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Molecular genetic characterization of retinoblastoma tumors lacking RB1 mutations Kennett, Jennifer Yvonne 2012

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MOLECULAR GENETIC CHARACTERIZATION OF RETINOBLASTOMA TUMORS LACKING RB1 MUTATIONS  by  Jennifer Yvonne Kennett  B.Sc., Simon Fraser University, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  The Faculty Of Graduate Studies  (Pathology and Laboratory Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)     December 2012  © Jennifer Yvonne Kennett, 2012 ii  Abstract  Retinoblastoma is a rare childhood cancer of the retina and is the most common intraocular tumor in children. Classically, retinoblastoma results from biallelic loss of the RB1 tumor suppressor gene. As with other cancer types, dysregulation of a single gene alone is not considered sufficient for complete transformation to malignancy. Frequent regions of genetic alteration harbouring additional genes, implicated in retinoblastoma oncogenesis and progression, include chromosomes 1q, 2p, 6p, 13q and 16q. Sensitive molecular genetic screening techniques are capable of identifying RB1 mutations in 98% of unilateral retinoblastoma tumors. The remaining 2% harbour no identifiable RB1 inactivating alterations, and therefore molecular interrogation of these cases would likely reveal alternative genetic events driving retinoblastoma tumorigenesis in the absence of RB1 inactivation. Towards this objective, in this thesis work, I describe genetic alterations identified by tiling path array comparative genomic hybridization in a rare sample set composed of 23 RB1+/+ tumors. In addition to gene disruption by copy number alteration, mechanisms of gene disruption resulting in no overall change in copy number or change in copy number with allelic imbalance were also investigated utilizing genome-wide SNP array analysis on five of the RB1+/+ tumors. The most striking recurrent genetic alteration identified in retinoblastoma tumors lacking RB1 inactivating mutations, was focal high-level MYCN amplification, which occurred at a frequency of approximately 48%. The MYCN amplified RB1+/+ tumors also exhibited a statistically significant lower proportion of their genome affected by genomic instability when compared with the RB1-/- tumors. In a subset of five matched tumor and blood normal samples the occurrence of copy neutral LOH was explored, although none was observed. Allele specific copy number analysis identified instances of allelic imbalance, including all five MYCN amplifications. Amplification of MYCN may represent a rare and novel alternate mechanism of retinoblastoma tumorigenesis. This work provides insight into the role of mutational events driving tumorigenesis in retinoblastoma in the absence of RB1 inactivation.   iii  Preface  Chapters 2 and 3 are based on work conducted with Dr. Brenda Gallie, Diane Rushlow, and Stephanie Yee of The Ontario Cancer Institute and The Toronto Western Hospital Research Institute. I was responsible for performing array CGH experiments, generating copy number profiles, identifying frequently altered regions and genes including identifying MYCN amplification as the most frequent event and defining the minimal region of amplification, as well as performing statistical and pathway analyses for all of the data presented in this thesis. I am co-second author of a manuscript in preparation describing the findings presented in this thesis, in addition to QmPCR, histology, immunohistochemistry and functional work conducted by Diane Rushlow et al.  This research was approved by the British Columbia Cancer Agency Research Ethics Board – Clinical, Ethics Certificate R05-1072.    iv  Table of Contents  Abstract .................................................................................................................................... ii  Preface ..................................................................................................................................... iii  Table of Contents ................................................................................................................... iv  List of Tables ......................................................................................................................... vii  List of Figures ....................................................................................................................... viii  List of Abbreviations ............................................................................................................. ix  Acknowledgements ............................................................................................................... xii  Dedication ............................................................................................................................. xiii  1    Chapter: Introduction ...................................................................................................... 1  1.1  Retinoblastoma ..................................................................................................................... 1  1.1.1  Incidence and occurrence ................................................................................................. 1  1.1.2  Diagnosis and treatment ................................................................................................... 2  1.1.3  Hereditary and nonhereditary forms of retinoblastoma .................................................... 2  1.2  Retinoblastoma genetics ....................................................................................................... 3  1.2.1  Discovery of tumor suppressor class of genes .................................................................. 3  1.2.2  RB1 gene and pRB ............................................................................................................ 3  1.2.3  Cell models of retinoblastoma .......................................................................................... 5  1.2.4  Mouse models of retinoblastoma ...................................................................................... 6  1.2.5  Copy number alterations and cancer ................................................................................. 7  1.2.6  Array comparative genomic hybridization ....................................................................... 7  1.2.7  Genotyping arrays ............................................................................................................. 8  1.2.8  Common genetic alterations in retinoblasoma in addition to RB1 ................................... 9  1.2.9  Gain of 1q ....................................................................................................................... 10  1.2.10  Gain of 2p and MYCN amplification .......................................................................... 11  1.2.11  Gain of 6p ................................................................................................................... 11  1.2.12  Copy number alterations on 13q ................................................................................ 12  1.2.13  Loss of 16q ................................................................................................................. 12  1.2.14  Retinoblastoma molecular genetic testing .................................................................. 13  v  1.3  Hypotheses and research questions ..................................................................................... 14  1.3.1  Research questions.......................................................................................................... 14  1.3.2  First hypothesis ............................................................................................................... 14  1.3.3  Second hypothesis .......................................................................................................... 14  1.3.4  Third hypothesis ............................................................................................................. 15  2    Chapter: Materials and Methods .................................................................................. 16  2.1  Samples ............................................................................................................................... 16  2.1.1  Sample DNA ................................................................................................................... 16  2.1.2  RB1 gene mutation status ................................................................................................ 16  2.1.3  Incidence of RB1+/+ retinoblastoma across the four testing sites .................................... 17  2.2  Whole-genome sub-megabase resolution tiling path aCGH analysis ................................. 17  2.2.1  Array CGH data analysis ................................................................................................ 18  2.2.2  Segmentation and alteration detection criteria ................................................................ 19  2.2.3  Identification of frequent regions of copy number alteration ......................................... 19  2.2.4  Definition of gains and high-level amplifications .......................................................... 20  2.2.5  Determination of proportion of the genome altered ....................................................... 21  2.2.6  Statistical comparison of data means between two groups ............................................. 22  2.2.7  Network/ pathway level analysis of altered genes .......................................................... 22  2.3  Affymetrix genome-wide SNP 6.0 array Genotyping ......................................................... 23  2.3.1  Affymetrix genome-wide human SNP 6.0 array genotyping ......................................... 23  2.3.2  Copy number profiling and genotyping of five RB1+/+ tumor samples .......................... 23  2.3.3  Determination of regions of copy number alteration ...................................................... 24  2.3.4  Determination of regions of allelic imbalance ................................................................ 24  2.3.5  Determination of regions of cnLOH ............................................................................... 24  3    Chapter: Results.............................................................................................................. 25  3.1  Whole genome segmental copy number profiling .............................................................. 25  3.2  Identification of frequent regions of altered copy number status ........................................ 26  3.2.1  RB1+/+ .............................................................................................................................. 29  3.2.2  RB1-/- ............................................................................................................................... 30  3.2.3  RB1+/- .............................................................................................................................. 31  3.2.4  Frequency plot comparison between RB1+/+ MYCNA and RB1-/- tumors ........................ 31  3.3  High resolution detection of 2p amplification and the MYCN oncogene ............................ 32  3.3.1  Minimal overlapping amplicon on chromosome 2p ....................................................... 33  vi  3.4  All high-level amplifications identified including chromosomes 1q, 2p and 21q .............. 35  3.5  Comparison of the overall proportion of the genome altered between RB1+/+, RB1+/-, and RB1-/- tumors ..................................................................................................................................... 38  3.5.1  Comparison of copy number altered genes between RB1+/+, RB1+/-, and RB1-/- tumors . 40  3.6  Pathway/ network level analysis ......................................................................................... 41  3.6.1  Canonical pathways and top gene functions ................................................................... 41  3.6.2  Gene association networks ............................................................................................. 42  3.7  Genome wide SNP 6.0 analysis of five RB1+/+ tumors ....................................................... 44  3.7.1  Copy number alterations identified using oligonucleotide resolution SNP profiling ..... 45  3.7.2  Genomic regions affected by allelic imbalance .............................................................. 48  3.7.3  Detection of copy neutral allelic imbalance by allele specific copy number analysis .... 54  3.8  MYCNA and age of diagnosis .............................................................................................. 54  4    Chapter: Discussion and Conclusions ........................................................................... 55  4.1  Novel high level MYCNA identified in RB1+/+ retinoblastoma tumors ................................ 55  4.2  RB1+/+ tumors display a lesser proportion of their genome affected by genomic instability than RB1-/- tumors ............................................................................................................................ 55  4.3  Genes identified within regions of frequent copy number alteration .................................. 56  4.4  Pathway/ network level analysis ......................................................................................... 57  4.5  Genomic regions affected by multiple mechanisms of genetic alteration (copy number change, allelic imbalance, and combination) ................................................................................... 58  4.6  RB1+/- retinoblastoma tumors .............................................................................................. 58  4.7  Allele specific amplification of MYCN ............................................................................... 59  4.8  Conclusions ......................................................................................................................... 59  4.8.1  Characterization of retinoblastoma tumors with known RB1 mutation status ................ 59  4.8.2  Possible future research directions ................................................................................. 60  4.8.3  Significance .................................................................................................................... 61  References .............................................................................................................................. 62  Appendices ............................................................................................................................. 73  Appendix A Additional aCGH data ................................................................................................. 73  A.1  Genomic regions of gain and loss identified by aCGH for all 48 samples and two cell lines …………………………………………………………………………………………73  A.2  Retinoblastoma cell line karyograms ............................................................................ 125  A.3  Top significantly disrupted canonical pathways and networks .................................... 127  vii  List of Tables  Table 3.1    All discreet high level amplicons identified. ....................................................... 36  Table 3.2    Genomic regions of copy number alteration detected by SNP array. .................. 46  Table 3.3    Genomic regions affected by allelic imbalance (AI). .......................................... 49  Table A.1    aCGH alterations by sample (RB1+/+). ................................................................ 73  Table A.2    aCGH alterations by sample (RB1-/-). ................................................................. 95  Table A.3    aCGH alterations by sample (RB1+/-). ............................................................... 111  Table A.4    Top significantly disrupted canonical pathways in RB1+/+ tumors. .................. 127  Table A.5    Top significantly disrupted canonical pathways in RB1-/- tumors. ................... 128  Table A.6    Top significant gene association networks disrupted in RB1+/+ tumors. .......... 129  Table A.7    Top significant gene association networks disrupted in RB1-/- tumors. ............ 131  viii  List of Figures  Figure 1.1   Function of pRB under normal cellular conditions........................................... 5  Figure 2.1   Overview of aCGH methodology ................................................................... 18  Figure 2.2  Low-level gain compared with high-level amplification ................................ 21  Figure 3.1   Whole genome karyogram of RB1+/+ retinoblastoma tumor, RB522 ............. 26  Figure 3.2   Overall RB1+/+ frequency plot ......................................................................... 27  Figure 3.3   RB1-/- frequency plot ....................................................................................... 28  Figure 3.4   RB1+/- frequency plot ...................................................................................... 29  Figure 3.5   Frequency plot comparison between RB1+/+ MYCNA and RB1-/- tumors ........ 32  Figure 3.6   2p multiple alignment ..................................................................................... 34  Figure 3.7   High-level amplifications in addition to MYCN ............................................. 37  Figure 3.8   Comparison of genomic instability between tumor groups ............................ 39  Figure 3.9   Venn diagram of frequently altered genes ...................................................... 41  Figure 3.10      IPA derived RB1-/- gene association network involving RB1 ......................... 43  Figure 3.11      IPA derived RB1+/+ gene association network involving MYCN .................... 44  Figure 3.12      MYCN amplification with allelic imbalance ................................................... 52  Figure 3.13      Detection of both copy number and allele specific copy number (ASCN) ..... 53  Figure A.1      Weri-Rb-1 cell line karyogram ...................................................................... 125  Figure A.2      Y79 cell line karyogram ................................................................................ 126    ix  List of Abbreviations  aCGH   array-based comparative genomic hybridization AI   Allelic imbalance ASCN   Allele specific copy number BAC   Bacterial artificial chromosome  bps   Basepairs BASP1   Brain abundant, membrane attached signal protein 1 CCD   Charge-coupled device CDH11  Cadherin 11, type 2, OB-cadherin (osteoblast) CDH13  Cadherin 13, H-cadherin (heart) cdk   Cyclin Dependant Kinase CENPF  Centromeres protein F, 350/400kDa (mitosin) CGH   Comparative genomic hybridization cnLOH  Copy neutral LOH CRABP2  Cellular retinoic acid binding protein 2 dCTP   deoxycytidine triphosphate DDX1   DEAD (Asp-Glu-Ala-Asp) box polypeptide 1 DEK   DEK oncogene DNA   Deoxyribonucleic acid DNMT3A  DNA (cytosine-5-)-methyltransferase 3 alpha DP   Differentiation regulated transcription factor protein Ds-DNA  Double-stranded DNA EGFR   Epidermal growth factor receptor E2F1   E2F transcription factor 1 E2F3   E2F transcription factor 3 FOXC1  Forkhead box C1 G   Gain GS   Genomics Suite ID2 Inhibitor of DNA binding 2, dominant negative helix-loop-helix protein x  IPA   Ingenuity Pathway Analysis IRF2BP2  Interferon regulatory factor 2 binding protein 2 kb   Kilobases kd   Kilo-dalton KIF14   Kinesin family member 14 KRAS   v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog LOH   Loss of heterozygosity L   Loss Mb   Megabases MDM4   Mdm4 p53 binding protein homolog (mouse) MLPA   Multiplex ligation-dependent probe amplification MT   Mitochondrial DNA Myc   V-myc myelocytomatosis viral oncogene homolog MYCN V-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian) MYCNA  MYCN gene amplification NAG   N-acetylglutamate synthase NBPF11  neuroblastoma breakpoint family, member 11 ng   Nanogram NTRK1  Neurotrophic tyrosine kinase, receptor, type 1 OSGIN1  Oxidative stress induced growth inhibitor 1 PCR   Polymerase chain reaction P14ARF   CDKN2A alternate reading frame p53   Tumor protein p53 p107   retinoblastoma-like 1 (RBL1) (p107) QmPCR  Quantitative multiplex PCR pRB   Retinoblastoma protein encoded by RB1 Rb1   Mouse retinoblastoma gene RB1   Retinoblastoma susceptibility gene RB1-/-   RB1 homozygous mutant RB1+/-   RB1 heterozygous mutant xi  RB1+/+   RB1 wildtype RNA   Ribonucleic acid SMRT   Sub megabase resolution tiling set SNP   Single Nucleotide Polymorphism TF   Transcription factor TSG   Tumor suppressor gene UPD   Uniparental disomy WT1   Wilms tumor 1 µm   Micrometer   xii  Acknowledgements  I am most grateful to my supervisor, Dr. Wan Lam, for the many opportunities he’s provided me with and his continued guidance and support of my studies.  I would like to thank the members my supervisory committee, Dr. Mladen Korbelik, Dr. Calum MacAulay, Dr. Cathie Garnis for their guidance throughout my thesis project.  Thank you to the members of the Lam Lab (both past and present), especially Emily Vucic, Kelsie Thu, Dr. Raj Chari, Larissa Pikor, and Dr. Ashleen Shadeo for their thoughtful discussions, technical expertise and valuable feedback.  I would also like to thank Dr. Brenda Gallie and the members of her lab for their collaboration on this as well as other projects, especially Stephanie Yee and Diane Rushlow for their technical expertise and valuable intellectual discussions. xiii  Dedication  This thesis is dedicated to my family and friends.  1  1    Chapter: Introduction  1.1 Retinoblastoma  1.1.1 Incidence and occurrence Retinoblastoma is a rare childhood cancer originating from the developing retina, although the exact cell of origin remains unidentified. It is the most common intraocular tumor occurring in children and represents approximately 3% of all childhood cancers between the ages of 0 – 14 years (Luthra and Donaldson 1997). The incidence rates during 1988-1997 in Europe were between 44.2 and 67.9 per million births (MacCarthy, Draper et al. 2006).  Incidence rates are relatively similar between North America, Europe and Australia (Shields and Shields 2004). The disease can develop as one tumor (unifocal) in one eye (unilateral) or multiple tumors (multifocal) in one or both eyes (bilateral). A rare third form of retinoblastoma also exists, which is known as trilateral retinoblastoma. The term trilateral retinoblastoma is used to describe patients who develop an independent pineoblastoma, a malignant brain tumor, in addition to retinoblastoma. Although retinoblastoma can occur at any age, 95% of cases present in children under the age of five, with two thirds of these occurring under the age of two, and in some cases tumors can even develop before birth (Goddard, Kingston et al. 1999; Butros, Abramson et al. 2002). Retinoblastoma is clearly a cancer which relies on hijacking important developmental pathways. The average age of diagnosis for bilateral and unilateral cases is estimated at 7 and 24 months respectively (Balmer 2002). Worldwide, it is estimated there are approximately five to eight thousand new cases diagnosed each year (Abramson 2005). Retinoblastoma is responsible for approximately 3000 cancer-related childhood deaths worldwide each year (Kivela 2009). The mortality rate in developing countries is considerably higher than in developed countries and can be as high as 70% in regions of Africa (Kivela 2009). In Canada however, the current survival rate exceeds 98%; which is largely attributable to improvements in early detection and treatment (Canadian Cancer Society 2008).   2  1.1.2 Diagnosis and treatment The two most common presenting clinical features of retinoblastoma are leukocoria (56.2%) and strabismus (23.6%) (Abramson, Frank et al. 1998). Leukocoria is an abnormal white reflection from the retina of the eye, and can be detected in photographs, under casual observation or during routine eye examination. Leukocoria is associated with more advanced disease; whereas strabismus is a condition which involves improper alignment of the eyes, and is always associated with macular involvement (Abramson, Beaverson et al. 2003). Early detection of retinoblastoma, while still localized to the eye, results in a high cure rate in the developed world (Zhao, Dimaras et al. 2011). Multiple treatment options including enucleation, external beam radiotherapy, focal treatments, and chemotherapy, alone or in combinations, are available depending on the size, location and number of tumors (Balmer, Zografos et al. 2006). Enucleation is still the treatment of choice for advanced cases to prevent escape of the tumor along the optic nerve to the brain, which is most often fatal (Shields and Augsburger 1981; Albert 1987).  Enucleated eyes are then evaluated histopathologically to identify the presence of features indicative of metastatic risk, helping to guide further treatment and observation strategies.  Pathological features such as involvement of the optic nerve and vitreous seeding, tumor cells floating within the vitreous cavity, suggest an unfavorable prognosis for eye-preservation (Kaneko and Suzuki 2003). The first priority when treating retinoblastoma is patient survival, followed by globe salvation and preservation of visual function. Focal treatment options include cryotherapy, laser photocoagulation, thermotherapy, and brachytherapy (Shields and Shields 2004). Chemoreduction may be used to reduce the size of tumors prior to their focal removal, however recent evidence suggests this strategy may mask the severity and use valuable time in certain advanced cases that would otherwise have been saved by enucleation (Zhao, Dimaras et al. 2011). Survivors of retinoblastoma experience life-long adverse effects as a result of either the cancer itself or from treatment (Canadian Cancer Society 2004).  1.1.3 Hereditary and nonhereditary forms of retinoblastoma Retinoblastoma can be heritable or nonheritable. The hereditary form of the disease is characterized by a positive family history, multifocal tumors or the presence of an identified germline mutation. Germline mutations may either be inherited or may arise in utero at the 3  time of conception, which is termed sporadic retinoblastoma. Children with bilateral retinoblastoma account for approximately 40% of overall cases, while the remaining 60% are affected unilaterally (Lohmann and Gallie 1993). All bilateral cases and approximately 15% of unilateral cases result from the heritable form of retinoblastoma (Lohmann, Gerick et al. 1997). Patients with the heritable predisposition for retinoblastoma are at an increased risk of additional cancers later in life (Eng, Li et al. 1993). Due to the genetic nature of retinoblastoma, studies to use targeted molecular therapies as a treatment option are under development (Huang, Yee et al. 1988; Xu 2003; Chevez-Barrios, Chintagumpala et al. 2005).  1.2 Retinoblastoma genetics  1.2.1 Discovery of tumor suppressor class of genes In 1971, Alfred Knudson developed his “two-hit” hypothesis for cancer initiation from observations of bilateral versus unilateral retinoblastoma and age of onset, which predicted that two mutational events are rate-limiting for tumor formation (Knudson 1971). At the time of Knudson’s hypothesis, chromosome 13q14 was thought to contain the retinoblastoma causing gene which was later identified by Friend et al in 1986 as the first tumor suppressor gene (TSG) retinoblastoma susceptibility gene (RB1) (Friend, Bernards et al. 1986; Fung, Murphree et al. 1987; Lee, Bookstein et al. 1987). Retinoblastoma results from the loss of function of both alleles of the RB1 gene which encodes the retinoblastoma protein, pRB; this is the paradigm for TSGs. The RB1 gene was first identified through the study of retinoblastoma, but was subsequently found to be important in many additional human cancers including osteosarcoma, small cell lung carcinoma, prostate, bladder, and other sporadic cancers (Friend, Bernards et al. 1986; Goodrich 2006)  1.2.2 RB1 gene and pRB The pRB protein, encoded by the RB1 tumor suppressor gene on chromosome 13q14.1-q14.2, has a multitude of important cellular functions in vivo. This 105 kilo-dalton (kd) nuclear phosphoprotein is an important negative regulator of cell cycle proliferation (Huang, Yee et al. 1988; Goodrich, Wang et al. 1991; Qin, Chittenden et al. 1992; Herrera, Sah et al. 1996; Kaelin 1999; Classon, Salama et al. 2000). It also plays a role in the 4  stabilization of constitutive heterochromatin to maintain overall chromatin structure thereby regulating gene expression (Brehm and Kouzarides 1999; Harbour and Dean 2000; Harbour and Dean 2001). Complicating the elucidation of the precise tumor suppressing role of pRB, is its involvement in a multitude of cellular processes in addition to cell proliferation including DNA replication, differentiation, and apoptosis (Szekely, Jiang et al. 1992; Lee, Hu et al. 1994; Morgenbesser, Williams et al. 1994; Nevins 2001). The pRB protein belongs to a family of related mammalian ‘pocket proteins’ including p107 and p130 which all contain a pocket for the functional binding of other proteins. One of the first cellular targets of pRB to be identified was the E2F1 transcription factor (TF) (Nevins 1992; Dyson 1998). Several other members of the E2F family of TFs have subsequently been found to interact with pRB (Chellappan, Hiebert et al. 1991; Kaelin, Pallas et al. 1991). Most E2F proteins form heterodimeric complexes, with a differentiation regulated transcription factor protein (DP), that bind DNA in a sequence specific manner to control the expression of genes responsible for regulating cell cycle progression, such as cyclins. Under normal conditions pRB is bound to and inhibits E2F-mediated transcriptional activity, and upon phosphorylation undergoes a conformational change thereby releasing the E2F TF allowing target gene transcription (see Figure 1.1). The retinoblastoma protein then becomes transiently inactivated during progression of a normal cell through the cell cycle. This control mechanism is disrupted in cancer cells leading to a loss of cell cycle control, as well as an increase in genomic instability (Weinberg 1995).  5  pRB E2F DP PpRB DP DNA E2F target gene transcription E2F Transient process during cell  cycle progression Phosphorylation by Cyclin/ cdk  complexes  Figure 1.1  Function of pRB under normal cellular conditions. Function of pRB under normal cellular conditions. Under normal conditions pRB is bound to and inhibits E2F mediated transcription of target genes. Most E2F family proteins form heterodimeric complexes with DP proteins which bind DNA in a sequence dependent manner to control gene expression. Upon phosphorylation, by Cyclin/ cdk complexes, pRB undergoes a conformational change releasing the E2F TF complex and enabling the transcription of genes responsible for regulating cell cycle progression, such as cyclins  1.2.3 Cell models of retinoblastoma Cell lines provide an important model for the study of the driving mechanisms behind cancers. Continuously growing retinoblastoma cell lines first became available in the early 70s. Two of the first and most commonly studied retinoblastoma cell lines are Y79 and Weri- Rb-1, both of which are RB1-/-. The Y79 retinoblastoma cell line was originally derived from the unilateral retinoblastoma tumor of a 2.5 year old female patient with familial history of 6  retinoblastoma. The Y79 cell line contains a focal amplification of the V-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian) (MYCN) oncogene on chromosome 2p and exhibits non-adherent growth in culture (Reid, Albert et al. 1974). Weri-Rb-1 by contrast, is derived from a spontaneous retinoblastoma, does not contain amplification of MYCN (although chromosome 2p is gained) and exhibits adherent growth (McFall, Sery et al. 1977).  1.2.4 Mouse models of retinoblastoma Dysregulation of normal pRB function and disruption of pRB related cell-cycle regulatory pathways are frequent and important events in tumorigenesis. Mouse model systems developed to study retinoblastoma development driven by RB1 inactivation have shown RB1 to be essential for mouse development, as RB1-/- mice do not survive beyond day 15 of gestation (Clarke, Maandag et al. 1992; Jacks, Fazeli et al. 1992; Lee, Chang et al. 1992). To circumvent RB1 knockout lethality, various chimeric mouse models that inactivate pRB at different times throughout development in different populations of cells have been developed. Unlike in humans, Rb1 heterozygous mice fail to develop retinoblastoma (Clarke, Maandag et al. 1992; Jacks, Fazeli et al. 1992; Lee, Chang et al. 1992). A conditional knock- out mouse model of retinoblastoma was subsequently developed, but required the inactivation of at least two additional tumor suppressor genes; p107 and p53 (Zhang, Schweers et al. 2004). Chimeras and conditional knock-out mutants have contributed much to the study of the molecular genetics of retinoblastoma progression as well as the role of pRB in murine retinal development. However, it is unclear why p107 or p130 inactivation is required for retinoblastoma development in addition to RB1 inactivation in mice. Both proteins are closely related members of the pocket protein family to pRB and have been shown to share some level of functional compensation, which could potentially be different between mice and humans. It is also unclear if the additional genetic alterations which contribute to retinoblastoma progression in mice are shared with humans. It has been shown that loss of pRB function can activate the p53 pathway, through p14ARF, which acts to suppress cell growth, thus inactivating p53 (Bates, Phillips et al. 1998). Differences in the level of interaction between pRB and p53 pathways between mice and humans may explain 7  part of why p53 inactivation is needed in the mouse model to generate retinoblastoma which occurs at similar frequency and aggressiveness to human retinoblastoma.  1.2.5 Copy number alterations and cancer The hallmarks of cancer are defined as acquired functional capabilities by cancer cells that allow them to survive, proliferate, and disseminate during the course of tumorigenesis (Hanahan and Weinberg 2011). Genomic instability is one mechanism that can lead to the genetic changes enabling these cells to fulfill the hallmarks of cancer (Hanahan and Weinberg 2011). Epigenetic deregulation affecting cancer genes by mechanisms such as DNA methylation or Histone modifications can also contribute to the cellular changes acquired by cancer cells (Hanahan and Weinberg 2011). Alterations in gene copy number and structure are a prominent mechanism by which function of key cancer related genes are affected. Identifying those events that drive or are causal to tumorigenesis is key to the discovery of biologically relevant biomarkers and therapeutic targets. Gene expression level analyses alone cannot separate causal from reactive expression (Chari, Thu et al. 2010). Changes conserved at the DNA level are more likely to represent key causal events in cancer development, as they are heritable and subject to selection. Alterations identified at the DNA level potentially represent causal events in cancer development. For example, amplification is a prevalent mechanism of oncogene activation across multiple cancer types (Lockwood, Chari et al. 2008). Therefore, assessment of recurrent chromosomal aberrations has great potential to improve our understanding of tumor development (Albertson, Collins et al. 2003). Furthermore, the overall percent genome altered for a particular tumor type has also been reported to correlate with clinical features, for example, the correlation between number of copy number alterations and poor prognosis in gastric cancer, and may represent an important genomic parameter to consider in genome wide copy number alteration studies (Rossi, Klersy et al. 2011).  1.2.6 Array comparative genomic hybridization Recurrent regions of copy number alteration are frequently observed in most cancers including retinoblastoma. Early genome wide techniques to detect such regions included conventional comparative genomic hybridization (CGH) using metaphase chromosome 8  spreads as the hybridization targets, with a low resolution of approximately 5-10 megabases (Mb). Advances in whole genomic profiling techniques such as array CGH (aCGH) over the last 20 years have greatly improved the level of coverage and resolution of detectable copy number alterations in the genome for use in the study of human genetics and cancer using DNA clones of various sizes (Pinkel, Segraves et al. 1998; Pollack, Perou et al. 1999; Ishkanian, Malloff et al. 2004).  All CGH based techniques are sensitive to physical changes in copy number and can detect altered ploidy status, gain or loss of individual chromosomes or portions of chromosomes and unbalanced structural rearrangements between two genomic DNA samples (Albertson, Collins et al. 2003).  Arrays comprised of bacterial artificial chromosome (BAC) clones, in a tiling path manner, provide the most comprehensive genome coverage and robust low-noise hybridizations, however, their resolution is limited to about 50 kilobases (kb) at the most and they are unable to detect balanced translocation events and unbalanced translocations smaller than the detection threshold (Carter 2007). Commercially available oligonucleotide arrays such as the Affymetrix Single Nucleotide Polymorphism (SNP) 6.0 genotyping array provide copy number and genotype information using approximately 1.8 million oligonucleotide probes, to obtain a median inter-marker distance overall of less than 700 base pairs (bps). The Affymetrix SNP array contains approximately 946,000 copy number detection probes, and more than 906,600 SNP probes chosen to maximize genomic coverage and detection of genetic variation. Oligonucleotide arrays however, require approximately twice the amount of high quality, double stranded DNA per experiment compared to BAC aCGH. Tucker et al. provide a detailed review of the advantages and disadvantages of different platforms for the detection of copy number variants including tiling path BAC aCGH and Affymetrix SNP 6.0 arrays (Tucker, Montpetit et al. 2011).  1.2.7 Genotyping arrays Loss of heterozygosity (LOH) can occur at a particular locus heterozygous for a mutant and a normal allele when the normal allele is lost and the remaining allele undergoes gene conversion, somatic recombination, loss of the other allele, or loss of one allele followed by reduplication or by mutation followed by aberrant promoter methylation of the remaining allele (Tuna, Knuutila et al. 2009). LOH can be associated with copy number 9  changes (such as loss of one copy) or can be copy number neutral (cnLOH) (also referred to as uniparental disomy (UPD) or gene conversion). One of the limitations of non-SNP based array platforms is their inability to detect cnLOH. LOH and, more specifically, cnLOH are known to occur in retinoblastoma as mechanisms of inactivation of the RB1 gene (Zhu, Dunn et al. 1992). Recently advances in SNP genotyping techniques have begun to allow routine genome wide assessment of LOH, including cnLOH and copy number variation in various cancer types, and have been used to identify novel chromosomal features of potential importance to disease biology (Mullighan, Goorha et al. 2007; Ching, Naidu et al. 2011; Kryh, Caren et al. 2011; Cheung, Rogic et al. 2012). These types of studies allow the assessment of cnLOH to be explored on a genome-wide scale in the context of cancer, and will help define the contribution of this fundamental genetic mechanism to disease biology.  1.2.8 Common genetic alterations in retinoblasoma in addition to RB1 In addition to inactivating mutations of both normal RB1 alleles retinoblastomas exhibit recurrent genetic alterations. The most frequent chromosomal alterations detected by comparative genomic hybridization are gains of chromosomes 1q, 6p and losses on 16q (Mairal, Pinglier et al. 2000; Chen, Gallie et al. 2001; Herzog, Lohmann et al. 2001; Lillington, Kingston et al. 2003; van der Wal, Hermsen et al. 2003; Zielinski, Gratias et al. 2005). Other recurrent alterations seen at lower frequencies include gain of 2p and 19q, in addition to both gains and losses on 13q (Zielinski, Gratias et al. 2005). Retinoma is a benign non-progressive retinal lesion highly associated with retinoblastoma (Gallie, Ellsworth et al. 1982). There is clinical and histopathological evidence to suggest retinoma is a benign precursor variant of retinoblastoma (Gallie, Ellsworth et al. 1982; Margo, Hidayat et al. 1983). It has been shown that retinomas exhibit loss of both alleles of the RB1 gene and lack pRB expression (Dimaras, Khetan et al. 2008). Retinomas also exhibit low level genomic instability whereas retinoblastomas exhibit greater genomic instability and altered expression of additional oncogenes and tumor suppressor genes (Bowles, Corson et al. 2007; Corson and Gallie 2007). Unlike in humans, Rb1 heterozygous mice fail to develop retinoblastoma, and knock-out mouse models of retinoblastoma required the inactivation of at least two additional genes; p107 and p53 to recapitulate the aggressiveness and invasiveness reflective of human retinoblastoma (Clarke, Maandag et al. 1992; Jacks, Fazeli et al. 1992; Lee, Chang et al. 10  1992) (Zhang, Schweers et al. 2004). While mutational inactivation of RB1 can initiate tumorigenesis, additional mutational events affecting other genes are likely important in driving retinoblastoma progression (Bowles, Corson et al. 2007; Corson and Gallie 2007). Thus it is important to identify these additional genes, which may be associated with clinical features. Candidate genes identified through CGH and aCGH analysis include the oncogenes KIF14, MYCN, E2F3, DEK, and tumor suppressor gene CDH11, on chromosomes 1q, 2p, 6p, and 16q, respectively (Chen, Gallie et al. 2001). These recurrent regions and candidate genes will be discussed in further detail below.  1.2.9 Gain of 1q Gain of chromosome 1q31-1q32 is one of the most frequent genetic alterations observed in CGH studies of retinoblastomas occurring at a frequency of approximately 50% (Mairal, Pinglier et al. 2000; Chen, Gallie et al. 2001; Herzog, Lohmann et al. 2001; Lillington, Kingston et al. 2003; van der Wal, Hermsen et al. 2003; Zielinski, Gratias et al. 2005). Gain of a second region at 1q21 has also been reported.  Corson et al. identified KIF14 as a candidate oncogene in this region and found its expression to be disregulated in multiple cancer types in addition to retinoblastoma (Corson, Huang et al. 2005). Gain of 1q and overexpression of KIF14 is also associated with later age at time of diagnosis (Herzog, Lohmann et al. 2001; Gratias, Schuler et al. 2005).  The association of 1q gain with clinical parameters would suggest that 1q gain plays a functional role in retinoblastoma tumor biology (Gratias, Schuler et al. 2005). It is possible that more than one gene located on chromosome 1 may be involved in retinoblastoma tumorigenesis, underlying possible selection of the large number of whole chromosome or chromosome arm gains observed in retinoblastoma tumors.  KIF14 is a member of the kinesin superfamily of microtubule- dependent motor proteins that play an essential role in intracellular transport and cell division (Nakagawa, Wang et al. 1997). In retinoblastoma, the Mdm4 p53 binding protein homolog (mouse) (MDM4) gene, located at chromosome 1q32, is within the region of frequent gain and has been shown to be amplified and overexpressed (Laurie, Donovan et al. 2006; Guo, Pajovic et al. 2008). MDM4 is a member of the MDM family of genes and is involved in negative regulation of the p53 pathway.  11  1.2.10 Gain of 2p and MYCN amplification Copy number gain of 2p, and commonly the entire 2p arm, is a frequent genomic event seen in approximately 30% of retinoblastomas across several CGH studies (Chen, Gallie et al. 2001; Lillington, Kingston et al. 2003; van der Wal, Hermsen et al. 2003; Zielinski, Gratias et al. 2005; Corson and Gallie 2007). The main region at chromosome 2p24-25 contains the V-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian) (MYCN) gene. MYCN is a member of the MYC family of transcription factors. Amplification of MYCN (MYCNA) is most notably associated with neuroblastoma, where it is associated with aggressiveness of the disease and resistance to therapy (Brodeur, Seeger et al. 1984; Seeger, Brodeur et al. 1985). In addition, MYCN has been found to be amplified in approximately 3% of primary retinoblastoma tumors (Bowles, Corson et al. 2007). Due to its amplification and overexpression in neuroblastoma as well as in the Y79 and RB522 retinoblastoma cell lines, the MYCN transcription factor has been assumed to be the target oncogene of this gain. Additional genes have been found to be co-amplified with MYCN in neuroblastoma including DDX1 (Godbout and Squire 1993), NAG (Wimmer, Zhu et al. 1999), and ID2 (Chen, Gallie et al. 2001; Lillington, Goff et al. 2002), however the potential biological importance of genes co-amplified on 2p remains largely unknown.  1.2.11 Gain of 6p Squire et al. initially identified isochromosome 6p through karyotyping as a chromosomal abnormality frequent in retinoblastoma tumors but not observed commonly in other tumor types (Squire, Phillips et al. 1984). Across conventional CGH studies, 6p gain has been reported at a frequency of approximately 44-54% in retinoblastoma (Mairal, Pinglier et al. 2000; Chen, Gallie et al. 2001; Herzog, Lohmann et al. 2001; Chen, Pajovic et al. 2002). The minimal region of gain has been localized to 6p22 and candidate genes implicated based on expression analyses include the DEK oncogene (DEK) and E2F transcription factor 3 (E2F3) (Mairal, Pinglier et al. 2000; Chen, Gallie et al. 2001; Herzog, Lohmann et al. 2001; Chen, Pajovic et al. 2002; Orlic, Spencer et al. 2006). Both genes are known to be amplified in a number of other cancer types including leukemia, melanoma, and brain (Feber, Clark et al. 2004; Kappes, Scholten et al. 2004; Carro, Spiga et al. 2006). DEK is a ubiquitous phosphorylated nuclear protein with roles in chromatin architecture and 12  remodeling (Kappes, Burger et al. 2001; Waldmann, Eckerich et al. 2002; Waldmann, Scholten et al. 2004). E2F3 is also a part of the RB1 pathway and is one of the E2F transcription factors regulated by pRB involved in transcribing genes important for promoting cellular proliferation. This emphasizes the fundamental importance of deregulation of the RB1 pathway in retinoblastoma as multiple hits to the pathway frequently co-occur.  1.2.12 Copy number alterations on 13q Copy number alterations in addition to those affecting the RB1 have been identified on chromosome 13. Loss of 13q spanning this region was reported at a frequency of approximately 21% by early karyotyping studies (Potluri, Helson et al. 1986). In a summary of five CGH studies, gains and losses involving 13q were found to occur at frequencies of approximately 16% and 12% respectively (Corson and Gallie 2007). The minimal region of loss was identified as 13q14-q21 and 13q14-q22 by Mairal et al. and Lillington et al. respectively (Mairal, Pinglier et al. 2000; Lillington, Kingston et al. 2003). A minimal region of gain was identified at 13q32-13q34 (Chen, Gallie et al. 2001). No candidate genes to date have been identified on 13q outside of RB1.  1.2.13 Loss of 16q Loss of all or parts of chromsosome16q is a frequent event in retinoblastoma, reported at a frequency of approximately 30% across a summary of CGH studies (Corson and Gallie 2007). The minimal region of loss has been localized to 16q22, encompassing candidate tumor suppressor genes, CDH11 and CDH13, both of which belong to the cadherin superfamily of genes (Chen, Gallie et al. 2001; Marchong, Chen et al. 2004; Gratias, Rieder et al. 2007; Gustmann, Klein-Hitpass et al. 2011). Marchong et al. showed expression of normal levels of CDH13 in retinoblastoma tumors and cell lines making it unlikely to be the target gene (Marchong, Chen et al. 2004). However, decreased expression of CDH11 was shown to correlate with loss of 16q and is a strong potential target (Marchong, Chen et al. 2004). CDH11 is an integral membrane protein involved in calcium-dependent cell adhesion, cell aggregation, and migration, and has been associated as a tumor suppressor gene in other cancers such as osteosarcoma, breast and gastric carcinomas (Vleminckx, Vakaet et al. 1991; 13  Takeichi and Abe 2005) (Oka, Shiozaki et al. 1993; Jankowski, Bruton et al. 1997; Kashima, Kawaguchi et al. 1999; Pishvaian, Feltes et al. 1999).  1.2.14  Retinoblastoma molecular genetic testing Approximately 15% of unilateral cases result from the heritable form of retinoblastoma making it clinically important to identify patients with germline mutations. Identification of children with RB1 germline mutations can allow focus of costly and invasive clinical surveillance on those patients at greatest risk for developing additional eye tumors (Rushlow, Piovesan et al. 2009). The wide range of possible RB1 mutation types include: point mutations, gross deletions and insertions, splice mutations, gene rearrangements and promoter hypermethylation, requiring a multi-level approach to molecular genetic screening (Rushlow, Piovesan et al. 2009). Sensitive molecular techniques including quantitative multiplex polymerase chain reaction (QmPCR) of RB1's 27 exons and promoter, sequencing of the core promoter, 27 exons and exon flanking intronic regions, and methylation specific PCR of the promoter are employed in clinical testing centers around the world (Rushlow, Piovesan et al. 2009). RB1 mutations can be identified using these techniques in 98% of retinoblastoma tumors, however the remaining 2% comprise a unique subset in which no mutation can be found, and are referred to as RB1+/+ (Richter, Vandezande et al. 2003). The 23 RB1+/+ tumors used in this study were collected from four retinoblastoma screening centers in: Toronto, Germany, France and New Zealand by Dr. Brenda Gallie et al. at the Toronto Western Research Institute (Rushlow, Kennett et al. 2012). These tumors provide a unique opportunity to assess the alternate genetic events that lead to retinoblastoma formation in the absence of RB1 mutations. Understanding the role additional mutational events play in retinoblastoma tumorigenesis will lend greater insight into the biology underlying retinoblastoma, provide additional early screening techniques and possibly opportunities for targeted therapy for patients with RB1+/+ tumors.     14  1.3 Hypotheses and research questions  1.3.1 Research questions The goal of this work is to decipher the alternate mechanisms for the development of retinoblastoma in the absence of RB1 mutations.  i) Using high resolution aCGH techniques can we identify both known and novel copy number aberrations in a small set of tumors with known RB1 mutations? ii) What genetic alterations which may be important for tumorigenesis are specific to the RB1+/+ subset of retinoblastoma tumors? iii) Can SNP profiling of a subset of the RB1+/+ tumors identify regions/genes of interest in addition to those identified by copy number alteration alone (affected by mechanisms of cnLOH or allelic imbalance)? iv) What is the potential role of these disrupted genes in retinoblastoma tumorigenesis?  1.3.2 First hypothesis RB1+/+ tumorigenesis is initiated by mechanisms other than RB1 tumor suppressor gene inactivation. As a consequence, their genomes harbor novel chromosome aberrations. We hypothesize that identifying recurrent copy number alterations within these tumors will allow the identification of important regions which may harbor known or novel oncogenes or tumor suppressor genes that contribute to retinoblastoma tumor initiation in the presence of wild type pRB.  1.3.3 Second hypothesis RB1 inactivation is associated with genomic instability and has been correlated with clinical parameters such as outcome or age of diagnosis in both gastric cancer and retinobastoma (Zheng, Flesken-Nikitin et al. 2002; Zheng and Lee 2002) (Herzog, Lohmann et al. 2001; Sampieri, Amenduni et al. 2009). Since RB1+/+ tumors develop in the presence of wildtype pRB, we hypothesize that they may consequently harbor less genomic instability than retinoblastoma tumors containing RB1 inactivating mutations. Therefore, in addition to identifying specific genetic alterations, we will also investigate the instability in the RB1+/+ 15  retinoblastoma tumor genomes. A more stable genome may indicate that the copy number alterations we identify are being strongly selected for and more likely to represent causal events in tumorigenesis rather than as a consequence of overall genomic instability.  1.3.4 Third hypothesis Allele specific copy number alteration is an important mechanism of gene deregulation in multiple cancer types.  Both allele specific copy number (ASCN) alteration and cnLOH are known mechanisms of inactivation of the RB1 TSG in retinoblastoma (Zhu, Dunn et al. 1992). It is likely that these mechanisms are not unique to inactivation of the RB1 gene and may be involved in deregulation of additional genes involved in retinoblastoma tumorigenesis. In addition to investigating genes deregulated by copy number alteration, by tiling path aCGH, we will investigate the role of ASCN alteration and cnLOH in gene deregulation in retinoblastoma on a whole genome scale by SNP array profiling and genotyping a small subset of five RB1+/+ MYCN amplified samples and their paired blood normal references. In this manner, we will identify genes, in addition to RB1, affected by these mechanisms which are not detectable by copy number profiling alone.  16  2    Chapter: Materials and Methods  2.1  Samples  2.1.1 Sample DNA DNA from 48 primary retinoblastoma tumors of patients with sporadic unilateral retinoblastoma was collected from four genetic screening centers including; Retinoblastoma Solutions, Toronto, Canada; Department of Molecular Pathology, Canterbury Health Laboratories, Christchurch, New Zealand; Service de Génétique Oncologique, Institut Curie and Université Paris Descartes, Paris, France; and Institut für Humangenetik, Universitätsklinikum, Essen, Germany (Table 2.1).  For Toronto samples, DNA was extracted using the PuregeneTM DNA extraction kit (Qiagen, Mississauga, ON).  In addition, genomic DNA from two human retinoblastoma cell lines (Weri-Rb-1 and Y79) was obtained from Dr. Brenda Gallie at Toronto Western Hospital Research Institute, Toronto, Ontario, Canada.  2.1.2 RB1 gene mutation status Patient samples were initially submitted to Retinoblastoma Solutions as clinical samples for RB1 mutation detection. As such they were screened for RB1 mutations by Qm- PCR and sequencing of all 27 exons and the core promoter (sequencing also included the 25 exon-flanking intronic nucleotides) to detect copy number alterations. As well as promoter methylation analysis to identify RB1 promoter hypermethylation, and in some cases allele- specific PCR to identify low-level mosaicism of 11 recurrent RB1 nonsense mutations (Richter, Vandezande et al. 2003; Rushlow, Piovesan et al. 2009). Tumor samples from the additional retinoblastoma testing centers also had RB1 gene mutation testing performed including sequence analysis, either QmPCR or Multiplex Ligation-dependant Probe Amplification (MLPA) (MRC-Holland) to detect copy number alterations, RB1 promoter methylation analysis, and detection of LOH at RB1 by microsatellite analysis (Raizis, Schmitt et al. 1995; Stirzaker, Millar et al. 1997; Raizis, Clemett et al. 2002; Schouten, McElgunn et al. 2002; Houdayer, Gauthier-Villars et al. 2004; Schuler, Weber et al. 2005; Mitter, Rushlow et al. 2009). 17  2.1.3 Incidence of RB1+/+ retinoblastoma across the four testing sites The 48 primary tumors used in this study are a subset of 1023 primary unilateral retinoblastomas which were tested for RB1 gene mutations across the four testing sites. Due to similar frequencies of occurrence of RB1+/+ tumors between testing centers, tumors from across all four sites were pooled together to make up the RB1+/+ tumor set described (Rushlow, Kennett et al. 2012). Genomic DNA from 23 of the 26 RB1+/+ tumors comprise part of the 48 primary tumors samples used in this study.  2.2 Whole-genome sub-megabase resolution tiling path aCGH analysis Whole genome tiling path aCGH was used to identify copy number alterations genome-wide in 48 primary unilateral retinoblastoma tumors, which included 11 RB1-/-, 14 RB1+/- and 23 RB1+/+, as well as the two retinoblastoma cell lines. See Figure 2.1 for an overview of aCGH methodology. The array platform, comprised of 26,363 genomic segments represented by BAC clones, was manufactured at BC Cancer Research Centre, as previously described (Watson, deLeeuw et al. 2004; Ishkanian, Malloff 2004; Watson, deLeeuw et al. 2007). The Sub- megabase resolution tiling path (SMRT) array can detect alterations as small as 50 kb in size (Coe, Ylstra et al. 2007). Array hybridization was performed as previously described (Kennett, Watson et al. 2008). Briefly, 300 nanograms (ng) of sample and reference (single male) genomic DNA were differentially labelled with Cyanine-3 and Cyanine-5 dCTPS using the BioPrime DNA labelling system (Invitrogen, Burlington, Ontario, Canada). Labelled sample and reference DNA were pooled and allowed to competitively co-hybridize to the array. After an incubation period the unbound samples were washed from the slide and the array was imaged as described next. 18  Magnified view of a single grid of array elements spotted in duplicate (ex. BAC clones) Cell Line Karyogram Chr. 1 Normalized log2 signal intensity ratio plotted against chromosomal position for each array element 5. Visualization 2. Competitive Co-hybridization 1. DNA Labelling 3. Washing and 4. Imaging 6. Data Analysis Background normalization, breakpoint detection, alteration calling and comparisons across multiple samples Differentially labelled sample and reference DNA 0 1q12 1q44 1p31.2 1p36.33 1q32.1 1p13.3 Focal amplifications Gain Loss   Figure 2.1  Overview of aCGH methodology. This figure shows the basic flow scheme (steps 1 through 6) of a typical aCGH experiment using BAC clones as an example of spotted array elements.  Gain, loss and amplifications are demonstrated in a typical cell line karyogram in step 5. Potential data analysis steps are briefly mentioned in step 6.  This protocol is described in detail elsewhere (Kennett, Watson et al. 2008).  2.2.1 Array CGH data analysis Array CGH images were obtained using the Array-WoRx charge-coupled device (CCD)-based scanner (Applied Precision, Issaquah, WA) at a resolution of 10 µm with median intensity channel normalization. Image analysis was performed with the Softworx software suite (Applied Precision). Raw data was normalized for spatial and printing intensity bias with CGH Normalize suite (Khojasteh, Lam et al. 2005). Data was imported 19  into SeeGH (Chi, DeLeeuw et al. 2004) and SIGMA2 (Chari, Coe et al. 2008). Both programs allow electronic representation and visualization of log2 signal intensity ratios of sample to reference at each array element. All replicate spots with standard deviation above 0.1 or signal-to-noise ratios of less than three were excluded from further analysis. These data are vertically aligned to chromosomal position allowing multiple alignment and copy number annotation. Clones were aligned by chromosomal positions as per the human RefSeq hg18 March 2006 (NCBI36/hg18) genome assembly.  2.2.2 Segmentation and alteration detection criteria The segmentation and calling algorithm FACADE was utilized to segment the data and determine the alterations for each sample (Coe, Chari et al. 2010). Segmentation is the process of identifying putative breakpoints within aCGH data that define regions of equal copy number. Calling of array data, assigns states to the segmented regions of gain, loss or neutral, which is useful for downstream analysis. Breakpoint boundaries for segmental alterations were determined by the end sequence position of the BAC clone on either side of the breakpoint. When a BAC clone exhibited a ratio that was an intermediate value of the two flanking copy number ratio levels, the clone was considered to contain the breakpoint. When such a clone did not exist, the two flanking clones were considered to encompass the breakpoint event. Breakpoints at the centromeres were indiscernible due to repetitive DNA content. Only regions containing two or more overlapping clones in agreement were called to reduce false positives due to hybridization artifacts. The FACADE ‘3 level output’ option was used to identify segments having less than two copies, greater than two copies, and two copies as lost, gained or neutral respectively.  2.2.3 Identification of frequent regions of copy number alteration Regions of frequent differential copy number were identified as follows: (1) FACADE calls for each array element of +1 (copy number increase), 0 (neutral), or -1 (copy number loss) were generated as described in Section 2.2.2, (2) values for missing array elements removed by quality control filtering, as described in Section 2.2.1, were inferred based on the status of neighbouring clones within a 10 Mb window, (3) individual elements were then merged into genomic regions if copy number status was identical across all 20  samples within a group, (4) this list of genomic regions was then filtered based on the number of clones within each region and those consisting of less than two clones were removed from further analyses. Frequency plots to visualize the occurrence of copy number alterations within a group of samples were generated by loading the list of genomic regions with call values for each sample into SeeGH software.  DNA copy number alterations can encompass both driver genes defined as genes providing some selectable advantage to the cancer, and passenger genes defined as those genes altered due to proximity to the driver gene and that have little or no effect on the cancer (Stratton, Campbell et al. 2009). In order to reduce the inclusion of passenger genes in our subsequent gene analysis, altered gene lists were sorted by frequency of occurrence, with more interest placed on the genes within regions at the highest alteration frequency.  2.2.4 Definition of gains and high-level amplifications DNA gain is defined as an increase in DNA copy number of large chromosomal regions or intact chromosomes, due to aneuploidy or unbalanced translocations (Lengauer, Kinzler et al. 1998).  In contrast, DNA amplification refers to a focal increase in copy number of at least five copies of a DNA segment 0.5-10 Mb in size (Myllykangas, Himberg et al. 2006). Figure 2.2 shows a comparison between tumors with varying levels of gain and amplification. Direct thresholding of aCGH data using a log2 ratio of 1.0 represents both a commonly used and stringent criteria for defining amplified chromosome segments in array data of this type (Lockwood, Chari et al. 2008). High-level amplifications were defined in this dataset as two or more contiguous clones having a log2 signal intensity ratio equal to or greater than 1.0.  Whereas gains were defined as two or more contiguous clones having a log2 signal intensity ratio equal to or greater than 0.2 and less than 1.0.  21    Figure 2.2 Low-level gain compared with high-level amplification. Multiple alignment of chromosome 2 across three retinoblastoma tumors displaying broad low-level gain of all of chromsome 2, gain of 2p, and a high-level focal amplification at 2p24.3-2p24.2, as indicated by the respective highlighted boxes.  Sample order: T48 (RB1+/-), T19 (RB1+/-), and E7 (RB1+/+).  2.2.5 Determination of proportion of the genome altered The segmental alterations calls identified by FACADE were used to determine the proportion of the genome altered. Gaps between array elements were inferred by the status of neighbouring clones within 10 Mb. The sex chromosomes were removed from this analysis due to the sample set being comprised of both males and females hybridized to a male 22  genomic reference DNA sample. Segmental alteration calls for each sample were filtered by a minimum number of clones altered within each region of two or more. The number of bps altered, both gained and lost, were summed for each sample from the list of segmental alteration calls. In addition, the number of discrete alterations were calculated for each sample.  In order to investigate whether there was a difference between MYCNA and non- MYCNA tumors, the RB1+/+ tumors were assessed all together as well as separated based on MYCN amplification status.  2.2.6 Statistical comparison of data means between two groups Unpaired t-tests were performed to compare the means of the data, for the proportion of the genome altered, between two groups where appropriate. Unpaired t-tests were performed using a two-tailed distribution and two sample unequal variance.  2.2.7 Network/ pathway level analysis of altered genes Genes were mapped to regions of altered copy number status using the RefSeq HG18 March 2006 mapping build. In order to enrich our gene list for driver genes and reduce the inclusion of passenger genes only those genes altered within a minimal common region across multiple samples at a frequency of 25% of greater were included in the network/ pathway analysis. The RB1 mutation status was included in the list of altered genes in RB1-/- cases, regardless of copy number status. Datasets containing gene identifiers and corresponding alteration directions (+1 for a gain, and -1 for a loss) for the RB1+/+ and RB1-/- tumor groups were uploaded to the Ingenuity Pathway Analysis (IPA) bioinformatics software (Ingenuity Systems, www.ingenuity.com). IPA is a curated database and analysis program that identifies canonical pathways that were most significantly enriched in or between large datasets and generates networks of molecules from the dataset based on their connectivity from information contained within the Ingenuity Knowledge Base. Pathway enrichment analysis is performed by a right-tailed Fisher’s exact test based on the number of genes annotated, number of genes represented in the input dataset and the total number of genes being assessed in the experiment. In this study, a pathway was deemed significant if the p-value for enrichment was < or = to 0.05 (adjusted for multiple comparisons using Benjamini-Hochberg multiple testing correction). In addition to pathways, IPA generates 23  networks as a graphical representation of the interrelationships between molecules in the dataset. Networks are given a network score based on the negative exponent of the right- tailed Fisher’s exact test result, which represents the likelihood that the molecules that are part of the network would be found there by random chance.  2.3 Affymetrix genome-wide SNP 6.0 array Genotyping  2.3.1 Affymetrix genome-wide human SNP 6.0 array genotyping SNP arrays provide a high density platform for high-throughput genome-wide analyses of both copy number and genotypic alterations. The Affymetrix Genome-Wide Human SNP Array 6.0 contains approximately 946,000 non-polymorphic copy number oligonucleotide probes in addition to approximately 906,600 SNP probes. In addition to detecting large copy number alteration events the SNP array platform provides increased resolution of focal copy number alterations and breakpoints. The SNP array is thus used for the detection of copy number variations at extremely high resolution as well as LOH, which can occur in the absence of an alteration in copy number such as with cnLOH. SNP arrays are now commonly used to study both copy number and allelic imbalances in a number of different cancer types such as lung, breast, and neuroendocrine cancers. SNP array analysis was performed on five of the RB1+/+ MYCNA tumors initially profiled by tiling path aCGH.  2.3.2 Copy number profiling and genotyping of five RB1+/+ tumor samples Matching patient normal blood DNA was obtained for five of the 23 RB1+/+ primary unilateral retinoblastomas for which there was sufficient DNA material remaining after profiling by tiling path aCGH. These five tumors and paired reference samples were assessed for DNA quality and double-stranded DNA (dsDNA) content prior to analysis on the Affymetrix Genome-Wide Human SNP 6.0 array platform. Five hundred ng of genomic DNA from each tumor and paired reference sample were prepared and hybridized to the Affymetrix Genome-Wide Human SNP 6.0 array platform following manufacturer’s instructions. The raw data, stored in CEL files, was processed using the Affymetrix Genotyping Console (version 3.0.2) to generate .chp files, using the birdseed v2 genotyping algorithm (Korn, Kuruvilla et al. 2008). 24  2.3.3 Determination of regions of copy number alteration CEL files were imported into Partek Genomics Suite (GS) 6.5 using the recommended default parameters. To determine total copy number, paired copy number intensities were calculated for each sample from the intensity values in the tumor compared to its paired blood reference. The Partek GS Genomic Segmentation method was used to analyze the paired copy number intensities using the default parameters with the exception of changing the minimum number of markers to 50. To account for dips in frequency where one region is split into two, adjacent regions less than 1 Mb apart, having the same copy number status were merged into a single region.  2.3.4 Determination of regions of allelic imbalance The CEL files and .chp files were imported into Partek GS 6.5 as stated in Section 2.2.3. In Partek GS the allele specific copy number (ASCN) analysis was used to identify regions of allelic imbalance (AI) in each sample. The ASCN analysis generates a proportion score for each probe, with a score of 0 representing equal allelic intensities. Allelic imbalance regions were defined as having an imbalance proportion of equal to or greater than 0.15 as recommended by Partek GS for high quality array data. Similar to copy number alteration regions, adjacent probes, less than 1 Mb apart, with frequencies greater than the threshold were merged into one region.  2.3.5 Determination of regions of cnLOH The ASCN data was combined with total copy number data from the segmentation analysis, to identify regions of cnLOH. A region was classified as cnLOH if the region displayed AI in the absence of a change in the overall copy number.        25  3    Chapter: Results  3.1 Whole genome segmental copy number profiling Regions of copy number alteration were detected throughout the genomes of all 48 primary unilateral retinoblastoma samples and two retinoblastoma cell lines analyzed by whole genome tiling path aCGH. The high resolution of this array allowed for the precise determination of breakpoints and detection of focal regions of copy number change (Coe, Ylstra et al. 2007). Alterations detected ranged in size from a focal gain of approximately 50 kb on chromosome 7 in the RB1+/+ tumor T5, to large whole chromosome gains and losses observed in multiple samples.  Figure 3.1 demonstrates the identification of segmental copy number gains, losses and high level amplification within a single sample; RB1+/+ tumor, RB522. This tumor sample contains losses on chromosomes 11p, 17p and 18q, as well as gains on chromosomes 1q, 7q, 11q, and 15q. In addition to the gains and losses there was a high-level amplification identified on chromosome 2p, encompassing the MYCN oncogene. Karyograms of two commonly used retinoblastoma cell lines; Weri-Rb-1 and Y79 are also included as Figures A.1 and A.2. 26    Figure 3.1  Whole genome karyogram of RB1+/+ retinoblastoma tumor, RB522. This figure demonstrates the identification of segmental copy number gains, losses and high level amplification within a single sample. It is a visualization of the log2 signal intensity ratio of sample to reference at each array element, vertically aligned with chromosomal position as per the human RefSeq hg18 March 2006 (NCBI36/hg18) genome assembly. Log2 ratio lines at -1, -0.5, 0, 0.5 and 1 are shown. All replicate spots with a standard deviation above 0.1 or signal-to-noise ratios of less than three were excluded from further analysis. This tumor sample contains losses on chromosomes 11p, 17p and 18q, highlighted in green as well as gains on chromosomes 1q, 7q, 11q, and 15q and a high level amplification identified on chromosome 2p, encompassing the MYCN oncogene, highlighted in red.  3.2 Identification of frequent regions of altered copy number status We generated frequency plots for each tumor group to visualize the frequency of copy number alterations occurring across multiple tumor samples. Sample groups were based on their RB1 gene mutation status and included 23 RB1+/+ tumors (Figure 3.2), 11 RB1-/- tumors 27  (Figure 3.3), and 14 RB1+/- tumors (Figure 3.4). First, genomic segments of altered copy number status were detected using the FACADE breakpoint detection and calling algorithm described in chapter two. Second, the regions of altered copy number were summed across all samples within each group and displayed as a frequency diagram using SeeGH software (Figures 3.2, 3.3 and 3.4). The complete list of all copy number alterations detected in each sample is included as Supplementary Table 1.    Figure 3.2  Overall RB1+/+ frequency plot. This figure displays the frequency of alterations identified across 23 RB1+/+ samples. Frequencies of gain are displayed in red to the right of the chromosome, while frequencies of loss are displayed to the left in green.  The ratio lines for reference are located at +0.5 and - 0.5 and represent a frequency of gain or loss respectively in 50% of the total samples.  The most frequent chromosomal events in the RB1+/+ tumors is gain of 2p, 6p, and 1q, with the 2p gain being the most frequent event. 28    Figure 3.3  RB1-/- frequency plot. The frequency of alterations identified across 11 RB1-/- samples.  Frequencies of gain are displayed in red to the right of the chromosome, while frequencies of loss are displayed to the left in green.  The ratio lines for reference are located at +0.5 and -0.5 and represent a frequency of gain or loss respectively at 50% of the total samples.  The most frequent chromosomal events in the RB1-/- tumors is gain of 1q, 6p, loss of 16q, and both gains and losses on 13q, all commonly reported alterations of retinoblastoma. 29   Figure 3.4  RB1+/- frequency plot. The frequency of alterations identified across the 13 RB1+/- tumors.  Frequencies of gain are displayed in red to the right of the chromosome, while frequencies of loss are displayed to the left in green.  The ratio lines for reference are located at +0.5 and -0.5 and represent a frequency of gain or loss respectively at 50% of the total samples.  The most frequent chromosomal events in the RB1+/- tumors are gain of 1q, 2p, 6p, and 20, as well as losses on 13q and 16q.  3.2.1 RB1+/+ A major focus of this study was to identify novel regions of altered copy number status within the RB1+/+ retinoblastoma tumors. Figure 3.2 shows the frequency plot of the copy number alterations identified within the 23 RB1+/+ tumors. The most frequent copy number alteration event identified in these RB1+/+ tumors is copy number increase on chromosome 2p occurring in 15 tumors in total. Of these 15 RB1+/+ tumors, four exhibited broad low-level gains, while 11 showed high level amplification of 2p24.2-25.1, the region containing the known oncogene MYCN (MYCNA). The number of altered bps detected per 30  sample ranged from 27 Mb to 436 Mb. The most frequent regions of copy number increase were identified on chromosomes 1, 2p, 5p, 15q, 6p and 17p. The most frequent regions of copy number loss were observed on chromosomes 3p, 11, 16q, 8q and 22q. Multiple regions within 1q, such as 1q42.3 display higher frequency of copy number gain than the majority of the 1q arm. One of these regions of gain was observed in ten tumors at 1q42.1 and contains one gene, interferon regulatory factor 2 binding protein 2 (IRF2BP2), a novel target gene of p53 (Koeppel, van Heeringen et al. 2009). Other genes within regions of frequent copy number increase include brain abundant, membrane attached signal protein 1 (BASP1) on chromosome 5p15.1, which has been shown to be differentially expressed in human cancer cell lines and may play a role in transcriptional co-suppression for the Wilms’ tumor suppressor protein (WT1). Interestingly, Wilms’ tumor, a kidney cancer which occurs in children also displays frequent gain of MYCN and occasional amplification. Another gene gained on chromosome 1q23.1 in nine of the RB1+/+ tumors, cellular retinoic acid binding protein (CRABP2) shows overexpression in a wide variety of cancer types and its expression has been shown to correlate strongly with that of MYCN in Wilms’ tumor.  3.2.2 RB1-/- Figure 3.3 shows the frequency plot summarizing the frequency of detected copy number alterations across 11 RB1-/- retinoblastoma tumors. Previously reported regions of common copy number alteration were found to be frequently altered in these 11 RB1-/- tumors, demonstrating the ability to detect known copy number alterations using our breakpoint detection settings. All regions of gain or loss at 13q14 identified by Gallie et al. through mutational analysis were confirmed by aCGH analysis (Rushlow, Kennett et al. 2012). The most frequent regions of copy number increase include 1q, 6, 11 and 18q. The most frequent regions of copy number loss occur on chromosomes 1p, 8q, 12q, 16q, and 17p. Additionally chromosome 13 frequently exhibits both increase and decrease of genomic material, often with multiple rearrangements within a single tumor sample. The number of altered bps detected per sample ranged from 35.4 Mb to 1054.5 Mb. The 6p arm is frequently gained in RB1-/- tumors, a region at 6p25.3 is gained at slightly higher frequency and contains the gene forkhead box 1 (FOXC1). This gene is a member of the FOX family of genes which are disrupted in several cancer types (Katoh 2004). 31  3.2.3 RB1+/- Figure 3.4 shows the frequency plot summarizing detected copy number alterations across 14 RB1+/- retinoblastoma tumors. Similar to the RB1-/- tumors, regions of frequent copy number increase included 1q, 2p, 6p, and chromosome 20. Frequent regions of copy number decrease included chromosomes 13, and 16q. However, altered copy number regions detected on chromosomes 1p, 6q, 10q, 17p, 18, and 19 were not observed to occur frequently in the RB1+/- tumors. The total number of altered bps detected per sample ranged from 32.6 Mb to 932.8 Mb. Only one out of 14 RB1+/- tumors, sample number T33, displayed amplification of 2p24.3 encompassing the MYCN gene.  3.2.4 Frequency plot comparison between RB1+/+ MYCNA and RB1-/- tumors To investigate whether tumors harbouring RB1 inactivation or MYCN amplification may have arisen by different mechanisms and consequently share few genomic similarities we performed a frequency plot comparison between the 11 RB1+/+ MYCNA tumors and 11RB1-/- tumors. Figure 3.5 shows the copy number alteration regions of overlap between the RB1+/+ MYCN amplified and RB1-/- tumor groups. The main regions of overlap, which occur less frequently in the RB1+/+ group are gain of 1q, 14q21.3-q terminus, and loss of 8p21.2 –p terminus, 16q and 17p. While many alterations are group specific, such as copy number increase of 2p, 6p, 17q, and loss of 1q, 8, 16p, and 20q. 32  -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 -.5 .5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22  Figure 3.5  Frequency plot comparison between RB1+/+ MYCNA and RB1-/- tumors. A comparison of frequency plots between each group showing event frequencies for each group and their overlap.  In this figure RB1+/+ MYCNA tumors are represented in fuschia, while RB1-/- tumors are represented in blue.  Common regions of overlap between the two groups are represented in yellow.  Frequencies of gain and loss are displayed to the right and left of the chromosome, respectively.  There are fewer frequent alterations in the RB1+/+ MYCNA group and four main regions of overlap between the groups, gain of 1q, 8p21.2 – p terminus, 14q21.3 – q terminus and loss of 16q, indicating that these groups of tumors share few common alterations.  3.3 High resolution detection of 2p amplification and the MYCN oncogene The most striking and frequent event identified in the RB1+/+ tumors is a high-level amplification of a focal region on chromosome 2p. A total of 11 out of 23 RB1+/+ primary unilateral retinoblastoma tumors, one RB1+/- retinoblastoma tumor (T33), one RB1-/- retinoblastoma tumor (RB381) , and one RB1-/- retinoblastoma cell line displayed high-level 33  amplification of chromosome 2p24.2-25.1 when analyzed by tiling path aCGH. Additionally, 12 tumors, two RB1-/-, four RB1+/+ and six RB1+/- showed broad gain of 2p, including MYCN; However using a stringent criteria of ten copies, Rushlow et al. did not report any MYCN amplifications by QmPCR in 93 RB1-/- tumors (of which the 11 RB1-/- used in this thesis work were a subset), (Rushlow, Kennett et al. 2012). However, MYCN copy number did reach upwards of nine copies in one RB1-/- tumor, which is significantly lower than the range of 33 to 121 copies for the MYCN amplification seen in RB1+/+ tumors (Rushlow, Kennett et al. 2012). Furthermore, using the criteria outlined in the current thesis work, we report a significantly higher proportion of 2p24 amplification (47% in RB1+/+ tumors) than the estimated frequency of occurrence reported in the literature of 3%, as described by Bowles et al. (pvalue = 0.000187; two-tailed, unpaired, unequal variance t-test) (Bowles, Corson et al. 2007). However in other studies, the frequency of MYCN amplification has been reported as anywhere from 0-30% across various CGH studies (Lee, Murphree et al. 1984; Squire, Goddard et al. 1986; Sakai, Tanooka et al. 1988; Choi, Lee et al. 1993; Doz, Peter et al. 1996; Mairal, Pinglier et al. 2000; Lillington, Goff et al. 2002; Zielinski, Gratias et al. 2005). Part of this discrepancy may result from variance between study sizes and criteria for defining amplifications, platform detection sensitivity, and importantly, RB1 gene mutation status, which was often not defined.  3.3.1 Minimal overlapping amplicon on chromosome 2p The ability of tiling path aCGH to detect alterations with a resolution of approximately 50kb allowed for high resolution identification of the minimal region of amplification on chromosome 2p. High level amplifications on chromosome 2p 24.2-25.1 were aligned to identify the minimal common region of overlap between 14 MYCNA samples, which include: 11 RB1+/+ primary retinoblastoma tumors, one RB1+/- primary retinoblastoma tumor, one RB1-/- primary retinoblastoma tumor as well as the MYCNA retinoblastoma cell line, Y79. Using the primary retinoblastoma tumors to identify the minimal common region, the boundaries were defined by tumors T33 and P2 (Figure 3.6). The size of the minimal amplicon is approximately 537 kb and spans the bps between 15895098 and 16432161, of the BAC clones N0451A14 and N0062M03, respectively. The MYCN gene (located on 34  chromosome 2p from bps 15998134 to 16004580), is the only gene contained within the minimal region of amplification. 2p 25 .1 2p 24 .3 2p 24 .2 2p 24 .1 P5 E2 6 C1 RB 52 2 Y7 9 T1 4 T5 T2 5 T4 2 E4 E7 P2 RefSeq  (NCBI36/hg18)  Genes MYCN DDX1 NAG FAM49A T3 3 RB 38 1  Figure 3.6  2p multiple alignment. Multiple alignment of eleven primary RB1+/+ retinoblastoma tumors, one RB1+/- and one RB1-/- tumor displaying high level 2p24.1-25.1 amplification allowed identification of the minimal overlapping region (highlighted in blue) and the genes contained within.  The upper and lower boundaries of the minimal region were defined by tumors T33 and P2, respectively. The minimal amplicon is approximately 537 kb in size and spans the base pair positions from 15895098 to 16432161.  MYCN is the only gene contained within the minimal region of amplification. The RB1-/- retinoblastoma cell line, Y79 also displayed MYCN amplification and was included for comparison.   35  3.4 All high-level amplifications identified including chromosomes 1q, 2p and 21q High level amplifications were identified in 11 RB1+/+ samples, including an additional 2p amplification to MYCN in one of the RB1+/+ samples, as well as in three RB1-/- tumors and two RB1+/- tumors. All amplifications identified were non-overlapping (with the exception of the MYCN amplicon) and involved only chromosomes 1q, 2p, and 21q in the RB1+/+ samples (detailed in Table 3.1). Primary tumor T42 contained more than one amplicon on chromosome 2p, and another tumor sample (T5) contained a 1q amplicon within a gain of the chromosome arm, and an amplicon on 21q in addition to the 2p MYCN amplification (shown in Figure 3.7). Amplicons in the RB1-/- tumors occurred on chromosomes 1q, 2p, 13 and 12q. Only two tumors within the RB1+/- group displayed amplifications, which included chromosomes 1q32.1, 2p and 13q. All amplifications in the RB1-/- and RB1+/- tumors were non-overlapping, with the exception of the 1q32.1 amplification in RB1+/- tumor, E27 and RB1-/- tumor, RB2052. The minimal shared region of amplification at 1q32.1 contained 50 genes, including the MDM4 gene which has been previously implicated in retinoblastoma development (Corson and Gallie 2007; Sampieri, Mencarelli et al. 2008). MDM4 encodes a nuclear protein containing a p53 binding domain and binds the p53 tumor suppressor protein, inhibiting its activity; subsequently MDM4 is overexpressed in many human cancers. Amplifications spanning this gene ranged in size from 0.28 Mb to 6.5 Mb. The number of genes contained within the amplified regions is listed in table 3.1, as well as any candidate target genes with previous implications in retinoblastoma or cancer. Interestingly, a 1q41 gain was reported by Gratias et al., but they focused on CENPF gene, which was not located within or near the boundaries of the 1q41 amplification we identified in RB1+/+ tumor T5 (Gratias, Schuler et al. 2005).         36  Table 3.1    All discreet high level amplicons identified. Status Sample  Chr Start Clone Start (bp) End Clone End (bp) Size (Mb) # of genes Known Oncogenes RB1+/+ T5 1q41 N0282E08 217613871 N0514C19 219396428 1.78 10 2p24.3 N0451A14 15703698 N0065N17 16825910 1.12 2 MYCN 21q22.3 M2410F14 44478602 M2053G20 44754475 0.28 8 T42 2p25.1 N0487B06 11436647 N0641J22 11754223 0.32 3 2p24.3- 2p24.2 N0571E19 14565626 N0427M01 17376543 2.81 5 MYCN T14 2p24.3-24.2 N0451A14 15703698 M2123N14 18028443 2.33 6 MYCN T25 2p24.3 N0231J10 14410329 N0701F16 16915017 2.51 5 MYCN E4 2p24.3- 2p24.2 N0619O15 15175256 N0325P07 17748109 2.57 6 MYCN E7 2p24.3- 2p24.2 N0619O15 15175256 N0631D03 17177195 2.00 4 MYCN P2 2p25.1- 2p24.3 N0686G09 9935380 N0062M03 16432161 6.50 26 MYCN P5 2p24.3- 2p24.2 N0541K19 15467410 N0554B24 18350885 2.88 8 MYCN E26 2p24.3- 2p24.2 N0196C08 13032063 N1007E02 17123259 4.09 5 MYCN C1 2p24.3- 2p24.2 N0723F23 14978618 M2305P22 18851300 3.87 9 MYCN RB522 2p24.3- 2p24.2 N0091E09 14051860 N0554B24 18350885 4.30 8 MYCN RB1-/- RB2052 1q32.1 N0165E10 200361154 N0595K11 205125953 4.77 71 MDM4 13q13.3 N0162F21 35606696 N0289J04 36164519 0.56 5 13q13.3 N0791D19 37743294 N0718A20 38322375 0.58 2 13q31.3 N0093L24 90298089 N0487A02 91073134 0.78 1 GPC5 RB381 2p25.2- 2p25.1 N0129I01 5022521 N0517E08 9267587 4.25 8 2p24.3- 2p24.2 N0541K19 15467410 N0419K22 18088207 2.62 8 MYCN    37  Status Sample  Chr Start Clone Start (bp) End Clone End (bp) Size (Mb) # of genes Known Oncogenes RB1-/- T23 12q24.31- 12q24.32 N0387F15 122580551 N0006I24 125366514 2.79 18 12q24.32 N0016J23 125921569 N0337L12 128699668 2.78 3 E27 1q32.1 N0783D13 201524037 N0421E17 204708300 3.18 50 MDM4 *13q RB1+/- T33 2p24.3- 2p24.2 N9463P22 16001272 N1007E02 17123259 1.12 2 MYCN *Tumor E27 contains many rearrangements including too many high level amplification events on chromosome 13q to list T5*E27RB2052 1q41 1q32.3 1q32.1 1q32.2 1q42.11 1q31.3 Genes: SLC30A10, EPRS, BPNT1, IARS2, RAB3GAP2, MARK1, C1orf115, MOSC2, MOSC1, HLX1 A B T42 2p21 2p25.1 2p24.3 2p24.2 2p12 2p13.3 2p13.1 Genes: E2F6, GREB1, NTSR2 C T5* 21q22.3 Genes: ICOSLG, DNMT3L, AIRE, PFKL, C21orf2, TRPM2, LRRC3, C21orf29, KRTAP10-1, - 2, -3, -5, -6, -9, -10, -11, KRTAP12-3, -2, -1, UBE2G2 50 Genes: including MDM4  Figure 3.7  High-level amplifications in addition to MYCN. Normalized log2 signal intensity ratios were plotted against chromosomal position using SeeGH software. A log2 signal intensity ratio of 0 represents equivalent copy number between the reference and sample DNA. Vertical lines denote log2 ratios of -1, 0 and +1, 38  with increases in copy number to the right and decreases in copy number to the left of the centre line. Each red, green or black dot represents a single BAC clone on the array. (A) aCGH profiles of two RB1-/-, tumors and an RB1+/+ MYCNA tumor, showing chr 1q32.1 and 1q41 amplicons. (B) aCGH profile for one RB1+/+ MYCNA tumor showing one additional 2p amplicon. (C) aCGH profile for an RB1+/+ MYCNA tumor showing chr 21q22.3 amplicon. *denotes the same sample. Orange boxes highlight the region(s) amplified within each sample. The blue box highlights the minimal region of overlap between samples sharing the same amplicon. Genes located in each amplified region are listed.  The gene names highlighted in bold denote a previous implication for a potential role in cancer.  3.5 Comparison of the overall proportion of the genome altered between RB1+/+, RB1+/-, and RB1-/- tumors Comparisons were performed between different tumor subgroups based on gene status (RB1+/+, RB1+/+ MYCNA, RB1-/-, and RB1+/+ without MYCNA, RB1+/-). Figure 3.8 shows the group comparison results of a) number of alterations and b) the number of bps altered. There was no statistically significant difference between any of the groups when comparing the total number of alterations as determined by FACADE algorithm described in chapter 2. However, a comparison between the total number of bps altered between these tumor groups showed a significantly lower number of bps altered in the RB1+/+ group when compared to the RB1-/- group (pvalue = 0.0109; unpaired t-test), Figure 3.8 b. There was no statistically significant difference in either the number of alterations or the number of bps altered between the RB1+/+ tumors with and without MYCN amplifications (pvalue = 0.771 and pvalue = 0.908; unpaired t-test).         39  (p = 0.1335; unpaired t-test) N um be r o f A lte ra tio ns A.  Comparison of the number of alterations between tumor groups   B. Comparison of the number of bps altered between tumor groups (p = 0.0109; unpaired t-test) N um be r o f b as e pa irs  a lte re d  40  Figure 3.8  Comparison of genomic instability between tumor groups. Comparisons of two different indicators of genomic instability were performed between different tumors grouped by gene status (RB1-/-, RB1+/+, RB1+/+ MYCNA, and RB1+/+ w/o MYCNA).  The results are displayed as box plots with the box representing the upper and lower quartiles, the tips of the bars show the minimum and maximum values and the median is depicted by the line within each box. A. Comparison of the overall numbers of alterations between tumor groups.  There was no statistically significant difference between the RB1+/+ and RB1-/- tumor groups (pvalue = 0.1335; unpaired t-test).  B. Comparison of the number of bps altered between tumor groups.  Significantly less bps were altered in the RB1+/+ tumors than the RB1-/- tumors (pvalue = 0.0109; unpaired t-test).  3.5.1 Comparison of copy number altered genes between RB1+/+, RB1+/-, and RB1-/- tumors Regions of copy number alteration containing 763 (514 gained, 249 lost) genes, 1037 (978 gained, 59 lost) genes and 1285 (1062 gain, 223 lost) genes were identified as frequently altered in RB1+/+ MYCNA, RB1+/+ without MYCN amplification and RB1-/- samples respectively. A Venn diagram comparison of these frequently altered genes between RB1-/-, RB1+/+ with and without MYCN amplification groups is shown in Figure 3.9. There were a total of 109 frequently altered genes common to all three groups. As expected from the number of bps identified as altered in section 3.5 the greatest number of genes frequently altered were identified in the RB1-/- tumor group, while the RB1+/+ without MYCNA had fewer genes altered and the RB1+/+ MYCNA had the lowest.   41  RB1‐/‐ RB1+/+ MYCNA RB1+/+ Without MYCNA 1285  763 1037 151 292 227 109  Figure 3.9  Venn diagram of frequently altered genes. Comparisons of the frequently altered genes between RB1-/-, RB1+/+ without MYCNA and RB1+/+ MYCNA tumors groups.  All numbers shown indicate numbers of genes altered at a frequency of 25% or greater within each group.  3.6 Pathway/ network level analysis  3.6.1 Canonical pathways and top gene functions In an effort to interpret the large list of copy number alterations in a biological context, the Ingenuity Pathway Analysis (IPA) program was utilized. Frequently altered gene lists for the RB1-/- and RB1+/+ tumor groups were uploaded separately into IPA to identify canonical pathways enriched within each dataset. Appendix Tables A4 and A5 contain the 42  top 10 canonical pathways disrupted in each dataset (RB1+/+ and RB1-/-). Top biological functions of the genes disrupted by copy number in RB1-/- tumors include cancer (p = .001- .016), embryonic development (p = .001- .016), cell death (p = .001- .016), and cell growth and proliferation (p = .001- .014). While the top biological functions of genes disrupted by copy number in RB1+/+ tumors include cancer (p = .001- .037), cellular growth and proliferation (p = .001-0.037), cellular development (p = .001- .037), and embryonic development (p = .001- .037).  3.6.2 Gene association networks Appendix Tables A6 and A7 contain the complete IPA gene association network results. Networks significantly enriched in each group are presented below. Networks are given network scores in IPA, the higher the score the lower the probability of finding the molecules observed in a given network by chance. Among the top gene association networks generated by IPA, from the RB1-/- dataset was a network with functions involving tissue morphology, cellular assembly and organization, DNA replication, recombination and repair. This network contains 29 focus molecules, including RB1, DEK and the E2F family genes (Figure 3.10). The top gene association network generated from the RB1+/+ gene list having the highest network score of 50, involved 27 focus molecules including the genes ID2 and CRABP2. The top network functions include skeletal and muscular system development and function, tissue morphology, and molecular transport. The next network, shown in Figure 3.11, generated from the RB1+/+ gene list had a network score of 45, involving 25 focus molecules and contains MYCN, NBPF11, NTRK1, and DNMT3A.Top functions of the genes in this network include cellular movement, cancer and tissue development. 43  Inhibits Binding only Acts on Inhibits and acts on A A A A B B B B   Figure 3.10  IPA derived RB1-/- gene association network involving RB1. Top networks generated by IPA using the RB1-/- gene set. Genes in frequent regions of gain or loss are coloured red or green, respectively.  Molecules not identified by colour are not altered in the dataset, but are used by IPA to generate the association network.   44  Inhibits Binding only Acts on Inhibits and acts on A A A A B B B B   Figure 3.11  IPA derived RB1+/+ gene association network involving MYCN. This figure shows one of the top networks generated by IPA using the RB1+/+ gene set involving MYCN. Genes in frequent regions of gain or loss are coloured red or green, respectively.  Molecules not identified by colour are not altered in the dataset, but are used by IPA to generate the association network.  3.7 Genome wide SNP 6.0 analysis of five RB1+/+ tumors SNP array experiments were performed to analyze, in parallel, tumor and normal genomic DNA from the same patients for five of the RB1+/+ cases.  45  3.7.1 Copy number alterations identified using oligonucleotide resolution SNP profiling SNP array profiling allows the identification of alterations in copy number at oligonucleotide probe resolution. A total of 75 copy number alterations were identified by SNP array copy number profiling encompassing 675 Mb and 386 Mb within regions of gain and loss, respectively (see Table 3.2). On average, 76% of tiling path aCGH copy number regions overlapped with the SNP copy number regions, with the lowest percent overlap in sample T5 (62%) and the highest in tumor T14 (82%). All five tumors examined by SNP array analysis contained MYCN amplification. All five MYCN amplicon breakpoints were refined by SNP copy number analysis, and are highlighted in yellow in Table 3.2. However, none of the five MYCN amplifications refined by SNP copy number profiling here were limiting in determining the minimal region of amplification in Figure 3.2. In Table 3.2 MT stands for mitochondrial DNA and the MYCN amplification is highlighted yellow for each sample.      46  Table 3.2    Genomic regions of copy number alteration detected by SNP array. Sample  Chromosome Start (bp) End (bp) Copy Number Status Length (kb) Mean* RB522 1 143591598 246815829 Amplification 102834 2.43 2 14199472 18410001 4211 17.01 2 88913210 89219698 306 3.50 7 123528211 150253930 35010 2.92 11 77729342 122914228 45185 2.90 14 105472642 105818073 345 3.50 2 47047732 47126711 Deletion 79 1.57 2 220046095 220147466 101 1.49 3 12685032 28430324 15745 1.45 3 50125786 50627811 502 1.61 3 101092395 101227474 135 0.88 6 33999530 34200703 201 1.61 6 111799222 111922162 123 1.37 6 166593831 170892931 4299 1.34 10 133604579 135055267 1451 1.66 11 188510 32396356 32171 1.01 11 63523303 63648374 125 1.49 12 3151214 3293962 143 1.60 15 39886460 39995153 109 1.51 16 83741349 84260113 519 1.51 17 526 18224251 18224 1.02 22 18407442 18814233 407 1.61 X 152285302 152456516 171 1.52 T45 2 2784 11597833 Amplification 11595 2.55 2 14630878 17362239 2732 22.21 2 20999949 224189680 203190 2.52 7 38269814 38344875 75 2.80 14 21570034 22099267 529 2.49 14 45613409 106356483 60615 3.05 22 47391269 47474598 83 2.59 MT 410 16150 16 2.52 X 12583078 12647343 64 5.60 Y 2710438 57427648 54717 2.37 2 11597833 14611236 Deletion 3013 1.09 2 17445973 20999949 3554 1.10 2 224189680 242738130 18548 1.43 X 10452961 12583078 2130 1.27 47   Sample  Chromosome bp Start bp End Copy Number Status Length (kb) Mean* T25 2 14568303 16749334 Amplification 2181 17.90 7 38258932 38318607 60 2.62 7 141976433 142210199 234 2.46 14 21462905 22017432 555 2.37 14 34085601 34389209 304 2.85 19 41910 8122910 8081 2.81 MT 410 16150 16 2.65 16 83868980 88815037 Deletion 4946 1.09 T5 1 131939195 247191012 Amplification 115252 2.71 2 15682120 16680100 998 21.15 2 38277757 38306658 29 3.13 2 141873734 142210199 336 2.59 2 21500741 22029795 529 2.62 2 69401585 69760144 359 2.51 7 410 16150 16 2.76 7 13087736 14653556 Deletion 1566 1.06 11 14653556 15682120 1029 1.34 14 16680100 16872936 193 1.31 16 16872936 17703572 831 1.09 16 65391790 134449983 69058 1.69 16 48232686 69401585 21169 1.55 MT 69760144 88815037 19055 1.54 T14 2 15706592 18017505 Amplification 2311 14.47 4 170175614 191261905 21056 2.94 5 77555727 77700664 145 3.06 6 112020688 112140376 120 2.89 7 38273603 38323840 50 2.69 7 142144512 142202756 58 2.58 14 21785489 22016671 231 2.59 14 56206175 56393004 187 3.10 MT 410 16150 16 2.62 6 65436733 65497247 Deletion 61 1.44 7 146422974 146743335 320 1.11 8 141863716 141959097 95 1.39 11 188510 46692220 46459 1.24 11 55057448 63709288 8652 1.12 11 64897105 134449983 69553 1.25 16 47251614 88815037 41563 1.12 48  *The Mean column refers to the mean copy number status of all SNP probes in the copy number alteration indicated The highlighted regions in the table identify amplifications containing MYCN on 2p  3.7.2 Genomic regions affected by allelic imbalance Allele specific copy number information was derived as described in Section 2.3.4. By combining somatic and germline analysis, LOH was detected in cases where loss of chromosomal material did not occur, providing allele specific intensity information for amplifications. In this study, there was no change in overall copy number or ASCN for chromosome 8 between the tumor and its matched normal (Figure 3.12a). However, in Figure 3.12b there is an overall single copy loss of the majority of chromosome 16q with an associated loss of a single allele in the ASCN. All five tumors assessed by allele specific copy number analysis contained regions of AI. In total, 80 regions of AI were identified ranging from 8 to 14 regions per sample (Table 3.3). The smallest and the largest regions of AI identified were 31kb on chromosome 6 (RB522) and 242692kb on chromosome 3 (T42), respectively. Figure 3.13 shows both the total copy number plot and the ASCN plot of the five MYCN amplifications as determined by SNP array. All five of the MYCN amplifications in the tumors genotyped by SNP array were observed (inferred) to be allele specific, however it is unknown from this analysis if there is a difference between the alleles.             49  Table 3.3    Genomic regions affected by allelic imbalance. Sample Chromosome Bp Start Bp End Copy Number Status Length (kb) Mean* RB522  1 143591598 246815829 Amplification 102834 2.43 2 14199472 18410001 4211 17.01 2 88913210 89219698 306 3.5 7 123528211 150253930 35010 2.92 11 77729342 122914228 45185 2.9 14 105472642 105818073 345 3.5 2 47047732 47126711 Deletion 79 1.57 2 220046095 220147466 101 1.49 3 12685032 28430324 15745 1.45 3 50125786 50627811 502 1.61 3 101092395 101227474 135 0.88 6 33999530 34200703 201 1.61 6 111799222 111922162 123 1.37 6 166593831 170892931 4299 1.34 10 133604579 135055267 1451 1.66 11 188510 32396356 32171 1.01 11 63523303 63648374 125 1.49 12 3151214 3293962 143 1.6 15 39886460 39995153 109 1.51 16 83741349 84260113 519 1.51 17 526 18224251 18224 1.02 22 18407442 18814233 407 1.61 X 152285302 152456516 171 1.52 T45 2 2784 11597833 Amplification 11595 2.55 2 14630878 17362239 2732 21.99 2 20999949 224189680 203190 2.52 7 38269814 38344875 75 2.8 14 21570034 22099267 529 2.49 14 45613409 106356483 60615 3.05 22 47391269 47474598 83 2.59 MT 410 16150 16 2.52 X 12583078 12647343 64 5.6 Y 2710438 57427648 54717 2.37 2 11597833 14611236 Deletion 3013 1.09 2 17445973 20999949 3554 1.1 2 224189680 242738130 18548 1.43 X 10452961 12583078 2130 1.27 50  Sample Chromosome Start (bp) End (bp) Copy Number Status Length (kb) Mean* T25 2 14568303 16749334 Amplification 2181 17.9 7 38258932 38318607 60 2.62 7 141976433 142210199 234 2.46 14 21462905 22017432 555 2.37 14 34085601 34389209 304 2.85 19 41910 8122910 8081 2.81 MT 410 16150 16 2.65 16 83868980 88815037 Deletion 4946 1.09 T5              1 131939195 247191012 Amplification 115252 2.71 2 15682120 16680100 998 21.15 2 38277757 38306658 29 3.13 2 141873734 142210199 336 2.59 2 21500741 22029795 529 2.62 2 69401585 69760144 359 2.51 7 410 16150 16 2.76 7 13087736 14653556 Deletion 1566 1.06 11 14653556 15682120 1029 1.34 14 16680100 16872936 193 1.31 16 16872936 17703572 831 1.09 16 65391790 134449983 69058 1.69 16 48232686 69401585 21169 1.55 MT 69760144 88815037 19055 1.54 T14                2 15706592 18017505 Amplification 2311 14.47 4 170175614 191261905 21056 2.94 5 77555727 77700664 145 3.06 6 112020688 112140376 120 2.89 7 38273603 38323840 50 2.69 7 142144512 142202756 58 2.58 14 21785489 22016671 231 2.59 14 56206175 56393004 187 3.1 MT 410 16150 16 2.62 6 65436733 65497247 Deletion 61 1.44 7 146422974 146743335 320 1.11 8 141863716 141959097 95 1.39 11 188510 46692220 46459 1.24 11 55057448 63709288 8652 1.12 11 64897105 134449983 69553 1.25 16 47251614 88815037 41563 1.12 51  *The Mean column refers to the mean copy number status of all SNP probes in the copy number alteration indicated The highlighted regions in the table identify amplifications containing MYCN on 2p               52  C o p y   n u m b e r C o p y   n u m b e r 4 0 3 0 2 0 1 0 0 4 0 3 0 2 0 1 0 0 Total copy num ber A llele specific copy num ber RB522 T5 T25 T14 T45 RB522 T5 T25 T14 T45Major alleles RB522 T5 T25 T14 T45Minor alleles 2p24.32p25.1 2p24.2 2p24.1 53  Figure 3.12  MYCN amplification with allelic imbalance. Total copy number and allele specific copy number plots of chromosome 2p25.1 to 2p24.1 for five MYCN amplified RB1+/+ tumors (RB522, T45, T25, T5, and T14).  Plots of the allele specific copy number are shown on the top and total copy number is shown on the bottom. Smoothed points (5) for each sample are connected.  The minimal region altered in these five samples is bounded by T14 and T5 (highlighted by the blue bar).   Figure 3.13  Detection of both copy number and allele specific copy number (ASCN). Plots for total copy number and allele specific copy number are displayed above and below, respectively, for each example (A and B).  The total copy number plot represents the sum of the allele specific copy numbers for each array element.  In the allele specific copy number 54  plot, the major allele is represented by red data points and the minor allele is represented by blue data points.  (A) A tumor with neutral total copy number and no allelic imbalance of chromosome 8. (B) A tumor with a large region of copy number loss and allelic imbalance is evident on chromosome 16q.  No examples of an alteration in allele specific copy number without a change in overall copy number (cnLOH/ UPD) were observed.  3.7.3 Detection of copy neutral allelic imbalance by allele specific copy number analysis No regions of cnLOH were identified by comparing the regions identified by ASCN analysis (see Table 3.3) with total copy number analysis (see Table 3.2) in any of the five RB1+/+ samples assessed. Although regions of AI were detected, all 80 AI regions corresponded with an alteration in total copy number. (Table 3.2 and 3.3)  3.8 MYCNA and age of diagnosis The median age of diagnosis for children with unilateral sporadic RB1-/- was 24.0 months (Rushlow, Kennett et al. 2012). The median age of diagnosis for the 11 children with RB1+/+ MYCNA disease analyzed in this thesis work was 4.5 months within a range of 1 to 16 months (Rushlow, Kennett et al. 2012). RB1+/+ MYCNA tumors demonstrate a significantly younger age of diagnosis than their RB1-/- counterparts (pvalue < 10-3; unpaired t-test). This suggests that MYCN amplification may be a useful marker for detection in younger patients.            55  4    Chapter: Discussion and Conclusions  4.1 Novel high level MYCNA identified in RB1+/+ retinoblastoma tumors High-level focal amplification of 2p24.2-25.1 was identified as the most frequent copy number alteration in the RB1+/+ tumors, identified in nearly 50% of these tumors. This thesis work has shown that MYCN is likely the major target gene of the 2p amplification in RB1+/+ retinoblastoma, although other genes in close proximity are frequently co-amplified. Rushlow et al found that MYCNA is associated with overexpression in RB1+/+ tumors (Rushlow, Kennett et al. 2012). MYCN is a member of the myc family of oncogenic TFs amplified in numerous cancer types. Other myc family protein members include c-Myc, and L-Myc. The myc family of genes is involved in regulating key cellular processes including proliferation and differentiation (Mukherjee, Morgenbesser et al. 1992). No other myc family members were found to be amplified in any of the retinoblastoma samples analyzed. The role of MYCNA has best been studied in the context of neuroblastoma, as it is altered in approximately 20% of neuroblastoma tumors and is correlated with poor prognosis. The myc family of proteins bind to DNA influencing gene transcription. Studies to identify transcription targets have yielded numerous RNA and microRNAs and other transcription factors regulated by MYCN. In addition, there are large numbers of MYCN binding sites throughout the genome, many intergenic which have been suggested to play a role in maintaining euchromatin structure in addition to regulating expression of specific genes (Cotterman, Jin et al. 2008). Thus it is difficult to speculate by what mechanism MYCNA may be driving retinoblastoma progression in the absence of RB1 inactivation. Overexpression of MYCN in a neuroblastoma mouse model was capable of initiating the sequence of events leading to neuroblastoma formation in mice, which could be augmented by also inactivating RB1. This suggests that MYCN and RB1 may have overlapping and complimentary tumorigenic functions under certain conditions (Weiss, Aldape et al. 1997).  4.2 RB1+/+ tumors display a lesser proportion of their genome affected by genomic instability than RB1-/- tumors Our analysis has revealed RB1+/+ tumors display smaller regions of copy number alteration but at similar frequencies of alteration when compared with RB1-/- tumors. RB1+/+ 56  MYCNA retinoblastoma tumors had both a significantly lower number of proportion of their genome altered when looking at the number of bps altered as well as fewer frequently altered genes. Interestingly, neuroblastoma tumors with MYCNA have been shown to display a unique genomic signature of 1p deletion, 17q gain but few other genomic rearrangements (Mosse, Diskin et al. 2007). By comparison the genomes of MYCNA retinoblastoma tumors don’t display 1p deletion or 17q gain but have similarly few genomic rearrangements.  In contrast, loss of the RB1 gene has been associated with increasing genomic instability in retinoma and retinoblastoma (Bowles, Corson et al. 2007; Corson and Gallie 2007). The genomes of RB1+/+ tumors without MYCNA displayed an intermediate number of frequently altered genes and were similar to the MCYNA tumors in both the number of alterations and the number of bps altered. These results suggest that RB1+/+ tumors overall exhibit less disruption of bps and genes than their RB1-/- counterpart, and may therefore be less affected by genomic instability. This difference in relative genomic instability might suggest each group may arise through distinct genetic mechanisms from independent genomic backgrounds.  4.3 Genes identified within regions of frequent copy number alteration One of the purposes of this thesis was to identify the most frequent regions of copy number alteration to narrow down genes of potential importance within each tumor group. We identified previously reported frequent regions of copy number alteration in retinoblastoma as frequent in both RB1-/- and RB1+/+ tumors analyzed, for example the 1q gain of KIF14 and MDM4. In addition we identified frequent regions of copy number alteration unique to the RB1+/+ tumors, containing genes such as IRF2BP2 which may play a role in RB1+/+ tumorigenesis in the absence of RB1 gene inactivation. We also identified genes altered frequently in both RB1+/+ tumors and RB1-/- tumors such as the gene CRABP2 also located on chromosome 1 at 1q23.1 which has been shown to be overexpressed in retinoblastoma tumors compared to normal retinas (Mallikarjuna, Sundaram et al. 2010). In addition to being overexpressed in a wide variety of cancers including neuroblastoma, retinoblastoma and Wilms’ tumor. Wilms’ tumor is a childhood kidney cancer in which MYCN is frequently gained and occasionally amplified (Williams, Al-Saadi et al. 2011). In Wilms’ tumor, CRABP2 expression strongly correlates with MYCN expression (Gupta, 57  Kessler et al. 2008). It would be interesting to examine whether CRABP2 expression shows a similar correlation in retinoblastoma. In addition to identifying known and novel regions of frequent alteration within larger regions of common copy number alterations, we also identified more focal regions of novel frequent copy number alteration in the RB1+/+ tumors, such as BASP1 on 5p15.1, which has been implicated in directing cell fate during differentiation, in other cancer types and it has been suggested to play a role as a TSG although our data suggest it may play an oncogenic role in retinoblastoma. The work presented in this thesis, has demonstrated our ability to identify both previously known and novel genes frequently altered at the copy number level. Further investigation into the possible effects of these copy number alterations on expression would be the logical next step. Further work would need to be done to see whether copy number gain correlates with increased expression, as well as cell model work to explore the function of these novel genes in retinoblastoma cells.  4.4 Pathway/ network level analysis It is highly likely that deregulation of multiple genes is involved in retinoblastoma tumorigenesis. Pathway and network level analysis using IPA can add context to the large numbers of genes frequently altered in the RB1-/- and RB1+/+ tumor sets. Top canonical pathways significantly enriched in both gene sets included key cancer related functions and contained known oncogenes and tumor suppressor genes. Networks are another way of identifying known interactions between altered genes which may lead to generation of novel hypotheses. One of the most significant gene networks in the RB1-/- tumors involved the RB1 gene as well as other well known genes important for retinoblastoma tumorigenesis including DEK and members of the E2F gene family (Figure 3.10). Loss of the two TSGs RB1 and OSGIN1 from the network can both lead to uncontrolled cell growth and promote tumor formation. Altered copy number of histone family genes may also act to promote tumorigenesis by altering gene expression activity within a cell. Gain of DEK is associated with multiple cancer types and it has been suggested that one of its functions is to inhibit p53-driven tumor suppressor pathways thereby promoting cell survival (Wise-Draper, Allen et al. 2006). This network shows how 58  many of these genes could function together to promote tumorigenesis, some of which we know to be important for retinoblastoma. In the RB1+/+ gene dataset, one of the most significant networks involved MYCN. It included genes such as DNMT3A, which is involved in de novo methylation, normally occurring during development rather than maintenance methylation in somatic cells. As in other cancers, its reactivation may alter gene expression in favour of tumor development and progression (Zhang, Zhu et al. 2012). NBPF11, one of the few genes lost in the RB1+/+ network has previously been identified as a potential TSG which frequently undergoes LOH in both neuroblastoma and colon cancer. The biological relevance of these pathways to retinoblastoma development and progression represent promising areas of further investigation.  4.5 Genomic regions affected by multiple mechanisms of genetic alteration (copy number change, allelic imbalance, and combination) As retinoblastoma is known to undergo cnLOH at the RB1 locus as a mechanism of TSG inactivation, this led us to investigate whether cnLOH was a prevalent mechanism in the RB1+/+ subset of tumors. However, in our small sample set of five MYCNA RB1+/+ tumors we were unable to identify any occurrences of cnLOH, suggesting that this mechanism may not play a significant role in gene activation or inactivation in these tumors, although a larger cohort is clearly needed to rule out the possibility of cnLOH in RB1+/+ tumors.  4.6 RB1+/- retinoblastoma tumors The RB1+/- group of retinoblastoma tumors may represent a group of tumors from mixed origins. This may be due to the inclusion of tumors which arose via similar mechanisms to either the RB1+/+ and RB1-/- tumors or an intermediate. Some tumors are likely RB1-/- without an identified second mutation and some may represent RB1+/+ tumors which have acquired a subsequent RB1 mutation. The RB1+/- tumors showed a similar proportion of the genome altered as the RB1+/+ tumors, one showed amplification of MYCN and six of the tumors showed moderate gain. It is possible RB1+/- tumor T33 represents an RB1+/+ tumor, based on its early age of diagnosis which may have secondarily developed a mutation in one of the RB1 alleles 59  4.7 Allele specific amplification of MYCN We observed allele specific amplification of MYCN in all five tumor samples assessed by the SNP array. However, as the SNP 6 array is designed to assess SNPs, which often occur outside of genes, no SNP probes occur within the MYCN gene itself. Therefore, surrounding probe information was used to infer preferential amplification of the entire region. If there was a difference between the MYCN alleles in either the gene itself or its regulatory elements, this could represent a selective force with the potential to drive preferential amplification of one allele. In lung cancer allelic imbalance has been shown both to occur equally between mutant and wildtype cases, for example with KRAS, and preferentially for only the mutant case, as in EGFR (Soh, Okumura et al. 2009). Interestingly unlike the frequent MYCNA seen in cancer, Feingold syndrome, a microcephaly entity is primarily caused by MYCN loss of function either by gene deletion or coding-sequence mutation.  This information suggests that mutations in the coding or non-coding regulatory elements of MYCN may exist and would likely be highly cell type and context specific. Further investigation would be of interest to search for potential activating mutations in the MYCN gene in the context of retinoblastoma.  4.8 Conclusions  4.8.1 Characterization of retinoblastoma tumors with known RB1 mutation status Approximately 2% of retinoblastoma tumors lack RB1 mutations, despite the long standing dogma that two-hit inactivation that the TSG RB1 is the initiating event in retinoblastoma tumorigenesis. This thesis work genetically characterizes these newly identified RB1+/+ tumors on a whole-genome scale. High-level amplification of a minimal region on chromosome 2p, containing only the MYCN oncogene, was identified as the most prevalent genetic event in these tumors. This raises the possibility that high-level MYCN oncogene amplification may be capable of driving retinoblastoma tumorigenesis in the absence of RB1 inactivation.  In response to the first hypothesis, both previously reported and novel regions of copy number alteration were identified within the RB1-/-, RB1+/-, and RB1+/+ tumors including key cancer related genes. In addition, the genomes of RB1+/+ tumors appear to be more stable than RB1-/- tumor genomes, by both number of bps and number of genes 60  altered, confirming the second hypothesis.  RB1+/+ tumors share some genomic alterations with RB1-/- tumors, but also display recurrent distinct alterations suggesting they may develop via partially or fully independent genetic mechanisms. It is also possible RB1+/+ and RB1-/- tumors may arise from different cell types within the retina. We were able to identify the most frequently altered regions in RB1+/+ tumors although many common copy number alterations in retinoblastoma involve whole chromosomes or the entire chromosome arm, making candidate gene identification difficult. In an attempt to add some context to the large amount of data generated, pathway and network analysis was performed to identify both canonical pathways and interactions between the genes frequently altered. To address the third hypothesis, although no cnLOH was detected, allelic imbalance affecting multiple genes including the MYCN amplification was identified using the SNP 6.0 arrays, which would have otherwise gone undetected by tiling path aCGH alone.  4.8.2 Possible future research directions This research suggests that reanalyzing previous and new retinoblastoma datasets in light of the information that not all retinoblastomas may be caused by RB1 inactivation will likely identify additional cases of RB1+/+ MYCNA retinoblastoma and expand upon our current understanding of the biology of these tumors. Further work to elucidate through which biological pathways retinoblastoma develops may improve our understanding of how different genetic mechanisms can lead to similar tumors, specifically in children. Cell model work is especially important in studying retinoblastoma and pediatric cancer research due to the relative rarity of patients and therefore low sample availability. Knowing the genetic background of retinoblastoma tumor cell lines is important for predicting and interpreting results of manipulation experiments. Cell lines such as Y79 and Weri-Rb-1 provide useful systems for studying gene expression changes in response to perturbations to genes or pathways to study consequences of gene interactions. As with the MYCNA neuroblastoma mouse models, is would be interesting to determine whether MYCNA is sufficient to drive tumor development or what additional steps are required in formation of a mouse retinoblastoma model. Treating MYCN amplified retinoblastoma cell lines with newly developed MYCN inhibitors, to test their sensitivity would be another interesting potential cell model experiment. 61  Although the RB1 TSG has been shown to exhibit cnLOH, the five RB1+/+ tumor and matched blood normal DNA we analyzed by SNP array demonstrated no cnLOH, suggesting that it does not represent a significant mechanism of gene inactivation. Although our sample set was very small, cnLOH may represent a significant mechanism in other types of retinoblastoma such as the RB1+/- tumors or RB1+/+ tumors without MYCN amplification. Despite the lack of cnLOH, SNP array analysis identified an allelic imbalance in all five MYCNA RB1+/+ tumors examined. Sequencing the MYCN gene and promoter regions to identify any differences between the amplified and non amplified alleles would be interesting to determine if preferential allelic amplification is occurring or if it is just a mechanistic consequence of the amplification itself. It would be interesting to see if cnLOH occurs in RB1+/+ tumors without MYCN amplification. Methylation and expression analysis would also nicely complement the genetic copy number data generated by this study, but is dependent on the availability of appropriate and sufficient material.  4.8.3 Significance Characterization of this unique cohort of RB1+/+ tumors has identified MYCN as a potential diagnostic target by QmPCR for clinical screening in retinoblastoma tumors presenting at an early age. The identification of MYCNA and a lack of RB1 mutation may have implications for genetic counseling as MYCNA is not a germline mutation event. This would suggest that young patients with MYCNA RB1+/+ retinoblastoma may not require aggressive monitoring strategies for secondary tumors and familial screening of relatives may similarly not be necessary. MYCN amplification can currently only be detected after the tumor has been removed, unlike RB1 mutation detection from blood. MYCN targeted therapeutic strategies are being developed for the treatment of other cancer types and may be useful in treating retinoblastoma patients with MYCN amplified tumors which have metastasized. This research emphasizes the need to question even long-standing paradigms for understanding the world around us.    62  References  Canadian Cancer Society (2004): Canadian Cancer Statistics 2004. Toronto, Canada. Canadian Cancer Society/National Cancer Institute of Canada (2008): Canadian Cancer Statistics 2008. Toronto, Canada. Abramson, D. H. (2005). "Retinoblastoma in the 20th century: past success and future challenges the Weisenfeld lecture." Invest Ophthalmol Vis Sci 46(8): 2683-2691. Abramson, D. H., K. Beaverson, et al. (2003). "Screening for retinoblastoma: presenting signs as prognosticators of patient and ocular survival." Pediatrics 112(6 Pt 1): 1248- 1255. Abramson, D. H., C. M. Frank, et al. (1998). "Presenting signs of retinoblastoma." J Pediatr 132(3 Pt 1): 505-508. 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ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E4 G 1 16646685 17141712 495 E19 G 1 16646685 17086056 439 E4 G 1 44122639 44370880 248 E19 G 1 52091543 52349308 258 E4 G 1 88987745 89217390 230 E19 G 1 88444733 88711100 266 E4 G 1 232727671 233010148 282 E19 G 1 142533622 144288841 1755 E4 G 2 14978618 17715047 2736 E19 G 1 149858970 151131174 1272 E4 G 2 24892640 25572843 680 E19 G 1 152795955 153230241 434 E4 G 2 121240000 122362789 1123 E19 G 1 158010337 158996174 986 E4 G 3 49081051 49782857 702 E19 G 1 159680000 160677252 997 E4 G 3 198661730 198819853 158 E19 G 1 162887424 163728359 841 E4 G 5 125838199 126214785 377 E19 G 1 164082233 165430178 1348 E4 G 6 158979778 159379225 399 E19 G 1 173228930 174094669 866 E4 G 7 97475547 98965520 1490 E19 G 1 174639051 175287621 649 E4 G 7 100027063 102036700 2010 E19 G 1 179071822 180163431 1092 E4 G 7 105069977 105602595 533 E19 G 1 182037344 182693973 657 E4 G 7 127398355 127812452 414 E19 G 1 199215670 199653218 438 E4 G 7 127848914 129934305 2085 E19 G 1 199831132 203411834 3581 E4 G 7 131677611 132059225 382 E19 G 1 204118116 208088220 3970 E4 G 7 141206417 142434257 1228 E19 G 1 208781386 209169786 388 E4 G 7 148316114 149799209 1483 E19 G 1 214852240 215267049 415 E4 G 9 45908108 46284566 376 E19 G 1 218273469 218795330 522 E4 G 9 91235105 91864006 629 E19 G 1 219082723 220169719 1087 E4 G 11 61823795 62111938 288 E19 G 1 222753539 223064465 311 E4 G 11 67511350 68042929 532 E19 G 1 226230204 228803178 2573 E4 G 12 2717303 2975966 259 E19 G 1 232727671 233105801 378 E4 G 14 73121067 73575089 454 E19 G 1 235641020 236172564 532 E4 G 14 94620036 95405991 786 E19 G 2 41662 1218689 1177 E4 G 14 96434336 97482889 1049 E19 G 2 1642610 3489907 1847 E4 G 14 98532350 103624847 5092 E19 G 2 3903894 11755093 7851 E4 G 14 104222750 104589570 367 E19 G 2 12063690 23881796 11818 E4 G 15 30328715 30881934 553 E19 G 2 25273765 26864767 1591 74  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E4 G 15 72441859 73217136 775 E19 G 2 27134515 43220834 16086 E4 G 15 76894500 77184857 290 E19 G 2 44824074 52869360 8045 E4 G 16 30539947 31120743 581 E19 G 2 53351511 53831046 480 E4 G 16 33207279 33463485 256 E19 G 2 54256708 55105184 848 E4 G 16 82567035 82963492 396 E19 G 2 56202060 62301010 6099 E4 G 16 88443716 88573088 129 E19 G 2 63797391 70677309 6880 E4 G 17 510963 1628597 1118 E19 G 2 70684434 82714465 12030 E4 G 17 37016754 37707027 690 E19 G 2 83467797 89405301 5938 E4 G 17 56678248 57089085 411 E19 G 4 3645088 4065877 421 E4 G 17 69307909 69931286 623 E19 G 4 67582165 67861860 280 E4 G 20 54443015 54747849 305 E19 G 4 109386058 109668156 282 E4 G 20 60809452 61325197 516 E19 G 5 57285917 57537297 251 E4 G 21 20794703 21052440 258 E19 G 5 85900497 86163409 263 E4 G 21 43761584 44194069 432 E19 G 5 95697117 95852946 156 E4 G 22 21035693 21445636 410 E19 G 5 118943200 119294214 351 E4 L 1 24642063 24852567 211 E19 G 6 73242 45175251 45102 E4 L 2 76560762 77046290 486 E19 G 6 45494917 58849697 13355 E4 L 2 91608685 91680249 72 E19 G 7 57163665 57368235 205 E4 L 2 109113179 109402865 290 E19 G 7 83422963 83649776 227 E4 L 2 158728287 159062951 335 E19 G 10 49935163 50237413 302 E4 L 3 26243462 26651493 408 E19 G 10 87854528 88147135 293 E4 L 3 100412584 100932125 520 E19 G 10 97978547 98240450 262 E4 L 3 190572982 190793756 221 E19 G 11 60661723 60880212 218 E4 L 5 49479231 49913062 434 E19 G 12 51140849 51492452 352 E4 L 5 115399904 115637029 237 E19 G 12 52090606 52327387 237 E4 L 6 58352741 58849697 497 E19 G 12 66859847 67063558 204 E4 L 6 62000083 63102316 1102 E19 G 12 108154779 108581445 427 E4 L 6 64287458 65557020 1270 E19 G 12 114113748 114520437 407 E4 L 6 77454026 77857011 403 E19 G 12 121949281 122155980 207 E4 L 6 86423400 87841029 1418 E19 G 13 43581707 43926080 344 E4 L 6 93274524 93823839 549 E19 G 13 55372389 55655559 283 E4 L 6 95517378 96361943 845 E19 G 13 108629457 109193347 564 E4 L 6 113443711 113802827 359 E19 G 13 110600643 112590314 1990 E4 L 6 160616018 161039688 424 E19 G 14 93699417 94182499 483 E4 L 7 9862060 10928986 1067 E19 G 15 21973970 22386839 413 E4 L 7 11969246 12542008 573 E19 G 15 28106102 28734907 629 E4 L 7 18699485 20220381 1521 E19 G 15 77142292 77855965 714 E4 L 7 57765880 58017280 251 E19 G 18 42657928 42803873 146 E4 L 7 61061239 62176519 1115 E19 G 22 30095758 30360524 265 E4 L 7 66502015 66754917 253 E19 L 3 78394977 78583892 189 E4 L 7 87945541 88499287 554 E19 L 6 160616018 161039688 424 E4 L 7 127648906 127924442 276 E19 L 7 1542959 1737551 195 E4 L 8 16092289 16348466 256 E19 L 11 97278670 97609941 331 E4 L 8 43185803 43912178 726 E19 L 11 125166505 125470718 304 E4 L 8 86699540 87055819 356 E19 L 15 19842977 20071155 228 E4 L 8 132297428 132772696 475 E19 L 16 20943307 21212616 269 E4 L 8 143780694 144006276 226 E19 L 16 33730527 34016785 286 75  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E4 L 9 67812022 68761941 950 E19 L 18 16659756 17277836 618 E4 L 9 90861578 91085013 223 E19 L 22 40739833 41198248 458 E4 L 9 105583846 105969389 386 E22 G 1 143665914 144162829 497 E4 L 9 129150841 129545178 394 E22 G 1 157111728 157249573 138 E4 L 10 41947396 42224061 277 E22 G 3 141103332 141471148 368 E4 L 10 113323636 113623272 300 E22 G 4 134269189 134522870 254 E4 L 11 38072737 38323517 251 E22 G 4 190667654 190733475 66 E4 L 11 58901926 59283911 382 E22 G 5 568397 1013978 446 E4 L 11 97278670 97609941 331 E22 G 5 17284044 17553934 270 E4 L 12 36302222 36786132 484 E22 G 5 71207958 71428448 220 E4 L 12 77277923 77556391 278 E22 G 6 53635140 53820956 186 E4 L 13 73742673 74123958 381 E22 G 7 57163665 57368235 205 E4 L 16 33659117 34016785 358 E22 G 7 141707706 142316803 609 E4 L 17 7088883 7457682 369 E22 G 8 58157391 58393591 236 E4 L 19 50398622 50652469 254 E22 G 9 122056354 122361130 305 E7 G 1 1058909 2545127 1486 E22 G 15 21973970 22386839 413 E7 G 1 21717568 22285488 568 E22 G 15 28106102 28734907 629 E7 G 1 232830875 233010148 179 E22 G 15 30237319 30597338 360 E7 G 2 15182883 17177195 1994 E22 G 16 33280312 33741546 461 E7 G 2 64982297 65322374 340 E22 G 19 47784067 48626174 842 E7 G 3 121682380 121964439 282 E22 G 20 44119293 44472487 353 E7 G 3 137801269 138197862 397 E22 L 5 127204007 127468573 265 E7 G 3 198661730 198819853 158 E22 L 6 13223270 13524298 301 E7 G 4 189018797 189277190 258 E22 L 6 74106063 74476174 370 E7 G 6 213736 1923096 1709 E22 L 7 1542959 1737551 195 E7 G 6 4800956 5673594 873 E22 L 7 61087267 61270796 184 E7 G 6 6705438 7834700 1129 E22 L 7 64729861 65014606 285 E7 G 6 10144863 11419717 1275 E22 L 7 77228817 77535943 307 E7 G 6 23259241 23609362 350 E22 L 11 97278670 97609941 331 E7 G 6 25059720 26850182 1790 E22 L 11 118808228 119073431 265 E7 G 6 30758621 32151955 1393 E22 L 12 32108495 32334573 226 E7 G 6 32872724 36488793 3616 E22 L 14 30416622 31059479 643 E7 G 6 36804059 38315288 1511 E22 L 14 49100288 49484060 384 E7 G 6 39096919 39555660 459 E22 L 16 20943307 21212616 269 E7 G 6 39928065 44057109 4129 E22 L 19 32504109 32613567 109 E7 G 6 158979778 159379225 399 E22 L 21 33637551 34149277 512 E7 G 8 58157391 58393591 236 E22 L 22 17053965 17206783 153 E7 G 9 132643829 132999756 356 E22 L 22 40935061 41198248 263 E7 G 9 136245273 136359804 115 C2 G 1 3519965 3748249 228 E7 G 10 69245834 69639428 394 C2 G 1 16646685 17086056 439 E7 G 11 47710156 48122594 412 C2 G 1 120249993 121077638 828 E7 G 12 48413812 49295862 882 C2 G 1 142533622 144162829 1629 E7 G 12 54361649 54722693 361 C2 G 1 144594746 145133682 539 E7 G 12 97265837 97621020 355 C2 G 1 145511311 145872489 361 E7 G 13 113932258 114093192 161 C2 G 1 149858970 153230241 3371 E7 G 14 68087188 68444627 357 C2 G 1 154247819 157619715 3372 E7 G 14 89221786 90956790 1735 C2 G 1 158010337 158996174 986 76  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E7 G 14 94620036 95405991 786 C2 G 1 159680000 160677252 997 E7 G 14 97186900 104589570 7403 C2 G 1 161016469 161777223 761 E7 G 15 20302216 20627698 325 C2 G 1 162887424 167445692 4558 E7 G 16 923071 2131094 1208 C2 G 1 168402600 171187941 2785 E7 G 17 623188 962070 339 C2 G 1 173228930 174094669 866 E7 G 17 34574333 34955272 381 C2 G 1 174639051 176344936 1706 E7 G 17 55870907 56233848 363 C2 G 1 177764373 178479748 715 E7 G 17 75566340 75862414 296 C2 G 1 179462543 181045400 1583 E7 G 18 25034674 25284594 250 C2 G 1 182037344 182693973 657 E7 G 19 1331124 2630211 1299 C2 G 1 195889657 196341623 452 E7 G 20 5506268 5961085 455 C2 G 1 196594358 198833601 2239 E7 G 20 44981867 45202369 221 C2 G 1 199215670 209169786 9954 E7 G 22 16245214 16594536 349 C2 G 1 212028920 215373858 3345 E7 L 2 91303216 91680249 377 C2 G 1 218406885 220609576 2203 E7 L 2 95774261 96130285 356 C2 G 1 222704527 223152230 448 E7 L 2 96783694 96834810 51 C2 G 1 223993446 233391573 9398 E7 L 2 161249510 161442145 193 C2 G 1 234814970 236172564 1358 E7 L 3 80918434 81176295 258 C2 G 1 237944944 240324819 2380 E7 L 3 163846727 164196155 349 C2 G 1 242294867 244296856 2002 E7 L 3 190572982 190793756 221 C2 G 2 91608685 91680249 72 E7 L 4 7507478 7829767 322 C2 G 2 113424819 113704783 280 E7 L 4 144726984 144928389 201 C2 G 3 52725023 52967443 242 E7 L 5 26139638 26409556 270 C2 G 3 114828518 115117759 289 E7 L 6 58352741 58849697 497 C2 G 4 134269189 134522870 254 E7 L 6 79956399 80258421 302 C2 G 5 568397 1013978 446 E7 L 7 11969246 12200051 231 C2 G 5 16358405 16598168 240 E7 L 7 87945541 88433327 488 C2 G 5 134308327 134619053 311 E7 L 8 7035076 8212432 1177 C2 G 7 57163665 57368235 205 E7 L 8 86699540 87055819 356 C2 G 8 7035076 8212432 1177 E7 L 8 88151299 88551969 401 C2 G 8 58157391 58393591 236 E7 L 8 115440593 115731250 291 C2 G 11 60661723 60880212 218 E7 L 9 66254996 67409630 1155 C2 G 11 132051142 132389201 338 E7 L 9 67803916 70224724 2421 C2 G 13 43481375 43926080 445 E7 L 10 41947396 42224061 277 C2 G 15 18473375 20053874 1580 E7 L 11 96953808 97518886 565 C2 G 15 21973970 22386839 413 E7 L 11 102841892 103697609 856 C2 G 15 28106102 28734907 629 E7 L 11 105035952 106669861 1634 C2 G 15 30237319 30597338 360 E7 L 11 109773406 110120305 347 C2 G 17 20185620 20448380 263 E7 L 11 111537832 113144857 1607 C2 G 19 61714137 62000777 287 E7 L 11 114281321 117260839 2980 C2 L 3 75285656 75515882 230 E7 L 11 118950133 121132876 2183 C2 L 3 78394977 78583892 189 E7 L 11 122460473 127141998 4682 C2 L 4 8979402 9291412 312 E7 L 11 127686192 128232929 547 C2 L 6 13223270 13524298 301 E7 L 11 129706418 134433506 4727 C2 L 7 1499748 1737551 238 E7 L 12 59227752 59583586 356 C2 L 7 77228817 77535943 307 E7 L 15 37191685 37629456 438 C2 L 11 125166505 125470718 304 E7 L 16 15040819 15407666 367 C2 L 12 32108495 32334573 226 77  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E7 L 18 15026396 15320434 294 C2 L 12 53976376 54173283 197 E7 L 19 50398622 50652469 254 C2 L 16 28918888 29239140 320 E7 L 22 17053965 17206783 153 C2 L 16 47457492 50618041 3161 E7 L 22 18401409 19036350 635 C2 L 16 50650921 51434198 783 P2 G 1 9725864 9983505 258 C2 L 16 52550335 56200067 3650 P2 G 1 26950696 27234980 284 C2 L 16 60759769 63642008 2882 P2 G 1 157848491 158603002 755 C2 L 16 64289064 67577655 3289 P2 G 2 8123122 16432161 8309 C2 L 16 69643219 70905058 1262 P2 G 2 28278420 28756000 478 C2 L 16 71288486 73055634 1767 P2 G 2 91608685 91680249 72 C2 L 16 75492204 78760401 3268 P2 G 2 120629322 121861830 1233 C2 L 16 79508959 88573088 9064 P2 G 2 241955282 242720443 765 C2 L 18 25034674 25284594 250 P2 G 3 62915894 63393838 478 C2 L 19 4648531 4966988 318 P2 G 4 27244185 27690451 446 C2 L 19 32504109 32613567 109 P2 G 5 85900497 86206359 306 C2 L 22 17053965 17206783 153 P2 G 5 86432253 86737136 305 E2 G 1 617 3000997 3000 P2 G 5 106184345 106509625 325 E2 G 1 9725864 9983505 258 P2 G 6 82918123 83251931 334 E2 G 1 17239175 19020943 1782 P2 G 7 28368924 28712572 344 E2 G 1 36428818 37442774 1014 P2 G 7 132216136 132737649 522 E2 G 1 145425752 145734554 309 P2 G 8 58157391 58393591 236 E2 G 1 149747994 150399374 651 P2 G 8 101972208 102297846 326 E2 G 1 153361602 155397482 2036 P2 G 9 113559615 113848887 289 E2 G 1 158010337 158739744 729 P2 G 9 138368718 138503912 135 E2 G 1 176619795 176971757 352 P2 G 10 75055 538407 463 E2 G 1 182699897 183127629 428 P2 G 10 28180758 28445060 264 E2 G 1 199091008 203475222 4384 P2 G 10 69245834 69639428 394 E2 G 1 205931802 206903358 972 P2 G 10 73363600 74407034 1043 E2 G 1 223536766 224717804 1181 P2 G 11 118808228 119073431 265 E2 G 1 228205983 229467946 1262 P2 G 12 54120030 54512007 392 E2 G 1 241997888 242642629 645 P2 G 12 62920426 63310916 390 E2 G 1 245436183 245747506 311 P2 G 13 113000386 113414382 414 E2 G 2 925973 1404470 478 P2 G 14 23602843 23978395 376 E2 G 2 1831108 2663054 832 P2 G 15 19970520 20224003 253 E2 G 2 2815042 3266649 452 P2 G 15 28106102 28937648 832 E2 G 2 7369112 7828054 459 P2 G 15 82870937 83217528 347 E2 G 2 8295224 11025497 2730 P2 G 16 1341140 2346105 1005 E2 G 2 15598791 16914958 1316 P2 G 16 70168706 70579581 411 E2 G 2 19690629 21011770 1321 P2 G 16 80253332 80497048 244 E2 G 2 24892640 25572843 680 P2 G 17 623188 962070 339 E2 G 2 28214990 28865200 650 P2 G 17 24695117 25127880 433 E2 G 2 84979184 86465542 1486 P2 G 17 40215472 41357984 1143 E2 G 2 96074237 96378723 304 P2 G 17 41755471 42728503 973 E2 G 2 119880768 121861830 1981 P2 G 17 45294025 45685773 392 E2 G 5 70263 1562826 1493 P2 G 17 54544587 55060070 515 E2 G 5 172799092 173131927 333 P2 G 17 71250082 73457154 2207 E2 G 6 5423679 5673594 250 P2 G 17 73871046 74851642 981 E2 G 6 30758621 32151955 1393 78  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) P2 G 17 75249971 78615238 3365 E2 G 6 32872724 36862995 3990 P2 G 19 3128263 4966988 1839 E2 G 6 39096919 39555660 459 P2 G 19 5172606 6694652 1522 E2 G 6 40472908 44268248 3795 P2 G 19 17824690 18030848 206 E2 G 6 139650310 140003204 353 P2 G 21 46485627 46927776 442 E2 G 6 157097294 159379225 2282 P2 G 22 37632987 37982948 350 E2 G 6 167951793 169148970 1197 P2 G 22 39513597 40036674 523 E2 G 7 2038803 2407575 369 P2 L 1 74725261 74916322 191 E2 G 7 20168369 20427128 259 P2 L 1 180445938 180878805 433 E2 G 7 97475547 100193425 2718 P2 L 2 159197634 159555327 358 E2 G 7 155193677 155735685 542 P2 L 3 30195993 30479678 284 E2 G 7 156529437 157364621 835 P2 L 3 44821716 45416063 594 E2 G 7 158404469 158818116 414 P2 L 5 62884026 63168836 285 E2 G 8 28931963 29117855 186 P2 L 6 160616018 161039688 424 E2 G 8 144140067 145740218 1600 P2 L 8 304176 4307888 4004 E2 G 9 129488250 131906000 2418 P2 L 8 5294980 11933605 6639 E2 G 9 132643829 132999756 356 P2 L 8 12725608 13280883 555 E2 G 9 136245273 136359804 115 P2 L 8 17799615 19423330 1624 E2 G 10 132891454 133271394 380 P2 L 8 20179288 22320777 2141 E2 G 10 133771689 135065876 1294 P2 L 8 22768625 24986910 2218 E2 G 11 47194406 47629247 435 P2 L 8 25235119 27192114 1957 E2 G 11 64048806 65814442 1766 P2 L 8 40646088 40940717 295 E2 G 11 67511350 68042929 532 P2 L 8 86699540 87055819 356 E2 G 12 122604516 124234371 1630 P2 L 9 43012161 43277572 265 E2 G 12 130673016 131701464 1028 P2 L 9 67322055 68460734 1139 E2 G 13 28785074 29182576 398 P2 L 10 41947396 42224061 277 E2 G 13 32954484 33795089 841 P2 L 10 50596911 50869761 273 E2 G 13 110600643 114093192 3493 P2 L 10 77487773 77592293 105 E2 G 14 103814279 104995019 1181 P2 L 16 34750831 35136830 386 E2 G 15 27830649 29601956 1771 P2 L 17 26147289 26497031 350 E2 G 15 30441504 30966443 525 P2 L 19 8934066 9026570 93 E2 G 15 62717015 63392257 675 P2 L 19 55439223 55707294 268 E2 G 15 71764685 72141641 377 P2 L 19 63182936 63654245 471 E2 G 15 76894500 77640945 746 P2 L 20 10711991 11003593 292 E2 G 15 98989163 100309437 1320 P5 G 1 155943888 156178456 235 E2 G 16 44231 1111069 1067 P5 G 1 160338731 160677252 339 E2 G 16 49932229 50295218 363 P5 G 1 232727671 233105801 378 E2 G 16 87260490 87542642 282 P5 G 1 246424085 246944046 520 E2 G 17 0 1628597 1629 P5 G 2 15467410 18682260 3215 E2 G 17 7516212 9162774 1647 P5 G 3 21893268 22110528 217 E2 G 17 27213580 28028278 815 P5 G 5 17284044 17553934 270 E2 G 17 29749613 30490642 741 P5 G 6 19267498 19549037 282 E2 G 17 36547206 44892002 8345 P5 G 6 158979778 159379225 399 E2 G 17 57842004 58351860 510 P5 G 8 58157391 58393591 236 E2 G 17 69307909 73230854 3923 P5 G 14 42674393 42924774 250 E2 G 17 73330751 78615238 5284 P5 G 14 94478987 94732235 253 E2 G 18 62103149 62501278 398 P5 G 15 28106102 28734907 629 E2 G 19 1331124 7667777 6337 79  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) P5 G 15 30237319 30597338 360 E2 G 19 16184022 18500927 2317 P5 G 15 58631770 59207810 576 E2 G 19 52983968 55753488 2770 P5 L 1 9725864 9983505 258 E2 G 20 815899 2441070 1625 P5 L 2 203627906 204011558 384 E2 G 20 3546156 4125081 579 P5 L 3 27391786 27658854 267 E2 G 20 29486920 29933148 446 P5 L 4 140187025 140521672 335 E2 G 20 43980265 44352061 372 P5 L 7 72652466 73120941 468 E2 G 20 45499042 47958110 2459 P5 L 7 84569635 84758710 189 E2 G 20 54011886 57461196 3449 P5 L 7 134815645 135104307 289 E2 G 20 58741174 62435964 3695 P5 L 9 37692836 37940151 247 E2 G 21 20794703 21052440 258 P5 L 10 69883760 70127694 244 E2 G 21 44701920 45428172 726 P5 L 11 97278670 97609941 331 E2 G 21 45569817 46927776 1358 P5 L 11 110813193 111560892 748 E2 G 22 16245214 16911630 666 P5 L 12 27658181 27873830 216 E2 G 22 17053965 18549230 1495 P5 L 12 115508956 115883865 375 E2 G 22 28360809 30166633 1806 P5 L 12 117025244 117328738 303 E2 G 22 35408079 41130527 5722 P5 L 14 49100288 49484060 384 E2 G 22 41150627 49534385 8384 P5 L 15 56648904 57266624 618 E2 L 1 5415955 5868138 452 P5 L 15 91086545 91292369 206 E2 L 1 195691334 196060227 369 P5 L 17 2442274 2767054 325 E2 L 1 196414499 197270713 856 P5 L 17 38288035 38502716 215 E2 L 2 55836372 56202049 366 P5 L 20 29297109 29933148 636 E2 L 2 96783694 96834810 51 E26 G 1 16646685 17086056 439 E2 L 2 109113179 109402865 290 E26 G 1 142533622 144162829 1629 E2 L 2 158728287 159062951 335 E26 G 1 145511311 145872489 361 E2 L 2 176134627 176383225 249 E26 G 1 149858970 151131174 1272 E2 L 2 210844152 211656085 812 E26 G 1 152160475 153230241 1070 E2 L 3 5346982 5765115 418 E26 G 1 154247819 157619715 3372 E2 L 3 18886339 19184259 298 E26 G 1 158010337 158487941 478 E2 L 3 23277687 23709450 432 E26 G 1 159680000 160677252 997 E2 L 3 26243462 27403349 1160 E26 G 1 161016469 161777223 761 E2 L 3 28659721 30479678 1820 E26 G 1 163384213 167381584 3997 E2 L 3 37346489 37800287 454 E26 G 1 169651394 171187941 1537 E2 L 3 68278236 68785539 507 E26 G 1 173228930 174094669 866 E2 L 3 69383522 71190722 1807 E26 G 1 174639051 177140954 2502 E2 L 3 78394977 79284456 889 E26 G 1 177764373 178479748 715 E2 L 3 83375589 83859392 484 E26 G 1 179462543 181045400 1583 E2 L 3 88478268 90318890 1841 E26 G 1 181292771 181831357 539 E2 L 3 95948765 96367298 419 E26 G 1 182037344 182204185 167 E2 L 3 100412584 100932125 520 E26 G 1 187861212 188252302 391 E2 L 3 103643599 104222284 579 E26 G 1 195889657 196341623 452 E2 L 3 105677908 105966136 288 E26 G 1 199215670 208789026 9573 E2 L 3 110593448 111713202 1120 E26 G 2 1438693 3489907 2051 E2 L 3 113133331 113548487 415 E26 G 2 3903894 9407162 5503 E2 L 3 132236080 132563562 327 E26 G 2 10828736 11755093 926 E2 L 3 166061278 169039899 2979 E26 G 2 13120558 17177195 4057 E2 L 3 188927793 189175790 248 E26 G 2 20739093 23881796 3143 E2 L 4 7507478 7829767 322 80  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E26 G 2 29024702 31254640 2230 E2 L 4 13328765 13703700 375 E26 G 2 36371522 37433458 1062 E2 L 4 18407291 19814983 1408 E26 G 2 39691420 42547144 2856 E2 L 4 22415257 23718996 1304 E26 G 2 44824074 46285231 1461 E2 L 4 63527542 64522776 995 E26 G 2 47479400 48415259 936 E2 L 4 65613252 68082157 2469 E26 G 2 50448852 51101232 652 E2 L 4 68980297 71380753 2400 E26 G 2 54385409 55105184 720 E2 L 4 78267102 78501811 235 E26 G 2 56202060 60777537 4575 E2 L 4 96537113 97073537 536 E26 G 2 64170018 68144882 3975 E2 L 4 98579930 99945045 1365 E26 G 2 73670503 75142707 1472 E2 L 4 107706636 108057925 351 E26 G 2 217405254 218556881 1152 E2 L 4 111775984 113738861 1963 E26 G 5 17284044 17553934 270 E2 L 4 120125759 121956556 1831 E26 G 5 61430081 61656696 227 E2 L 4 128037982 128508036 470 E26 G 5 85532315 85754045 222 E2 L 4 158607566 159783789 1176 E26 G 5 86471625 86737136 266 E2 L 5 50839218 51198402 359 E26 G 6 26747302 27094077 347 E2 L 5 66790934 67144270 353 E26 G 6 158979778 159379225 399 E2 L 5 75006893 75588705 582 E26 G 7 57163665 57368235 205 E2 L 5 92624264 92873101 249 E26 G 7 83422963 83649776 227 E2 L 5 107679112 107863907 185 E26 G 7 125777779 126485305 708 E2 L 5 122816388 123306424 490 E26 G 7 141622813 141981029 358 E2 L 5 152030340 152413822 383 E26 G 8 58157391 58393591 236 E2 L 6 57940311 58849697 909 E26 G 8 133939247 135765484 1826 E2 L 6 62000083 63102316 1102 E26 G 8 145725741 146208093 482 E2 L 6 149900596 150405281 505 E26 G 10 28063255 28308281 245 E2 L 7 11526253 11842183 316 E26 G 10 48361074 49216977 856 E2 L 7 61061239 62176519 1115 E26 G 12 114113748 114564982 451 E2 L 8 47568877 47901818 333 E26 G 15 18272238 19353919 1082 E2 L 8 60811916 61320284 508 E26 G 15 20588181 20967788 380 E2 L 8 62881808 63455889 574 E26 G 15 21973970 22386839 413 E2 L 8 64395580 64915161 520 E26 G 15 28106102 28734907 629 E2 L 8 71613225 72050929 438 E26 G 15 30006525 30597338 591 E2 L 8 77791301 78175516 384 E26 G 16 33280312 33741546 461 E2 L 8 92229960 92957538 728 E26 G 17 623188 962070 339 E2 L 8 116696615 117061503 365 E26 G 21 20794703 21052440 258 E2 L 8 119798295 120752324 954 E26 G 21 28557641 28948912 391 E2 L 8 143780694 144006276 226 E26 L 2 1042562 1404470 362 E2 L 9 937155 1901823 965 E26 L 2 12063690 12541039 477 E2 L 9 7418505 9455003 2036 E26 L 2 18946602 20796325 1850 E2 L 9 14270695 14729902 459 E26 L 3 78394977 78583892 189 E2 L 9 66254996 66661410 406 E26 L 3 114814923 115117759 303 E2 L 9 67812022 68974225 1162 E26 L 3 170074359 170242091 168 E2 L 9 82142726 82817108 674 E26 L 3 198952699 199276959 324 E2 L 9 90861578 91085013 223 E26 L 4 8979402 9291412 312 E2 L 9 124184461 124626662 442 E26 L 6 13223270 13577224 354 E2 L 10 32785206 33316028 531 E26 L 6 76244437 76653060 409 E2 L 10 37069462 37447817 378 E26 L 6 160616018 161039688 424 E2 L 11 48090650 51464991 3374 81  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E26 L 7 1499748 1737551 238 E2 L 11 54712078 55479236 767 E26 L 7 84569635 84758710 189 E2 L 11 57506078 58635415 1129 E26 L 8 764689 2926879 2162 E2 L 11 62532845 62994414 462 E26 L 8 3329536 4564649 1235 E2 L 11 96953808 97518886 565 E26 L 8 5449846 6551195 1101 E2 L 12 33000844 34082900 1082 E26 L 8 6741630 11933605 5192 E2 L 12 61666609 62334361 668 E26 L 8 14316570 15321874 1005 E2 L 12 85650592 86993917 1343 E26 L 8 17007797 18508401 1501 E2 L 12 89788969 90323411 534 E26 L 11 69544401 70008693 464 E2 L 14 27311281 28580622 1269 E26 L 11 76815285 77212623 397 E2 L 14 38967497 40587410 1620 E26 L 11 97154277 97609941 456 E2 L 14 47542813 48244496 702 E26 L 12 32108495 32334573 226 E2 L 15 79676203 79953718 278 E26 L 13 51777911 51987426 210 E2 L 16 29181450 29392366 211 E26 L 15 19842977 20071155 228 E2 L 16 34133153 34490212 357 E26 L 16 20943307 21212616 269 E2 L 16 45866013 46748378 882 E26 L 16 28918888 29239140 320 E2 L 16 49649051 50083079 434 E26 L 16 50079561 56200067 6121 E2 L 16 52550335 52874597 324 E26 L 16 62466723 63724558 1258 E2 L 17 21762537 22154806 392 E26 L 16 64289064 73888135 9599 E2 L 17 22287134 22595733 309 E26 L 16 75492204 78760401 3268 E2 L 17 73158376 73381889 224 E26 L 16 78991561 79358404 367 E2 L 18 18245092 18758389 513 E26 L 16 79508959 84688207 5179 E2 L 18 23662330 24490809 828 E26 L 16 85109626 88573088 3463 E2 L 18 64918775 65354592 436 E26 L 17 810744 2937807 2127 E2 L 19 23775088 24178465 403 E26 L 17 4272135 5025418 753 E2 L 19 50398622 50652469 254 E26 L 17 7288226 14613783 7326 E2 L 20 12243506 12915543 672 E26 L 17 16857007 17246408 389 E2 L 20 21691719 22141882 450 E26 L 17 32864722 33700089 835 E28 G 1 5872056 6741408 869 E26 L 18 25034674 25284594 250 E28 G 1 9725864 9983505 258 E26 L 19 32504109 32613567 109 E28 G 1 52141199 53007168 866 E26 L 21 33637551 33992274 355 E28 G 1 63512406 63808021 296 E26 L 22 40935061 41198248 263 E28 G 1 81845355 82146337 301 C1 G 1 116637869 116921215 283 E28 G 1 91947505 92353749 406 C1 G 1 155943888 156178456 235 E28 G 1 148114990 149276757 1162 C1 G 1 162887424 163728359 841 E28 G 1 188722502 189134568 412 C1 G 1 170814706 171187941 373 E28 G 1 204625506 204975414 350 C1 G 1 173228930 174094669 866 E28 G 2 7542268 8181289 639 C1 G 1 174639051 175287621 649 E28 G 2 24892640 26124952 1232 C1 G 1 179462543 179933730 471 E28 G 2 27134515 27528584 394 C1 G 1 200319051 200612614 294 E28 G 2 47752592 48098425 346 C1 G 1 202255347 203411834 1156 E28 G 2 55550156 55858798 309 C1 G 1 206141166 208088220 1947 E28 G 2 84979184 85274881 296 C1 G 1 232727671 233105801 378 E28 G 2 203627906 204180576 553 C1 G 2 14810177 18795403 3985 E28 G 3 48536891 49782857 1246 C1 G 2 36371522 36670189 299 E28 G 3 103294167 103630934 337 C1 G 2 46355058 46727455 372 E28 G 3 128806897 128962916 156 C1 G 2 71611728 72107211 495 E28 G 3 151447608 151897698 450 82  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) C1 G 2 141973184 142221989 249 E28 G 3 155644236 156012945 369 C1 G 2 145486871 145902063 415 E28 G 3 186383958 186768122 384 C1 G 2 159150060 159359345 209 E28 G 4 91190679 91570695 380 C1 G 2 207584984 208085679 501 E28 G 4 124735415 125231515 496 C1 G 3 141103332 141588859 486 E28 G 4 140187025 140521672 335 C1 G 5 68951921 69239886 288 E28 G 4 170275508 170915624 640 C1 G 6 158979778 159379225 399 E28 G 5 57340044 57607852 268 C1 G 7 131677611 132059225 382 E28 G 5 78205814 78691735 486 C1 G 7 141571927 143131253 1559 E28 G 5 81026543 81370252 344 C1 G 7 148451510 149799209 1348 E28 G 5 85900497 86206359 306 C1 G 7 150112723 151323222 1210 E28 G 5 180284904 180610179 325 C1 G 7 153229741 153591526 362 E28 G 6 73242 39555660 39482 C1 G 7 154182039 155735685 1554 E28 G 6 39928065 45175251 5247 C1 G 7 156304214 158818116 2514 E28 G 6 45632263 57181727 11549 C1 G 8 58157391 58393591 236 E28 G 6 147305808 147798957 493 C1 G 11 60661723 60880212 218 E28 G 7 44733999 45119616 386 C1 G 11 132424829 133314615 890 E28 G 7 104656015 105001474 345 C1 G 11 133659897 134433506 774 E28 G 7 156780334 157071420 291 C1 G 14 42674393 42924774 250 E28 G 7 157782375 158185681 403 C1 G 15 18272238 19353919 1082 E28 G 8 21708071 23077984 1370 C1 G 15 28106102 28734907 629 E28 G 8 58157391 58393591 236 C1 G 15 30237319 30597338 360 E28 G 8 144602260 145876186 1274 C1 G 15 41491024 41851765 361 E28 G 9 36088595 36539166 451 C1 G 15 86274969 86865897 591 E28 G 9 124565721 125173997 608 C1 G 17 623188 962070 339 E28 G 9 126474147 126805467 331 C1 G 18 60562274 65581946 5020 E28 G 9 138899424 139826635 927 C1 G 18 66303540 67415521 1112 E28 G 10 5764881 6282946 518 C1 L 1 8634114 9057702 424 E28 G 10 104813634 105172292 359 C1 L 1 10826432 14457649 3631 E28 G 11 50991 1776138 1725 C1 L 1 15428321 16167785 739 E28 G 11 3573460 3835784 262 C1 L 1 19297297 20082385 785 E28 G 11 10327230 10641310 314 C1 L 1 24642063 24852567 211 E28 G 11 47194406 47550302 356 C1 L 1 26424669 27049576 625 E28 G 11 57344375 57551130 207 C1 L 2 18946602 19624269 678 E28 G 11 60661723 61724402 1063 C1 L 3 114828518 115117759 289 E28 G 11 118808228 119073431 265 C1 L 4 30307070 33114191 2807 E28 G 12 52090606 52382298 292 C1 L 4 33836648 35289377 1453 E28 G 12 56288136 56704759 417 C1 L 4 35567290 35995977 429 E28 G 12 66689909 67063558 374 C1 L 4 36170117 37236400 1066 E28 G 12 108154779 108581445 427 C1 L 4 140187025 140521672 335 E28 G 12 118937352 121720520 2783 C1 L 5 80392139 80686277 294 E28 G 13 59352443 59638075 286 C1 L 7 50158388 50617602 459 E28 G 14 19149713 19485285 336 C1 L 8 86620595 87055819 435 E28 G 14 34144592 34709087 564 C1 L 9 96096087 96322935 227 E28 G 14 83790255 84134919 345 C1 L 10 88056805 88348196 291 E28 G 15 21973970 22386839 413 C1 L 11 97278670 97609941 331 E28 G 15 28106102 28734907 629 C1 L 12 110370511 110713626 343 E28 G 15 28918531 29261049 343 83  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) C1 L 14 49100288 49484060 384 E28 G 15 88088780 88498667 410 C1 L 15 36223897 36476298 252 E28 G 16 44231 1901486 1857 C1 L 16 20943307 21212616 269 E28 G 16 2425578 3680499 1255 C1 L 16 33659117 34016785 358 E28 G 16 29195478 29881418 686 C1 L 17 810744 2767054 1956 E28 G 16 79375882 79829612 454 C1 L 17 3216580 4200549 984 E28 G 17 24695117 25127880 433 C1 L 17 4687532 5025418 338 E28 G 17 69307909 69931286 623 C1 L 17 5418209 9192837 3775 E28 G 17 76498848 78036762 1538 C1 L 17 9276838 13532268 4255 E28 G 19 1331124 6359433 5028 C1 L 17 14415762 16804346 2389 E28 G 19 9105583 11860892 2755 C1 L 17 17035866 20236821 3201 E28 G 19 40402918 41316776 914 C1 L 19 55554013 55707294 153 E28 G 19 53658159 54819578 1161 RB522 G 1 16646685 17086056 439 E28 G 19 57249575 57517224 268 RB522 G 1 96864050 97110652 247 E28 G 20 3729010 4125081 396 RB522 G 1 142533622 153692169 11159 E28 G 20 26104051 26233197 129 RB522 G 1 154247819 157619715 3372 E28 G 20 34785437 35853341 1068 RB522 G 1 158010337 158996174 986 E28 G 20 48465589 49157995 692 RB522 G 1 159680000 160677252 997 E28 G 22 43225305 43569976 345 RB522 G 1 161016469 167381584 6365 E28 L 1 4220468 4552378 332 RB522 G 1 168402600 171187941 2785 E28 L 1 5504245 5868138 364 RB522 G 1 173228930 174094669 866 E28 L 1 83905739 84192189 286 RB522 G 1 174639051 176344936 1706 E28 L 2 79166395 79443127 277 RB522 G 1 177945800 181571274 3625 E28 L 2 83394849 83668734 274 RB522 G 1 182037344 185060852 3024 E28 L 2 91608685 91680249 72 RB522 G 1 196594358 227844070 31250 E28 L 2 94689898 95017226 327 RB522 G 1 228205983 233440847 5235 E28 L 2 103819868 104173214 353 RB522 G 1 233834184 242043970 8210 E28 L 2 159197634 159555327 358 RB522 G 1 243223806 244296856 1073 E28 L 2 211144204 211423562 279 RB522 G 1 245724640 246944046 1219 E28 L 3 78394977 78583892 189 RB522 G 2 8123122 8616192 493 E28 L 3 96819091 97228441 409 RB522 G 2 14051860 18347973 4296 E28 L 3 114906026 115117759 212 RB522 G 2 66790913 67153990 363 E28 L 3 140772863 141053191 280 RB522 G 2 102712967 103110650 398 E28 L 3 155328424 155471419 143 RB522 G 3 43597989 43934882 337 E28 L 3 163846727 164196155 349 RB522 G 3 49579895 49782857 203 E28 L 4 8979402 9463330 484 RB522 G 3 52725023 52967443 242 E28 L 4 141770131 142199996 430 RB522 G 3 129385338 129987663 602 E28 L 4 157491341 158162936 672 RB522 G 3 141103332 141588859 486 E28 L 5 63219983 63557236 337 RB522 G 4 1110971 1847477 737 E28 L 5 80232861 80567551 335 RB522 G 4 8073005 8427133 354 E28 L 5 89942881 90441374 498 RB522 G 4 8617326 8780507 163 E28 L 5 170231354 170505998 275 RB522 G 4 9282726 9841887 559 E28 L 5 174498664 174812270 314 RB522 G 4 49189866 49416673 227 E28 L 6 149876461 150732866 856 RB522 G 4 138441550 138878456 437 E28 L 6 169542240 169662983 121 RB522 G 5 12704139 13007203 303 E28 L 6 170655612 170880179 225 RB522 G 5 17284044 17553934 270 E28 L 7 8197110 8445226 248 RB522 G 5 86471625 86737136 266 E28 L 7 57627188 58017280 390 84  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) RB522 G 5 134145577 134619053 473 E28 L 7 88304685 88632733 328 RB522 G 5 168095482 168363001 268 E28 L 7 158536720 158818116 281 RB522 G 5 170936139 171218064 282 E28 L 8 86699540 87055819 356 RB522 G 6 73242 3890080 3817 E28 L 8 144157451 144660691 503 RB522 G 6 11402225 11936033 534 E28 L 9 67803916 68460734 657 RB522 G 6 39096919 39555660 459 E28 L 10 38650109 39116217 466 RB522 G 6 158979778 159379225 399 E28 L 10 41947396 42224061 277 RB522 G 7 83422963 83649776 227 E28 L 10 50519291 50869761 350 RB522 G 7 125777779 127812452 2035 E28 L 10 77487773 78116466 629 RB522 G 7 128232689 132521810 4289 E28 L 10 88945287 89583299 638 RB522 G 7 132692815 138997094 6304 E28 L 11 39773284 40041456 268 RB522 G 7 140395448 145320187 4925 E28 L 11 50230022 51421925 1192 RB522 G 7 147385243 151584725 4199 E28 L 13 71226333 71704903 479 RB522 G 7 152768219 156313401 3545 E28 L 14 32846307 33407161 561 RB522 G 7 156529437 158818116 2289 E28 L 14 86151504 86629578 478 RB522 G 8 36943 179853 143 E28 L 15 18272238 18543505 271 RB522 G 8 6515253 6907559 392 E28 L 15 91211800 91567261 355 RB522 G 8 58157391 58393591 236 E28 L 18 12806854 13066991 260 RB522 G 11 60661723 60880212 218 E28 L 19 63079488 63476569 397 RB522 G 11 78123405 93639762 15516 E28 L 20 10980066 11362367 382 RB522 G 11 94101838 97448007 3346 E28 L 20 26009233 26227775 219 RB522 G 11 99935229 106974429 7039 E28 L 21 22979811 23404186 424 RB522 G 11 108149766 124646260 16496 E20 G 1 5795529 6741408 946 RB522 G 12 12941004 13750814 810 E20 G 1 78738027 79075345 337 RB522 G 12 51140849 51672891 532 E20 G 1 143665914 143923232 257 RB522 G 12 107204119 107495724 292 E20 G 1 204625506 204975414 350 RB522 G 12 130310595 130519529 209 E20 G 2 41662 1218689 1177 RB522 G 13 58396143 58861408 465 E20 G 2 1438693 2178036 739 RB522 G 14 19149713 19360529 211 E20 G 2 2444142 4615940 2172 RB522 G 14 42674393 42924774 250 E20 G 2 4831746 13719545 8888 RB522 G 14 85052158 85476972 425 E20 G 2 15716583 23881796 8165 RB522 G 14 93699417 95801714 2102 E20 G 2 24892640 31254640 6362 RB522 G 14 96434336 99232080 2798 E20 G 2 36371522 39527609 3156 RB522 G 15 18473375 20338507 1865 E20 G 2 41880246 42508730 628 RB522 G 15 41491024 41851765 361 E20 G 2 42852543 44472090 1620 RB522 G 15 71764685 72164226 400 E20 G 2 44824074 48731932 3908 RB522 G 16 20137044 20628545 492 E20 G 2 50085577 51219186 1134 RB522 G 16 33280312 33741546 461 E20 G 2 54239177 55438372 1199 RB522 G 17 623188 962070 339 E20 G 2 60376763 62598530 2222 RB522 G 17 56678248 57089085 411 E20 G 2 64300715 66156086 1855 RB522 G 17 74090777 74851642 761 E20 G 2 66790913 71135781 4345 RB522 G 19 47284211 47405380 121 E20 G 3 49937934 50482968 545 RB522 G 20 61041281 61325197 284 E20 G 3 155644236 156012945 369 RB522 G 22 14410042 14647683 238 E20 G 4 7937374 8780507 843 RB522 L 1 25351676 25650204 299 E20 G 4 54822121 55235676 414 RB522 L 2 91608685 91680249 72 E20 G 5 19701106 20176670 476 RB522 L 2 109113179 110005502 892 E20 G 5 50423152 50897952 475 85  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) RB522 L 3 36111427 36427114 316 E20 G 5 85900497 86206359 306 RB522 L 4 140187025 140521672 335 E20 G 5 95697117 95852946 156 RB522 L 5 68311858 69102023 790 E20 G 6 73242 20763610 20690 RB522 L 6 77071746 77347942 276 E20 G 6 21122117 24801727 3680 RB522 L 8 86620595 87055819 435 E20 G 6 25059720 29123783 4064 RB522 L 10 64553287 64995092 442 E20 G 6 29457137 39555660 10099 RB522 L 11 476030 23649364 23173 E20 G 6 39928065 44815780 4888 RB522 L 11 24020342 32382584 8362 E20 G 6 45632263 53956516 8324 RB522 L 12 106476666 106770408 294 E20 G 6 56250565 57181727 931 RB522 L 12 117025244 117328738 303 E20 G 7 48717014 49076562 360 RB522 L 17 0 695461 695 E20 G 8 49137279 49417714 280 RB522 L 17 1542071 4200549 2658 E20 G 8 54928152 55191935 264 RB522 L 17 4687532 5025418 338 E20 G 9 36088595 36774290 686 RB522 L 17 5418209 18110985 12693 E20 G 9 96551213 97039243 488 RB522 L 18 16659756 17277836 618 E20 G 9 138368718 138919624 551 RB522 L 19 55554013 55707294 153 E20 G 10 24794390 25038334 244 T42 G 1 27020386 27234980 215 E20 G 11 2055967 3183753 1128 T42 G 2 7267176 9407162 2140 E20 G 11 60661723 60880212 218 T42 G 2 14468368 17494103 3026 E20 G 11 79872816 80194218 321 T42 G 2 22667637 23428639 761 E20 G 12 19062093 19403874 342 T42 G 2 30874076 31142648 269 E20 G 12 31699805 31917245 217 T42 G 2 36371522 36799901 428 E20 G 12 52090606 52327387 237 T42 G 2 44563993 45938104 1374 E20 G 12 115064470 115347630 283 T42 G 2 97026359 98604893 1579 E20 G 12 129125980 129776292 650 T42 G 2 132821463 134996036 2175 E20 G 13 58515152 58861408 346 T42 G 2 140471942 140728943 257 E20 G 13 63561919 64068098 506 T42 G 2 201110343 201426850 317 E20 G 14 64383439 64555988 173 T42 G 2 222493290 222928085 435 E20 G 15 20302216 20627698 325 T42 G 3 108016085 108230341 214 E20 G 15 21973970 22386839 413 T42 G 4 8655779 9129871 474 E20 G 15 30006525 30597338 591 T42 G 5 68951921 70342024 1390 E20 G 15 62785585 63049771 264 T42 G 5 180195851 180444136 248 E20 G 15 75603709 75885169 281 T42 G 6 62000083 62065546 65 E20 G 15 83040659 83217528 177 T42 G 6 158818189 159379225 561 E20 G 16 4441455 4947778 506 T42 G 9 10178412 10618299 440 E20 G 16 88443716 88573088 129 T42 G 11 60661723 60880212 218 E20 G 17 623188 1865526 1242 T42 G 11 131887972 132151114 263 E20 G 17 7205406 8079234 874 T42 G 13 63561919 64068098 506 E20 G 17 16624310 17035446 411 T42 G 14 49448838 57610292 8161 E20 G 17 74307357 74959077 652 T42 G 14 58300168 62987077 4687 E20 G 18 25034674 25284594 250 T42 G 14 63455662 71665768 8210 E20 G 19 2928774 3354214 425 T42 G 14 71880540 87964669 16084 E20 G 19 5172606 8832847 3660 T42 G 14 88433473 106318374 17885 E20 G 19 10040796 11860892 1820 T42 G 15 30006525 30597338 591 E20 G 19 18121106 18643264 522 T42 G 17 623188 962070 339 E20 G 19 18873669 19630711 757 T42 G 18 42657928 42840984 183 E20 G 20 4484372 5049159 565 T42 G 19 8629145 8832847 204 E20 G 20 31716051 32417420 701 86  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T42 G 20 33618231 33841512 223 E20 G 22 43225305 43569976 345 T42 G 21 19095584 19368540 273 E20 L 1 12724432 13569886 845 T42 L 1 25351676 25650204 299 E20 L 1 104202532 104657658 455 T42 L 2 11750125 13079174 1329 E20 L 2 42339545 42688302 349 T42 L 2 14137447 14464191 327 E20 L 2 211144204 211423562 279 T42 L 2 17417308 18347973 931 E20 L 3 44892353 45416063 524 T42 L 2 18946602 21011770 2065 E20 L 3 78394977 78583892 189 T42 L 2 229910358 230310424 400 E20 L 3 114906026 115117759 212 T42 L 2 237043334 239819601 2776 E20 L 5 12026212 12326896 301 T42 L 2 239997825 241063385 1066 E20 L 5 20112325 20653881 542 T42 L 3 140772863 141053191 280 E20 L 5 30331484 30610845 279 T42 L 6 13223270 13577224 354 E20 L 5 98983920 99378879 395 T42 L 7 150155467 150543808 388 E20 L 6 57571522 57829665 258 T42 L 8 58157391 58393591 236 E20 L 6 103057262 103422050 365 T42 L 8 86724325 87055819 331 E20 L 6 149900596 150732866 832 T42 L 10 4260820 4547435 287 E20 L 6 160616018 161039688 424 T42 L 12 47207929 47466311 258 E20 L 7 1499748 1737551 238 T42 L 15 19842977 20071155 228 E20 L 7 18699485 19259911 560 T42 L 15 21945545 22386839 441 E20 L 7 50158388 50617602 459 T42 L 19 32504109 32613567 109 E20 L 7 57627188 58017280 390 T5 G 1 16646685 17086056 439 E20 L 7 61061239 62176519 1115 T5 G 1 142533622 145133682 2600 E20 L 7 158298447 158687174 389 T5 G 1 145511311 147083700 1572 E20 L 8 6741630 8080568 1339 T5 G 1 150310886 158487941 8177 E20 L 8 43185803 43912178 726 T5 G 1 158758725 163430309 4672 E20 L 8 85434907 87055819 1621 T5 G 1 174639051 176344936 1706 E20 L 8 91756147 92017226 261 T5 G 1 179462543 187248945 7786 E20 L 10 38650109 39116217 466 T5 G 1 187522092 188584062 1062 E20 L 10 50596911 50869761 273 T5 G 1 189269177 189714992 446 E20 L 10 54832926 55256917 424 T5 G 1 190496532 194595123 4099 E20 L 10 77487773 77592293 105 T5 G 1 196195485 214702922 18507 E20 L 11 3414002 3661668 248 T5 G 1 215473404 215816541 343 E20 L 11 10665713 10755802 90 T5 G 1 216564705 220169719 3605 E20 L 11 34772104 35267094 495 T5 G 1 221391322 242043970 20653 E20 L 11 50230022 51302042 1072 T5 G 1 243722287 244912908 1191 E20 L 11 125166505 125470718 304 T5 G 1 246181048 246944046 763 E20 L 12 32108495 32334573 226 T5 G 2 5090758 5891801 801 E20 L 12 46772772 47154491 382 T5 G 2 15467410 16651868 1184 E20 L 13 41211420 41424564 213 T5 G 2 21054372 21296906 243 E20 L 16 33659117 33927835 269 T5 G 2 51035350 51768164 733 E20 L 18 12806854 13135104 328 T5 G 2 78140908 78929009 788 E20 L 18 15026396 15320434 294 T5 G 2 87512069 87846427 334 E20 L 19 55554013 55707294 153 T5 G 2 103493167 104173214 680 E20 L 20 939429 2441070 1502 T5 G 2 144430193 144816425 386 P4 G 1 16646685 17086056 439 T5 G 2 145486871 145902063 415 P4 G 1 116927827 117385135 457 T5 G 2 191147109 191549049 402 P4 G 1 143665914 143791318 125 T5 G 2 222240742 222928085 687 P4 G 1 232830875 233105801 275 87  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T5 G 3 82058696 83273329 1215 P4 G 3 75285656 75515882 230 T5 G 3 85873782 86189057 315 P4 G 4 8617326 8780507 163 T5 G 3 109747007 109971516 225 P4 G 5 17284044 17553934 270 T5 G 3 114906026 115117759 212 P4 G 5 49960569 50290883 330 T5 G 3 130702173 131070392 368 P4 G 5 71207958 71428448 220 T5 G 3 165585035 165967707 383 P4 G 5 134642615 134881601 239 T5 G 3 188200702 188562337 362 P4 G 6 73242 346084 273 T5 G 3 190572724 190902546 330 P4 G 14 19149713 19485285 336 T5 G 4 8073005 8427133 354 P4 G 15 21973970 22386839 413 T5 G 4 13472033 13770492 298 P4 G 15 28106102 28734907 629 T5 G 4 48771496 49210770 439 P4 G 15 30006525 30597338 591 T5 G 4 117123171 117838327 715 P4 G 16 33280312 33741546 461 T5 G 4 126043717 126776025 732 P4 G 20 53300231 54270330 970 T5 G 4 151664080 152203871 540 P4 G 22 17053965 17206783 153 T5 G 5 17284044 17553934 270 P4 L 3 78394977 78583892 189 T5 G 5 51281449 51628988 348 P4 L 3 114828518 115117759 289 T5 G 5 86471625 86737136 266 P4 L 4 8979402 9291412 312 T5 G 5 97652683 97856979 204 P4 L 6 13223270 13524298 301 T5 G 5 180195851 180444136 248 P4 L 12 32108495 32334573 226 T5 G 6 48501263 48963512 462 P4 L 15 19884809 20071155 186 T5 G 6 49184078 49461459 277 P4 L 18 12806854 13135104 328 T5 G 6 54592590 54729884 137 P4 L 22 40935061 41198248 263 T5 G 6 65932130 66468315 536 E24 G 1 16638721 17086056 447 T5 G 6 79052729 79753932 701 E24 G 1 120249993 120932201 682 T5 G 6 81288115 81664223 376 E24 G 1 142533622 144162829 1629 T5 G 6 152028464 152461187 433 E24 G 1 144594746 144873033 278 T5 G 7 15542301 15793197 251 E24 G 1 145425752 145872489 447 T5 G 7 61087267 61270796 184 E24 G 1 156649526 157113477 464 T5 G 7 86477382 86755032 278 E24 G 1 158010337 158283641 273 T5 G 7 114491027 115586861 1096 E24 G 1 160338731 160677252 339 T5 G 8 18242281 18508401 266 E24 G 1 161558031 161777223 219 T5 G 8 28931963 29222390 290 E24 G 1 162887424 163728359 841 T5 G 8 83316594 83767100 451 E24 G 1 173658230 174094669 436 T5 G 8 106904387 107311076 407 E24 G 1 174639051 176344936 1706 T5 G 8 113870788 114193060 322 E24 G 1 179462543 179933730 471 T5 G 9 9588884 10246475 658 E24 G 1 199215670 199653218 438 T5 G 9 24798764 25832442 1034 E24 G 1 202255347 203411834 1156 T5 G 9 72806252 73010767 205 E24 G 1 204118116 205439437 1321 T5 G 10 15913133 16440716 528 E24 G 1 206141166 207970850 1830 T5 G 10 61498616 62052694 554 E24 G 1 212028920 212449749 421 T5 G 11 100189595 100588796 399 E24 G 1 214753241 215373858 621 T5 G 11 100820386 101077028 257 E24 G 1 232727671 233105801 378 T5 G 12 17318075 18090825 773 E24 G 1 243881127 244296856 416 T5 G 12 36302222 36432239 130 E24 G 2 121240000 121633928 394 T5 G 12 73200372 73777803 577 E24 G 4 108599229 108797097 198 T5 G 12 77766046 78269342 503 E24 G 5 16358405 16598168 240 T5 G 12 83504953 83977622 473 E24 G 5 17284044 17553934 270 88  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T5 G 12 86487712 86754944 267 E24 G 8 144158852 144660691 502 T5 G 12 89886535 90205587 319 E24 G 13 100030217 101730172 1700 T5 G 12 90278355 90618336 340 E24 G 13 102377421 102768830 391 T5 G 12 128150230 128548133 398 E24 G 13 106484325 107155648 671 T5 G 13 59084555 59396464 312 E24 G 13 108629457 112845578 4216 T5 G 13 64317369 64544139 227 E24 G 13 113492713 114093192 600 T5 G 13 67175227 67681892 507 E24 G 14 19149713 19485285 336 T5 G 13 82307672 82609458 302 E24 G 14 21763281 22006855 244 T5 G 14 26401540 27384263 983 E24 G 14 105584423 105967733 383 T5 G 14 41518987 42281787 763 E24 G 18 42657928 42803873 146 T5 G 14 42674393 42924774 250 E24 L 4 140187025 140521672 335 T5 G 14 70137529 70215232 78 E24 L 10 48361074 49013085 652 T5 G 14 80658075 80879562 221 E24 L 11 97278670 97609941 331 T5 G 14 99356742 99584727 228 E24 L 12 92113258 92516893 404 T5 G 16 21939143 22187488 248 E24 L 13 62267126 62577460 310 T5 G 16 52309608 52599483 290 E24 L 16 20943307 21212616 269 T5 G 16 64588814 65053676 465 E24 L 22 31240461 32005256 765 T5 G 17 48684140 49205759 522 E24 L 22 32217850 36515461 4298 T5 G 18 5362621 5746803 384 E24 L 22 36928815 37572224 643 T5 G 18 36430587 36743158 313 E24 L 22 41907119 49534385 7627 T5 G 18 43069061 43436781 368 E6 G 1 1058909 3000997 1942 T5 G 18 61022046 61554258 532 E6 G 1 8127005 9983505 1857 T5 G 18 69281624 69613151 332 E6 G 1 26424669 29453636 3029 T5 G 19 7749446 7984147 235 E6 G 1 39063444 40504667 1441 T5 G 21 19365844 19698905 333 E6 G 1 44687143 45325761 639 T5 G 21 44616528 45041136 425 E6 G 1 232727671 233546093 818 T5 G 21 45569817 45747046 177 E6 G 1 245436183 245747506 311 T5 G 22 36007572 36324767 317 E6 G 2 9985433 11755093 1770 T5 L 1 30216330 30537929 322 E6 G 2 24892640 25572843 680 T5 L 1 65050275 65530390 480 E6 G 2 84979184 85274881 296 T5 L 1 178395125 178621310 226 E6 G 2 208050137 208489071 439 T5 L 2 17341117 17715047 374 E6 G 2 232967040 233991801 1025 T5 L 2 51706280 52051295 345 E6 G 3 121682380 121964439 282 T5 L 3 44892353 45303025 411 E6 G 5 1019971 1562826 543 T5 L 3 73471190 73915690 445 E6 G 5 175623077 176701325 1078 T5 L 3 152832716 153205949 373 E6 G 6 41352064 43237228 1885 T5 L 3 190306876 190600506 294 E6 G 7 6038742 6960042 921 T5 L 5 37052109 37473658 422 E6 G 7 57194816 57562822 368 T5 L 5 103018513 103426734 408 E6 G 7 71946110 72215337 269 T5 L 7 24242808 24445001 202 E6 G 7 72809108 73534009 725 T5 L 8 7035076 7289755 255 E6 G 7 100027063 102036700 2010 T5 L 8 75167545 75553560 386 E6 G 7 129173245 129934305 761 T5 L 8 89314331 89669378 355 E6 G 8 144602260 146208093 1606 T5 L 10 124792962 126788208 1995 E6 G 9 33783560 34331118 548 T5 L 10 127834059 128349027 515 E6 G 9 35583889 36539166 955 T5 L 11 129706418 130119560 413 E6 G 9 129488250 132999756 3512 T5 L 12 79917287 80564119 647 E6 G 9 133976381 136359804 2383 89  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T5 L 15 57743715 58409873 666 E6 G 9 138368718 139959845 1591 T5 L 15 73569812 74091082 521 E6 G 11 50991 1875933 1825 T5 L 16 58148238 58608110 460 E6 G 11 47194406 47629247 435 T5 L 16 71288486 72135811 847 E6 G 11 61823795 62236904 413 T5 L 16 84535576 85564330 1029 E6 G 11 62944653 67116241 4172 T5 L 17 2534851 2937807 403 E6 G 11 69117677 69880011 762 T5 L 18 22079376 22624097 545 E6 G 11 72553832 73412975 859 T5 L 19 45512392 45693347 181 E6 G 11 118055345 118758082 703 T5 L 20 42615749 42799481 184 E6 G 12 119183787 122155980 2972 T5 L 20 55362290 55807791 446 E6 G 14 22869703 23236087 366 T25 G 1 50760069 51272846 513 E6 G 15 30328715 30597338 269 T25 G 2 14410329 16914958 2505 E6 G 15 39080446 39391363 311 T25 G 2 23828557 24216814 388 E6 G 15 72775397 73217136 442 T25 G 3 78049335 78310037 261 E6 G 16 44231 3680499 3636 T25 G 3 190572724 190793756 221 E6 G 16 11419973 12014547 595 T25 G 4 65966121 66436409 470 E6 G 16 29306982 29880154 573 T25 G 5 17284044 17553934 270 E6 G 16 30539947 31499638 960 T25 G 5 97652683 97856979 204 E6 G 16 87260490 88573088 1313 T25 G 5 105950837 106368860 418 E6 G 17 0 1628597 1629 T25 G 5 180195851 180444136 248 E6 G 17 1604505 2767054 1163 T25 G 6 54803239 55088907 286 E6 G 17 3295734 4904795 1609 T25 G 7 61061239 62784594 1723 E6 G 17 16857007 17246408 389 T25 G 8 18242281 18508401 266 E6 G 17 26042085 26307306 265 T25 G 8 96606103 96811332 205 E6 G 17 43090007 45085833 1996 T25 G 10 16290075 16607824 318 E6 G 17 45153711 46634199 1480 T25 G 10 28063255 28308281 245 E6 G 17 54447833 54973480 526 T25 G 10 118147658 118360397 213 E6 G 17 69977768 73230854 3253 T25 G 11 22289940 22485886 196 E6 G 17 73871046 78615238 4744 T25 G 11 131887972 132151114 263 E6 G 18 3167304 3472372 305 T25 G 12 32399702 32687563 288 E6 G 18 11684403 12100695 416 T25 G 12 36302222 36432239 130 E6 G 18 16897445 18303681 1406 T25 G 12 130568951 130978591 410 E6 G 18 25034674 25284594 250 T25 G 15 18473375 18717253 244 E6 G 19 74843 1095508 1021 T25 G 15 19087319 19353919 267 E6 G 19 1331124 6694652 5364 T25 G 15 30237319 30597338 360 E6 G 19 10458166 11737906 1280 T25 G 16 20943307 21212616 269 E6 G 19 16184022 18643264 2459 T25 G 16 64588814 64837478 249 E6 G 19 50652470 52308679 1656 T25 G 17 623188 962070 339 E6 G 19 53243799 55203857 1960 T25 G 17 55638613 56233848 595 E6 G 20 54443015 54747849 305 T25 G 18 20449482 20735586 286 E6 G 20 61386952 62435964 1049 T25 G 18 42657928 43072296 414 E6 G 21 20794703 21052440 258 T25 G 19 8934066 9026570 93 E6 G 21 43899532 44194069 295 T25 G 19 23835271 24178465 343 E6 G 21 46246247 46694679 448 T25 G 19 55554013 55707294 153 E6 G 22 17475778 18674825 1199 T25 G 21 20794703 21052440 258 E6 G 22 35061869 41802041 6740 T25 L 1 87202895 87588045 385 E6 G 22 43225305 46032632 2807 T25 L 2 46355058 46727455 372 E6 L 1 100794896 101075341 280 90  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T25 L 2 87057872 87638682 581 E6 L 1 167301416 167654916 354 T25 L 2 120934194 121441961 508 E6 L 1 171178733 171743135 564 T25 L 2 134784717 135096043 311 E6 L 2 1438693 1649373 211 T25 L 2 217757668 218352598 595 E6 L 2 77054044 77302660 249 T25 L 3 38457066 38837528 380 E6 L 2 97026359 97280716 254 T25 L 3 78394977 78583892 189 E6 L 2 109113179 109402865 290 T25 L 3 129385338 129741914 357 E6 L 2 209283920 209965965 682 T25 L 3 140772863 141053191 280 E6 L 3 26243462 27041194 798 T25 L 4 3362479 3610639 248 E6 L 3 80918434 81176295 258 T25 L 5 15782020 16253318 471 E6 L 3 89945475 90318890 373 T25 L 5 134308327 135430819 1122 E6 L 3 110593448 111097910 504 T25 L 6 94071515 94355791 284 E6 L 4 191006683 191137774 131 T25 L 6 169888230 170479234 591 E6 L 5 49479231 49913062 434 T25 L 7 149177480 150001617 824 E6 L 5 75006893 75588705 582 T25 L 7 158404469 158818116 414 E6 L 5 100253676 100665725 412 T25 L 8 764689 1142225 378 E6 L 5 170231354 170505998 275 T25 L 9 82833412 83098662 265 E6 L 5 180093032 180343927 251 T25 L 9 95274007 95599862 326 E6 L 6 57874178 58849697 976 T25 L 10 77123718 77578274 455 E6 L 6 62000083 62603705 604 T25 L 10 79607481 81420249 1813 E6 L 6 122778393 123049799 271 T25 L 10 110633487 111047441 414 E6 L 6 149900596 150732866 832 T25 L 10 124792962 126455020 1662 E6 L 8 47568877 47901818 333 T25 L 10 129620434 131698871 2078 E6 L 8 77791301 78175516 384 T25 L 10 132635360 135279687 2644 E6 L 8 86699540 87055819 356 T25 L 11 6270497 6580309 310 E6 L 8 90405349 90822242 417 T25 L 11 114281321 114600734 319 E6 L 9 66254996 66661410 406 T25 L 11 118980810 119354299 373 E6 L 9 67803916 70224724 2421 T25 L 11 132051142 132915120 864 E6 L 11 54712078 55479236 767 T25 L 12 34127668 34451327 324 E6 L 11 62532845 62994414 462 T25 L 12 100290871 100633658 343 E6 L 11 93414925 93936247 521 T25 L 12 112894158 113314551 420 E6 L 11 96953808 97518886 565 T25 L 13 110703658 112948798 2245 E6 L 12 69844305 70114686 270 T25 L 16 75550721 75802088 251 E6 L 14 38847479 39960099 1113 T25 L 16 83873509 88336665 4463 E6 L 15 79676203 79953718 278 T25 L 17 2534851 2937807 403 E6 L 16 33659117 34016785 358 T25 L 18 25034674 25284594 250 E6 L 17 22287134 22595733 309 T25 L 18 36149440 36407233 258 E6 L 17 73158376 73381889 224 T25 L 18 44137937 44552924 415 E6 L 18 15026396 15320434 294 T25 L 20 47317703 47816133 498 E6 L 18 40025831 40393746 368 T25 L 22 17053965 17206783 153 E6 L 18 63741633 63919160 178 T14 G 1 33572793 33943747 371 E6 L 19 23835271 24178465 343 T14 G 2 15716583 18181769 2465 E6 L 19 50398622 50652469 254 T14 G 2 214907768 215352824 445 E6 L 20 4968909 5343849 375 T14 G 3 190572724 190793756 221 E21 G 1 145425752 145872489 447 T14 G 4 48771496 49210770 439 E21 G 1 150080115 156658752 6579 T14 G 4 171943081 172140052 197 E21 G 1 157848491 159891049 2043 T14 G 4 177422553 177909585 487 E21 G 1 160338731 160958046 619 91  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T14 G 4 183340483 184029894 689 E21 G 1 166264720 167381584 1117 T14 G 4 185082952 185401436 318 E21 G 1 176574921 176971757 397 T14 G 4 187161436 187685225 524 E21 G 1 179640235 181831357 2191 T14 G 4 190853246 191137774 285 E21 G 1 181937518 182204185 267 T14 G 5 17284044 17553934 270 E21 G 1 198461529 201431856 2970 T14 G 5 20897033 21399027 502 E21 G 1 201646252 208088220 6442 T14 G 5 41237134 41668526 431 E21 G 1 209696870 212332499 2636 T14 G 5 69602994 70066836 464 E21 G 1 223536766 228168309 4632 T14 G 5 180093032 180444136 351 E21 G 1 228205983 229467946 1262 T14 G 7 57163665 57368235 205 E21 G 1 229883839 231685446 1802 T14 G 7 64972395 65209849 237 E21 G 1 232727671 233391573 664 T14 G 7 109009814 109465769 456 E21 G 1 242294867 242642426 348 T14 G 8 12999086 13429882 431 E21 G 1 242997307 244387464 1390 T14 G 8 92381128 92957538 576 E21 G 2 1438693 2793742 1355 T14 G 9 139979344 140185705 206 E21 G 2 7542268 8825151 1283 T14 G 10 50596911 50869761 273 E21 G 2 9985433 11755093 1770 T14 G 12 85650592 86068176 418 E21 G 2 15716583 16914958 1198 T14 G 12 130568951 130978591 410 E21 G 2 19690629 20796325 1106 T14 G 14 85765244 86154997 390 E21 G 2 23361965 23881796 520 T14 G 15 18674575 20053874 1379 E21 G 2 28214990 28865200 650 T14 G 15 50899677 51145331 246 E21 G 2 42999225 43518222 519 T14 G 15 59960618 60314021 353 E21 G 2 45282682 46285231 1003 T14 G 15 86073780 86865897 792 E21 G 2 54031551 55105184 1074 T14 G 17 27961446 28214642 253 E21 G 2 71611728 72007879 396 T14 G 18 42657928 43436781 779 E21 G 2 94986258 95863637 877 T14 G 19 47912047 48626174 714 E21 G 3 139134656 139386613 252 T14 G 19 55554013 55707294 153 E21 G 3 143938661 144222024 283 T14 G 20 24766816 25046218 279 E21 G 3 150646482 151003446 357 T14 G 20 40126629 40500600 374 E21 G 4 417247 1847477 1430 T14 L 1 116637869 116921215 283 E21 G 4 5927785 6293125 365 T14 L 2 29678589 30015687 337 E21 G 5 17284044 17553934 270 T14 L 2 223727102 224009643 283 E21 G 5 55199515 55485623 286 T14 L 3 145364190 145595605 231 E21 G 5 63789206 64142333 353 T14 L 4 142631188 142828372 197 E21 G 5 82802679 83034279 232 T14 L 4 172058782 172379103 320 E21 G 5 95697117 95852946 156 T14 L 5 61657012 61943229 286 E21 G 5 116123966 116386009 262 T14 L 5 62959058 63386658 428 E21 G 6 73242 5789420 5716 T14 L 7 7376245 7947720 571 E21 G 6 6362577 14506540 8144 T14 L 7 16589721 16636674 47 E21 G 6 14674050 18121480 3447 T14 L 9 97212464 97444301 232 E21 G 6 21356155 21654555 298 T14 L 11 50991 1776138 1725 E21 G 6 25059720 32151955 7092 T14 L 11 2197942 5839559 3642 E21 G 6 33017597 38660643 5643 T14 L 11 7926129 13369379 5443 E21 G 6 39096919 39555660 459 T14 L 11 14183583 16057348 1874 E21 G 6 39928065 45175251 5247 T14 L 11 16945105 20006238 3061 E21 G 6 45494917 46734973 1240 T14 L 11 30243665 35665813 5422 E21 G 6 51704583 53956516 2252 T14 L 11 44571231 44925353 354 E21 G 7 44818259 45119616 301 92  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T14 L 11 68457614 71041286 2584 E21 G 7 73680992 73956925 276 T14 L 11 72553832 73412975 859 E21 G 7 124677369 124943364 266 T14 L 11 74847555 78921469 4074 E21 G 7 127398355 127812452 414 T14 L 11 97154277 97534210 380 E21 G 8 106601289 107311076 710 T14 L 11 102841892 103303951 462 E21 G 8 120711365 121163538 452 T14 L 11 104388385 106138665 1750 E21 G 8 140294522 141002709 708 T14 L 11 109535513 113295360 3760 E21 G 9 123326445 123648944 322 T14 L 11 114281321 131598665 17317 E21 G 11 1321707 1660253 339 T14 L 11 132051142 132915120 864 E21 G 11 57344375 57551130 207 T14 L 12 42172642 42629204 457 E21 G 11 63321142 64621654 1301 T14 L 12 59957940 60195470 238 E21 G 12 32254703 32687563 433 T14 L 12 79773979 80088388 314 E21 G 12 71994675 72180449 186 T14 L 12 102225696 102559169 333 E21 G 13 102618440 102958785 340 T14 L 12 117025244 117328738 303 E21 G 14 19497166 19718160 221 T14 L 15 39035666 39391363 356 E21 G 14 76307165 76655057 348 T14 L 16 47457492 48858380 1401 E21 G 14 89811265 90956790 1146 T14 L 16 50079561 52510034 2430 E21 G 15 18898414 19133581 235 T14 L 16 53615738 59512370 5897 E21 G 15 21973970 22386839 413 T14 L 16 60515970 66485262 5969 E21 G 15 26275701 26597300 322 T14 L 16 68719438 69159549 440 E21 G 15 28106102 28691954 586 T14 L 16 70168706 73945290 3777 E21 G 15 39724582 40237983 513 T14 L 16 75370384 75802088 432 E21 G 15 71764685 72403143 638 T14 L 16 75886028 85564330 9678 E21 G 15 73336790 73793805 457 T14 L 16 87165550 87542642 377 E21 G 15 75603709 75885169 281 T14 L 17 2534851 2937807 403 E21 G 15 96709108 97132862 424 T14 L 17 35065361 35304654 239 E21 G 16 52079281 52360249 281 T14 L 18 25034674 25284594 250 E21 G 17 6383241 7387494 1004 T14 L 19 17227985 17558373 330 E21 G 17 36547206 38155435 1608 T14 L 19 20479187 20771666 292 E21 G 17 39434414 40139868 705 T14 L 19 32504109 32613567 109 E21 G 17 45294025 45685773 392 T14 L 22 17053965 17206783 153 E21 G 17 56558187 57247862 690 P3 G 1 52663511 53007168 344 E21 G 17 65201407 65563337 362 P3 G 1 78738027 79075345 337 E21 G 17 69307909 73457154 4149 P3 G 1 177764373 178479748 715 E21 G 17 73871046 74851642 981 P3 G 2 7750863 8181289 430 E21 G 17 75249971 78615238 3365 P3 G 2 198492712 198759383 267 E21 G 18 20449482 21010506 561 P3 G 2 241714149 242006836 293 E21 G 18 42657928 42803873 146 P3 G 3 49579895 49782857 203 E21 G 19 1331124 2196817 866 P3 G 6 147323170 147837918 515 E21 G 19 13709724 15301063 1591 P3 G 7 72809108 73201519 392 E21 G 19 63435376 63654245 219 P3 G 7 151676150 151985718 310 E21 G 20 3729010 4125081 396 P3 G 8 54928152 55339325 411 E21 G 20 60809452 61325197 516 P3 G 9 25318379 25715548 397 E21 G 21 44152494 44522210 370 P3 G 9 35286076 35798430 512 E21 G 22 37376687 38610512 1234 P3 G 9 130791422 131615932 825 E21 L 2 35571545 35837286 266 P3 G 11 60661723 60880212 218 E21 L 2 75857509 77204381 1347 P3 G 11 131887972 132389201 501 E21 L 2 78140908 81001459 2861 93  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) P3 G 12 1526627 1719982 193 E21 L 2 82120028 83668734 1549 P3 G 12 12243842 12729565 486 E21 L 2 145486871 146254326 767 P3 G 13 57053680 57436388 383 E21 L 2 179449929 179831272 381 P3 G 13 59352443 59638075 286 E21 L 2 211144204 211656085 512 P3 G 14 24926668 25366361 440 E21 L 3 78394977 78583892 189 P3 G 15 21945545 22386839 441 E21 L 3 117931539 118057362 126 P3 G 15 28106102 29121290 1015 E21 L 3 140772863 141053191 280 P3 G 15 30237319 30597338 360 E21 L 4 132667102 133645430 978 P3 G 15 32897394 33198054 301 E21 L 4 180427322 181366822 940 P3 G 15 91086545 91292369 206 E21 L 5 66790934 67144270 353 P3 G 16 1341140 1901486 560 E21 L 5 95842127 96089798 248 P3 G 17 4303684 4700666 397 E21 L 6 67459297 67668616 209 P3 G 17 35490927 35997410 506 E21 L 7 1499748 1737551 238 P3 G 19 51436748 51896236 459 E21 L 7 11969246 12200051 231 P3 G 19 60306236 60867589 561 E21 L 7 61087267 62176519 1089 P3 L 1 104420480 104657658 237 E21 L 7 87945541 88632733 687 P3 L 1 106860717 107290858 430 E21 L 8 36095236 36516657 421 P3 L 2 1042562 1404470 362 E21 L 8 43185803 43912178 726 P3 L 2 59571563 59886525 315 E21 L 8 63189500 63663884 474 P3 L 2 199463802 199805543 342 E21 L 8 86196327 87055819 859 P3 L 2 211144204 211540445 396 E21 L 8 117969492 118356842 387 P3 L 3 30099494 30364799 265 E21 L 10 41947396 42224061 277 P3 L 3 75285656 75515882 230 E21 L 10 66355758 66994232 638 P3 L 3 78394977 78583892 189 E21 L 10 77389763 77592293 203 P3 L 3 107037366 107517939 481 E21 L 11 3414002 3661668 248 P3 L 3 140772863 141053191 280 E21 L 11 18783635 19003338 220 P3 L 4 108039548 108580318 541 E21 L 11 47479617 47860662 381 P3 L 5 49479231 49636937 158 E21 L 11 84053362 84505710 452 P3 L 5 62884026 63168836 285 E21 L 11 97084524 97518886 434 P3 L 5 102114463 102519831 405 E21 L 12 32108495 32334573 226 P3 L 6 67474215 67751642 277 E21 L 12 36302222 36432239 130 P3 L 6 149900596 150732866 832 E21 L 12 103566212 104021895 456 P3 L 6 170655612 170880179 225 E21 L 14 40026915 40587410 560 P3 L 7 1499748 1737551 238 E21 L 16 33659117 35136830 1478 P3 L 7 121931947 122282009 350 E21 L 18 16659756 16944006 284 P3 L 8 36186658 36516657 330 E21 L 18 56366046 56761467 395 P3 L 8 43185803 43912178 726 E21 L 18 57739648 58026453 287 P3 L 8 85434907 85823834 389 E21 L 19 8934066 9026570 93 P3 L 11 40510250 40852607 342 E21 L 19 55554013 55707294 153 P3 L 11 46058617 46407944 349 E21 L 21 23153285 23404186 251 P3 L 11 47474531 47860662 386 T3 G 1 5116964 5290233 173 P3 L 11 49344159 49626739 283 T3 G 1 120047377 120331938 285 P3 L 11 50230022 51192828 963 T3 G 3 48134752 48468265 334 P3 L 11 97154277 97518886 365 T3 G 5 68905030 69102023 197 P3 L 12 62057653 62430133 372 T3 G 5 82709486 82952978 243 P3 L 12 106476666 106770408 294 T3 G 5 143388623 143726339 338 P3 L 12 107204119 107495724 292 T3 G 5 180195851 180444136 248 94  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) P3 L 13 64317369 64544139 227 T3 G 7 57163665 57368235 205 P3 L 14 64454982 64964479 509 T3 G 7 64972395 65209849 237 P3 L 17 22287134 22595733 309 T3 G 11 131887972 132151114 263 P3 L 17 65056159 65563337 507 T3 G 12 29727999 29929154 201 P3 L 18 12806854 13135104 328 T3 G 13 63561919 64068098 506 P3 L 19 910519 1369051 459 T3 G 14 19149713 19485285 336 P3 L 19 8934066 9026570 93 T3 G 14 104967556 105600462 633 P3 L 19 32504109 32613567 109 T3 G 15 18473375 20053874 1580 P3 L 19 55554013 55707294 153 T3 G 15 21973970 22386839 413       T3 G 15 30237319 30597338 360       T3 G 15 36223897 36476298 252       T3 G 16 33280312 33584068 304       T3 L 2 210209170 210467608 258       T3 L 2 211144204 211349074 205       T3 L 3 78394977 78583892 189       T3 L 3 140772863 141053191 280       T3 L 4 8979402 9291412 312       T3 L 4 48771496 49210770 439       T3 L 5 80392139 80686277 294       T3 L 6 13223270 13524298 301       T3 L 6 160616018 161039688 424       T3 L 7 143515150 143839638 324       T3 L 8 86699540 87055819 356       T3 L 9 82833412 82953738 120       T3 L 10 4158992 4425972 267       T3 L 13 51777911 52002865 225       T3 L 19 8629145 8784792 156       T3 L 19 32504109 32613567 109             95  Table A.2    aCGH alterations by sample (RB1-/-). ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T23 G 1 220760289 221225248 465 T66 G 1 27020386 27234980 215 T23 G 2 6372092 6738494 366 T66 G 1 142652783 142998578 346 T23 G 2 120641847 121052681 411 T66 G 1 150229090 155733368 550 T23 G 3 21893268 22110528 217 T66 G 1 155801127 158283641 248 T23 G 3 141103332 141588859 486 T66 G 1 158758725 160677252 191 T23 G 5 568397 1013978 446 T66 G 1 161016469 163072819 205 T23 G 5 17284044 17553934 270 T66 G 1 165155625 169658465 450 T23 G 5 20897033 21399027 502 T66 G 1 174041164 175146843 110 T23 G 5 49960569 50670962 710 T66 G 1 177864963 178479748 615 T23 G 6 73242 346084 273 T66 G 1 179640235 183513118 387 T23 G 6 15729721 16332107 602 T66 G 1 189269177 189714992 446 T23 G 6 19638959 24574853 4936 T66 G 1 190304054 191386589 108 T23 G 6 27295136 27628148 333 T66 G 1 196799511 209169786 123 T23 G 6 33848331 35024860 1177 T66 G 1 210772211 215373858 460 T23 G 6 103346894 103604028 257 T66 G 1 216039670 220609576 457 T23 G 6 170655612 170880179 225 T66 G 1 222704527 227325091 462 T23 G 7 57163665 57368235 205 T66 G 1 228205983 229034632 829 T23 G 11 134225567 134433506 208 T66 G 1 229174289 234815927 564 T23 G 12 71994675 72180449 186 T66 G 1 242089298 245339706 325 T23 G 12 112894158 116351108 3457 T66 G 3 142345971 142646405 300 T23 G 12 122604516 125418667 2814 T66 G 3 175445960 175738257 292 T23 G 12 125926009 126964150 1038 T66 G 4 143031922 143345850 314 T23 G 12 127185807 128548133 1362 T66 G 5 34094987 34371608 277 T23 G 12 130113790 131701464 1588 T66 G 5 180195851 180444136 248 T23 G 14 19149713 19485285 336 T66 G 6 73242 36488793 364 T23 G 15 21973970 22386839 413 T66 G 6 36620128 58849697 222 T23 G 16 33400710 33741546 341 T66 G 7 141962686 142316803 354 T23 G 16 50551653 50792425 241 T66 G 11 57344375 57551130 207 T23 G 18 1946123 2506229 560 T66 G 14 53626221 55640667 201 T23 G 18 7039213 8637511 1598 T66 G 14 60182950 64555988 437 T23 G 18 10433893 11111665 678 T66 G 14 65111104 71263950 615 T23 G 18 31135911 35657079 4521 T66 G 14 73806697 77513223 370 T23 G 18 42007182 44723913 2717 T66 G 14 77836558 80558546 272 T23 G 18 45570558 46140574 570 T66 G 14 80600513 86154997 555 T23 G 18 69281624 72148783 2867 T66 G 14 86623012 95405991 878 T23 G 18 72745519 76098439 3353 T66 G 14 96121656 103750538 762 T23 G 19 36871853 37049593 178 T66 G 14 104967556 106318374 135 T23 G 22 14410042 14660949 251 T66 G 15 30237319 30539910 303 T23 L 1 62245042 62523933 279 T66 G 16 64588814 65053676 465 T23 L 1 152139606 152376962 237 T66 G 17 10094466 10520549 426 T23 L 4 69910765 70332437 422 T66 G 17 33264100 33700089 436 T23 L 8 86196327 87506471 1310 T66 G 18 42657928 42803873 146 T23 L 8 134535936 134863249 327 T66 G 22 17053965 17206783 153 T23 L 9 29711074 30043324 332 T66 L 1 617 3000997 300 T23 L 9 113299231 113848887 550 T66 L 1 4924403 5088349 164 96  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T23 L 11 97278670 97534210 256 T66 L 1 17486898 19020943 153 T23 L 11 125166505 125470718 304 T66 L 1 29953410 30239890 286 T23 L 12 32108495 32334573 226 T66 L 1 104001576 104300583 299 T23 L 13 47570684 47823050 252 T66 L 2 88921446 89405301 484 T23 L 13 59352443 59961173 609 T66 L 2 223605559 224009643 404 T23 L 13 66263513 66805219 542 T66 L 3 150185459 150424655 239 T23 L 13 67175227 67681892 507 T66 L 4 5574550 5959685 385 T23 L 13 70429098 73462825 3034 T66 L 4 49189866 49416673 227 T23 L 13 87396186 87732535 336 T66 L 5 80319034 80686277 367 T23 L 13 92154455 92774084 620 T66 L 5 137083206 137340623 257 T23 L 15 19884809 20071155 186 T66 L 5 143610226 143913187 303 T23 L 19 32504109 32613567 109 T66 L 8 86620595 87055819 435 T53 G 1 36466785 37675114 1208 T66 L 12 120601206 120855003 254 T53 G 1 46604573 48257830 1653 T66 L 13 36678499 40124114 344 T53 G 1 142533622 153559241 11026 T66 L 13 41211420 43127842 191 T53 G 1 155029936 160677252 5647 T66 L 13 43481375 45768530 228 T53 G 1 161016469 167445692 6429 T66 L 13 46168557 46488557 320 T53 G 1 168471887 171525137 3053 T66 L 13 48654459 52002865 334 T53 G 1 173228930 174053395 824 T66 L 13 54559086 54932279 373 T53 G 1 174639051 176344936 1706 T66 L 13 57053680 57436388 383 T53 G 1 177945800 178479748 534 T66 L 13 58515152 61033077 251 T53 G 1 179462543 184426068 4964 T66 L 16 10586248 11089099 503 T53 G 1 196195485 198714418 2519 T66 L 16 12340398 12607349 267 T53 G 1 199539859 201882048 2342 T66 L 16 26891980 29392366 250 T53 G 1 202916221 220609576 17693 T66 L 16 53615738 54216885 601 T53 G 1 222704527 223152230 448 T66 L 16 63381801 63762681 381 T53 G 1 224564745 225431615 867 T66 L 16 64289064 64554473 265 T53 G 1 226511813 233440847 6929 T66 L 16 64890123 65208073 318 T53 G 1 240316624 242491480 2175 T66 L 16 71288486 72135811 847 T53 G 1 245724640 246944046 1219 T66 L 16 76619437 77228585 609 T53 G 2 41662 317294 276 T66 L 16 77371695 77830699 459 T53 G 2 5541877 6228194 686 T66 L 16 79053536 79572749 519 T53 G 2 29403359 29872762 469 T66 L 16 79901920 85564330 566 T53 G 2 36442569 36996317 554 T66 L 17 0 5025418 502 T53 G 2 46355058 46727455 372 T66 L 17 9663047 9930706 268 T53 G 2 71611728 72279682 668 T66 L 18 23570175 23897445 327 T53 G 2 73898940 74604122 705 T4 G 1 617 1487588 148 T53 G 2 91021063 91590359 569 T4 G 1 2341648 3000997 659 T53 G 2 113143567 114143148 1000 T4 G 1 3204885 3514176 309 T53 G 2 120934194 121633928 700 T4 G 1 12343280 12747440 404 T53 G 2 128716306 129315130 599 T4 G 1 27020386 27330241 310 T53 G 2 179059362 179486309 427 T4 G 1 29573058 30009232 436 T53 G 2 218193818 218637368 444 T4 G 1 74438002 74628468 190 T53 G 2 236568755 238194747 1626 T4 G 1 101933924 102562274 628 T53 G 3 46140 2390404 2344 T4 G 1 106221897 106419881 198 T53 G 3 2804349 6226254 3422 T4 G 1 154928108 155216620 289 T53 G 3 13499970 14924624 1425 T4 G 1 155661462 155849211 188 97  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T53 G 3 72602189 72944366 342 T4 G 1 161558031 161940069 382 T53 G 3 75285656 75515882 230 T4 G 1 186283205 189025452 274 T53 G 3 108872278 109483457 611 T4 G 1 196656854 197078724 422 T53 G 3 114906026 115117759 212 T4 G 1 206753691 207207021 453 T53 G 3 124106796 125664529 1558 T4 G 1 242997307 243354271 357 T53 G 3 126980939 128752028 1771 T4 G 2 41662 696156 654 T53 G 3 129122714 131723522 2601 T4 G 2 21930354 22694417 764 T53 G 3 141082476 141588859 506 T4 G 2 47000363 47697639 697 T53 G 4 3362479 4065877 703 T4 G 2 51035350 51219186 184 T53 G 4 9051500 9841887 790 T4 G 2 83394849 83776592 382 T53 G 4 119557416 119899496 342 T4 G 2 103819868 104173214 353 T53 G 4 188225705 188555694 330 T4 G 2 128716306 129127951 412 T53 G 5 1684158 2928706 1245 T4 G 2 131247060 131497926 251 T53 G 5 7229957 7549647 320 T4 G 2 145673807 146453724 780 T53 G 5 17284044 17553934 270 T4 G 2 162918102 163117738 200 T53 G 5 67361489 67899255 538 T4 G 2 210136808 210467608 331 T53 G 5 68951921 70342024 1390 T4 G 2 211840216 212109960 270 T53 G 5 85900497 86206359 306 T4 G 2 218406423 218904083 498 T53 G 5 132432815 133318206 885 T4 G 2 240644071 241242608 599 T53 G 5 134308327 137208902 2901 T4 G 3 79291730 80556786 126 T53 G 5 139059617 139415354 356 T4 G 3 82058696 84433393 237 T53 G 5 163102487 163429773 327 T4 G 3 86469401 86941710 472 T53 G 5 175953307 176299886 347 T4 G 3 97140154 98067151 927 T53 G 5 180195851 180715161 519 T4 G 3 106883718 107517939 634 T53 G 6 73242 13658686 13585 T4 G 3 108495168 108929373 434 T53 G 6 14319218 14506540 187 T4 G 3 152666909 153108469 442 T53 G 6 15450735 20622576 5172 T4 G 3 161145124 161433890 289 T53 G 6 22866262 24238752 1372 T4 G 3 166015273 167091431 107 T53 G 6 25059720 27094077 2034 T4 G 3 177833733 178060147 226 T53 G 6 29457137 34703255 5246 T4 G 3 190595564 190902546 307 T53 G 6 35048432 42527171 7479 T4 G 4 598877 3080362 248 T53 G 6 43065666 44815780 1750 T4 G 4 3362479 3610639 248 T53 G 6 45632263 49681191 4049 T4 G 4 8073005 8427133 354 T53 G 6 51704583 52998900 1294 T4 G 4 31100819 34976979 387 T53 G 6 53635140 54076882 442 T4 G 4 65923063 66303313 380 T53 G 6 55796805 56001013 204 T4 G 4 115770483 118383130 261 T53 G 6 58230371 58849697 619 T4 G 4 126893902 127864345 970 T53 G 6 64983144 65308099 325 T4 G 4 130811298 131769087 958 T53 G 6 167951793 170880179 2928 T4 G 4 132864128 133645430 781 T53 G 7 1803168 2241382 438 T4 G 4 134084101 135735630 165 T53 G 7 76182168 76393428 211 T4 G 4 137051803 137853745 802 T53 G 7 126130514 126748077 618 T4 G 4 151664080 151911392 247 T53 G 7 131677611 132059225 382 T4 G 4 160913179 161136288 223 T53 G 7 157061042 158818116 1757 T4 G 4 163153267 165271726 211 T53 G 8 7035076 8212432 1177 T4 G 5 568397 1724268 115 T53 G 8 9929219 11424992 1496 T4 G 5 3426806 3867261 440 T53 G 8 36772374 37157205 385 T4 G 5 9850585 10296924 446 98  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T53 G 8 41586944 41975651 389 T4 G 5 12026212 13368700 134 T53 G 8 58157391 58393591 236 T4 G 5 17853590 19851640 199 T53 G 9 37935190 46275010 8340 T4 G 5 20876004 21826908 951 T53 G 9 67270003 70788998 3519 T4 G 5 28137140 28541732 405 T53 G 9 71333871 72221392 888 T4 G 5 51375482 51628988 254 T53 G 9 77122888 77684326 561 T4 G 5 54892344 55411390 519 T53 G 9 79136921 83098662 3962 T4 G 5 82384788 82601181 216 T53 G 9 83221598 89657240 6436 T4 G 5 85289117 85576510 287 T53 G 9 90481774 95197578 4716 T4 G 5 86471625 86737136 266 T53 G 9 95419404 98300736 2881 T4 G 5 91109216 91477358 368 T53 G 9 99552250 103520670 3968 T4 G 5 103436481 105722711 228 T53 G 9 105583846 106714988 1131 T4 G 5 107352814 107724919 372 T53 G 9 107633128 112014037 4381 T4 G 5 113017391 113857732 840 T53 G 9 115724200 126805467 11081 T4 G 6 73242 7910433 783 T53 G 9 127104199 137949835 10846 T4 G 6 11164609 16365288 520 T53 G 10 48181069 50621676 2441 T4 G 6 23408467 48878004 254 T53 G 10 72583189 73288590 705 T4 G 6 49184078 49461459 277 T53 G 10 76667248 81420249 4753 T4 G 6 51175372 54076882 290 T53 G 10 113577597 114056756 479 T4 G 6 55105411 55556955 452 T53 G 10 129620434 133477631 3857 T4 G 6 66804980 67412533 608 T53 G 11 11106340 11619989 514 T4 G 6 83651714 84033730 382 T53 G 11 17293901 17904451 611 T4 G 6 95000985 96251641 125 T53 G 11 19248489 19798627 550 T4 G 6 100652068 101234842 583 T53 G 11 60261715 60880212 618 T4 G 6 113284541 113626936 342 T53 G 11 79995579 80272391 277 T4 G 6 117035497 117412065 377 T53 G 11 111537832 112922707 1385 T4 G 6 128957546 129480026 522 T53 G 11 114934071 116279817 1346 T4 G 6 164485720 165232186 746 T53 G 11 129706418 130938010 1232 T4 G 7 1183592 2116944 933 T53 G 11 133980136 134433506 453 T4 G 7 9300329 10126382 826 T53 G 12 6223 156640 150 T4 G 7 11760038 12019653 260 T53 G 12 1526627 2655195 1129 T4 G 7 12814477 13607390 793 T53 G 12 70588953 71253094 664 T4 G 7 15542301 15793197 251 T53 G 12 103377461 103757127 380 T4 G 7 24830990 25439319 608 T53 G 12 121949281 122155980 207 T4 G 7 88524373 88740787 216 T53 G 13 17918001 18578218 660 T4 G 7 93841024 94092002 251 T53 G 13 21089625 21693822 604 T4 G 7 117127748 118527467 140 T53 G 13 23083059 39713370 16630 T4 G 7 119361791 120589006 122 T53 G 13 41967454 44523135 2556 T4 G 7 154455963 154975687 520 T53 G 13 44927806 45109681 182 T4 G 8 16092289 16638707 546 T53 G 13 45408174 51467864 6060 T4 G 8 28931963 29222390 290 T53 G 13 70429098 74860115 4431 T4 G 8 78448239 78685129 237 T53 G 13 87156308 97308084 10152 T4 G 8 93781529 94497499 716 T53 G 13 98251198 109977945 11727 T4 G 8 96724583 97118768 394 T53 G 13 110600643 114093192 3493 T4 G 8 103932260 104167305 235 T53 G 15 18272238 20338507 2066 T4 G 8 113870788 114193060 322 T53 G 15 21805449 22386839 581 T4 G 8 117067679 117371007 303 T53 G 15 34937367 35184622 247 T4 G 8 121448112 122162585 714 99  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T53 G 15 55550826 56641093 1090 T4 G 8 135225625 135540266 315 T53 G 15 58811890 59207810 396 T4 G 8 142694183 145876186 318 T53 G 15 67006694 68746547 1740 T4 G 9 24526829 24949266 422 T53 G 15 70693862 71172800 479 T4 G 9 29711074 30004618 294 T53 G 15 71764685 72403143 638 T4 G 9 30251881 30734359 482 T53 G 15 75603709 77640945 2037 T4 G 9 36804527 37437556 633 T53 G 16 14634005 15414812 781 T4 G 9 104230595 105634144 140 T53 G 16 46484333 46868750 384 T4 G 9 120574755 120905046 330 T53 G 17 27877766 30238813 2361 T4 G 9 132730134 133208844 479 T53 G 17 41103988 41357984 254 T4 G 9 137669516 138503912 834 T53 G 18 32179153 32536391 357 T4 G 10 3631141 4728854 109 T53 G 18 33246557 34397042 1150 T4 G 10 9907230 10715992 809 T53 G 18 43778413 44723913 946 T4 G 10 55462156 57087918 162 T53 G 18 70635327 71854573 1219 T4 G 10 84248928 84618005 369 T53 G 19 47912047 48626174 714 T4 G 10 104813634 105172292 359 T53 G 20 22146196 25235340 3089 T4 G 10 116116186 116299949 184 T53 G 20 28034381 28251024 217 T4 G 10 133771689 135065876 129 T53 G 20 57063146 61192343 4129 T4 G 11 50991 3280020 322 T53 G 20 61845302 62435964 591 T4 G 11 16278066 16447510 169 T53 G 21 13879419 14303412 424 T4 G 11 24250583 24571510 321 T53 G 21 45667514 46296399 629 T4 G 11 43332487 44361069 102 T53 G 22 28845092 29262259 417 T4 G 11 63724994 65814442 208 T53 L 1 617 11929541 11929 T4 G 11 67845569 69880011 203 T53 L 1 12724432 16620917 3896 T4 G 11 75861350 76088566 227 T53 L 1 17217972 21457860 4240 T4 G 11 98093765 98789266 696 T53 L 1 25351676 25650204 299 T4 G 11 134144181 134433506 289 T53 L 1 26465691 27049576 584 T4 G 12 23626836 23877346 251 T53 L 1 27084577 28189085 1105 T4 G 12 57923758 59149642 122 T53 L 1 174146398 174644590 498 T4 G 12 72171596 72601663 430 T53 L 1 233378012 233846155 468 T4 G 12 73177214 73413076 236 T53 L 2 25523248 25753042 230 T4 G 12 77219422 78114570 895 T53 L 2 36936339 37365420 429 T4 G 12 82532979 83712894 118 T53 L 2 47752592 48098425 346 T4 G 12 87593424 88097028 504 T53 L 2 54031551 55858798 1827 T4 G 12 130673016 130978591 306 T53 L 2 84979184 85274881 296 T4 G 13 57053680 57788215 735 T53 L 2 91608685 91680249 72 T4 G 13 61360375 63426814 206 T53 L 2 98900266 99340892 441 T4 G 13 63561919 64544139 982 T53 L 2 112291685 113206293 915 T4 G 13 83509678 83894529 385 T53 L 2 120082855 120612421 530 T4 G 13 87025230 88958211 193 T53 L 2 128133786 128794430 661 T4 G 13 93005803 93325237 319 T53 L 2 159891003 160506021 615 T4 G 13 103611212 104580216 969 T53 L 2 169863522 170458376 595 T4 G 13 105444244 106278034 834 T53 L 2 176359968 176588985 229 T4 G 13 111007205 113901932 289 T53 L 2 182718234 183145980 428 T4 G 14 19149713 19485285 336 T53 L 2 197782980 198337752 555 T4 G 14 26401540 27384263 983 T53 L 2 201419219 202193137 774 T4 G 14 64135811 65175772 104 T53 L 2 202673686 203715728 1042 T4 G 14 69785420 70215232 430 100  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T53 L 3 50670818 51228685 558 T4 G 14 77649096 78016279 367 T53 L 3 139761363 140244152 483 T4 G 14 99356742 99584727 228 T53 L 3 142657253 143956290 1299 T4 G 14 103592318 104701663 110 T53 L 3 154054634 154412636 358 T4 G 15 35738718 36476298 738 T53 L 3 170842335 171665102 823 T4 G 15 57895577 58297865 402 T53 L 3 182089134 183176951 1088 T4 G 15 93269408 93588984 320 T53 L 3 184091384 185039705 948 T4 G 16 44231 2131094 208 T53 L 3 196335425 198168305 1833 T4 G 16 21821763 22187488 366 T53 L 4 53766377 54116603 350 T4 G 16 50018681 50295218 277 T53 L 4 64081058 64375660 295 T4 G 16 52929680 53291453 362 T53 L 4 67413317 67604634 191 T4 G 16 66095316 66700366 605 T53 L 4 76601966 77271252 669 T4 G 17 778421 2767054 198 T53 L 4 78196277 78417632 221 T4 G 17 26144828 26734130 589 T53 L 4 89233079 89804528 571 T4 G 17 48435632 49357882 922 T53 L 4 128952512 129390725 438 T4 G 17 76603108 78615238 201 T53 L 4 140187025 140521672 335 T4 G 18 3288399 4172772 884 T53 L 4 151848353 152580921 733 T4 G 18 24452290 26635705 218 T53 L 5 36939489 37473658 534 T4 G 18 34214266 35380105 116 T53 L 5 54439135 55149649 711 T4 G 18 36512729 37308847 796 T53 L 5 61657012 61943229 286 T4 G 18 43250315 44029213 779 T53 L 5 125838199 126214785 377 T4 G 18 60693813 62501278 180 T53 L 5 150080197 150187832 108 T4 G 18 65196792 65424827 228 T53 L 5 159195585 159600148 405 T4 G 18 74125368 74382157 257 T53 L 5 171301660 171827544 526 T4 G 19 1331124 9419100 808 T53 L 5 178692833 179173835 481 T4 G 19 10650427 11482607 832 T53 L 6 76244437 76653060 409 T4 G 19 12587922 19630711 704 T53 L 7 5585965 6688617 1103 T4 G 19 41542240 41714071 172 T53 L 7 61087267 61797715 710 T4 G 19 48791653 55707294 691 T53 L 7 64729861 65136155 406 T4 G 19 57626931 59231132 160 T53 L 7 72452483 75447342 2995 T4 G 20 815899 4125081 330 T53 L 7 84446843 84758710 312 T4 G 20 6122936 8318803 219 T53 L 7 94209394 94652765 443 T4 G 20 8539983 11810672 327 T53 L 7 117369099 117931065 562 T4 G 20 15934583 16944382 101 T53 L 7 134704640 134984794 280 T4 G 20 22338389 24692601 235 T53 L 8 22007157 22460920 454 T4 G 20 37476876 43463770 598 T53 L 8 38745689 39395199 650 T4 G 20 45278601 47958110 268 T53 L 8 54928152 55339325 411 T4 G 20 50602592 51422726 820 T53 L 8 95411183 96094250 683 T4 G 20 53153655 61537434 838 T53 L 8 100098076 100648473 550 T4 G 20 61776383 62435964 660 T53 L 8 111875825 112118072 242 T4 G 21 14257878 14732426 475 T53 L 9 35985129 36774290 789 T4 G 21 19095584 21052440 195 T53 L 9 126774264 127153760 379 T4 G 21 24522262 25202097 680 T53 L 10 64411573 64995092 584 T4 G 21 27436191 27790782 355 T53 L 10 69354590 69965801 611 T4 G 21 44616528 45747046 113 T53 L 10 70260473 70459411 199 T4 G 22 17195002 17535932 341 T53 L 10 73239331 76708097 3469 T4 G 22 35691590 36324767 633 T53 L 10 126352795 126746888 394 T4 L 1 66480499 66848029 368 101  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T53 L 11 3573460 4099388 526 T4 L 1 83984048 84252716 269 T53 L 11 69544401 70008693 464 T4 L 2 27854604 28097537 243 T53 L 11 72852680 73412975 560 T4 L 2 151040712 151352772 312 T53 L 11 107109972 107894392 784 T4 L 2 187976679 188355962 379 T53 L 11 131887972 132151114 263 T4 L 2 201759280 202038728 279 T53 L 12 712002 1552778 841 T4 L 3 58033558 58812691 779 T53 L 12 7750091 8160827 411 T4 L 3 74689512 75815846 112 T53 L 12 47207929 47466311 258 T4 L 3 78788532 79284456 496 T53 L 12 47664568 48197129 533 T4 L 3 134068627 135020184 952 T53 L 12 91463507 92516893 1053 T4 L 4 3027675 3386296 359 T53 L 12 93408240 94098702 690 T4 L 4 75764600 78342846 257 T53 L 12 111244517 111622215 378 T4 L 4 78343948 79851451 150 T53 L 12 117025244 117328738 303 T4 L 4 168349638 168799583 450 T53 L 12 119676721 121965492 2289 T4 L 4 191006683 191137774 131 T53 L 12 131682527 132289534 607 T4 L 5 85532315 85754045 222 T53 L 13 19098344 19558501 460 T4 L 5 101825141 102287074 462 T53 L 13 57621677 58001888 380 T4 L 5 122361093 122701240 340 T53 L 14 20593740 21126835 533 T4 L 5 178125654 178591229 466 T53 L 14 34291680 35020664 729 T4 L 5 180331881 180715161 383 T53 L 14 48748451 50329715 1581 T4 L 7 95014255 95529637 515 T53 L 14 57764251 58150647 386 T4 L 7 158404469 158818116 414 T53 L 14 62114710 64952073 2837 T4 L 8 10276809 10616585 340 T53 L 14 66358138 80558546 14200 T4 L 8 86558071 87055819 498 T53 L 14 80831828 84340902 3509 T4 L 8 96300993 96756614 456 T53 L 15 32897394 33198054 301 T4 L 10 34146294 34496649 350 T53 L 15 48336894 49146558 810 T4 L 10 57886282 58405377 519 T53 L 15 56648904 57929589 1281 T4 L 11 30684238 31011553 327 T53 L 15 63341491 64164137 823 T4 L 12 33932542 34306741 374 T53 L 15 69961317 70600888 640 T4 L 15 58811890 59271378 459 T53 L 15 91086545 91292369 206 T4 L 15 73569812 73949331 380 T53 L 16 48586222 52599483 4013 T4 L 16 21188371 21763928 576 T53 L 16 52778760 55140874 2362 T4 L 16 46085157 50124383 403 T53 L 16 56049631 77830699 21781 T4 L 16 54179429 54559961 381 T53 L 16 77920768 88204460 10284 T4 L 16 70754647 72135811 138 T53 L 17 964480 2937807 1973 T4 L 16 79901920 82488856 258 T53 L 17 5120725 5420944 300 T4 L 17 38869035 39241864 373 T53 L 17 24264614 26307306 2043 T4 L 18 15026396 15320434 294 T53 L 17 37543886 38502716 959 T4 L 18 52833221 53677902 845 T53 L 17 46400485 46774257 374 T4 L 21 27730967 28080965 350 T53 L 17 53797993 57521098 3723 T4 L 22 24374944 24638104 263 T53 L 17 61469371 61864837 395 RB381 G 2 5090758 9226809 413 T53 L 17 63118029 63366540 249 RB381 G 2 15716583 18041019 232 T53 L 17 64736904 65427062 690 RB381 G 5 568397 1013978 446 T53 L 18 607831 1114347 507 RB381 G 5 180195851 180444136 248 T53 L 18 16659756 18900408 2241 RB381 G 7 73855619 74244009 388 T53 L 18 21843355 22489038 646 RB381 G 7 77705921 78212652 507 T53 L 18 23570175 23897445 327 RB381 G 7 80993980 81349702 356 102  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T53 L 18 25034674 25284594 250 RB381 G 7 82142679 82396484 254 T53 L 18 52327118 52822764 496 RB381 G 7 86097224 86461550 364 T53 L 18 58393970 58835881 442 RB381 G 7 91419826 93841472 242 T53 L 19 11828363 12483235 655 RB381 G 7 94475862 96533845 205 T53 L 19 41542240 43446499 1904 RB381 G 7 98287386 99431439 114 T53 L 19 51436748 51896236 459 RB381 G 7 102927908 103353609 426 T53 L 19 53383082 54181552 798 RB381 G 7 104955948 106766151 181 T53 L 20 34485436 35235053 750 RB381 G 7 106999071 108529189 153 T53 L 21 17603596 18289926 686 RB381 G 7 110167913 112008877 184 T53 L 22 16103207 16594536 491 RB381 G 7 114006676 115691288 168 T53 L 22 28520754 28911819 391 RB381 G 7 115927393 118090772 216 T53 L 22 34376492 34801814 425 RB381 G 7 124762143 127651400 288 T53 L 22 35061869 35449188 387 RB381 G 7 128850276 131378563 252 T45 G 1 92445707 92944794 499 RB381 G 7 131677611 136034008 435 T45 G 2 3214213 3489907 276 RB381 G 7 136860444 139822331 296 T45 G 2 35640496 35914637 274 RB381 G 7 140577096 144249366 367 T45 G 2 108347119 109056951 710 RB381 G 7 145723772 151584725 586 T45 G 3 45246741 45581671 335 RB381 G 8 58157391 58393591 236 T45 G 3 109856425 110182616 326 RB381 G 11 60661723 60880212 218 T45 G 4 48771496 49210770 439 RB381 G 14 19149713 19485285 336 T45 G 4 174746383 175167828 421 RB381 G 15 18898414 19353919 456 T45 G 5 17284044 17553934 270 RB381 G 15 30237319 30597338 360 T45 G 5 20897033 21399027 502 RB381 L 1 10299936 10690103 390 T45 G 5 43267631 43566931 299 RB381 L 2 9249270 15370826 612 T45 G 5 45748775 46151067 402 RB381 L 3 39737594 39987069 249 T45 G 5 80446929 81024602 578 RB381 L 4 48771496 49210770 439 T45 G 5 89734694 90044835 310 RB381 L 5 80392139 80686277 294 T45 G 5 102223029 102675072 452 RB381 L 8 7035076 8080568 104 T45 G 5 113712200 114011512 299 RB381 L 8 85161425 85465689 304 T45 G 5 118943200 119181190 238 RB381 L 8 86620595 87055819 435 T45 G 6 68167956 70205640 2038 RB381 L 10 71871522 72583104 712 T45 G 6 70800143 73482416 2682 RB381 L 13 45408174 46121194 713 T45 G 6 75184917 75503002 318 RB381 L 13 100632225 100888613 256 T45 G 6 84548248 85244397 696 RB381 L 19 8031324 8784792 753 T45 G 6 85390617 92660145 7270 RB381 L 19 55554013 55707294 153 T45 G 6 93357664 93727522 370 T55 G 1 120249993 120618526 369 T45 G 6 95939610 100686131 4747 T55 G 1 142533622 144710230 217 T45 G 6 104733720 105325375 592 T55 G 1 150229090 150543034 314 T45 G 6 106006097 106319178 313 T55 G 1 155029936 155733368 703 T45 G 6 108918904 114694182 5775 T55 G 1 164883009 165430178 547 T45 G 6 116520215 120222369 3702 T55 G 1 170049227 170578971 530 T45 G 6 122111262 127248750 5137 T55 G 1 179640235 180447764 808 T45 G 6 129868081 132831924 2964 T55 G 1 202525981 203577171 105 T45 G 6 135381781 139666375 4285 T55 G 1 211445419 212449749 100 T45 G 6 141913543 143058980 1145 T55 G 1 221606180 222040987 435 T45 G 6 145043003 147473656 2431 T55 G 2 58638970 58825808 187 T45 G 6 148646458 149659594 1013 T55 G 2 91021063 91302356 281 103  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T45 G 6 150702917 152461187 1758 T55 G 3 104552828 104714426 162 T45 G 6 152721626 170694605 17973 T55 G 3 139398053 139636565 239 T45 G 7 81155139 81349702 195 T55 G 5 25660260 25955183 295 T45 G 7 82142679 82396484 254 T55 G 5 26098550 26409556 311 T45 G 7 153637511 153901567 264 T55 G 5 63707477 64017810 310 T45 G 8 58157391 58393591 236 T55 G 5 134642615 134881601 239 T45 G 10 118147658 118360397 213 T55 G 7 57163665 57368235 205 T45 G 11 30835652 31082171 247 T55 G 8 58157391 58393591 236 T45 G 12 36302222 37321303 1019 T55 G 11 60661723 60880212 218 T45 G 12 60897220 61267390 370 T55 G 11 131887972 132151114 263 T45 G 13 57253289 57548957 296 T55 G 12 11435998 11800632 365 T45 G 15 30328715 30597338 269 T55 G 12 31917246 32219514 302 T45 G 16 87724052 88014087 290 T55 G 12 36302222 36734317 432 T45 G 17 49519884 49813633 294 T55 G 14 48439624 48680312 241 T45 G 17 67503237 69931286 2428 T55 G 15 28106102 28734907 629 T45 G 17 74307357 76516243 2209 T55 G 15 30237319 30539910 303 T45 G 17 77978981 78615238 636 T55 G 16 31969232 32197825 229 T45 G 18 9594546 9961693 367 T55 G 19 55138786 55462387 324 T45 G 18 42657928 42840984 183 T55 G 21 19095584 19368540 273 T45 G 19 55554013 55707294 153 T55 L 2 131247060 131497926 251 T45 G 20 35841263 38408361 2567 T55 L 3 78394977 78583892 189 T45 G 20 39114015 43811931 4698 T55 L 3 140772863 141053191 280 T45 G 20 45619372 47640221 2021 T55 L 3 189707676 190076997 369 T45 G 20 55869603 61192343 5323 T55 L 3 193362761 193832711 470 T45 G 21 16889256 17422704 533 T55 L 7 61087267 61270796 184 T45 G 21 38358843 38767124 408 T55 L 7 153637511 153901567 264 T45 L 1 617 3000997 3000 T55 L 9 129896705 130218614 322 T45 L 1 3204885 7755859 4551 T55 L 10 95411040 95815130 404 T45 L 1 8127005 8563040 436 T55 L 16 64289064 64837478 548 T45 L 1 10604300 12202620 1598 T55 L 16 76619437 77228585 609 T45 L 1 12724432 16394945 3671 T55 L 19 32504109 32613567 109 T45 L 1 17597152 31725218 14128 T24 G 1 5116964 5290233 173 T45 L 1 32848185 38725638 5877 T24 G 1 200319051 204975414 465 T45 L 1 40481959 51336384 10854 T24 G 1 210168489 210730372 562 T45 L 1 53600993 60383238 6782 T24 G 1 245190021 245747506 557 T45 L 1 61064196 61556855 493 T24 G 2 166365143 166700143 335 T45 L 1 63017988 63922590 905 T24 G 3 45308484 45581671 273 T45 L 1 73596099 74034168 438 T24 G 3 108872278 109166176 294 T45 L 1 74725261 74916322 191 T24 G 3 150124226 150335909 212 T45 L 1 76163853 76565297 401 T24 G 4 13423 3610639 359 T45 L 1 84608789 85259121 650 T24 G 4 4002548 6293125 229 T45 L 1 87291697 88542802 1251 T24 G 4 6978715 8692829 171 T45 L 1 94749395 96313767 1564 T24 G 4 14689269 20121820 543 T45 L 1 99141579 99454277 313 T24 G 4 30307070 31397944 109 T45 L 1 107478600 107958124 480 T24 G 4 39634648 43297704 366 T45 L 1 109426617 117819048 8392 T24 G 4 73760771 79705627 594 T45 L 2 134654310 134996036 342 T24 G 4 90275255 91011295 736 104  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T45 L 2 142035883 142422251 386 T24 G 4 108168108 111872612 370 T45 L 3 18708675 18905397 197 T24 G 4 119871532 122880766 300 T45 L 3 75285656 75515882 230 T24 G 4 141770131 142828372 105 T45 L 3 108430837 108693369 263 T24 G 4 154806629 156270118 146 T45 L 3 173127512 173537697 410 T24 G 4 182505813 184029894 152 T45 L 4 3645088 4065877 421 T24 G 4 186859136 188408487 154 T45 L 5 62191844 62519576 328 T24 G 4 190667654 190953845 286 T45 L 8 47102600 47541900 439 T24 G 5 55064491 55411390 347 T45 L 8 78197078 78685129 488 T24 G 5 82709486 82952978 243 T45 L 8 86699540 87055819 356 T24 G 5 85900497 86206359 306 T45 L 8 102139242 102624962 486 T24 G 5 172799092 173016342 217 T45 L 8 105361095 105797445 436 T24 G 6 73242 5437064 536 T45 L 8 132754059 134115508 1361 T24 G 6 12035857 12513884 478 T45 L 8 140178271 141422749 1244 T24 G 6 13508273 18231811 472 T45 L 9 82833412 82953738 120 T24 G 6 29457137 38315288 885 T45 L 10 101496919 102554000 1057 T24 G 6 39096919 44815780 571 T45 L 10 105427275 106912787 1486 T24 G 6 45632263 46448077 816 T45 L 10 108937968 109743759 806 T24 G 6 51798982 52237235 438 T45 L 10 112505977 115170450 2664 T24 G 6 73071029 74115777 104 T45 L 10 115390767 116822855 1432 T24 G 6 90177196 92909352 273 T45 L 10 117527909 118176252 648 T24 G 6 94703006 95299063 596 T45 L 10 118242031 118611337 369 T24 G 6 134723355 135274812 551 T45 L 10 119109301 123350993 4242 T24 G 6 142505116 144140048 163 T45 L 10 123939148 128535967 4597 T24 G 6 154044907 170880179 168 T45 L 10 129057100 135279687 6223 T24 G 7 57163665 57368235 205 T45 L 11 29591189 29810959 220 T24 G 7 64972395 65209849 237 T45 L 11 96545676 96833884 288 T24 G 7 152147266 152594870 448 T45 L 11 132551773 132915120 363 T24 G 8 23528659 24305391 777 T45 L 12 115722300 116073342 351 T24 G 8 73539189 74846711 130 T45 L 15 18674575 18891255 217 T24 G 9 138899424 139193163 294 T45 L 15 25855573 26564241 709 T24 G 11 60661723 60880212 218 T45 L 15 75603709 75885169 281 T24 G 11 131887972 132151114 263 T45 L 15 92398433 93718264 1320 T24 G 12 29727999 29929154 201 T45 L 16 44231 1750189 1706 T24 G 12 103519284 104707178 118 T45 L 16 4961896 9954815 4993 T24 G 12 126169055 126509298 340 T45 L 16 53615738 56056872 2441 T24 G 13 21320075 24477693 315 T45 L 16 60759769 63762681 3003 T24 G 13 35606696 36031506 425 T45 L 16 64289064 65812064 1523 T24 G 13 37671793 38416673 745 T45 L 16 66600107 67097726 498 T24 G 13 63561919 64068098 506 T45 L 16 69534056 70151844 618 T24 G 13 90250611 91073177 823 T45 L 16 71288486 87542642 16254 T24 G 13 110703658 114093192 339 T45 L 17 1882737 2937807 1055 T24 G 15 36223897 36476298 252 T45 L 17 7205406 12810358 5605 T24 G 15 72959418 73401643 442 T45 L 17 13230426 14235312 1005 T24 G 17 27245834 27571984 326 T45 L 17 14696523 17035446 2339 T24 G 17 71010525 71279315 269 T45 L 17 17372997 17865872 493 T24 G 18 182061 15060997 148 T45 L 17 20438273 21191548 753 T24 G 18 16659756 54911494 382 105  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T45 L 18 5680355 6146022 466 T24 G 18 56063594 76098439 200 T45 L 18 18121223 18535826 415 T24 G 19 74843 7984147 790 T50 G 1 16646685 17086056 439 T24 G 19 9090400 13472945 438 T50 G 1 85694718 86053387 359 T24 G 19 13844286 15928714 208 T50 G 1 142533622 144162829 1629 T24 G 19 16184022 24178465 799 T50 G 1 154668788 155733368 1065 T24 G 19 32839254 34943389 210 T50 G 1 157581141 158487941 907 T24 G 19 35186754 56396446 212 T50 G 1 158845477 163685206 4840 T24 G 19 56430305 63654245 722 T50 G 1 175300547 178811659 3511 T24 G 20 60369 734327 674 T50 G 1 179640235 180447764 808 T24 G 20 815899 24411323 235 T50 G 1 182037344 182693973 657 T24 G 20 29954191 35971325 601 T50 G 1 190304054 191386589 1083 T24 G 20 36528233 39479846 295 T50 G 1 200361154 208949372 8588 T24 G 20 39677527 44130755 445 T50 G 1 209520699 212332499 2812 T24 G 20 44493987 61192343 166 T50 G 1 213258610 213891474 633 T24 G 21 31898817 38767124 686 T50 G 1 214367650 214702922 335 T24 G 21 39766436 46927776 716 T50 G 1 216417887 217242640 825 T24 L 1 167301416 167753878 452 T50 G 1 217480858 219481944 2001 T24 L 2 16254527 16432161 178 T50 G 1 220456289 220938014 482 T24 L 3 78394977 78583892 189 T50 G 1 221391322 221739224 348 T24 L 3 140772863 141334635 562 T50 G 1 242089298 242703195 614 T24 L 3 162633116 163227623 595 T50 G 2 25634157 25895046 261 T24 L 4 3645088 4065877 421 T50 G 3 165087143 165392754 306 T24 L 4 6406037 7071120 665 T50 G 4 41670052 41922725 253 T24 L 4 10371953 10941759 570 T50 G 5 4943436 5437991 495 T24 L 4 82568192 84598755 203 T50 G 5 7370486 7983116 613 T24 L 4 115659364 116177484 518 T50 G 5 20897033 21399027 502 T24 L 4 118592935 119487920 895 T50 G 5 27918183 28306347 388 T24 L 4 141100509 141832678 732 T50 G 5 28982664 29258816 276 T24 L 4 142795200 148963622 616 T50 G 5 69210855 70180160 969 T24 L 4 152699346 153069035 370 T50 G 5 78205814 78877668 672 T24 L 4 159111257 159988962 878 T50 G 5 80446929 81024602 578 T24 L 5 93450342 93830506 380 T50 G 5 95697117 96195619 499 T24 L 6 95354742 95609774 255 T50 G 5 97652683 97856979 204 T24 L 6 103346894 103604028 257 T50 G 5 112408899 112878659 470 T24 L 7 24242808 24491213 248 T50 G 5 145266747 145667242 400 T24 L 7 55784611 56234811 450 T50 G 6 213736 19606867 19393 T24 L 7 101876389 102207473 331 T50 G 6 20072259 43638171 23566 T24 L 7 108283569 108768829 485 T50 G 6 43995870 45546354 1550 T24 L 7 126932281 127397229 465 T50 G 6 45777468 49967039 4190 T24 L 7 143515150 143839638 324 T50 G 6 50826897 55723924 4897 T24 L 7 153572414 153780253 208 T50 G 6 63037007 63309019 272 T24 L 8 764689 2798932 203 T50 G 7 61087267 62176519 1089 T24 L 8 3329536 3634216 305 T50 G 7 101876389 102207473 331 T24 L 8 17007797 19423330 241 T50 G 7 111855955 112555864 700 T24 L 8 19765603 23579445 381 T50 G 7 153637511 153901567 264 T24 L 8 32968313 34237587 126 T50 G 8 8242248 8972061 730 T24 L 8 52621388 53720032 109 106  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T50 G 10 118147658 118360397 213 T24 L 8 57031842 58555824 152 T50 G 10 128335445 128787784 452 T24 L 8 75167545 76418032 125 T50 G 11 60661723 60880212 218 T24 L 8 77458893 78033463 575 T50 G 12 36302222 36432239 130 T24 L 8 86699540 87055819 356 T50 G 13 20698521 26017210 5319 T24 L 8 87744061 94497499 675 T50 G 13 27142461 39713370 12571 T24 L 8 97285991 98111425 825 T50 G 13 40460256 43127842 2668 T24 L 8 103542930 105219912 167 T50 G 13 43481375 45109681 1628 T24 L 8 105636430 109065071 342 T50 G 13 51492647 52938369 1446 T24 L 8 116012122 126818082 108 T50 G 13 83509678 83894529 385 T24 L 8 133072743 133464258 392 T50 G 13 89110421 98902701 9792 T24 L 8 140919525 141422749 503 T50 G 13 105166636 113901932 8735 T24 L 8 141840700 143165362 132 T50 G 14 74831170 75200717 370 T24 L 9 1302960 1745895 443 T50 G 14 89221786 91985996 2764 T24 L 9 3762511 4605818 843 T50 G 14 94478987 95405991 927 T24 L 9 38500449 38736668 236 T50 G 14 97332470 98569435 1237 T24 L 9 82833412 83098662 265 T50 G 15 50959420 51145331 186 T24 L 9 113006323 113222716 216 T50 G 18 42657928 42840984 183 T24 L 10 477678 3496279 301 T50 G 19 55554013 55707294 153 T24 L 10 4260820 12011805 775 T50 L 1 3204885 3670395 466 T24 L 10 13548200 21627134 807 T50 L 1 18152027 19020943 869 T24 L 10 22227856 32041152 981 T50 L 1 41399038 42076304 677 T24 L 10 34351870 39116217 476 T50 L 1 47813577 48194648 381 T24 L 10 43318759 61160076 178 T50 L 1 71623262 72069166 446 T24 L 10 65100222 66994232 189 T50 L 1 87291697 87588045 296 T24 L 10 72339683 102554000 302 T50 L 2 5746343 6228194 482 T24 L 10 103279334 108738304 545 T50 L 2 23512042 23881796 370 T24 L 10 110133250 110889891 757 T50 L 2 71611728 72107211 495 T24 L 10 112801345 117118223 431 T50 L 2 134654310 134996036 342 T24 L 10 117429331 135279687 178 T50 L 2 163641233 164029300 388 T24 L 11 39250444 39661602 411 T50 L 2 217405254 218352598 947 T24 L 11 55463571 55923540 460 T50 L 2 238603267 241063385 2460 T24 L 11 84557237 85007763 451 T50 L 3 18708675 18905397 197 T24 L 11 89012345 89476176 464 T50 L 3 43774937 44281588 507 T24 L 11 130977965 131329903 352 T50 L 3 53469006 53880921 412 T24 L 11 132051142 132389201 338 T50 L 3 108872278 109166176 294 T24 L 12 92435964 92696372 260 T50 L 3 128108621 128622314 514 T24 L 13 24483411 26526090 204 T50 L 3 129385338 129741914 357 T24 L 13 27727371 28177459 450 T50 L 3 131422673 131723522 301 T24 L 13 36678499 37673052 995 T50 L 4 49189866 49416673 227 T24 L 13 38419312 39713370 129 T50 L 4 77946571 78342846 396 T24 L 13 46168557 56269250 101 T50 L 4 109386058 109668156 282 T24 L 13 70057505 73606932 354 T50 L 7 2864230 3184062 320 T24 L 13 78513431 90250492 117 T50 L 7 57297100 57589990 293 T24 L 13 90997052 97825675 682 T50 L 7 140395448 140575191 180 T24 L 13 103017055 108808111 579 T50 L 8 156973 2612270 2455 T24 L 13 110362947 110764162 401 T50 L 8 3171492 5600558 2429 T24 L 14 98083684 98569435 486 107  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T50 L 8 8938573 11424992 2486 T24 L 15 24553837 27477714 292 T50 L 8 87208452 87407061 199 T24 L 15 46199533 47809868 161 T50 L 9 9067399 9654318 587 T24 L 15 82472074 85278339 280 T50 L 9 82833412 82953738 120 T24 L 15 85411106 87781613 237 T50 L 10 3631141 4728854 1098 T24 L 15 89632877 93718264 408 T50 L 10 8851508 9117982 266 T24 L 15 94546822 100309437 576 T50 L 10 48181069 50237413 2056 T24 L 16 44231 1032612 988 T50 L 10 51903179 52337214 434 T24 L 16 15040819 15414812 374 T50 L 10 79776333 80241593 465 T24 L 16 33866283 35136830 127 T50 L 10 130431595 132473339 2042 T24 L 16 45548262 46605357 105 T50 L 10 133771689 134669886 898 T24 L 16 47457492 51434198 397 T50 L 13 48069306 48404010 335 T24 L 16 52309608 52599483 290 T50 L 13 49595011 50888467 1293 T24 L 16 53615738 55091064 147 T50 L 13 71226333 71728231 502 T24 L 16 61056663 61730602 674 T50 L 16 48106458 48638074 532 T24 L 16 70754647 73114581 236 T50 L 16 49406045 50618041 1212 T24 L 16 77175405 78760401 158 T50 L 16 54410314 55091064 681 T24 L 16 78813031 79773936 961 T50 L 16 69643219 70151844 509 T24 L 16 79901920 82488856 258 T50 L 16 71288486 72036034 748 T24 L 19 15874684 16219285 345 T50 L 16 75370384 75899230 529 T24 L 20 39427063 39747527 320 T50 L 16 77371695 77830699 459 T17 G 4 142292357 142586186 294 T50 L 16 77920768 79572749 1652 T17 G 7 57163665 57368235 205 T50 L 16 79901920 82307174 2405 T17 G 11 60661723 60880212 218 T50 L 16 84535576 85732272 1197 T17 G 11 96833936 97154680 321 T50 L 17 2534851 2937807 403 T17 G 11 131887972 132151114 263 T50 L 17 4687532 5025418 338 T17 G 12 36302222 36432239 130 T50 L 17 5418209 7457682 2039 T17 G 12 69844305 70114686 270 T50 L 17 9487785 12620614 3133 T17 G 15 18674575 19353919 679 T50 L 17 13230426 14235312 1005 T17 G 15 30328715 30597338 269 T50 L 17 14415762 15759518 1344 T17 G 15 46039586 46542062 502 T50 L 17 17534762 17865872 331 T17 G 18 42657928 42840984 183 T50 L 19 8629145 8832847 204 T17 G 19 23835271 24178465 343 T50 L 20 44493987 44796823 303 T17 L 2 134784717 135096043 311 T50 L 20 56245366 57461196 1216 T17 L 3 78394977 78583892 189 T50 L 20 61041281 61537434 496 T17 L 3 140772863 141053191 280 T50 L 22 15930939 16096955 166 T17 L 7 20168369 20487247 319 T50 L 22 16853741 17206783 353 T17 L 8 7035076 7289755 255 T50 L 22 45774295 47743222 1969 T17 L 8 136577260 137000216 423 T34 G 1 160338731 160677252 339 T17 L 8 139282254 140847408 156 T34 G 1 162887424 163728359 841 T17 L 9 82833412 82953738 120 T34 G 1 164996988 167445692 2449 T17 L 11 79463518 80194218 731 T34 G 1 167684245 168121001 437 T17 L 11 127563703 128090809 527 T34 G 1 173228930 174053395 824 T17 L 17 13230426 14235312 100 T34 G 1 174639051 177140954 2502 T17 L 19 8629145 8832847 204 T34 G 1 177945800 178479748 534 T11 G 1 3553533 3811527 258 T34 G 1 179071822 181045400 1974 T11 G 1 11435982 11755347 319 T34 G 1 181937518 183023220 1086 T11 G 1 16646685 17086056 439 108  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T34 G 1 185070352 186433554 1363 T11 G 1 24934692 25334144 399 T34 G 1 198807950 203411834 4604 T11 G 1 120249993 121077638 828 T34 G 1 204678374 209169786 4491 T11 G 1 142533622 145133682 260 T34 G 1 211445419 215373858 3928 T11 G 1 145357581 145734554 377 T34 G 1 216039670 216754904 715 T11 G 1 146778005 147083700 306 T34 G 1 217480858 220609576 3129 T11 G 1 149747994 151131174 138 T34 G 1 220922499 222040987 1118 T11 G 1 152333831 156488812 415 T34 G 1 222772446 223152230 380 T11 G 1 159680000 160677252 997 T34 G 1 226511813 229467946 2956 T11 G 1 161016469 161777223 761 T34 G 1 229883839 233058352 3175 T11 G 1 163384213 171187941 780 T34 G 1 234037398 236445764 2408 T11 G 1 174639051 176344936 170 T34 G 1 241399361 241887674 488 T11 G 1 177945800 181045400 310 T34 G 1 242101448 242491480 390 T11 G 1 182037344 183769234 173 T34 G 6 160807593 161039688 232 T11 G 1 196594358 208088220 114 T34 G 7 78657435 79091309 434 T11 G 1 211445419 213891474 244 T34 G 9 115724200 117541359 1817 T11 G 1 214852240 215373858 522 T34 G 9 118654542 120225709 1571 T11 G 1 218273469 220609576 233 T34 G 9 121736149 122361130 625 T11 G 1 221391322 221739224 348 T34 G 9 124820910 125614093 793 T11 G 1 223882340 233440847 955 T34 G 9 127622217 128420787 799 T11 G 1 233834184 236328485 249 T34 G 16 45866013 46868750 1003 T11 G 1 239216214 241090397 187 T34 L 1 3056267 3670395 614 T11 G 1 242089298 242491480 402 T34 L 1 4138943 7449191 3310 T11 G 2 41662 696156 654 T34 L 1 10604300 11929541 1325 T11 G 2 1438693 6391789 495 T34 L 1 12724432 16394945 3671 T11 G 2 6648268 12541039 589 T34 L 1 17730518 23183947 5453 T11 G 2 14520907 21856943 733 T34 L 1 26950696 28189085 1238 T11 G 2 27134515 31254640 412 T34 L 1 29953410 30239890 286 T11 G 2 39691420 40675772 984 T34 L 3 80918434 81176295 258 T11 G 2 41706139 42547144 841 T34 L 6 62016102 62350605 335 T11 G 2 44824074 47789368 296 T34 L 7 61094178 61797715 704 T11 G 2 54554030 55438372 884 T34 L 8 156973 2926879 2770 T11 G 2 58126468 60943837 281 T34 L 8 3171492 13280883 10109 T11 G 2 66790913 72007879 521 T34 L 8 20179288 20966050 787 T11 G 2 73109249 74467228 135 T34 L 8 22611007 24112444 1501 T11 G 2 84721136 89405301 468 T34 L 8 32739754 33295340 556 T11 G 2 94986258 95863637 877 T34 L 8 39644485 43912178 4268 T11 G 2 97249568 98980797 173 T34 L 8 47010496 47901818 891 T11 G 2 101985939 108262692 627 T34 L 8 57031842 59599553 2568 T11 G 2 108761390 113816728 505 T34 L 8 62082431 63859487 1777 T11 G 2 118601009 120128215 152 T34 L 8 65367828 66128051 760 T11 G 2 132821463 134595150 177 T34 L 8 86724325 87055819 331 T11 G 2 135989619 137582864 159 T34 L 8 91826785 94331876 2505 T11 G 2 147842811 148157606 315 T34 L 8 97299998 98317863 1018 T11 G 2 148963859 150051708 108 T34 L 8 101868947 104530779 2662 T11 G 2 152902602 153748650 846 T34 L 8 116410840 120193443 3783 T11 G 2 162516066 163117738 602 T34 L 8 123818629 125734397 1916 T11 G 2 175041480 178341522 330 109  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T34 L 8 126801789 129476219 2674 T11 G 2 191477009 192734438 125 T34 L 8 133072743 134244619 1172 T11 G 2 202017847 202836206 818 T34 L 8 134988076 135520671 533 T11 G 2 207584984 208489071 904 T34 L 8 135753903 137000216 1246 T11 G 2 217405254 220909658 350 T34 L 8 137364884 141332829 3968 T11 G 2 226834062 228081471 124 T34 L 8 143710403 143916668 206 T11 G 2 229763361 229961863 199 T34 L 12 2519891 2655195 135 T11 G 2 230702740 234828785 412 T34 L 12 13450373 14142029 692 T11 G 2 236163199 236728809 566 T34 L 12 36302222 36786132 484 T11 G 2 237173612 241063385 389 T34 L 16 5170141 7586817 2417 T11 G 4 8979402 9841887 862 T34 L 16 9148951 9954815 806 T11 G 4 172753517 173131406 378 T34 L 16 14003160 14454827 452 T11 G 5 1684158 1965402 281 T34 L 16 18936858 27865580 8929 T11 G 5 17284044 17553934 270 T34 L 16 47811144 48264112 453 T11 G 5 69602994 70066836 464 T34 L 16 48842296 56056872 7215 T11 G 5 95697117 95852946 156 T34 L 16 60255176 63762681 3508 T11 G 5 138859931 139250455 391 T34 L 16 64289064 64876753 588 T11 G 6 73242 3890080 381 T34 L 16 69787193 79467985 9681 T11 G 6 6231728 8075923 184 T34 L 16 80059470 85732272 5673 T11 G 6 11402225 11936033 534 T11 L 1 27020386 27234980 215 T11 G 6 13508273 14506540 998 T11 L 1 190137112 190351358 214 T11 G 6 33017597 44166592 111 T11 L 3 40052595 41220493 1168 T11 G 6 45494917 46849677 135 T11 L 3 42380990 42652925 272 T11 G 6 53456669 53956516 500 T11 L 3 43774937 45581671 1807 T11 G 6 87028414 87408200 380 T11 L 3 56682157 57209359 527 T11 G 6 89795423 91296384 150 T11 L 3 68278236 68785539 507 T11 G 6 110846002 111130824 285 T11 L 3 69383522 70910161 1527 T11 G 6 123929276 124385689 456 T11 L 3 73143638 73736485 593 T11 G 6 149457230 149977460 520 T11 L 3 188713968 190600506 1887 T11 G 6 159737023 161039688 130 T11 L 3 193362761 195170190 1807 T11 G 6 163376951 164236656 860 T11 L 4 54787033 55371595 585 T11 G 6 167790639 168933714 114 T11 L 4 84575660 85692474 1117 T11 G 7 42475 2407575 236 T11 L 4 97791526 98324578 533 T11 G 7 3167180 5446129 227 T11 L 4 108865061 109473917 609 T11 G 7 6038742 8246051 220 T11 L 4 115659364 116742407 1083 T11 G 7 11281922 12075944 794 T11 L 4 125632333 125947507 315 T11 G 7 13834918 15851570 201 T11 L 4 130534517 131928006 1393 T11 G 7 33604992 34018904 414 T11 L 4 147902044 148530554 629 T11 G 7 57279942 57589990 310 T11 L 4 173045996 174144577 1099 T11 G 7 105553799 106086426 533 T11 L 4 181177641 185095200 3918 T11 G 7 127398355 127812452 414 T11 L 4 188522242 190387639 1865 T11 G 7 143515150 143839638 324 T11 L 5 29899094 30610845 712 T11 G 8 36943 179853 143 T11 L 5 68702646 69102023 399 T11 G 10 99476175 100248423 772 T11 L 8 7035076 7289755 255 T11 G 11 60661723 60880212 218 T11 L 9 23994 964142 940 T11 G 12 52090606 52327387 237 T11 L 9 937155 1498619 561 T11 G 13 23525346 28177459 465 T11 L 9 1415639 1901823 486 T11 G 13 29471954 33324334 385 110  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T11 L 9 1875733 2296356 421 T11 G 13 34965270 35422901 458 T11 L 9 2271469 2620657 349 T11 G 13 39330480 40124114 794 T11 L 9 2584552 3399803 815 T11 G 13 42504729 43926080 142 T11 L 9 3360032 3696642 337 T11 G 13 46168557 46488557 320 T11 L 9 3627891 4163314 535 T11 G 13 50689132 51467864 779 T11 L 9 4119797 13011570 8892 T11 G 13 62001010 62577460 576 T11 L 9 13490884 14138081 647 T11 G 13 93396760 101232430 783 T11 L 9 14568749 16325485 1757 T11 G 13 102377421 102837827 460 T11 L 9 16193562 16784841 591 T11 G 13 110600643 114093192 349 T11 L 9 16784842 17666004 881 T11 G 15 18674575 20053874 137 T11 L 9 20133898 21158464 1025 T11 G 15 45913031 46223231 310 T11 L 9 31530079 31868461 338 T11 G 15 66365409 66909639 544 T11 L 9 31810164 33355181 1545 T11 G 17 33264100 33700089 436 T11 L 9 35779611 36133758 354 T11 G 17 41755471 42239175 484 T11 L 9 39294215 40762683 1468 T11 G 17 68745974 69076047 330 T11 L 9 77684451 78480906 796 T11 G 18 42657928 42803873 146 T11 L 11 97278670 97609941 331 T11 G 18 62933102 63153508 220 T11 L 12 32108495 32334573 226 T11 G 19 32839254 36584144 374 T11 L 12 41471084 41979185 508 T11 G 19 37053061 37599399 546 T11 L 12 87593424 87952085 359 T11 G 19 44261521 44608085 347 T11 L 12 113919526 114162755 243 T11 G 19 45946143 49532531 358 T11 L 13 102814184 103787434 973 T11 G 19 61364524 63654245 229 T11 L 14 34291680 35020664 729 T11 G 20 60369 3341100 328 T11 L 14 35993653 36534612 541 T11 G 20 7516914 11951142 443 T11 L 14 51580716 52650444 1070 T11 G 20 15310602 26233197 109 T11 L 14 71515335 73019214 1504 T11 G 20 29954191 31357350 140 T11 L 14 73282483 73691608 409 T11 G 20 32552419 35971325 341 T11 L 14 95632850 97138734 1506 T11 G 20 36528233 49744292 132 T11 L 16 8317822 9816450 1499 T11 G 20 54011886 61192343 718 T11 L 16 12176022 16391845 4216 T11 G 21 21580499 21789792 209 T11 L 16 20943307 22020584 1077 T11 L 3 77228270 78310037 108 T11 L 16 25365870 26779984 1414 T11 L 3 78394977 78583892 189 T11 L 16 29181450 29392366 211 T11 L 3 114828518 115437585 609 T11 L 16 34133153 35136830 1004 T11 L 3 140772863 142716241 194 T11 L 16 46597919 47016802 419 T11 L 3 152957973 154220806 126 T11 L 16 60759769 61286942 527 T11 L 3 156012946 156952918 940 T11 L 17 73158376 73381889 224 T11 L 3 160202103 161251745 105 T11 L 18 10976351 11327785 351 T11 L 3 170074359 171101033 102 T11 L 18 23570175 23897445 327 T11 L 3 182483149 183176951 694 T11 L 19 8934066 9026570 93 T11 L 19 32423910 32613567 190 T11 L 19 55554013 55707294 153 T11 L 22 40935061 41198248 263 T11 L 22 45618914 47839586 2221 111  Table A.3    aCGH alterations by sample (RB1+/-). ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E25 G 1 16638721 17086056 447 T79 G 1 16646685 17086056 439 E25 G 1 232830875 233105801 275 T79 G 1 120249993 121077638 828 E25 G 4 108572846 108797097 224 T79 G 1 142533622 144873033 2339 E25 G 5 17284044 17553934 270 T79 G 1 145511311 145872489 361 E25 G 5 171952417 172226263 274 T79 G 1 147590048 148144657 555 E25 G 6 73242 346084 273 T79 G 1 148655012 153230241 4575 E25 G 6 62893855 63309019 415 T79 G 1 154247819 156300507 2053 E25 G 6 103346894 103604028 257 T79 G 1 158010337 158487941 478 E25 G 8 58157391 58393591 236 T79 G 1 159680000 160677252 997 E25 G 8 116773589 117061503 288 T79 G 1 161016469 161777223 761 E25 G 10 9174672 11386923 2212 T79 G 1 162887424 171525137 8638 E25 G 10 13548200 14520085 972 T79 G 1 173228930 174094669 866 E25 G 10 42112266 43201651 1089 T79 G 1 174639051 176053350 1414 E25 G 10 43318759 44600038 1281 T79 G 1 177864963 178479748 615 E25 G 10 45037616 50711049 5673 T79 G 1 179462543 181045400 1583 E25 G 10 62342913 63308545 966 T79 G 1 182037344 183361276 1324 E25 G 10 63597548 64220266 623 T79 G 1 199215670 209169786 9954 E25 G 10 70708535 71438469 730 T79 G 1 211445419 213185256 1740 E25 G 10 72033929 73288590 1255 T79 G 1 214172120 215373858 1202 E25 G 10 76667248 78143315 1476 T79 G 1 217480858 218795330 1314 E25 G 10 78319642 78833935 514 T79 G 1 219017537 220609576 1592 E25 G 10 79431219 85424122 5993 T79 G 1 222704527 223260778 556 E25 G 10 124978917 125259149 280 T79 G 1 224791361 233391573 8600 E25 G 11 46058617 46407944 349 T79 G 1 237294682 239002879 1708 E25 G 13 77747248 78216186 469 T79 G 1 242294867 244296856 2002 E25 G 13 90250611 91242980 992 T79 G 1 246424085 246944046 520 E25 G 13 92613317 93325237 712 T79 G 2 41662 9407162 9366 E25 G 13 95581707 98415699 2834 T79 G 2 10828736 13079174 2250 E25 G 13 98714149 99376982 663 T79 G 2 14520907 23881796 9361 E25 G 13 100030217 100888613 858 T79 G 2 27652475 31254640 3602 E25 G 13 101364859 102837827 1473 T79 G 2 36503134 38839346 2336 E25 G 13 105535345 107155648 1620 T79 G 2 38942295 39196527 254 E25 G 13 108629457 114093192 5464 T79 G 2 39691420 43518222 3827 E25 G 14 19149713 19485285 336 T79 G 2 44824074 47789368 2965 E25 G 15 20588181 20967788 380 T79 G 2 48847617 49273764 426 E25 G 15 27830649 28937648 1107 T79 G 2 50564941 51219186 654 E25 G 15 30237319 30597338 360 T79 G 2 53483127 53906745 424 E25 G 17 33264100 33700089 436 T79 G 2 54256708 55105184 848 E25 G 17 54544587 54973480 429 T79 G 2 58126468 60555094 2429 E25 G 17 55248203 55709933 462 T79 G 2 64350737 65026194 675 E25 G 17 60576372 61471405 895 T79 G 2 65474555 76133225 10659 E25 G 17 62228071 62594266 366 T79 G 2 83467797 88373278 4905 E25 G 17 67503237 69931286 2428 T79 G 2 88921446 91590359 2669 E25 G 17 71406761 71663462 257 T79 G 3 52725023 52967443 242 E25 G 17 74090777 74851642 761 T79 G 3 155644236 156012945 369 112  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E25 G 17 78249151 78615238 366 T79 G 3 190572724 190793756 221 E25 G 18 1946123 2270736 325 T79 G 4 109386058 109668156 282 E25 G 18 13210163 13643492 433 T79 G 4 140894330 142828372 1934 E25 G 18 26193974 26349693 156 T79 G 4 143195412 151471513 8276 E25 G 18 33328621 35657079 2328 T79 G 4 152158287 159988962 7831 E25 G 18 42226552 44723913 2497 T79 G 4 160281144 164670201 4389 E25 G 18 53512602 54477350 965 T79 G 4 164893174 185959726 21067 E25 G 18 58927478 59480384 553 T79 G 4 186022734 191013899 4991 E25 G 18 67604061 67966894 363 T79 G 5 17284044 17553934 270 E25 G 18 70635327 74815544 4180 T79 G 5 57285917 57537297 251 E25 G 18 75216360 76098439 882 T79 G 5 95697117 95852946 156 E25 G 19 12856233 13472945 617 T79 G 6 73242 10308635 10235 E25 G 19 15004017 16219285 1215 T79 G 6 10648344 19088319 8440 E25 G 19 21788967 22154696 366 T79 G 6 19480476 43925174 24445 E25 L 3 46140 1857205 1811 T79 G 6 43851257 44166592 315 E25 L 3 2804349 18905397 16101 T79 G 6 44117784 45175251 1057 E25 L 3 22209305 25600762 3391 T79 G 6 45494917 58849697 13355 E25 L 3 26527404 31335620 4808 T79 G 8 6515253 6907559 392 E25 L 3 36350375 41220493 4870 T79 G 8 58157391 58393591 236 E25 L 3 42380990 43363544 983 T79 G 9 73371288 73732453 361 E25 L 3 43774937 46115480 2341 T79 G 10 38983843 39116217 132 E25 L 3 48536891 50482968 1946 T79 G 11 60573488 60880212 307 E25 L 3 51228042 56176682 4949 T79 G 12 52090606 52327387 237 E25 L 3 57523638 58715235 1192 T79 G 12 66859847 67063558 204 E25 L 3 59349125 63205827 3857 T79 G 12 108154779 108581445 427 E25 L 3 63461865 68785539 5324 T79 G 12 114113748 114520437 407 E25 L 3 69900482 74025996 4126 T79 G 15 22796772 23152527 356 E25 L 3 76936762 78310037 1373 T79 G 15 28106102 28734907 629 E25 L 3 86143774 87362355 1219 T79 G 16 69365103 69777653 413 E25 L 4 77119217 78634666 1515 T79 G 16 83933107 84359546 426 E25 L 4 80523279 81600464 1077 T79 G 20 26104051 26233197 129 E25 L 4 82331002 83776911 1446 T79 G 20 44380338 44472487 92 E25 L 4 84756808 85692474 936 T79 G 20 55869603 56267785 398 E25 L 5 9117097 9336791 220 T79 L 2 91608685 91680249 72 E25 L 5 161447352 161731582 284 T79 L 3 36111427 36427114 316 E25 L 5 170424700 170635099 210 T79 L 3 39737594 39987069 249 E25 L 6 9303589 9524750 221 T79 L 4 132667102 133073640 407 E25 L 6 163246092 163450786 205 T79 L 5 103927370 104469666 542 E25 L 8 2059659 2279635 220 T79 L 5 104995250 107081530 2086 E25 L 8 86699540 87055819 356 T79 L 5 106909929 107306804 397 E25 L 10 477678 3904258 3427 T79 L 5 107235949 108154189 918 E25 L 11 121858210 122274860 417 T79 L 5 108502451 112557699 4055 E25 L 12 114960307 115230777 270 T79 L 5 112668515 113857732 1189 E25 L 13 52275633 52938369 663 T79 L 5 114700474 118294962 3594 E25 L 17 56824384 57089085 265 T79 L 5 129057000 134458770 5402 E1 G 1 6040269 6256800 217 T79 L 5 134568930 135240151 671 E1 G 1 7778452 11929541 4151 T79 L 5 135193500 141196500 6003 113  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E1 G 1 15896191 16620917 725 T79 L 5 141348434 155252853 13904 E1 G 1 17239175 17648568 409 T79 L 5 155766667 161647844 5881 E1 G 1 21717568 24615446 2898 T79 L 5 162634259 165246970 2613 E1 G 1 26424669 29453636 3029 T79 L 5 165508395 167079037 1571 E1 G 1 34892938 36369397 1476 T79 L 5 167024174 167819073 795 E1 G 1 39063444 41233201 2170 T79 L 5 167802449 168220953 419 E1 G 1 44122639 45325761 1203 T79 L 5 168095482 168709547 614 E1 G 1 51342022 53007168 1665 T79 L 5 168704419 179838343 11134 E1 G 1 108993704 110039385 1046 T79 L 7 72652466 72877032 225 E1 G 1 148535332 149813049 1278 T79 L 8 86558071 87055819 498 E1 G 1 152524737 155119104 2594 T79 L 11 97278670 97609941 331 E1 G 1 200877039 201431856 555 T79 L 13 46168557 46488557 320 E1 G 1 232955277 233546093 591 T79 L 13 47429131 47613957 185 E1 G 2 9985433 11150862 1165 T79 L 20 29297109 29909342 612 E1 G 2 24892640 25572843 680 T79 L 20 49568098 49715808 148 E1 G 2 26714284 28097537 1383 T48 G 1 143900677 144162829 262 E1 G 2 31916659 33205438 1289 T48 G 1 157848491 158487941 639 E1 G 2 55550156 55858798 309 T48 G 1 218033276 218461661 428 E1 G 2 96074237 96378723 304 T48 G 1 219229659 219532748 303 E1 G 2 202673686 203037744 364 T48 G 1 230214952 230585021 370 E1 G 2 203558341 204180576 622 T48 G 2 41662 317294 276 E1 G 2 218744125 219110147 366 T48 G 2 640765 1218689 578 E1 G 2 232967040 233991801 1025 T48 G 2 1438693 2663054 1224 E1 G 2 241955282 242720443 765 T48 G 2 2815042 8825151 6010 E1 G 3 12335374 12780009 445 T48 G 2 11750125 12541039 791 E1 G 3 46943432 48164871 1221 T48 G 2 12928689 15892772 2964 E1 G 3 49843917 50482968 639 T48 G 2 16361494 18347973 1986 E1 G 3 114814923 115059605 245 T48 G 2 18946602 23881796 4935 E1 G 3 121682380 121964439 282 T48 G 2 30764067 31915204 1151 E1 G 3 126071864 126244947 173 T48 G 2 36371522 36996317 625 E1 G 3 139855931 140244152 388 T48 G 2 59913866 60555094 641 E1 G 3 170977548 171510249 533 T48 G 2 65106400 67279330 2173 E1 G 3 198952699 199276959 324 T48 G 2 70345668 72279682 1934 E1 G 4 3705279 4065877 361 T48 G 2 86933421 91680249 4747 E1 G 4 38175656 40511826 2336 T48 G 2 109942316 114143148 4201 E1 G 5 350724 1562826 1212 T48 G 2 119008111 119748627 741 E1 G 5 10266635 10636898 370 T48 G 2 120626425 121633928 1008 E1 G 5 36939489 37473658 534 T48 G 2 128716306 131497926 2782 E1 G 5 53205455 53387793 182 T48 G 2 132821463 134595150 1774 E1 G 5 68311858 69037279 725 T48 G 2 138967268 139369283 402 E1 G 5 74566167 74865527 299 T48 G 2 164156982 164779204 622 E1 G 5 86471625 86737136 266 T48 G 2 199032194 199519884 488 E1 G 5 111925202 112249276 324 T48 G 2 216927309 218904083 1977 E1 G 5 126810683 127081020 270 T48 G 2 219925081 222928085 3003 E1 G 5 137318181 138329154 1011 T48 G 2 236568755 241063385 4495 E1 G 7 71800030 72164907 365 T48 G 3 26305164 26651493 346 E1 G 7 72809108 73534009 725 T48 G 3 106734450 106950425 216 114  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E1 G 7 99873579 101989129 2116 T48 G 3 108016085 108230341 214 E1 G 8 22867983 23232427 364 T48 G 3 134944834 135140571 196 E1 G 8 67828684 68608798 780 T48 G 4 48771496 49416673 645 E1 G 8 103387755 103959662 572 T48 G 5 70263 7150950 7081 E1 G 9 15176811 15509560 333 T48 G 5 7664722 10140637 2476 E1 G 9 20392848 20736915 344 T48 G 5 10597609 16847656 6250 E1 G 9 96096087 98512120 2416 T48 G 5 17284044 18012097 728 E1 G 9 113177037 114149576 973 T48 G 5 20897033 21549907 653 E1 G 9 123326445 128420787 5094 T48 G 5 21907045 22929763 1023 E1 G 9 128732487 140185705 11453 T48 G 5 32789563 36158483 3369 E1 G 10 21655748 22103168 447 T48 G 5 37446811 43279969 5833 E1 G 10 104813634 105172292 359 T48 G 5 50797452 54459262 3662 E1 G 11 50991 651922 601 T48 G 5 55064491 55411390 347 E1 G 11 3573460 4099388 526 T48 G 5 55718971 56366596 648 E1 G 11 6246775 6580309 334 T48 G 5 56587456 57537297 950 E1 G 11 46429333 47629247 1200 T48 G 5 58183784 61656696 3473 E1 G 11 47710156 48122594 412 T48 G 5 65479269 68369694 2890 E1 G 11 64048806 67116241 3067 T48 G 5 68951921 72139981 3188 E1 G 11 72553832 73412975 859 T48 G 5 72433622 72928146 495 E1 G 11 76815285 77212623 397 T48 G 5 72933899 75429647 2496 E1 G 11 82399450 82760983 362 T48 G 5 75354258 75669523 315 E1 G 11 118055345 118758082 703 T48 G 5 76889115 77472799 584 E1 G 12 7589386 8010390 421 T48 G 5 79041719 79525328 484 E1 G 12 47274158 49849518 2575 T48 G 5 180195851 180444136 248 E1 G 12 54296170 55617585 1321 T48 G 6 73242 20763610 20690 E1 G 12 108541669 110713626 2172 T48 G 6 106731286 106914529 183 E1 G 12 114851293 115132869 282 T48 G 7 57163665 57368235 205 E1 G 12 121469696 124234371 2765 T48 G 7 102001310 102640752 639 E1 G 12 131869058 132289534 420 T48 G 8 6515253 6907559 392 E1 G 13 29951709 30486781 535 T48 G 8 58157391 58393591 236 E1 G 13 42627655 42831764 204 T48 G 8 86196327 86435637 239 E1 G 13 54780783 55110752 330 T48 G 9 45908108 46458217 550 E1 G 13 90478077 90828221 350 T48 G 10 50596911 50869761 273 E1 G 13 97277801 99546821 2269 T48 G 11 118980810 119462934 482 E1 G 13 112897047 113901932 1005 T48 G 11 131887972 132151114 263 E1 G 14 30308385 31059479 751 T48 G 12 36090539 36311121 221 E1 G 14 72454326 73575089 1121 T48 G 12 111588013 112250545 663 E1 G 14 102640563 103624847 984 T48 G 13 63561919 64068098 506 E1 G 15 20302216 20792135 490 T48 G 13 90907034 101083925 10177 E1 G 15 73336790 73793805 457 T48 G 13 101364859 102690682 1326 E1 G 15 91086545 91292369 206 T48 G 13 102894434 109977945 7084 E1 G 16 1718470 2508230 790 T48 G 13 110362947 113901932 3539 E1 G 16 29306982 29880154 573 T48 G 14 103570986 103750538 180 E1 G 16 30539947 31120743 581 T48 G 15 18272238 18717253 445 E1 G 16 65200179 66485262 1285 T48 G 15 19087319 20224003 1137 E1 G 16 88032006 88573088 541 T48 G 15 30237319 30597338 360 E1 G 17 27213580 28028278 815 T48 G 16 31814669 32428987 614 115  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E1 G 17 30462926 45085833 14623 T48 G 16 33400710 33693171 292 E1 G 17 50943878 63366540 12423 T48 G 16 59038955 59260726 222 E1 G 17 65852467 73230854 7378 T48 G 18 42657928 42840984 183 E1 G 17 73330751 78615238 5284 T48 G 19 12587922 13472945 885 E1 G 18 16897445 18303681 1406 T48 G 19 15264050 16168320 904 E1 G 18 18745629 19695229 950 T48 G 19 32839254 34943389 2104 E1 G 18 25034674 25284594 250 T48 G 19 35186754 37230471 2044 E1 G 18 62103149 62837383 734 T48 G 19 39679939 40136267 456 E1 G 19 18013971 19781144 1767 T48 G 19 43448022 43949819 502 E1 G 19 54795121 55203857 409 T48 G 19 46693651 47430252 737 E1 G 20 2658810 4125081 1466 T48 G 19 47784067 48626174 842 E1 G 20 5506268 6145804 640 T48 G 19 50398622 50652469 254 E1 G 20 29486920 29909342 422 T48 G 19 55707295 56396446 689 E1 G 20 30430011 34853598 4424 T48 G 19 59799647 62794248 2995 E1 G 20 45219566 45756815 537 T48 G 20 60369 26233197 26173 E1 G 20 48069170 50228867 2160 T48 G 20 28034381 31357350 3323 E1 G 20 51597084 51966817 370 T48 G 20 32552419 62435964 29884 E1 G 20 54316042 54747849 432 T48 L 1 42399181 47483542 5084 E1 G 20 61386952 62435964 1049 T48 L 1 47658247 59669503 12011 E1 G 21 20794703 21052440 258 T48 L 1 59931165 60603939 673 E1 G 21 36306335 36850123 544 T48 L 1 62007293 62705908 699 E1 G 21 46391179 46927776 537 T48 L 1 63017988 68211732 5194 E1 G 22 17475778 18549230 1073 T48 L 1 68379704 69138390 759 E1 G 22 19054378 19736241 682 T48 L 1 74579113 76344381 1765 E1 G 22 28126621 30166633 2040 T48 L 1 77685641 90021480 12336 E1 G 22 30419219 30768051 349 T48 L 1 90930813 95323264 4392 E1 G 22 35061869 41661864 6600 T48 L 1 99921386 104094805 4173 E1 L 2 714656 944883 230 T48 L 1 106804596 107453098 649 E1 L 2 1438693 1649373 211 T48 L 1 108214875 116827699 8613 E1 L 2 3214213 3489907 276 T48 L 1 117081066 120078146 2997 E1 L 2 60112709 60403012 290 T48 L 3 129036625 129292335 256 E1 L 2 76560762 77347435 787 T48 L 3 197279380 198168305 889 E1 L 2 94689898 95017226 327 T48 L 4 39007380 39806180 799 E1 L 2 131538597 131894005 355 T48 L 4 64081058 64375660 295 E1 L 2 158728287 159062951 335 T48 L 4 113777516 114063996 286 E1 L 2 180385399 180842344 457 T48 L 4 128952512 129390725 438 E1 L 3 35484750 36234563 750 T48 L 4 151848353 152080415 232 E1 L 3 80918434 81176295 258 T48 L 5 75247038 75421795 175 E1 L 3 97715503 98211952 496 T48 L 5 95697117 95852946 156 E1 L 3 163846727 164196155 349 T48 L 5 172799092 173016342 217 E1 L 3 166701905 167210047 508 T48 L 5 176300084 176701325 401 E1 L 3 190572982 190902546 330 T48 L 6 20728596 58849697 38121 E1 L 4 22846486 23104480 258 T48 L 6 63255599 72853784 9598 E1 L 4 65613252 65926998 314 T48 L 6 73442278 79116376 5674 E1 L 4 68980297 70337363 1357 T48 L 6 84456091 89259710 4804 E1 L 4 76015896 76544458 529 T48 L 6 89365634 92119551 2754 E1 L 4 78040557 78823975 783 T48 L 6 93274524 93727522 453 116  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E1 L 4 142292357 142686744 394 T48 L 6 95000985 101234842 6234 E1 L 5 95697117 95992566 295 T48 L 6 105835894 106319178 483 E1 L 5 99124137 99378879 255 T48 L 6 106899458 149977460 43078 E1 L 5 170231354 170505998 275 T48 L 6 150084499 150405281 321 E1 L 6 58352741 58849697 497 T48 L 6 150414015 158417956 8004 E1 L 6 62000083 62977588 978 T48 L 6 159443190 170694605 11251 E1 L 6 73442278 73844143 402 T48 L 7 5691553 6688617 997 E1 L 6 85390617 85796999 406 T48 L 7 64207804 64485547 278 E1 L 6 149900596 150732866 832 T48 L 7 68323685 68727422 404 E1 L 6 160616018 161039688 424 T48 L 7 129173245 129771659 598 E1 L 7 10936621 11842183 906 T48 L 8 42528859 42880975 352 E1 L 7 48534377 48933660 399 T48 L 8 74696386 75318841 622 E1 L 7 66502015 66754917 253 T48 L 9 19026977 19373997 347 E1 L 7 81155139 81349702 195 T48 L 9 36214616 36774290 560 E1 L 8 85434907 85886119 451 T48 L 9 138571659 139070516 499 E1 L 8 86699540 87055819 356 T48 L 11 66670511 67116241 446 E1 L 8 132297428 132772696 475 T48 L 11 72852680 73412975 560 E1 L 9 43111687 44458663 1347 T48 L 11 132728882 133121207 392 E1 L 9 67270003 67409630 140 T48 L 12 7589386 8160827 571 E1 L 9 67803916 70224724 2421 T48 L 12 110370511 111395046 1025 E1 L 10 4260820 5235474 975 T48 L 12 117025244 117328738 303 E1 L 10 48181069 49216977 1036 T48 L 13 43986021 63637946 19652 E1 L 10 76667248 78484077 1817 T48 L 13 69889347 81142276 11253 E1 L 10 86396539 88170035 1773 T48 L 13 86479579 90923076 4443 E1 L 10 89987583 90397121 410 T48 L 14 34291680 35777003 1485 E1 L 10 95583121 96037340 454 T48 L 14 35993653 38968001 2974 E1 L 10 108801518 109288132 487 T48 L 14 44520774 45497527 977 E1 L 10 119281891 120267795 986 T48 L 14 49100288 49350925 251 E1 L 10 124978917 125357696 379 T48 L 14 63765831 64018070 252 E1 L 10 131945177 133477631 1532 T48 L 15 20302216 20627698 325 E1 L 11 15478026 15810241 332 T48 L 15 48336894 49146558 810 E1 L 11 47479617 47842883 363 T48 L 15 62141088 62552268 411 E1 L 12 38427897 38702944 275 T48 L 15 91086545 91292369 206 E1 L 13 49768827 51467864 1699 T48 L 16 18750161 19546524 796 E1 L 13 52402856 53318542 916 T48 L 16 51601593 51928256 327 E1 L 13 56242491 57788215 1546 T48 L 17 55205410 56647031 1442 E1 L 13 71226333 72149680 923 T48 L 17 59751854 60166708 415 E1 L 13 72702354 74860115 2158 T48 L 17 62517580 63701032 1183 E1 L 15 18272238 18543505 271 T48 L 19 32504109 32613567 109 E1 L 15 20762791 21207304 445 T19 G 1 3553533 3811527 258 E1 L 15 21805449 22655917 850 T19 G 1 27020386 27234980 215 E1 L 15 34634629 34933419 299 T19 G 1 120249993 121077638 828 E1 L 15 37191685 37846674 655 T19 G 1 142533622 144162829 1629 E1 L 15 55550826 56126615 576 T19 G 1 144594746 145133682 539 E1 L 16 33730527 34285558 555 T19 G 1 145584161 147083700 1500 E1 L 17 2938114 3371434 433 T19 G 1 150229090 153230241 3001 E1 L 17 16205935 17035446 830 T19 G 1 155029936 158487941 3458 117  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E1 L 17 21762537 22154806 392 T19 G 1 160338731 160677252 339 E1 L 17 22287134 22595733 309 T19 G 1 161016469 167445692 6429 E1 L 17 73158376 73381889 224 T19 G 1 170814706 171525137 710 E1 L 18 15026396 15320434 294 T19 G 1 174639051 176344936 1706 E1 L 18 61756829 62138970 382 T19 G 1 179462543 180447764 985 E1 L 19 47784067 48626174 842 T19 G 1 180659732 181045400 386 E1 L 19 50398622 50652469 254 T19 G 1 188010203 188453331 443 E1 L 19 55138786 55346714 208 T19 G 1 199091008 204975414 5884 E1 L 22 17053965 17206783 153 T19 G 1 205282499 208088220 2806 E1 L 22 18401409 19036350 635 T19 G 1 211445419 215373858 3928 E3 G 1 28120980 29453636 1333 T19 G 1 216039670 217850157 1810 E3 G 1 29953410 30162031 209 T19 G 1 218187008 218795330 608 E3 G 1 39063444 40504667 1441 T19 G 1 219017537 220609576 1592 E3 G 1 44687143 45130859 444 T19 G 1 222704527 223064465 360 E3 G 1 52663511 53007168 344 T19 G 1 224861037 233105801 8245 E3 G 1 151476033 155119104 3643 T19 G 1 242089298 242491480 402 E3 G 1 176574921 176971757 397 T19 G 2 41662 317294 276 E3 G 1 177764373 178479748 715 T19 G 2 714656 2663054 1948 E3 G 1 179640235 179933730 293 T19 G 2 2815042 8825151 6010 E3 G 1 181937518 182204185 267 T19 G 2 9512580 13154386 3642 E3 G 1 199539859 203475222 3935 T19 G 2 13269911 13575685 306 E3 G 1 204625506 204975414 350 T19 G 2 14137447 23881796 9744 E3 G 1 205931802 206903358 972 T19 G 2 26714284 27037976 324 E3 G 1 209299053 211282305 1983 T19 G 2 27515896 29595165 2079 E3 G 1 222335221 223152230 817 T19 G 2 30019511 31254640 1235 E3 G 1 223536766 224717804 1181 T19 G 2 36371522 41105817 4734 E3 G 1 230214952 231685446 1470 T19 G 2 41706139 42547144 841 E3 G 1 232727671 233546093 818 T19 G 2 44563993 47697639 3134 E3 G 1 241399361 241887674 488 T19 G 2 48847617 51219186 2372 E3 G 1 242089298 242642629 553 T19 G 2 54031551 55438372 1407 E3 G 1 245436183 245747506 311 T19 G 2 57719211 58137159 418 E3 G 2 8295224 9048252 753 T19 G 2 58638970 61582975 2944 E3 G 2 9985433 11025497 1040 T19 G 2 63994493 67943236 3949 E3 G 2 24892640 25572843 680 T19 G 2 68416702 69154160 737 E3 G 2 26714284 27037976 324 T19 G 2 70345668 73168364 2823 E3 G 2 31916659 33205438 1289 T19 G 2 73670503 82010715 8340 E3 G 2 55550156 55858798 309 T19 G 2 83467797 88331961 4864 E3 G 2 96151128 96842993 692 T19 G 2 88921446 91680249 2759 E3 G 3 9232089 9986408 754 T19 G 3 3179266 3667769 489 E3 G 3 49081051 50121242 1040 T19 G 3 130702173 131070392 368 E3 G 3 51996266 52619044 623 T19 G 5 17284044 17553934 270 E3 G 3 170977548 171510249 533 T19 G 5 20897033 21399027 502 E3 G 3 198952699 199276959 324 T19 G 5 59689279 59973593 284 E3 G 5 56264214 56679342 415 T19 G 5 86146718 86407148 260 E3 G 5 85422174 85713517 291 T19 G 6 73242 346084 273 E3 G 5 137318181 137920380 602 T19 G 6 662233 26126575 25464 E3 G 5 175623077 176701325 1078 T19 G 6 26395979 44815780 18420 118  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E3 G 6 73242 3553636 3480 T19 G 6 45632263 46448077 816 E3 G 6 4392553 5437064 1045 T19 G 6 46547724 56803717 10256 E3 G 6 6362577 7834700 1472 T19 G 6 58230371 58849697 619 E3 G 6 13508273 18231811 4724 T19 G 7 76182168 76393428 211 E3 G 6 19867614 23016737 3149 T19 G 8 11914818 12350857 436 E3 G 6 24229108 29123783 4895 T19 G 8 58157391 58393591 236 E3 G 6 29457137 32151955 2695 T19 G 11 60661723 60880212 218 E3 G 6 32872724 38315288 5443 T19 G 14 51180359 51608095 428 E3 G 6 39096919 39555660 459 T19 G 14 54110331 54510436 400 E3 G 6 39928065 44642460 4714 T19 G 14 60182950 62987077 2804 E3 G 6 90402399 90731676 329 T19 G 14 67008179 68444627 1436 E3 G 7 2038803 2407575 369 T19 G 14 74831170 82992497 8161 E3 G 7 4719283 6688617 1969 T19 G 14 83363672 85291082 1927 E3 G 7 66605756 67227917 622 T19 G 14 86623012 87817262 1194 E3 G 7 72809108 73534009 725 T19 G 14 88345764 94182499 5837 E3 G 7 98657508 102036700 3379 T19 G 14 94478987 97482889 3004 E3 G 7 129173245 129934305 761 T19 G 14 97754378 98569435 815 E3 G 8 144602260 146208093 1606 T19 G 14 105264727 106318374 1054 E3 G 9 19026977 19373997 347 T19 G 15 18473375 19917319 1444 E3 G 9 113177037 113848887 672 T19 G 15 20588181 20967788 380 E3 G 9 125895769 128420787 2525 T19 G 15 30006525 30539910 533 E3 G 9 128732487 132999756 4267 T19 G 16 6091158 6591299 500 E3 G 10 126236132 126557171 321 T19 G 16 33280312 33584068 304 E3 G 11 50991 2372103 2321 T19 G 19 8031324 8832847 802 E3 G 11 10327230 10641310 314 T19 G 19 59799647 59983377 184 E3 G 11 47194406 47629247 435 T19 L 1 100166580 100520827 354 E3 G 11 57164193 57354260 190 T19 L 3 175445960 175738257 292 E3 G 11 61823795 62515502 692 T19 L 5 139059617 139415354 356 E3 G 11 65808280 67116241 1308 T19 L 5 180195851 180444136 248 E3 G 11 107109972 107646360 536 T19 L 11 132728882 133121207 392 E3 G 12 5841370 7045503 1204 T19 L 13 47570684 48404010 833 E3 G 12 55654146 56198614 544 T19 L 13 52275633 52741383 466 E3 G 12 121469696 122996454 1527 T19 L 13 52597405 52938369 341 E3 G 14 73682195 74277120 595 T19 L 13 58025605 58861408 836 E3 G 14 98532350 100389009 1857 T19 L 13 76689469 77764260 1075 E3 G 15 57254835 57757359 503 T19 L 16 79667658 79901524 234 E3 G 15 72441859 73217136 775 T9 G 6 73242 346084 273 E3 G 16 3800374 4125558 325 T9 G 11 60661723 60880212 218 E3 G 16 54179429 54393514 214 T9 G 19 55554013 55707294 153 E3 G 17 510963 1432425 921 T9 L 4 3362479 3610639 248 E3 G 17 39961732 41722683 1761 T9 L 4 190667654 190953845 286 E3 G 17 43286090 46634199 3348 T9 L 5 55718971 56021821 303 E3 G 17 57842004 58351860 510 T9 L 5 60902209 62085860 1184 E3 G 17 69769995 73230854 3461 T9 L 5 62347735 62698196 350 E3 G 17 73330751 73695161 364 T9 L 5 67361489 67899255 538 E3 G 17 75566340 75862414 296 T9 L 5 70983047 72029339 1046 E3 G 17 76498848 78615238 2116 T9 L 5 72433622 72680299 247 119  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E3 G 18 25034674 25284594 250 T9 L 5 72933899 73837320 903 E3 G 19 1331124 5186670 3856 T9 L 5 75354258 75588705 234 E3 G 19 10430640 13371577 2941 T9 L 5 81524522 81697072 173 E3 G 19 16184022 18306025 2122 T9 L 5 88445542 88893940 448 E3 G 19 43206161 43719482 513 T9 L 5 95032442 95791871 759 E3 G 19 50652470 54706941 4054 T9 L 5 106882458 107135305 253 E3 G 19 54795121 55203857 409 T9 L 7 115766919 115974020 207 E3 G 20 43775966 44472487 697 T9 L 7 156780334 157782629 1002 E3 G 20 48069170 48483551 414 T9 L 8 135551492 135765484 214 E3 G 21 20794703 21052440 258 T9 L 13 39236837 39713370 477 E3 G 22 15930939 16096955 166 T9 L 13 40912907 42694359 1781 E3 G 22 17053965 17206783 153 T9 L 13 42627655 42831764 204 E3 G 22 26434947 26747513 313 T9 L 13 42832083 43127842 296 E3 G 22 35236505 41130527 5894 T9 L 14 105434666 105845800 411 E3 G 22 41150627 42198659 1048 T9 L 18 75216360 75531003 315 E3 L 1 60771032 61104212 333 T1 G 1 73684664 73883486 199 E3 L 1 246675462 246944046 269 T1 G 1 143900677 144162829 262 E3 L 2 25523248 25753042 230 T1 G 1 160735297 161015720 280 E3 L 2 77054044 77302660 249 T1 G 1 246424085 246741718 318 E3 L 2 96783694 97695429 912 T1 G 3 121556090 121848103 292 E3 L 2 131538597 131894005 355 T1 G 3 139398053 139636565 239 E3 L 2 161044707 161442145 397 T1 G 3 182708944 183039921 331 E3 L 2 193284705 193703377 419 T1 G 4 48771496 49210770 439 E3 L 2 195224267 195907912 684 T1 G 4 84397159 84777340 380 E3 L 3 3791057 4401633 611 T1 G 4 144726984 144928389 201 E3 L 3 88845493 90244571 1399 T1 G 5 17284044 17553934 270 E3 L 3 138266982 139234445 967 T1 G 5 25632061 25955183 323 E3 L 4 19191361 19814983 624 T1 G 5 75420049 75750313 330 E3 L 4 22846486 23104480 258 T1 G 5 138859931 139105069 245 E3 L 4 78267102 78823975 557 T1 G 6 104973405 105325375 352 E3 L 4 84891357 85692474 801 T1 G 8 78915837 79567870 652 E3 L 4 96537113 97073537 536 T1 G 11 60661723 60880212 218 E3 L 4 97896455 98480811 584 T1 G 11 61361854 61860041 498 E3 L 4 118973543 119393936 420 T1 G 11 77911332 78206818 295 E3 L 4 132667102 133073640 407 T1 G 11 96545676 96833884 288 E3 L 5 49479231 50093939 615 T1 G 12 67574005 67904215 330 E3 L 5 50839218 51198402 359 T1 G 12 80125405 80564119 439 E3 L 5 75006893 75588705 582 T1 G 15 26199536 26597300 398 E3 L 5 100253676 100665725 412 T1 G 16 21939143 22187488 248 E3 L 5 104100507 105046456 946 T1 G 17 5120725 5420944 300 E3 L 5 125401591 125696404 295 T1 G 17 54544587 54877132 333 E3 L 5 180093032 180343927 251 T1 G 18 11863172 12316092 453 E3 L 6 74510677 75644516 1134 T1 G 18 57821908 58047654 226 E3 L 6 77454026 77857011 403 T1 L 1 34421604 34697333 276 E3 L 6 86423400 87841029 1418 T1 L 1 222704527 222960993 256 E3 L 6 160616018 161039688 424 T1 L 1 243262497 243466817 204 E3 L 6 170655612 170880179 225 T1 L 2 6858150 7080915 223 120  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E3 L 7 48534377 48903100 369 T1 L 2 60553094 61048014 495 E3 L 8 43185803 43912178 726 T1 L 2 124195582 124430093 235 E3 L 8 47568877 47901818 333 T1 L 2 157294604 157467503 173 E3 L 8 60811916 61320284 508 T1 L 2 186879569 187224688 345 E3 L 8 62881808 63455889 574 T1 L 2 205347096 205689005 342 E3 L 8 72706992 73494710 788 T1 L 3 20821246 21090936 270 E3 L 8 86699540 87055819 356 T1 L 3 78394977 78583892 189 E3 L 9 11531920 11963059 431 T1 L 3 105928983 106173765 245 E3 L 9 120758160 121059699 302 T1 L 3 124714733 125016814 302 E3 L 10 32785206 33316028 531 T1 L 3 127309873 127565087 255 E3 L 10 100371823 100906136 534 T1 L 3 140668983 141334635 666 E3 L 11 29516501 29810959 294 T1 L 4 13423 256033 243 E3 L 11 48090650 48335007 244 T1 L 4 182808800 183027837 219 E3 L 11 50230022 51421925 1192 T1 L 5 197527 555524 358 E3 L 11 62532845 62994414 462 T1 L 5 12839471 13174647 335 E3 L 11 93414925 93936247 521 T1 L 5 55920336 56144127 224 E3 L 12 38427897 38702944 275 T1 L 5 72433622 72680299 247 E3 L 12 77871943 78081993 210 T1 L 5 93017881 93284274 266 E3 L 13 51074984 51467864 393 T1 L 5 98042952 98364209 321 E3 L 13 54559086 55373857 815 T1 L 6 42123312 42315751 192 E3 L 14 26520749 27084469 564 T1 L 6 75466376 76023862 557 E3 L 14 38967497 39556557 589 T1 L 6 78146017 78314751 169 E3 L 14 44763469 45817331 1054 T1 L 6 79822171 80031136 209 E3 L 15 34634629 34933419 299 T1 L 6 93016222 93431583 415 E3 L 15 79676203 79953718 278 T1 L 6 123300422 123547214 247 E3 L 16 33400710 34490212 1090 T1 L 6 143011827 143259770 248 E3 L 16 52550335 52874597 324 T1 L 6 159589768 159892720 303 E3 L 17 21762537 22154806 392 T1 L 7 52841765 53284451 443 E3 L 17 22287134 22595733 309 T1 L 7 154922742 155128400 206 E3 L 17 68744962 69076047 331 T1 L 8 8242248 8571704 329 E3 L 17 73158376 73381889 224 T1 L 8 69645093 69999863 355 E3 L 18 15026396 15320434 294 T1 L 9 68131743 68460734 329 E3 L 18 18385344 18758389 373 T1 L 9 120873426 121059699 186 E3 L 19 23835271 24178465 343 T1 L 10 29448208 29797600 349 E3 L 19 50398622 50652469 254 T1 L 10 116116186 116299949 184 E5 G 1 190957775 191386589 429 T1 L 10 118147658 118360397 213 E5 G 2 1642610 2663054 1020 T1 L 12 6511402 6754179 243 E5 G 2 3293984 3748630 455 T1 L 12 49598089 49987763 390 E5 G 2 3903894 4615940 712 T1 L 12 86487712 86754944 267 E5 G 2 9249270 11931175 2682 T1 L 12 95788252 96085223 297 E5 G 2 23512042 23881796 370 T1 L 12 99185936 99487301 301 E5 G 2 24892640 25450109 557 T1 L 12 114851293 115230777 379 E5 G 2 26714284 27284933 571 T1 L 13 42903254 43127842 225 E5 G 2 28214990 28756000 541 T1 L 13 46289605 46518008 228 E5 G 2 30764067 31254640 491 T1 L 13 46386689 47441575 1055 E5 G 2 42689036 43220834 532 T1 L 13 47901160 48242971 342 E5 G 2 73898940 74792844 894 T1 L 13 50026338 51042932 1017 121  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E5 G 2 79806185 80083233 277 T1 L 13 52541033 53095154 554 E5 G 2 87990794 88803463 813 T1 L 13 59352443 59638075 286 E5 G 2 185880300 186157143 277 T1 L 13 63233263 63637946 405 E5 G 3 9512372 9986408 474 T1 L 13 109502184 109838498 336 E5 G 5 17284044 17554876 271 T1 L 15 20217252 20338507 121 E5 G 6 43995870 44268248 272 T1 L 15 21290128 21703185 413 E5 G 6 154129550 154379516 250 T1 L 15 73769351 74357465 588 E5 G 7 30863988 31319741 456 T1 L 16 51601593 51928256 327 E5 G 7 64972395 65209849 237 T1 L 16 53058404 53364181 306 E5 G 9 138457104 138919624 463 T1 L 16 65720643 66067662 347 E5 G 10 103733984 104069156 335 T1 L 16 68343618 68959796 616 E5 G 13 93396760 102958785 9562 T1 L 16 79901920 80284468 383 E5 G 13 108359831 113901932 5542 T1 L 18 25034674 25284594 250 E5 G 15 19087319 20097557 1010 T1 L 18 69281624 69613151 332 E5 G 15 30237319 30597338 360 T1 L 19 7749446 7984147 235 E5 G 15 72889509 73217136 328 T76 L 6 13223270 13524298 301 E5 G 17 27213580 27812924 599 T76 L 2 211144204 211423562 279 E5 G 17 77390101 78036762 647 T76 L 5 11106805 11366567 260 E5 G 18 35421 3100070 3065 T76 L 6 151049955 151280704 231 E5 G 18 5467960 15060997 9593 T76 L 8 86699540 87055819 356 E5 G 18 16793577 18073471 1280 T76 L 12 45789177 46073952 285 E5 G 18 18745629 21117291 2372 T76 L 13 35996176 36510125 514 E5 G 18 21543717 22489038 945 T76 L 13 36816516 37223607 407 E5 G 18 75216360 76098439 882 T76 L 13 43481375 44009817 528 E5 G 19 10216515 10436184 220 T76 L 13 45408174 45768530 360 E5 G 20 42815688 43159048 343 T76 L 13 67175227 67681892 507 E5 G 21 43899532 44194069 295 T76 L 13 76862480 77225206 363 E5 L 1 1058909 1487588 429 T76 L 15 19884809 20071155 186 E5 L 1 5415955 5868138 452 T76 L 19 32504109 32613567 109 E5 L 1 12724432 13569886 845 T76 G 1 16646685 17086056 439 E5 L 1 55481887 56411469 930 T76 G 1 143665914 144162829 497 E5 L 1 59319690 59815749 496 T76 G 1 177293770 177908342 615 E5 L 1 60771032 61339873 569 T76 G 1 232830875 233105801 275 E5 L 1 67900421 69138390 1238 T76 G 5 4280335 5240403 960 E5 L 1 92317925 92612882 295 T76 G 5 17284044 17553934 270 E5 L 2 25382449 25753042 371 T76 G 5 62919710 63197515 278 E5 L 3 78394977 78583892 189 T76 G 5 68905030 70342024 1437 E5 L 3 81954578 82288232 334 T76 G 9 122514480 123029731 515 E5 L 3 126980939 127282195 301 T76 G 9 124184461 124491178 307 E5 L 3 190572724 190902546 330 T76 G 13 50689132 51467864 779 E5 L 4 764772 1020003 255 T76 G 13 66207174 66805219 598 E5 L 4 9608055 19814983 10207 T76 G 13 94004743 95306078 1301 E5 L 4 23247592 23876872 629 T76 G 13 96341247 96782355 441 E5 L 4 25775550 27257262 1482 T76 G 15 30237319 30597338 360 E5 L 4 28130866 29198517 1068 T72 G 1 16646685 17086056 439 E5 L 4 35485307 37370613 1885 T72 G 1 143219071 144162829 944 E5 L 4 45905935 47129172 1223 T72 G 1 155801127 156042705 242 122  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E5 L 4 52354875 54561183 2206 T72 G 1 232830875 233105801 275 E5 L 4 55204895 55856179 651 T72 G 5 20897033 21399027 502 E5 L 4 67582165 70337363 2755 T72 G 7 57163665 57368235 205 E5 L 4 73869033 76067534 2199 T72 G 11 118980810 119354299 373 E5 L 4 78343948 80379354 2035 T72 G 11 132051142 132389201 338 E5 L 4 82738643 83530160 792 T72 G 13 108629457 109102920 473 E5 L 4 91158111 91923787 766 T72 G 13 110600643 112948798 2348 E5 L 4 104154615 104907229 753 T72 G 14 19149713 19485285 336 E5 L 4 108540535 108797097 257 T72 G 15 21973970 22386839 413 E5 L 4 111625951 111926554 301 T72 G 15 22796772 23152527 356 E5 L 4 119871532 121956556 2085 T72 G 19 8629145 8784792 156 E5 L 4 137303256 137853745 550 T72 G 22 14410042 14660949 251 E5 L 4 139331093 140107223 776 T72 L 2 91608685 91680249 72 E5 L 4 145066326 147630673 2564 T72 L 3 78394977 78583892 189 E5 L 4 181177641 182980569 1803 T72 L 3 114828518 115117759 289 E5 L 5 174556369 174812270 256 T72 L 6 160616018 161039688 424 E5 L 6 13223270 13577224 354 T72 L 7 77228817 77535943 307 E5 L 7 24242808 24445001 202 T72 L 8 7035076 7289755 255 E5 L 7 88158926 88740787 582 T72 L 11 97278670 97609941 331 E5 L 7 156780334 157071420 291 T72 L 12 32108495 32334573 226 E5 L 8 4038043 4938784 901 T72 L 14 72215467 72636951 421 E5 L 8 43422554 43912178 490 T72 L 18 12806854 13135104 328 E5 L 8 46969582 48171893 1202 T72 L 19 32504109 32613567 109 E5 L 8 48324637 48787064 462 T72 L 22 40935061 41198248 263 E5 L 8 49937784 50475395 538 T20 G 17 54447833 54973480 526 E5 L 8 72706992 74263376 1556 T20 G 17 61469371 61961765 492 E5 L 8 76656394 77072172 416 T20 G 17 67376846 69931286 2554 E5 L 8 86699540 87055819 356 T20 G 18 42657928 42803873 146 E5 L 8 117853642 120752324 2899 T20 G 20 692082 1452295 760 E5 L 8 131773301 132059478 286 T20 G 20 52483586 52791277 308 E5 L 9 2271469 3399803 1128 T20 L 1 44277602 44370880 93 E5 L 9 8787144 9654318 867 T20 L 1 217124947 217262659 138 E5 L 9 18454415 18875253 421 T20 L 2 96785607 97054766 269 E5 L 9 42064305 46458217 4394 T20 L 2 120401666 120706743 305 E5 L 9 109623395 110355803 732 T20 L 2 140828781 141251152 422 E5 L 9 118654542 118945140 291 T20 L 2 143058946 143562739 504 E5 L 9 121713094 122361130 648 T20 L 2 169824122 170250237 426 E5 L 10 41947396 42224061 277 T20 L 2 226758143 227038326 280 E5 L 12 29727999 29929154 201 T20 L 3 24994819 25264504 270 E5 L 12 37754701 38138583 384 T20 L 3 36111427 36427114 316 E5 L 12 59227752 59583586 356 T20 L 3 37703118 37973246 270 E5 L 13 61014574 62048015 1033 T20 L 3 78394977 78583892 189 E5 L 13 62395584 64544139 2149 T20 L 4 3645088 4065877 421 E5 L 13 64861982 65274978 413 T20 L 4 29816475 30070593 254 E5 L 14 38847479 39263310 416 T20 L 4 62298232 62743878 446 E5 L 14 80635044 80879562 245 T20 L 4 104719821 105038688 319 E5 L 16 15040819 15407666 367 T20 L 4 138014420 138600123 586 123  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) E5 L 16 29181450 29392366 211 T20 L 5 22506952 22833632 327 E5 L 16 52550335 52874597 324 T20 L 5 27140678 27548165 407 E5 L 16 77920768 79358404 1438 T20 L 5 49479231 49636937 158 E5 L 17 22287134 22595733 309 T20 L 5 75160802 75421795 261 E5 L 17 68744962 69076047 331 T20 L 5 91292937 91664641 372 E5 L 19 50398622 50652469 254 T20 L 5 93244813 93709268 464 T20 G 1 16646685 17086056 439 T20 L 6 151040678 151280704 240 T20 G 1 62133505 62475327 342 T20 L 7 1499748 1737551 238 T20 G 1 92776245 93254333 478 T20 L 7 61087267 61797715 710 T20 G 1 142533622 145872489 3339 T20 L 7 84446843 84898403 452 T20 G 1 148925197 153230241 4305 T20 L 7 118629304 119269525 640 T20 G 1 154247819 155835711 1588 T20 L 7 145131434 145947929 816 T20 G 1 156649526 160677252 4028 T20 L 8 85161425 85626176 465 T20 G 1 161016469 161777223 761 T20 L 8 86558071 87055819 498 T20 G 1 162887424 167842836 4955 T20 L 8 120992957 122162585 1170 T20 G 1 170587093 171187941 601 T20 L 9 128256342 128682074 426 T20 G 1 173228930 174094669 866 T20 L 10 38983843 39116217 132 T20 G 1 174639051 176344936 1706 T20 L 10 41947396 48053466 6106 T20 G 1 176574921 178811659 2237 T20 L 10 47928933 49013085 1084 T20 G 1 179462543 181831357 2369 T20 L 10 48969280 50458757 1489 T20 G 1 186076660 186470859 394 T20 L 10 50519291 54498532 3979 T20 G 1 195455701 196149918 694 T20 L 10 54718352 58201767 3483 T20 G 1 198317928 215373858 17056 T20 L 10 60249701 60552579 303 T20 G 1 217329398 220609576 3280 T20 L 10 60520851 89428545 28908 T20 G 1 224861037 233105801 8245 T20 L 10 90654816 98693465 8039 T20 G 1 234814970 236029274 1214 T20 L 10 98960137 108738304 9778 T20 G 1 237944944 241203475 3259 T20 L 10 108937968 114878030 5940 T20 G 1 243223806 245038844 1815 T20 L 10 115215602 115832934 617 T20 G 2 925973 1218689 293 T20 L 10 115994387 118749875 2755 T20 G 2 5764382 9407162 3643 T20 L 10 119422575 127882581 8460 T20 G 2 11604683 12102751 498 T20 L 10 127834059 128595500 761 T20 G 2 12928689 13348548 420 T20 L 10 128961549 135279687 6318 T20 G 2 16361494 16914958 553 T20 L 11 25239852 25489221 249 T20 G 2 28067510 28504340 437 T20 L 11 100820386 101077028 257 T20 G 2 29024702 31254640 2230 T20 L 12 85650592 86068176 418 T20 G 2 36503134 36996317 493 T20 L 13 19646778 20414099 767 T20 G 2 72794626 73168364 374 T20 L 13 29471954 29739222 267 T20 G 2 165764127 165980904 217 T20 L 13 41563363 42494518 931 T20 G 2 234482908 234828785 346 T20 L 13 42472202 43127842 656 T20 G 3 49579895 49782857 203 T20 L 13 43481375 43792169 311 T20 G 4 8617326 8780507 163 T20 L 13 46386689 47441575 1055 T20 G 4 132469344 132802100 333 T20 L 13 53430317 55655559 2225 T20 G 4 139960125 140321282 361 T20 L 13 57621677 64356550 6735 T20 G 4 179461534 179886545 425 T20 L 13 64966450 68595839 3629 T20 G 5 12637462 13007203 370 T20 L 13 68841601 69301983 460 T20 G 5 17277342 17553934 277 T20 L 13 69468748 70603880 1135 T20 G 5 19832654 20176670 344 T20 L 13 70791339 81289277 10498 124  ID G/L Chr Start (bp) End (bp) Size (kb) ID G/L Chr Start (bp) End (bp) Size (kb) T20 G 5 49960569 50290883 330 T20 L 13 83509678 84537312 1028 T20 G 5 106909929 107306804 397 T20 L 13 84556448 87198713 2642 T20 G 6 73242 346084 273 T20 L 13 87575870 94267342 6691 T20 G 7 117127748 117527587 400 T20 L 13 94351464 96029356 1678 T20 G 8 80712778 81019111 306 T20 L 13 102618440 105229814 2611 T20 G 9 33336233 33775694 439 T20 L 13 105299403 106278034 979 T20 G 9 35583889 36033505 450 T20 L 13 106405817 107155648 750 T20 G 9 131137899 131452656 315 T20 L 15 19884809 20071155 186 T20 G 9 133976381 134847756 871 T20 L 15 99860521 100126412 266 T20 G 10 75055 5795590 5721 T20 L 16 33659117 33927835 269 T20 G 10 6112562 11008217 4896 T20 L 16 79901920 80284468 383 T20 G 10 13630717 14736903 1106 T20 L 17 2442274 3513298 1071 T20 G 10 20159003 21384213 1225 T20 L 17 9979758 13098904 3119 T20 G 11 18978846 19286458 308 T20 L 17 13230426 14103719 873 T20 G 11 60508638 60880212 372 T20 L 17 14560485 15759518 1199 T20 G 12 53976376 54173283 197 T20 L 17 17656740 20683824 3027 T20 G 12 71994675 72180449 186 T20 L 18 12806854 13135104 328 T20 G 14 19149713 19360529 211 T20 L 19 8934066 9026570 93 T20 G 15 21973970 23152527 1179 T20 L 19 32504109 32613567 109 T20 G 15 28106102 28734907 629 T20 L 19 36871853 37397010 525 T20 G 15 34937367 35376632 439 T20 L 19 41542240 41714071 172 T20 G 15 92651955 93031016 379 T20 L 19 55554013 55707294 153 T20 G 16 33280312 33741546 461 T20 L 19 58250784 58500199 249 T20 G 16 47189283 47753661 564 T20 L 21 20794721 21341020 546 T20 G 16 72964202 73144686 180 T20 L 21 36306335 36609093 303 T20 G 17 44818672 45685773 867 T20 L 22 40935061 41198248 263 T20 G 17 47498136 47977547 479 T20 G 17 51913051 53110374 1197             125  A.2 Retinoblastoma cell line karyograms   Figure A.1   Weri-Rb-1 cell line karyogram. Multiple copy number alterations can be seen on chromosome 13q including loss of the RB1 gene. Weri-Rb-1 contains a low-level gain of most of the 2p arm encompassing the MYCN gene among many others.  The Weri-Rb-1 genome contains many additional genetic alterations typical of tumor cell line genomes in general. 126  1 2 3 4 5 6 7 8 109 11 12 13 14 15 16 17 18 19 20 21 22 X Y -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1  Figure A.2   Y79 cell line karyogram. Y79 contains a focal amplification of approximately one Mb of DNA at 2p24.3 encompassing the MYCN gene. Additional alterations detected are relatively few for a cell line.            127  A.3 Top significantly disrupted canonical pathways and networks Tables A.4 and A.5 contain the additional details for the top ten most significantly disrupted canonical pathways in the RB1-/- and RB1+/+ frequently altered gene datasets. Tables A.6 and A.7 contain the additional details for the top ten gene association networks generated by IPA from the RB1-/- and RB1+/+ frequently altered gene datasets.  Table A.4    Top significantly disrupted canonical pathways in RB1+/+ tumors. Ingenuity Canonical Pathways -log(p-value)* Ratio Molecules Differential Regulation of Cytokine Production in Macrophages and T Helper Cells by IL-17A and IL-17F 1.68 1.11E-01 IL10, IL9 Role of Cytokines in Mediating Communication between Immune Cells 1.5 5.45E-02 IL10, IL20, IL24 Differential Regulation of Cytokine Production in Intestinal Epithelial Cells by IL-17A and IL-17F 1.48 8.7E-02 IL10, IL9 Glioma Invasiveness Signaling 1.44 5E-02 PIK3C2B,CD44 (includes EG:100330801), TIMP2 (includes EG:21858) Leukocyte Extravasation Signaling 1.42 3E-02 ROCK1, PIK3C2B, EZR, CD44 (includes EG:100330801), RASSF5, TIMP2 (includes EG:21858) CD40 Signaling 1.35 4.29E-02 PIK3C2B, IKBKE, MAPKAPK2 Maturity Onset Diabetes of Young (MODY) Signaling 1.35 6.25E-02 CACNA1E, CACNA1S IL-17A Signaling in Airway Cells 1.32 4.17E-02 PIK3C2B, IL19, IKBKE Amyotrophic Lateral Sclerosis Signaling 1.32 3.36E-02 PIK3C2B, CACNA1E, CACNA1S, GLUL NGF Signaling 1.3 3.45E-02 ROCK1, PIK3C2B, NTRK1, IKBKE * The –log(p-value) significance threshold for this dataset of 1.30 128  Table A.5    Top significantly disrupted canonical pathways in RB1-/- tumors. Ingenuity Canonical Pathways -log(p- value)* Ratio Molecules Antigen Presentation Pathway 17.4 5.12E-01 HLA-DMA, PSMB9, HLA-DOA, HLA-DQA1, HLA- DRB1, HLA-DMB, PSMB8, TAP1, HLA-DPA1, HLA-F, HLA-A, HLA-E, HLA-DRA, HLA-B, HLA- DOB, HLA-DQA2, HLA-G, HLA-DPB1, TAP2, TAPBP, HLA-DRB5, HLA-C Dendritic Cell Maturation 10.8 1.76E-01 FCGR3B, FCGR2C, HLA-DOA, NFKBIE, HLA- DQA1, LTB, HLA-DRB1, CD83, HLA-DMB, MAPK13, HLA-DQB1, TREM2, HLA-A, HLA-DRA, HLA-B, COL18A1, COL11A2, FCGR3A ,HLA-C, HLA-DMA, PIK3C2B, IL10, FCGR2A, IKBKE, DDR1, MAPK14, ZBTB12, LTA, FCER1G, HLA- DOB, IRF8, TNF, HLA-DRB5 Graft-versus-Host Disease Signaling 9.92 3.4E-01 HLA-DMA, HLA-DOA, HLA-DQA1, HLA-DRB1, HLA-DMB, HLA-DQB1, HLA-F, HLA-A, HLA-E, HLA-DRA, FCER1G, HLA-B, HLA-DOB, HLA-G, TNF, HLA-DRB5, HLA-C Autoimmune Thyroid Disease Signaling 9.59 2.79E-01 HLA-DMA, HLA-DOA, IL10, HLA-DQA1, HLA- DRB1, HLA-DMB, HLA-DQB1, HLA-F, HLA-A, HLA-E, HLA-DRA, FCER1G, HLA-B, HLA-DOB, HLA-G, HLA-DRB5, HLA-C Allograft Rejection Signaling 8.7 2.19E-01 HLA-DMA, HLA-DOA, IL10, HLA-DQA1, HLA- DRB1, HLA-DMB, HLA-DQB1, HLA-DPA1, HLA- F, HLA-A, HLA-E, HLA-DRA, HLA-B, FCER1G, HLA-DOB, HLA-DQA2, HLA-G, TNF, HLA-DPB1, HLA-DRB5, HLA-C OX40 Signaling Pathway 8.5 2.21E-01 HLA-DMA, HLA-DOA, TNFRSF4, NFKBIE, HLA- DQA1, HLA-DRB1, HLA-DMB, HLA-DQB1, HLA- DPA1, HLA-F, HLA-A, HLA-E, HLA-DRA, HLA-B, FCER1G, HLA-DOB, HLA-DQA2, HLA-G, HLA- DPB1, HLA-DRB5, HLA-C Type I Diabetes Mellitus Signaling 7.85 1.9E-01 HLA-DMA, HLA-DOA, NFKBIE, HLA-DQA1, HLA-DRB1, HLA-DMB, IKBKE, MAPK13, HLA- DQB1, HLA-F, RIPK1, MAPK14, HLA-A, HLA-E, LTA, HLA-DRA, HLA-B, FCER1G, HLA-DOB, HLA-G, TNF, HLA-DRB5, HLA-C Cytotoxic T Lymphocyte-mediated Apoptosis of Target Cells 7.45 2.21E-01 HLA-DMA,HLA-DOA,HLA-DQA1,HLA- DRB1,HLA-DMB,HLA-DQB1,HLA-DPA1,HLA- F,HLA-A,HLA-E,HLA-DRA,FCER1G,HLA-B,HLA- DOB,HLA-DQA2,HLA-G,HLA-DPB1,HLA- DRB5,HLA-C Crosstalk between Dendritic Cells and Natural Killer Cells 5.26 1.77E-01 NCR3, HLA-DRB1, LTB, CD83, TREM2, HLA-F, MICB, HLA-A, HLA-E, LTA, HLA-DRA, HLA-B, HLA-G, TNF, MICA, HLA-DRB5, HLA-C Altered T Cell and B Cell Signaling in Rheumatoid Arthritis 4.97 1.74E-01 HLA-DMA, HLA-DOA, SLAMF1, IL10, HLA- DQA1, HLA-DRB1, LTB, HLA-DMB, HLA-DQB1, IL17A, LTA, HLA-DRA, FCER1G, HLA-DOB, TNF, HLA-DRB5 *The –log(p-value) significance threshold for this dataset is 1.2  129  Table A.6    Top significant gene association networks disrupted in RB1+/+ tumors. ID Molecules in Network Score Molecules Top Functions 1 ATP1A2, ATP1A4, ATP1B1, ATP2B4, BASP1, CACNA1E, CACNA1S, Calmodulin, CCND3, CD5, CHI3L1, CRABP2, DNAH3, DYNLT1, ELF3, ETS, GLP1R, Gpcr, GPR37L1, ID2, IL9, IL10, IL24, MAPKAPK2, MYOG,Na-k-atpase, NTSR2, P38 MAPK, PTPN7, Rb, SRF, TH2 Cytokine, Tni, TNNI1, TNNT2 50 27 Skeletal and Muscular System Development and Function, Tissue Morphology, Molecular Transport 2 Actin,Akt,CD44 (includes EG:100330801), CD5L, Creb, DNMT3A, DUB, ERK1/2, Erm, EZR, FOXC1, FOXP4, FRS3, GLUL, Hsp90, IGSF8, IPO9, KIDINS220, KISS1, MED20, Mediator, MYCN, NBPF11 (includes others), NTRK1, ODC1, p85 (pik3r), PEA15, PHACTR1, PIAS2, PPP2R5D, RNA polymerase II, S100A11, TIMP2 (includes EG:21858), USP36, USP49 45 25 Cellular Movement, Cancer, Tissue Development 3 ADIPOR1, APOH, APPBP2, CYP3A4, FCAMR, FGB (includes EG:110135), FRK, HBP1, HDGF, HNF4A, HSD11B1, IRF6, KCNK5, KIF6, LBP, LPA, MLXIPL, MUC4, NRAS, NTHL1, PAPPA2, PDE4DIP, PEX6 (includes EG:117265), PPP1R12B, PTK7,  RASSF5, RNMTL1, RORA, RTCD1, SCFD1, SMAD4, TERF2IP, TLX2, TSNAX, TXNIP 25 16 Energy Production, Lipid Metabolism, Small Molecule Biochemistry 4 BCL6, BMI1, CCNE1, CDC5L, CDKN1C, CIAO1, CITED2, CYBASC3, FOXS1, G0S2, GRHL1, HDAC4, HNF4A, HOXC8, KIAA1377, KIF15, KLHDC3, LAD1, MDFI, MRPL2, MRPL24 (includes EG:295224), NBAS, NCOA2, PCGF2, PPARD, PPP1CA, PPP1R15B, PRCC, PRICKLE4, RNF2, RSPH3, RXRB, SRSF2, TPX2, XRCC5 23 15 Gene Expression, Skeletal and Muscular System Development and Function, Cellular Development 5 AR, BCAS3, BTF3, CCDC19, DDT, DSTYK, DYRK3, E2F6, ERP29, ESR1, GAL, GREB1, GUSB, H19, H2AFX, Histone h3, HYOU1, IKBKE, IMPDH2, KDM5B, KLHDC10, LGALS3BP, LMOD1, LSM12 (includes EG:124801), miR-193/miR-193b/miR-193a-3p, OIP5, PDIA6, PGC, PKP1, PRKCSH, RAB14, RPS9, SEC22B, T, TAGLN2 23 15 Embryonic Development, Organismal Development, Reproductive System Development and Function 6 ASL, C6orf105, CDCP1, DRD4, DUSP22, ELMO2, FAM40A, FAM40B, FLG, GLUL, GNMT, IRF2BP2, KCNJ3, KCNJ5, KCNJ6, KCNJ9, KLHL12, LAMA3, LAMB3, LAMC3, LECT2, mir-125,miR-429/miR- 200b/miR-200c, MKI67, NR3C1, NXN, PDCD10, PPARA, PPP2CA, Sfk, SIKE1, SRC, TNF, TRAF3IP3, ZP2 23 15 Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry 7 AQP5, ARRB1, BYSL, CD34, CDH1, CDH11, CTNNB1, FCRL6, HLA-DRA, HLA-DRB4, HLA- DRB5, HLA-DRB3 (includes others), LIMK2, MAPK3, MFGE8, MYBPH, MZF1, NKX2-1, NOTCH2NL, PEX6 (includes EG:117265), PIGM, PODXL, PPFIA4, PTCRA, PTPN7, ROCK1, RPL41, RPL7L1, RRM2, SFTPC, SMAD7, SMARCA4, SOX13, TFEB, TSPAN8 22 15 Cellular Movement, Cellular Development, Hair and Skin Development and Function 130  ID Molecules in Network Score Molecules Top Functions 8 ABL1, ABR, ARHGAP17, CD244, CNTNAP1, CRK, DDX41, DLG4, DUSP15, EFS, ERBB2, FYN, GNS, HEXA, ISG20L2, KCNB1, KCNF1, KCNG3, KCNH1, KCNJ10, KCNJ16, KCNS3, KCNV2, LAX1, LY9, NFASC, NOS1, NOS1AP, OPTC, PIK3R1, PRX, SH2D1B, SYTL3, TXK, ZDHHC3 20 14 Cellular Development, Nervous System Development and Function, Cellular Assembly and Organization 9 BUD13 (includes EG:215051), C4BPB, CAMK1G, Caspase, CASQ1, CLEC3B, CLTCL1, Creatine Kinase, CREBBP, DTX2, EP300, EZH2, HES6, HMGCS1, HOXB6, HOXD10, HPCAL1, HTT, KCNA1, MYLK2, OSTF1, PRELP, PTMA, RAB8A, RABIF, RBFOX3, REN, SCAMP5, SLC1A3, SYT2, T, TAF1B, Tdg (includes others), TRERF1, VSNL1 20 14 Amino Acid Metabolism, Carbohydrate Metabolism, Lipid Metabolism 10 ATAD3A/ATAD3B, C14orf166, C22orf28, CALR, CKAP4, DDX1, GJA5, HFE2, HRNR, IL19, IL20, IL24, IL20RA, IL20RB, IL22RA1, KLC4, LRRN2, MLH1, MYL6, NEO1, NONO, NOVA1, PEF1, PLEKHA6 ,RPS7, SNRPE, SNRPF, SNRPG, SRGAP2, UBAP2L, VCL, WDR61, YLPM1, YWHAZ, ZC3H11A 19 13 Cardiovascular Disease, Cell Cycle, Cell Morphology 131  Table A.7    Top significant gene association networks disrupted in RB1-/- tumors. ID Molecules in Network Score Molecules Top Functions 1 ABT1, ACSS3, ADPRHL1, ATAT1, BPHL, BTN2A1, C16orf61, C19orf42, C20orf111, C6orf108,  CDKAL1, FN3KRP, GMDS, GPC6, HIST1H2AH, HIST1H2BD, HMGN4, HNF4A, KLHDC3, LYRM4, MIPEP, MRPL2, MRPL24 (includes EG:295224), MUT, NOL7, PRICKLE4, PROZ, PRR3, RIOK1, SLMO2, TP53TG5, WDR46, ZNF193, ZNF318, ZSCAN16 53 35 Endocrine System Disorders, Gastrointestinal Disease, Immunological Disease 2 CDC5L, CDCP1, Creb, DPYSL4, FOXQ1, HMGA1, HSD17B2, HSD17B7, HSD17B8, HSD17B, INTS6, INTS7, KDM5B, KIAA0182, KLC4, LRRC47, MCM3, MGAT1, MPZ, NFYA, PDE4DIP, PPOX, PRCC, SDC4, SOX1, SOX4, Sox, SRF, SRPK1, SRSF3, TMEM14A, UBE2J2, YWHAB, ZFP57, ZMYND8 45 32 RNA Post- Transcriptional Modification, Drug Metabolism, Endocrine System Development and Function 3 CAP2, CCND3, CCNL2, CD83, CDK11A/CDK11B, CRABP2, CyclinD, DLL1, DTNBP1, EFNB2, ELK4, FOXC1, HES5, Hsp27, KISS1, MAPK13, MUTED, MYOG, NBPF11 (includes others), NOTCH4,P38 MAPK, PBX1, PBX2, PTPN7, REN, RNA polymerase II,RPS6KC1, SDHC, SKIV2L,TXNDC5, UBD, YOD1, ZDHHC7, ZFHX3, ZNF238 42 31 Gene Expression, Cellular Development, Cardiovascular Disease 4 ABHD16A, AMFR, ANKS1A, ATP5E, ATP5I, ATP6V1G2, ATPase, BTBD9, CPLX1, EBF3, F11R, H+- transporting two-sector ATPase, MCF2L, MLLT4, NUAK2, NUDT21, PEX6 (includes EG:117265), phosphoinositide phospholipase C, PLCG2, PVRL4, RNF5, RNF144B, RUNX2, RXRB, SGSM2, SMAD2, SPG20, TRIM27, UBE2T, Ubiquitin, UBR2 (includes EG:224826), WRNIP1, WWOX, ZNF423, ZRANB1 42 31 DNA Replication, Recombination, and Repair, Energy Production, Nucleic Acid Metabolism 5 ADA, CA2, CBX2, CDYL, CSNK2B, CTBP2, Ctbp, DAXX, EHMT2, FOXP4, HIC1, HIST1H3A (includes others), Histone h4, ID4, Interferon alpha, IRF6, IRF8, IRG, ISG15, JARID2, KDM1B, MDM4, MGMT, MSH5 (includes EG:17687), PHF1, POU5F1, PRDM16, PREX1, RING1, RNF8, SLPI, STK38, TCF7L2, TEAD3, TOP1 42 31 Gene Expression, Tissue Development, Cell Death 6 ACOT13, BPNT1, C6orf125, CLIC1, COX10 (includes EG:1352), COX4I1, Cytochrome c oxidase, DSTYK, DUB, EPRS, F7, FKBP5, GLO1, GLRX3, IKBKE, Immunoglobulin, MARK1, MED20, MRPL14, NADK, OGFOD1, PDCD2, PRL, SEC22B, SERINC3, SLC39A7, SSR1, TAGLN2, TOMM6, TOMM34, TOMM40L, USP10, USP21, USP49, XPO5 41 32 Endocrine System Development and Function, Infectious Disease, Lipid Metabolism 7 Cbp/p300, CDKN1A, CUL4A, DDB1, DTL, FOXF2, GAS6, Gcn5l, GLUL, GTF2H4, HIST1H2AD, HIST1H2BH/HIST1H2BO, Holo RNA polymerase II, LRRC16A, Mediator, PDE6B, PIAS2, POLR1C, RBBP5, RDBP, RNA polymerase I, SPIB, SUPT3H, TADA1, Taf, TAF10 (includes EG:216185), TAF11 (includes 38 29 Gene Expression, Cell- mediated Immune Response, Cellular 132  ID Molecules in Network Score Molecules Top Functions EG:309638), TAF1C, TAF4 (includes EG:100149942), TAF8 (includes EG:129685), TBP, TCHH, TH1L, TOR1AIP2, ZNF76 Development 8 ADORA1, CD48, G protein alpha, G protein alphai, G- protein beta, GABBR1, Gi-coupled receptor, GLP1R, GLP2R, GNAO1, GNAS, GNB1, Gpcr, GPR68, GPR110, GPR111, GPR115, GPR116, GPR37L1, GPSM3, GRM4, HCRTR2, HRH3, IL9, IL10, IL24, MAS1L, MBTPS1, NTSR1, PLC, RGS4, RGS5, SSTR4, TH2 Cytokine, ZDHHC3 36 28 Cell Signaling, Molecular Transport, Nucleic Acid Metabolism 9 BRD2, CCNA1, Cdk, CLPS, Cyclin A, Cyclin E, DEK, DYRK3, E2f, ETV3, ETV7, F10, HIST1H1A, HIST1H1B, HIST1H1C, HIST1H1D, HIST1H1E, HIST1H1T, HIST1H2AB/HIST1H2AE, HIST1H2BJ/HIST1H2BK, HIST1H4A (includes others), HIST2H2BE (includes others), Histone H1, Histone h3, L3MBTL1, MRPS18B, MYBL2, NFKBIL1, OSGIN1, PIM1, PPT2 (includes EG:100126133), RB1, TCF19, TFDP1, ZDHHC11 33 29 Tissue Morphology, Cellular Assembly and Organization, DNA Replication, Recombination, and Repair 10 20s proteasome, 26s Proteasome, AGER, Akt, ATXN1, BAG6, COPG2, GAS7, Hdac, HES4, HIVEP1, HSBP1, Hsp70, HSP, HSP90AB1, HSPA6, HSPA1A/HSPA1B, HSPA1L, IARS2, Immunoproteasome Pa28/20s, KLHL12, MYLK2, PACSIN1, POLH, PSMA7, PSMB1, PSMB8, PSMB9, PSMB, RIPK1, SLC17A1, STK4, TRIM38, WRAP73, ZHX3 32 27 Cellular Assembly and Organization, Cellular Compromise, Post- Translational Modification                  

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