A comprehensive analysis of common copy-number variations in the human genome.

Abstract

Segmental copy-number variations (CNVs) in the human genome are associated with developmental disorders and susceptibility to diseases. More importantly, CNVs may represent a major genetic component of our phenotypic diversity. In this study, using a whole-genome array comparative genomic hybridization assay, we identified 3,654 autosomal segmental CNVs, 800 of which appeared at a frequency of at least 3%. Of these frequent CNVs, 77% are novel. In the 95 individuals analyzed, the two most diverse genomes differed by at least 9 Mb in size or varied by at least 266 loci in content. Approximately 68% of the 800 polymorphic regions overlap with genes, which may reflect human diversity in senses (smell, hearing, taste, and sight), rhesus phenotype, metabolism, and disease susceptibility. Intriguingly, 14 polymorphic regions harbor 21 of the known human microRNAs, raising the possibility of the contribution of microRNAs to phenotypic diversity in humans. This in-depth survey of CNVs across the human genome provides a valuable baseline for studies involving human genetics.

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MANTLE CELL LYMPHOMA PATHOGENESIS


by

RONALD JOHN DELEEUW

B.Sc., the University of British Columbia, 2003


A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE FOR
DOCTOR OF PHILOSOPHY

in

THE FACULTY OF GRADUATE STUDIES
(Pathology and Laboratory Medicine)


THE UNIVERSITY OF BRITISH COLUMBIA
(Vancouver)



August 2009

© Ronald John deLeeuw, 2009

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Abstract
Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin’s lymphoma with a median patient
survival time of 3 years. Although the characteristic t(11;14)(q13;q32) is found in virtually all
cases, experimental evidence suggests that this event alone is insufficient to result in lymphoma
and secondary genomic alterations are required. Therefore, secondary genetic alterations have
been proposed as essential in MCL pathogenesis. Within this thesis I describe the creation of a
novel assay to determine segmental copy number alterations at a previously unprecedented
resolution. This new assay necessitated the development of new analytical software to
visualize and analyze the high density data sets created. The creation of this software is
described in detail. With these tools in place we assayed model genomes of MCL for recurrent
segmental copy number alterations. These recurrent regions were defined; however, among
these were copy number variations that appeared in both cases and controls. Investigation of
these natural copy number variations in this thesis revealed that the human genome has a
higher plasticity than previously appreciated. In fact, thousands of loci within the genome were
found to be variable in copy number that may influence sensory perception and possibly
disease susceptibility. I next investigated the genomes of MCL tumor samples to determine
which somatic copy number alterations are related to a poor clinical course. Among the
numerous loci that showed frequent copy number alterations in MCL genomes, many were
associated with poor patient outcome. Among these, the loss of 9p21 was a strong factor in
determining the clinical course of patients with MCL (P=0.0004). Three additional loci (4q13,
8q24, and 13q14) were combined with 9p21 to create a survival model that was very predictive
of patient outcome (P=5.87 x 10-6). Interestingly, a previously uncharacterized locus (4q13) was
within this survival model. Investigating this locus further revealed that the expression of two
genes (CCNG2 and CCNI) influences the overall survival of patients with MCL (P=0.0292 and
0.0201, respectively).
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Table of Contents
Abstract ......................................................................................................................................... ii 
Table of Contents ......................................................................................................................... iii 
List of Tables ............................................................................................................................... vii 
List of Figures ............................................................................................................................. viii 
List of Abbreviations ...................................................................................................................... x 
Acknowledgements ..................................................................................................................... xiii 
Dedication ................................................................................................................................... xv 
Co-Authorship Statement ........................................................................................................... xvi 
Chapter 1: Introduction ................................................................................................................. 1 
1.1 Lymphoma ........................................................................................................................... 1 
1.1.1. Definition ...................................................................................................................... 1 
1.1.2. Classification ................................................................................................................ 1 
1.1.3. Etiology ........................................................................................................................ 2 
1.1.4. Epidemiology ............................................................................................................... 3 
1.2 Mantle cell lymphoma .......................................................................................................... 4 
1.2.1. Classification ................................................................................................................ 4 
1.2.2. Clinical characteristics ................................................................................................. 4 
1.2.3. Genetic characteristics ................................................................................................ 5 
1.3 Genomic copy number alterations in cancer ....................................................................... 6 
1.3.1. Identification of copy number alterations in cancer ...................................................... 6 
1.3.2. Evolution of techniques to measure copy number alterations in cancer ...................... 6 
1.3.3. Natural copy number variation ..................................................................................... 8 
1.3.4. Other genetic alterations in cancer .............................................................................. 8 
1.4 Thesis theme ....................................................................................................................... 9 
1.5. Objectives and hypothesis ............................................................................................... 10 
1.6. Specific aims and thesis outline ....................................................................................... 10 
Aim 1: Development of tools for the comprehensive determination of copy number
alterations in MCL. ............................................................................................................... 10 
Aim 2: Determination of recurrent copy number alterations in MCL model genomes.......... 11 
Aim 3: Identification of natural copy number variation in the human genome. .................... 11 
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Aim 4: Determination of copy number alterations in MCL that correlate to a poor overall
survival. ................................................................................................................................ 12 
1.7 References ........................................................................................................................ 13 
Chapter 2: A tiling resolution DNA microarray with complete coverage of the human genome .. 19 
2.1 Introduction ........................................................................................................................ 20 
2.2 Results .............................................................................................................................. 21 
2.2.1 Array sensitivity ........................................................................................................... 21 
2.2.2 Array resolution compared to conventional CGH ....................................................... 21 
2.2.3 Comparison to previous array CGH ............................................................................ 22 
2.2.4 Identification of minute regions of alteration ............................................................... 23 
2.3 Discussion ......................................................................................................................... 23 
2.4 Methods ............................................................................................................................. 24 
2.4.1 BAC clone selection, preparation and validation ........................................................ 24 
2.4.2 Array production from BAC DNA ................................................................................ 24 
2.4.3 DNA labelling and hybridization .................................................................................. 25 
2.4.4 Array imaging and analysis ......................................................................................... 25 
2.5 References ........................................................................................................................ 31 
Chapter 3: SeeGH – A software tool for visualization of whole genome array comparative
genomic hybridization data ......................................................................................................... 34 
3.1 Introduction ........................................................................................................................ 35 
3.2 Software environment and information sources ................................................................ 36 
3.3 Results and discussion: ..................................................................................................... 37 
3.3.1 Overview of data flow ................................................................................................. 37 
3.3.2 Input requirements ...................................................................................................... 37 
3.3.3 Data filtering and storage ............................................................................................ 38 
3.3.4 Data presentation ....................................................................................................... 39 
3.3.4.1 Genomic view .......................................................................................................... 39 
3.3.4.2 Chromosome view ................................................................................................... 41 
3.3.4.3 Accessing previously entered data .......................................................................... 42 
3.4 Conclusions ....................................................................................................................... 42 
3.5 Availability and requirements ............................................................................................ 43 
3.6 References ........................................................................................................................ 50 
Chapter 4: Comprehensive whole genome array CGH profiling of mantle cell lymphoma model
genomes ..................................................................................................................................... 51 
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4.1 Introduction ........................................................................................................................ 52 
4.2 Results .............................................................................................................................. 53 
4.3 Discussion ......................................................................................................................... 60 
4.4 Materials and methods ...................................................................................................... 62 
4.4.1 Cell lines, culture conditions and DNA extraction ....................................................... 62 
4.4.2 Array CGH hybridization ............................................................................................. 62 
4.4.3 Array CGH analysis .................................................................................................... 63 
4.4.4 Expression microarray procedure ............................................................................... 64 
4.4.5 Fluorescent in-situ hybridization ................................................................................. 65 
4.5 References ........................................................................................................................ 72 
Chapter 5: A comprehensive analysis of common copy-number variations in the human genome
.................................................................................................................................................... 76 
5.1 Introduction ........................................................................................................................ 77 
5.2 Material and methods ........................................................................................................ 77 
5.2.1 DNA samples .............................................................................................................. 77 
5.2.2 BAC array CGH analysis ............................................................................................ 78 
5.2.3 CNV detection algorithm ............................................................................................. 79 
5.2.4 Determination of false-positive and false-negative rates ............................................ 80 
5.2.5 CNV association ......................................................................................................... 82 
5.2.6 Duplication analysis .................................................................................................... 83 
5.2.7 Clustering analysis ...................................................................................................... 83 
5.2.8 Sample diversity ......................................................................................................... 84 
5.2.9 Quantitative PCR ........................................................................................................ 84 
5.3 Results .............................................................................................................................. 84 
5.3.1 Identification of CNVs ................................................................................................. 84 
5.3.2 Genomic diversity within the sample population ......................................................... 87 
5.3.3 CNV-associated genes ............................................................................................... 88 
5.4 Discussion ......................................................................................................................... 89 
5.5 References ...................................................................................................................... 107 
Chapter 6: Deletion of 4q21 and low expression of CCNI and CCNG2 result in poor overall
survival in mantle cell lymphoma .............................................................................................. 110 
6.1 Introduction ...................................................................................................................... 111 
6.2 Methods ........................................................................................................................... 112 
6.3 Results ............................................................................................................................ 112 
v