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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
FATIM BINTI KHARUDDIN
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
FATIM BINTI KHARUDDIN
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
FATIM BINTI KHARUDDIN
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.
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Internal Examiner)
Shahrilnizam Abdullah
Dr.
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(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
Universiti Putra Malaysia
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
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Chairman)
Rozita binti Rosli
Associate Professor
Faculty of Medicine and Health Science
Universiti Putra Malaysia
(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
Universiti Putra Malaysia
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.
_______________________
FATIM BINTI KHARUDDIN
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