

Figure 2.1a Normal Retina 8

Figure 2.1b Retinoblastoma Tumor Develops on Retina 8

Figure 2.2 Retinoblastoma 9

Figure 2.3 Leukocoria 12

Figure 2.4 Patient with Unilateral Retinoblastoma 15

Figure 2.5 Untreated Retinoblastoma, Circa 1806 22

Figure 2.6a Knudson‟s „Two-Hit‟ Model for Retinoblastoma 23

Figure 2.6b Knudson‟s „Two-Hit‟ Hypothesis 24

Figure 2.7 Principles of Autosomal Dominant Inheritance 26 Figure 2.8 Ideogram Illustrating Human Chromosome 13 30

Figure 2.9 pRB Structure 36

Figure 2.10 Cellular Functions of pRb 37

Figure 2.11 Effects of Point Mutations in the Coding Sequence of a Gene 40 Figure 3.1 Schematic Representation of Experimental Design 71 Figure 4.1a Clustal Omega Alignment of RB1 Exon 23 Forward

Sequences of Normal Control and Patients

94 Figure 4.1b Clustal Omega Alignment of RB1 exon 23 Reverse

Sequences (in 5‟ to 3‟ Order) of Normal control and Patients

95 Figure 4.2 Nucleotide Sequence of a Fragment of RB1 exon 23 96

Figure 4.3 Clustal Omega Protein Sequence Alignment 97

Figure 4.4 RB1 Exon 23 Transition Mutation in Blood DNA of Bilateral Retinoblastoma Patient, RB-III

99 Figure 4.5 RB1 Exon 23 Transition Mutation in Blood DNA of

Bilateral Retinoblastoma Patient, RB-V

100

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LIST OF TABLES

Page

Table 2.1 Knudson‟s Two-Hit Hypothesis 25

Table 2.2 Characteristics of Autosomal Dominant Inheritance Pattern 27 Table 2.3 Cancer Syndromes Due to Tumor Suppressor Mutations 29

Table 2.4 Disease Genes Cloned by Gene Mapping 31

Table 2.5 Classes and Length of Genes 34

Table 2.6 Frequencies of Various Types of Retinoblastoma 42 Table 2.7 Distribution of Retinoblastoma by Type and Laterality 43 Table 2.8 Common Characteristics of Sporadic and Inherited

Retinoblastoma

43 Table 2.9 Genotype – Phenotype Correlates in Hereditary Rb 49 Table 2.10 Germ-Line Mutation in Association with Family History and

Tumor Type 52

Table 2.11 Uses of Genetic Tests 67

Table 2.12 Genetic Risks in Retinoblastoma 69

Table 3.1 Details of Rb Patients and Clinical Specimen Received 73

Table 3.2 PCR Master Mix Recipe 80

Table 3.3 Primer Sequences 81

Table 4.1 Description of Retinoblastoma Patients Enrolled in RB1 Mutation Screening Study

86 Table 4.2 Treatment Received by Patients with Retinoblastoma in

reference to the Laterality and Severity of Their Disease

86 Table 4.3 Quantity and Measurement of Purity of Genomic DNA

Extracted from Clinical Specimens of Patients and Parents

88

Table 4.4 RB1 Primer Sequences 91

Table 4.5 Summary of Mutation Analysis in Patients with Retinoblastoma

92

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LIST OF SYMBOLS AND ABBREVIATIONS

 to

A absorbance

as antisense

°C degree Celsius

ADA adenosine deaminase

AMD amplification mismatch detection ATF activating transcription factor

Arg arginine

BC before Christ

bp base pair(s)

BRCA1 breast cancer 1, early onset

CA California

cDNA complementary DNA

C-terminal carboxyl-terminal

cdk cyclin-dependent kinase

CpG – C – phosphate – G –

CTS computer tomography scan

ddH2O double-distilled water

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DEPC diethylpyrocarbonate

DNA deoxyribonucleic acid

DNase deoxyribonuclease

DGGE denaturant gradient gel electrophoresis dNTPs deoxynucleotide triphosphates

EBRT external beam radiation

EDTA ethylenediaminetetraacetic acid

et al. and others

E1A adenovirus early region 1A

g gram

G1 phase Gap 1 phase

HKL Hospital Kuala Lumpur

HUKM Hospital Universiti Kebangsaan Malaysia i.e. id est (Latin) / in example

Inc. Incorporated

IPS Institute of Postgraduate Studies

kb kilobase

kDa kilodaltons

LDL low density lipoprotein

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LOH loss of heterozygosity

M molar

MA Massachusetts

MEN multiple endocrine neoplasia

min minute

mA milliampere

mPCR multiplex polymerase chain reaction

mg milligram

ml millilitre

mM millimolar

mPCR multiplex polymerase chain reaction MRI magnetic resonance imaging

mRNA messenger ribonucleic acid

MO Missouri

n.d. no date

ng nanogram

nm nanometer

N-terminal amino-terminal

NY New York

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NaCl natrium chloride

OMIM Online Mendelian Inheritance in Man

ORF open reading frame

PCR polymerase chain reaction

p53 protein 53

PB Peripheral blood

pRb/ pRB RB1- encoded protein

PTC premature termination codon

PTT protein truncation test

q long arm of chromosome

13q14.2 region 1, band 4, sub-band 2 on long arm of chromosome 13

Rb / RB retinoblastoma

RB / RB1 retinoblastoma / retinoblastoma 1 gene

RBC red blood cell

RetCam retinal camera

RFLP restriction fragment length polymorphism

RNA ribonucleic acid

RNase ribonuclease

rpm revolutions per minute

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RT reverse transcriptase

SDS sodium dodecyl sulphate

se sense

secs seconds

SSC saline-sodium citrate

SSCP single strand conformation polymorphism S phase synthesis phase

Sp1 specificity protein 1

TBE tris borate EDTA

TE tris-EDTA

TP53 tumor protein 53

µl microliter

µg microgram

µM micro molar

UMMC University of Malaya Medical Centre uPCR uniplex polymerase chain reaction USA United Stated of America

UV ultraviolet

V volt

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VHL Von Hippel-Lindau

W watt

Xp short arm of the X chromosome

yr year

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