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UNIVERSITI PUTRA MALAYSIA

PROFILING OF DNA COPY NUMBER MICROABERRATIONS ASSOCIATED WITH CONGENITAL DISORDERS AMONG MALAYSIAN CHILDREN USING ARRAY COMPARATIVE GENOMIC HYBRIDISATION

FATIM BINTI KHARUDDIN

FPSK(m) 2010 32

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PROFILING OF DNA COPY NUMBER MICROABERRATIONS

ASSOCIATED WITH CONGENITAL DISORDERS AMONG MALAYSIAN

CHILDREN USING ARRAY COMPARATIVE GENOMIC

HYBRIDISATION

By


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirements for the Degree of Master of Science

October 2010

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment

of the requirement for the degree of Master of Science

PROFILING OF DNA COPY NUMBER MICROABERRATIONS

ASSOCIATED WITH CONGENITAL DISORDERS AMONG MALAYSIAN

CHILDREN USING ARRAY COMPARATIVE GENOMIC

HYBRIDISATION

By


October 2010

Chair: Abhimanyu Veerakumarasivam, PhD

Faculty: Faculty of Medicine and Health Sciences

Copy number gains or losses of various chromosomal regions, whole chromosomes

or subtelomeric rearrangements have been known to cause human mental retardation

syndromes and congenital malformations. This study characterised chromosomal

abnormalities associated with congenital disorders in Malaysian patients and

identified novel microdeletions or microduplications that correlate with the

phenotype. A high resolution method, array comparative genomic hybridization

(aCGH) was used. DNA was extracted from blood and then quantified and qualified

before proceeding with the technique. Samples with satisfactory DNA quality and

quantity were then chosen to be analysed with aCGH. The DNA was labelled,

combined, pre-hybridised, hybridised and scanned. The identification of genes

located in the region of duplication and deletion was conducted using ENSEMBL!.

The correlation between the genotype and phenotype was done using OMIM. One

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hundred patients were analysed in this study. Chromosome specific deletions and

duplications occurred most frequently in chromosomes 1 and 3. The reasons

probably are because these chromosomes are large and harbour many loci and genes

that are susceptible to changes. In this study, 496 clones harboured copy number

changes. Of this, 88 copy number changes were found in more than one individual.

A comparison of the most frequent changes associated with phenotypes was also

executed. For example, chromosome 17q21.31 was identified as major cause for

global delays observed in the patients within this study based on the comparitive

analysis, previous literature and CRHR1 and MAPT genes functions. Furthermore,

10 patients were selected for an in depth analysis to identify the possible causal

gene(s). Seven novel genes speculated to have significant effect on the phenotypes

were identified. Amongst them were FBN2 gene for abnormal pinna, FUZ for

holoprosencephaly, TMEM1 and ARFGEF2 for microcephaly, TM2D2 for

developmental delay, SH3GL2 for autism and INPP5A for severe psychomotor delay

and failure to thrive as observed in these patients. This study proves the importance

of aCGH as a tool that refines birth defects diagnosis. However, due to the high-

resolution output of the technique, the copy-number variable nature of the genome as

well as the massive gaps in the knowledge of many regions of the genome and gene

function; conclusive delineation of the genotype-phenotype correlation remains an

arduous task. Nonetheless, aCGH helps to provide better insight into the patient

diagnosis and helps greater understanding of the disease molecular pathogenesis.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

PEMPROFILAN MIKROPERUBAHAN SALINAN NOMBOR DNA DALAM

PENYAKIT KONGENITAL DI KALANGAN PESAKIT KANAK-KANAK

MALAYSIA DENGAN MENGGUNAKAN ‘ARRAY COMPARATIVE

GENOMIC HYBRIDISATION’

Oleh


Oktober 2010

Pengerusi: Abhimanyu Veerakumarasivam, PhD

Fakulti: Fakulti Perubatan dan Sains Kesihatan

Penambahan atau kehilangan pelbagai kawasan spesifik pada kromosom dan

penyusunan semula kromosom telah diketahui sebagai penyebab sindrom terencat

akal dan malformasi. Kajian ini mencirikan kecacatan kromosom dalam penyakit

kongenital di kalangan pesakit Malaysia dan mengenalpasti mikrodelesi dan

mikroduplikasi yang berhubungkait dengan fenotip. Satu kaedah yang mempunyai

resolusi tinggi ‘array comparative genomic hybridisation’ (aCGH) telah digunakan.

DNA telah diekstrak daripada sampel darah dan kemudian kualiti dan kuantitinya

diperiksa sebelum ujikaji dijalankan. Sampel-sampel yang memenuhi kriteria yang

telah ditetapkan dipilih untuk dianalisa dengan aCGH. Proses label, kombinasi,

hibridisasi awal dan hibridisasi serta imbasan dilakukan. Pengenalan gen-gen yang

terletak pada kawasan kromosom yang hilang atau bertambah dilakukan

menggunakan Ensembl!. Korelasi antara genotip dan fenotip dilakukan

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menggunakan OMIM. Kajian telah dijalankan ke atas 100 pesakit untuk dianalisis.

Kromosom yang paling kerap mengalami perubahan sama ada hilang atau bertambah

adalah 1 dan 3. Antara sebab yang mungkin ialah kerana kromosom-kromosom ini

adalah antara yang terbesar dan mempunyai banyak ‘loci’ dan gen-gen yang terdedah

kepada perubahan. Dalam kajian ini, 496 klon menunjukkan perubahan bilangan.

Terdapat 88 klon telah ditemui pada lebih daripada seorang individu. Perbandingan

di antara perubahan klon yang kerap berlaku yang berkait dengan fenotip juga

dijalankan. Sebagai contoh, kromosom 17q21.31 telah dikenal pasti sebagai

penyebab kepada terencat akal dalam kajian ini berdasarkan kepada perbandingan

analisis, kajian yang lepas dan juga fungsi gen-gen yang terlibat iaitu CRHR1 dan

MAPT. Selain itu, 10 pesakit dipilih untuk kajian analisis secara mendalam untuk

mengenalpasti gen-gen penyebab yang mungkin. Terdapat 7 gen-gen baru yang

dispekulasi mempunyai kesan signifikan ke atas fenotip iaitu gen FBN2 untuk bentuk

telinga yang tidak normal, FUZ untuk bentuk otak yang tidak normal, TMEM1 dan

ARFGEF2 untuk saiz kepala yang kecil, TM2D2 untuk masalah kelewatan

perkembangan kanak-kanak, SH3GL2 untuk autisme dan INPP5A untuk masalah

otot pergerakan yang diperhatikan pada pesakit-pesakit. Kajian ini membuktikan

kepentingan aCGH sebagai kaedah untuk pengdiagnosan kecacatan kelahiran.

Walaubagaimanapun, disebabkan oleh hasil resolusi tinggi dari teknik ini, variasi

semulajadi yang terdapat dalam genom dan juga pengetahuan yang kurang tentang

lokasi-lokasi genom dan fungsi gen, hubungkait antara genotip fenotip menjadi satu

tugas yang sukar. Namun, aCGH tetap membantu dalam memberi pengetahuan yang

mendalam untuk diagnosis pesakit dan dalam memahami tentang penyebab suatu

penyakit itu dari sudut molekular.

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ACKNOWLEDGEMENT

Alhamdulillah, thanks to God Almighty with His blessing I finally completed this

thesis for my Master of Science. First and foremost, I would like to express my

utmost gratitude to my supervisor, Dr. Abhimanyu Veerakumarasivam who has

guided, advised and encouraged me throughout this work. Special thanks goes to Dr.

Roziana Ariffin who has allowed me to learn and do experiment in her laboratory in

Genetic Department, Hospital Kuala Lumpur for her guidance and support is most

appreciated. I would also like to express my warm thanks to my other co-supervisors,

Prof. Madya Dr. Rozita bt. Rosli and Dr. Norshariza Nordin for the continuous

learning process I received during the time of my study.

My sincere thanks to Diyana who gave me the biggest help in conducting the

laboratory work. Her kindness and technical assistance has been a great value to this

study. A warm thanks to other labmates in HKL, Kathy, Suhaimi and Sasi who have

been very kind and supportive. I am also grateful to all my labmates in Medical

Genetics Laboratory, Fizi, Farhana, Marlini, Nadine, Wendy, Eunice, Low, Dr.Reza,

Razieh, Narges, Zahrah, Behnam, Keai Sinn, Pushpa, Aimi, Herson, Radha, Chan

and Chin for the great moments we had together and also for the encouragement and

concerned during the entire time of my study.

Lastly, I am deeply grateful to my parents, brothers and sisters who have never stop

encouraging and always been there for me whenever I need them. Without their

understanding and loving support, it will be impossible for me to finish this work.

Thanks a million.

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I certify that an Examination Committee has met on 28 October 2010 to conduct the

final examination of Fatim binti Kharuddin on her Master of Science thesis entitled

‘Profiling of DNA Copy Number Microaberrations Associated with Congenital

Disorders Among Malaysians Using Array Comparative Genomic Hybridisation’ in

accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and

Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee

recommends that the student be awarded the Master of Science

Members of the Examination Committee were as follows:

Chong Pei Pei

Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Chairman)

Sharmili Vidyadaran

Dr.



(Internal Examiner)

Shahrilnizam Abdullah

Dr.



(Internal Examiner)

Zilfalil Alwi

Associate Professor

School of Medical Sciences

Universiti Sains Malaysia

(External Examiner)

________________________

BUJANG KIM HUAT, PhD

Professor and Deputy Dean

School of Graduate Studies


Date:

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Abhimanyu Veerakumarasivam, PhD

Lecturer



(Chairman)

Rozita binti Rosli

Associate Professor

Faculty of Medicine and Health Science


(Member)

Shariza binti Nordin, PhD

Lecturer

Faculty of Medicine and Health Science

University Putra Malaysia

(Member)

__________________________

HASANAH MOHD.GHAZALI, PhD

Professor and Dean

School of Graduate Studies


Date:

DECLARATION

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I declare that the thesis is my original work except for quotations and citations which

have been duly acknowledged. I also declare that it has not been previously, and is

not concurrently, submitted for any other degree at Universiti Putra Malaysia or at

any other institution.

_______________________


Date: 28 October 2010

TABLE OF CONTENTS

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Page

ABSTRACT ii

ABSTRAK iv

ACKNOWLEDGEMENT vi

APPROVAL vii

DECLARATION ix

LIST OF TABLES xii

LIST OF FIGURES xiii

LIST OF ABBREVIATIONS xvi

CHAPTERS

1.0 INTRODUCTION 1

1.1 Objectives 2

2.0 LITERATURE REVIEW 4

2.1 Chromosomal rearrangements 4

2.2 Genetic Diseases 7

2.2.1 Examples of Genetic Diseases 9

2.3 Chromosomal disorders 16

2.3.1 Chromosomal microdeletions 16

2.3.2 Chromosomal microduplications 19

2.4 Karyotype 22

2.5 Conventional CGH 24

2.6 Fluorescence In Situ Hybridisation (FISH) 25

2.7 Array CGH (aCGH) 26

2.8 Copy Number Variations (CNVs) 31

3.0 MATERIALS & METHODS 34

3.1 Study Location 34

3.2 Subject Selection 34

3.3 Materials 34

3.3.1 Blood sample 34

3.4 Preparation of Nucleic Acid Materials 35

3.4.1 DNA Extraction 35

3.4.2 DNA Quantification and Quality Assessment 36

3.5 Array Comparative Genomic Hybridisation (aCGH) 38

3.5.1 Sample DNA 38

3.5.2 Reference DNA 39

3.5.3 DNA Labelling 39

3.5.4 Clean up, quantification and combination 42

3.5.5 Solution preparation 44

3.5.6 Pre-Hybridisation 46

3.5.7 Hybridisation 46

3.5.8 Image and Data Analysis 47

3.6 Limitations

48

4.0 RESULTS AND DISCUSSION 49

4.1 Subjects 49

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4.1.1 Ethnicity 49

4.1.2 Age 50

4.1.3 Sex 50

4.2 DNA Quality Assessment 51

4.3 Array Image 52

4.4 Whole genome profile 55

4.5 Chromosome profile 56

4.6 Chromosome changes analysis 57

4.6.1 Chromosome Specific Deletions and Duplications 57

4.6.2 Known Copy Number Variations (Deletions and

Duplications)

62

4.6.3 Novel Deletions and Duplications 64

4.6.4 Real Deletions and Duplications 66

4.7 The most frequent known CNVs with phenotype 69

4.8 The most frequent novel changes with phenotype 81

4.9 In depth analysis of selected individual patients 87

4.9.1 CG 262 87

4.9.2 CG 392 90

4.9.3 CG 320 94

4.9.4 CG 233 99

4.9.5 CG 125 104

4.9.6 CG 122 108

4.9.7 CG 247 114

4.9.8 CG 277 117

4.9.9 CG 293 121

4.9.10 CG 230 125

4.10 Comparison between karyotyping and aCGH 128

5.0 CONCLUSION 131

5.1 Recommendation for Future Studies 131

REFERENCES

135

APPENDIX 155

BIODATA OF STUDENT

160

LIST OF TABLES

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